Inducible gene expression system

Abstract

This disclosure relates to the fields of molecular biology and nucleic acid technology. This disclosure also relates to the treatment and prevention of diseases.

Description

[Technical Field]

[0001] This application claims priority to European Patent No. 23178358.0, filed on 9 June 2023, the contents and elements thereof being incorporated herein by reference for all purposes.

[0002] [Technical field] This disclosure relates to the fields of molecular biology and nucleic acid technology. This disclosure also relates to the treatment and prevention of diseases. [Background technology]

[0003] Gene therapy as a modality aims to improve disease-related phenotypes by utilizing both viral and nonviral delivery systems. Key efforts are focused on optimizing this therapy to be more efficient, non-immunogenic, and less toxic while enabling long-term expression of the target gene. One of the key challenges in achieving this objective is regulating the expression of the transgene to meet the required levels and to be expressed on demand.

[0004] For certain targets where constitutive and unrestricted expression leads to adverse events, regulated gene expression is necessary to make gene therapy a viable treatment modality. One example is Rett syndrome (RTT), an X-linked neurological disorder associated with severe motor impairment and shortened lifespan, occurring in 1 in 1,000 women. RTT is characterized by rapid regression following seemingly normal neurological and physical development in the early postnatal period, accompanied by intentional loss of motor skills and the onset of repetitive and autistic behaviors. 1,2In the rapidly progressing stage, children lose intentional hand movements and speech, experience motor impairments, develop respiratory problems, and may also develop autism-like characteristics and seizures. In later stages, further motor impairments begin, exemplified by severe physical disability, and many patients become dependent on wheelchairs. Current treatment options are limited to symptom control. Mutations in the MECP2 gene on the X chromosome, which encodes methyl-CpG binding protein 2, account for 95% of RTT cases. 3 MeCP2 is highly expressed in neurons and functions as a ubiquitous transcription regulator by binding to methylated DNA and recruiting protein partners and regulatory complexes to control transcriptional activity. MeCP2 modulates neuronal physiology and maintenance, and landmark studies have demonstrated that restoring MeCP2 protein levels dramatically reverses symptoms in mice. 4、5 .

[0005] Given that it is a monogenic disorder caused by a deficiency in MeCP2, a protein with multiple functions, gene therapy is one potential means of treating RTT. Studies in RTT mice have provided promising data showing that intravenous administration of adeno-associated virus serotype 9 (AAV9) expressing the wild-type (WT) MeCP2 gene reduces neurological dysfunction and extends lifespan. 5~8 Despite the promising potential of gene replacement therapy, high doses of MeCP2 via overexpression systems that rely on the use of ubiquitous promoters resulting in uncontrolled levels of transgenes can lead to phenotypic toxicity similar to that observed in duplication syndrome (a condition primarily affecting males, characterized by moderate to severe intellectual disability, and caused by duplication of the MeCP2 gene on the X chromosome). 9、10 The requirement to keep MeCP2 expression levels within a window for normal function remains one of the most challenging aspects to overcome. One solution would be to develop controlled transgene expression systems to overcome this limitation and make gene therapy a viable treatment option.

[0006] Inducible gene expression systems allow for reversibility and flexibility, enabling the manufacture of therapeutic drugs on demand and thereby avoiding overdose-related side effects. Such regulation can be achieved using small molecules that can induce (generate an ON switch) or suppress (an OFF switch) transgene expression. Of the existing inducible transcription gene regulation systems, the tetracycline (Tet) regulated system is the most widely used tool and can be used as either an ON or OFF switch. However, the Tet inducible system is bacterial-derived and requires the expression of regulatory proteins; for example, the ON switch component includes a tetracycline regulatory transactivator (tTA) consisting of bacterial tetR having the C-terminal domain of VP16 (virion protein 16) derived from herpes simplex virus. The required co-expression of these components further increases the size limitations of transgenes for AAV packaging and can cause both silencing and potential immunogenic effects when introduced into human tissue, hindering further clinical transition. 11、12 Another class of genetic switches derived from bacteria are riboswitches, which are RNA elements that can regulate gene expression in response to ligand binding, have a small genomic footprint, and are independent of other proteins in terms of activity. This makes these types of regulated gene expression an attractive alternative to protein-based expression regulatory systems. 13 However, riboswitches have a low dynamic range and high basal activity. 14 .

[0007] A novel class of small molecules that regulate SMN2 exon 7 splicing has recently been identified by screening using an SMN2 transcript-based reporter system (e.g., described in International Publication 2009 / 151546). The SMN2 reporter system has since been proposed to be used as a gene expression switch in which transgene expression is regulated by small molecule-controlled splicing of an expression cassette (Monteys et al. Nature (2021) 596:291-295). Known transgene expression systems derived from human SMN2 exons 6-8 are described, for example, in Zhang, et al., Gene Ther. (2001) 8:1532-1538, International Publication 2022 / 204471, Monteys et al. Nature (2021) 596:291-295, and International Publication 2021 / 163556.

[0008] However, these known SMN2-based switch systems have very long nucleotide sequences, limiting the size of the transgene that can be used under the system's control. That is, the size of the insert containing the coding sequence of the therapeutic polypeptide delivered as a switch and gene therapy is very often much larger than the packaging limits of vectors routinely used for gene therapy delivery, such as adeno-associated virus (AAV) vectors. [Overview of the Initiative]

[0009] In a first aspect, the disclosure provides a polynucleotide comprising the following from 5' to 3': (i) A first nucleotide sequence comprising a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in Sequence ID No. 222, but not comprising Sequence ID No. 2; (ii) A second nucleotide sequence comprising fewer than 1044 nucleotides, comprising a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 7 at its 5' end and a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in SEQ ID NO: 378 at its 3' end; (iii) A third nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in Sequence ID No. 226; (iv) A fourth nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in SEQ ID NO: 15 at its 5' end, and having at least 80% sequence identity with the nucleotide sequence described in SEQ ID NO: 16 at its 3' end; (v)(a) comprising a dinucleotide "GA", "TG", or "TT", or (b) comprising a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in SEQ ID NO: 19, wherein the nucleotide sequence contains "GA", "TG", or "TT" at positions corresponding to positions 1 and 2 of SEQ ID NO: 19, or (c) a fifth nucleotide sequence encoding the target polypeptide and containing "GA", "TG", or "TT" at positions 1 and 2; and (vi) If the fifth nucleotide sequence is the nucleotide sequence described in (v)(a) or (v)(b), then the sixth nucleotide sequence encoding the polypeptide of interest; Includes, The nucleotide sequence encoding the target polypeptide contains a start codon at the 5' end.

[0010] In a second aspect, the disclosure provides a polynucleotide comprising the following from 5' to 3': (i) A first nucleotide sequence comprising a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in SEQ ID NO: 1, but not comprising SEQ ID NO: 2; (ii) A second nucleotide sequence comprising fewer than 1044 nucleotides, comprising a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 7 at its 5' end and a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 8 at its 3' end; (iii) A third nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in Sequence ID No. 26, wherein the third nucleotide sequence contains "A" at the position corresponding to position 2 of Sequence ID No. 12; (iv) A fourth nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 15 at its 5' end and at least 80% sequence identity with SEQ ID NO: 16 at its 3' end; (v) a fifth nucleotide sequence consisting of (a) a trinucleotide "GAG" or (b) encoding the polypeptide of interest and containing "GAG" at positions 1-3; and (vi) If the fifth nucleotide sequence is the nucleotide sequence described in (v)(a), then the sixth nucleotide sequence encoding the target polypeptide. Includes, The nucleotide sequence encoding the target polypeptide contains a start codon at the 5' end.

[0011] In some embodiments of various aspects of this disclosure, when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion results in a polyribonucleotide substantially lacking a third nucleotide sequence.

[0012] In some embodiments, the first nucleotide sequence includes the nucleotide sequence described in SEQ ID NO: 222. In some embodiments, the first nucleotide sequence includes or consists of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 106, SEQ ID NO: 219, or SEQ ID NO: 220. In some embodiments, the first nucleotide sequence includes or consists of SEQ ID NO: 3.

[0013] In some embodiments, the second nucleotide sequence comprises SEQ ID NO: 7 at its 5' end and comprises SEQ ID NO: 8 or SEQ ID NO: 377 at its 3' end. In some embodiments, the second nucleotide sequence consists of less than 500 nucleotides. In some embodiments, the second nucleotide sequence comprises or consists of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 228. In some embodiments, the first nucleotide sequence comprises or consists of SEQ ID NO: 10.

[0014] In some embodiments, the third nucleotide sequence consists of the nucleotide sequence set forth in SEQ ID NO: 226. In some embodiments, the third nucleotide sequence consists of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 223, or SEQ ID NO: 224. In some embodiments, the third nucleotide sequence consists of the nucleotide sequence set forth in SEQ ID NO: 26. In some embodiments, the third nucleotide sequence consists of SEQ ID NO: 13 or SEQ ID NO: 27.

[0015] In some embodiments, the fourth nucleotide sequence comprises SEQ ID NO: 15 or SEQ ID NO: 379 at its 5' end and comprises SEQ ID NO: 16 at its 3' end. In some embodiments, the fourth nucleotide sequence consists of less than 500 nucleotides. In some embodiments, the fourth nucleotide sequence comprises or consists of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 227, or SEQ ID NO: 340. In some embodiments, the fourth nucleotide sequence comprises or consists of SEQ ID NO: 17.

[0016] In some embodiments, the fifth nucleotide sequence (a) consists of the dinucleotide "GA" or "TG", or (b) comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 19. In some embodiments, the fifth nucleotide sequence (a) consists of the dinucleotide "GA" or "TG", or (b) comprises or consists of SEQ ID NO: 20 or SEQ ID NO: 21.

[0017] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 111, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 164, and SEQ ID NO: 171. In some embodiments, the polynucleotide comprises at least 80% sequence identity with SEQ ID NO: 28 or SEQ ID NO: 29.

[0018] In some embodiments, the polynucleotide further comprises a promoter sequence 5' to the start codon.

[0019] In some embodiments, the polynucleotide further comprises a polyadenylation sequence 3' to the nucleotide sequence encoding the polypeptide of interest.

[0020] In some embodiments, the polynucleotide comprises an inverted terminal repeat (ITR) sequence at its 5' end and an ITR sequence at its 3' end.

[0021] The present disclosure also provides a vector comprising the polynucleotide according to the present disclosure. In some embodiments, the vector is an adeno-associated virus (AAV) vector.

[0022] The present disclosure also provides a pharmaceutical composition comprising the polynucleotide or vector according to the present disclosure and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0023] The present disclosure also provides a cell comprising the polynucleotide or vector according to the present disclosure. In some embodiments, the cell further comprises a splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

[0024] This disclosure also relates to a method for modifying cells to express a target polypeptide, (i) Introducing polynucleotides or vectors into cells according to the present disclosure, (ii) The present invention provides a method comprising contacting cells with a splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

[0025] This disclosure also provides a method for expressing a polypeptide of interest in cells, comprising contacting the cells according to this disclosure with a splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

[0026] The disclosure also provides a method for inhibiting the expression of a target polypeptide in cells, comprising contacting the cells according to the disclosure with a splicing modifier that promotes SMN2 exon 7 inclusion.

[0027] This disclosure also provides a method for modifying cells to express a polypeptide of interest, which includes introducing a polynucleotide or vector according to this disclosure into the cells.

[0028] This disclosure also provides polynucleotides, vectors, or pharmaceutical compositions according to this disclosure for use in methods of medical treatment or prevention.

[0029] The Disclosure also provides polynucleotides, vectors, or pharmaceutical compositions according to the Disclosure for use in treating or preventing diseases or conditions in which therapeutic or preventive benefits are obtained from increased expression levels of the polypeptide of interest.

[0030] This disclosure also provides the use of polynucleotides, vectors, or pharmaceutical compositions according to this disclosure in the manufacture of pharmaceuticals for treating or preventing diseases or conditions in which therapeutic or preventive benefits are obtained from increased expression levels of the polypeptide of interest.

[0031] The disclosure also provides methods for treating or preventing diseases or conditions in which therapeutic or preventive benefits are obtained from an increase in the expression level of a target polypeptide, including administering a polynucleotide, vector, or pharmaceutical composition according to the disclosure.

[0032] In some embodiments, treating or preventing a disease or symptom further includes administering a splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

[0033] In some embodiments, the disease or symptom is a disease or symptom characterized by a deficiency of the polypeptide of interest.

[0034] This disclosure also relates to a kit, (i) Polynucleotides, vectors or pharmaceutical compositions relating to this disclosure, (ii) A kit is provided which includes a splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplum. [Modes for carrying out the invention]

[0035] This disclosure relates to an inducible transgene expression system using small molecule splicing modifiers (SMSMs) based on the described mechanism of action of the Survival of Motor Neuron 2 (SMN2) splicing modifier risdiplam (and its variants).

[0036] The SMSM binding sites for SMN2 are two different sites within the SMN2 exon 7 pre-mRNA: the ESE2 region and the 5' splice donor site (5'ss), as previously described (Figure 2a). 15 The interaction between small molecules and mRNA-protein complexes is thought to be important for the high selectivity of compounds. This study suggested that both the ESE2 and 5'ss regions are required for the full activity of SMSM compounds in a dose-dependent manner. 15 Structural analysis using a compound (SMN-C5) from the same chemical class as risdiplam demonstrated that the drug selectively promotes the recognition of the weak 5' splice site of SMN2 exon 7 by U1 snRNP by stabilizing the unpaired adenine at the exon-intron junction within the RNA helix. 16 .

[0037] The present disclosure provides novel constructs derived from SMN2 exons 6 to 8 that provide SMSM-inducible regulation of transgene expression, which are suitable for use in chemically inducible regulation of gene therapy expression.

[0038] In particular, the present disclosure provides the following ON-switch constructs: (i) having a size that enables their application for SMSM-inducible expression of polypeptides delivered as gene therapy, (ii) minimizing unwanted expression of polypeptides in the absence of SMSM, and (iii) being highly responsive to SMSM and thus providing strong induction of polypeptide expression in the presence of SMSM, and (iv) minimizing / removing completely the number of foreign amino acids at the N-terminus of the expressed polypeptide. The present disclosure also provides the following OFF-switch constructs: (i) having a size that enables their application for SMSM-inducible expression of polypeptides delivered as gene therapy, (ii) maximizing the expression of polypeptides in the absence of SMSM, and (iii) being highly responsive to SMSM and thus minimizing the expression of polypeptides in the presence of SMSM, and (iv) minimizing / keeping to a minimum the number of foreign amino acids at the N-terminus of the expressed polypeptide.

[0039] Polynucleotides The aspects and embodiments of this disclosure relate to polynucleotides.

[0040] "Polynucleotide" refers to a polymer chain of multiple nucleotide monomers linked by monomer-to-monomer bonds, typically phosphodiester bonds (for example, in the case of polynucleotides formed from naturally occurring nucleotide monomers). Polynucleotides generally include oligonucleotides containing 50 nucleotides or less. Polynucleotides may be single-stranded or double-stranded (i.e., may include double helixes formed by hydrogen bonds between complementary nucleotides). Polynucleotides according to this disclosure may include, or may consist of, single-stranded DNA, double-stranded DNA, DNA which is a mixture of single-stranded and double-stranded regions, single-stranded RNA, double-stranded RNA, RNA which is a mixture of single-stranded and double-stranded regions, single-stranded molecules containing DNA and RNA, double-stranded molecules containing DNA and RNA, and molecules containing DNA and RNA having a mixture of single-stranded and double-stranded regions.

[0041] In some embodiments, the polynucleotide contains or consists of DNA. In some embodiments, the polynucleotide is a polydeoxyribonucleotide. In some embodiments, the polynucleotide contains or consists of RNA. In some embodiments, the polynucleotide is a polyribonucleotide.

[0042] In embodiments and representations where a polynucleotide in this disclosure is defined by reference to a given nucleotide sequence, and the given nucleotide sequence contains or consists of RNA and / or is a polyribonucleotide, it will be understood that the "T" for thymidine in such sequence is replaced with "U" for uracil.

[0043] This disclosure also envisions polynucleotides, for example, modified nucleotides in which the phosphonate and / or ribose and / or base of a deoxyribonucleotide or ribonucleotide are chemically altered. Nucleotide modifications envisioned in accordance with this disclosure include those described in Hu et al., Sig. Transduc. Tar. Ther. (2020) 5(101), which is incorporated herein by reference in its entirety.

[0044] Phosphonate modifications can be selected from phosphorothioates (e.g., Rp isomers, Sp isomers), phosphorodithioates, methylphosphonates, methoxypropylphosphonates, 5'-(E)-vinylphosphonates, 5'-methylphosphonates, (S)-5'-C-methyl phosphate-containing 5'-phosphorothioates, and peptide nucleic acid modifications. Ribose modifications can 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-allyl, 2'-deoxy, 2'-hydroxyl, 2'-arabino-fluoro, 2'-O-benzyl, 2'-O-methyl-4-pyridine, locked nucleic acids, (S)-cEt-BNA, tricyclo-DNA, PMO, unlocked nucleic acids, hexitol nucleic acids, and glycol nucleic acid modifications. Base modifications can 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'-phenylpyrrolocytosine, 2',4'-difluorotoluyl ribonucleoside, and 5'-nitroindole modifications.

[0045] In some embodiments, the modified nucleotides are 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'- The following may be selected: 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.

[0046] The nucleotide sequence of the polynucleotides disclosed herein The polynucleotides of this disclosure are defined herein by reference to their constituent nucleotide sequences.

[0047] It will be understood that the constituent nucleotide sequences of the polynucleotides provided herein are provided as partial sequences of the complete polynucleotide sequence.

[0048] In some aspects and embodiments, the constituent nucleotide sequences of the polynucleotide according to this disclosure are provided in a specific order within the polynucleotide sequence, for example, from 5' to 3'. For example, a first nucleotide sequence is provided 5' (i.e., upstream) of a second nucleotide sequence in relation to the polynucleotide sequence. Similarly, the second nucleotide sequence is 5' of a third nucleotide sequence, and so on.

[0049] The constituent nucleotide sequences of the polynucleotides of this disclosure are non-overlapping. In some embodiments, the constituent nucleotide sequences of a polynucleotide are provided in tandem with respect to the complete sequence of the polynucleotide. In some embodiments, the constituent nucleotide sequences of a polynucleotide are directly adjacent to each other (i.e., with respect to the complete sequence of the polynucleotide, the 5' nucleotide of another given nucleotide sequence follows immediately after the 3' nucleotide of a given nucleotide sequence). For example, in the polynucleotide of SEQ ID NO: 22, positions 1-45 form the first nucleotide sequence, positions 46-309 form the second nucleotide sequence, positions 310-364 form the third nucleotide sequence, positions 356-616 form the fourth nucleotide sequence, and positions 617 and 618 form the fifth nucleotide sequence.

[0050] First nucleotide sequence The first nucleotide sequence provided in this disclosure comprises or consists of a nucleotide sequence that is a variant of human SMN2 exon 6. A “variant” of a given reference nucleotide sequence comprises one or more differences compared to the reference nucleotide sequence. For example, a variant of a given reference nucleotide sequence may comprise one or more nucleotide insertions, deletions, or substitutions relative to the reference nucleotide sequence.

[0051] Therefore, in some embodiments, the first nucleotide sequence is not comprised of or does not include SEQ ID NO: 2. In some embodiments, the first nucleotide sequence comprises or consists of a nucleotide sequence having <100% sequence identity with SEQ ID NO: 2.

[0052] In some embodiments, the first nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in SEQ ID NO: 1. “The nucleotide sequence described in SEQ ID NO: 1” will be understood to refer to a nucleotide sequence that conforms to the consensus nucleotide sequence of SEQ ID NO: 1. Exemplary sequences that conform to the consensus nucleotide sequence of SEQ ID NO: 1 include SEQ ID NOs: 3, 4, 5, 6, and 106.

[0053] In some embodiments, the first nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 3 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%). In some embodiments, the first nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in SEQ ID NO: 4 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%). In some embodiments, the first nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity with sequence number 5 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%). In some embodiments, the first nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity with sequence number 6 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0054] In some embodiments, the first nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in SEQ ID NO: 1. Exemplary sequences that fit the consensus nucleotide sequence of SEQ ID NO: 1 include SEQ ID NOs: 219 and 220.

[0055] In some embodiments, the first nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in Sequence ID No. 222.

[0056] In some embodiments, the first nucleotide sequence includes "CAG" at positions 109-111 of SEQ ID NO: 2 (see, for example, positions 109-111 of SEQ ID NO: 6).

[0057] As described herein, the positions of nucleotide sequences that "correspond" to specified positions in a reference nucleotide sequence can be identified by sequence alignment of the target sequence to the reference sequence using sequence alignment software such as ClustalOmega (Soding, J.2005, Bioinformatics 21, 951-960). For example, positions 43-45 of SEQ ID NO: 3 will be understood to correspond to positions 109-111 of SEQ ID NO: 2. Similarly, positions 45-47 of SEQ ID NO: 4 will correspond to positions 109-111 of SEQ ID NO: 2.

[0058] In some embodiments, the first nucleotide sequence includes "CTG" at positions 109-111 of SEQ ID NO: 2 (see, for example, positions 109-111 of SEQ ID NO: 5).

[0059] In some embodiments, the first nucleotide sequence contains "GGC" at positions 64-66 of SEQ ID NO: 2 (see, for example, positions 64-66 of SEQ ID NO: 5 and 64-66 of SEQ ID NO: 6).

[0060] In some embodiments, the first nucleotide sequence contains "GGC" at positions 82-84 of SEQ ID NO: 2 (see, for example, positions 16-18 of SEQ ID NO: 3, positions 18-20 of SEQ ID NO: 4, and positions 82-84 of SEQ ID NO: 5).

[0061] In some embodiments, the first nucleotide sequence includes "GGC" at positions 64-66 of SEQ ID NO: 2 and "GGC" at positions 82-84 of SEQ ID NO: 2.

[0062] In some embodiments, the first nucleotide sequence includes "GTG" at positions 109-111 of SEQ ID NO: 2. In some embodiments, the first nucleotide sequence includes "TTG" at positions 109-111 of SEQ ID NO: 2. In some embodiments, the first nucleotide sequence includes "TAG" at positions 109-111 of SEQ ID NO: 2. In some embodiments, the first nucleotide sequence includes "CAG" at positions 39-41 of SEQ ID NO: 2. In some embodiments, the first nucleotide sequence includes "GGC" at positions 82-84 of SEQ ID NO: 2.

[0063] In some embodiments, the first nucleotide sequence consists of fewer than 111 nucleotides. In some embodiments, the first nucleotide sequence consists of <100 nucleotides, for example, one of <85 nucleotides, <80 nucleotides, <75 nucleotides, <70 nucleotides, <65 nucleotides, <60 nucleotides, <55 nucleotides, <50 nucleotides, or ≤45 nucleotides.

[0064] In some embodiments, the first nucleotide sequence consists of 45 nucleotides. In some embodiments, the first nucleotide sequence consists of 51 nucleotides. In some embodiments, the first nucleotide sequence consists of 81 nucleotides.

[0065] In some embodiments, the first nucleotide sequence includes or consists of: (i) a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in Sequence ID No. 1; (ii) containing "GGC" at positions corresponding to positions 82-84 of Sequence ID No. 2; and (iii) consisting of ≤45 nucleotides. Examples of such first nucleotide sequences include Sequence IDs No. 3, 4, and 106.

[0066] In some embodiments, the first nucleotide sequence includes or consists of: (i) a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in SEQ ID NO: 1; and (ii) containing "GGC" at positions 64-66 of SEQ ID NO: 2; and (iii) containing "GGC" at positions 82-84 of SEQ ID NO: 2; and (iv) containing "CTG" at positions 109-111 of SEQ ID NO: 2. An example of such a first nucleotide sequence is SEQ ID NO: 5.

[0067] In some embodiments, the first nucleotide sequence includes or consists of: (i) a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in SEQ ID NO: 1; and (ii) containing "GGC" at positions 64-66 of SEQ ID NO: 2; and (iii) containing "GGC" at positions 82-84 of SEQ ID NO: 2; and (iv) containing "CAG" at positions 109-111 of SEQ ID NO: 2. An example of such a first nucleotide sequence is SEQ ID NO: 6.

[0068] In some embodiments, the polynucleotides according to the present disclosure include, or comprise a nucleotide sequence (e.g., 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 with a nucleotide sequence selected from SEQ ID NOs. 3, 5, 6, 219, 220, and 230-237.

[0069] In some embodiments, the polynucleotides according to this disclosure do not contain the nucleotide sequence of SEQ ID NO: 2.

[0070] In some embodiments, the polynucleotides according to the disclosure include a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 3 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%), or a nucleotide sequence consisting thereof (e.g., a first nucleotide sequence). In some embodiments, the polynucleotides according to the disclosure include a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 5 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%), or a nucleotide sequence consisting thereof (e.g., a first nucleotide sequence). In some embodiments, the polynucleotides according to the disclosure include a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 6 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%), or a nucleotide sequence consisting thereof (e.g., a first nucleotide sequence). In some embodiments, the polynucleotides according to the disclosure include a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 219 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%), or a nucleotide sequence consisting thereof (e.g., a first nucleotide sequence). In some embodiments, the polynucleotides according to the present disclosure include a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 220 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%), or a nucleotide sequence consisting thereof (e.g., a first nucleotide sequence).

[0071] Second nucleotide sequence The second nucleotide sequence provided in this disclosure comprises or consists of a nucleotide sequence that is a variant of human SMN2 intron 6.

[0072] Therefore, in some embodiments, the second nucleotide sequence is not comprised of or does not include SEQ ID NO: 30. In some embodiments, the second nucleotide sequence comprises or consists of a nucleotide sequence having <100% sequence identity with SEQ ID NO: 30.

[0073] In some embodiments, the second nucleotide sequence includes a nucleotide sequence at its 5' end that has at least 80% sequence identity with sequence number 7 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0074] In some embodiments, the second nucleotide sequence includes 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 with the nucleotide sequence described in SEQ ID NO: 378 at its 3' end. It will be understood that "the nucleotide sequence described in SEQ ID NO: 378" refers to a nucleotide sequence that conforms to the consensus nucleotide sequence of SEQ ID NO: 378. Exemplary sequences that conform to the consensus nucleotide sequence of SEQ ID NO: 378 include SEQ ID NO: 8 and SEQ ID NO: 377.

[0075] In some embodiments, the second nucleotide sequence includes 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 with sequence number 8 at its 3' end. In some embodiments, the second nucleotide sequence includes 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 with sequence number 377 at its 3' end.

[0076] In some embodiments, the second nucleotide sequence includes a nucleotide sequence at its 5' end that has at least 80% sequence identity with sequence number 7 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%), and a nucleotide sequence at its 3' end that has at least 80% sequence identity with sequence number 8 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%). In some embodiments, the second nucleotide sequence includes a nucleotide sequence at its 5' end that has at least 80% sequence identity with sequence number 7 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%), and a nucleotide sequence at its 3' end that has at least 80% sequence identity with sequence number 377 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0077] As used herein, the "5'" end of a given nucleotide sequence refers to a region of the nucleotide sequence or a subsequence of such region formed by a nucleotide 5' (i.e., upstream in relation to the complete sequence of the given nucleotide sequence) relative to the midpoint of the given nucleotide sequence.

[0078] In a nucleotide sequence with an even number of nucleotides, the 5' region relative to the "midpoint" includes the nucleotide immediately preceding the midpoint. For example, in a nucleotide sequence consisting of 10 nucleotides, the 5' region relative to the midpoint consists of positions 1 through 5. Similarly, in a nucleotide sequence with an even number of nucleotides, the 3' region relative to the "midpoint" includes the nucleotide immediately following the midpoint. For example, in a nucleotide sequence consisting of 10 nucleotides, the 3' region relative to the midpoint consists of positions 6 through 10. In a nucleotide sequence with an odd number of nucleotides, the 5' region relative to the "midpoint" includes the nucleotide 5' (i.e., upstream) of the nucleotide supplied to the midpoint. For example, in a nucleotide sequence consisting of 9 nucleotides, the 5' region relative to the midpoint consists of positions 1 through 4. Similarly, in a nucleotide sequence with an odd number of nucleotides, the 3' region relative to the "midpoint" includes the nucleotide 3' (i.e., downstream) of the nucleotide supplied to the midpoint. For example, in a nucleotide sequence consisting of 9 nucleotides, the 3' region relative to the midpoint consists of positions 6 through 9.

[0079] As an example, in sequence number 9, which consists of 414 nucleotides, the sequence provided at the 5' end of sequence number 9 refers to the nucleotide sequence formed by positions 1 to 207 of sequence number 9, or a subsequence thereof.

[0080] Conversely, the "3'" end of a given nucleotide sequence refers to a region of the nucleotide sequence formed by nucleotides 3' (i.e., downstream in relation to the complete sequence of the given nucleotide sequence) relative to the midpoint of the given nucleotide sequence, or a subsequence of this region. For example, in SEQ ID NO: 9, the sequence provided at the 3' end of SEQ ID NO: 9 refers to the nucleotide sequence formed by positions 208 to 414 of SEQ ID NO: 9, or a subsequence thereof.

[0081] In some embodiments, a subsequence of a given nucleotide sequence provided at the 5' end of a 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 at 5' (i.e., upstream in relation to the complete sequence of the given nucleotide sequence) relative to the midpoint of the given nucleotide sequence. For example, the sequence provided at the 5' end of SEQ ID NO: 9 may include 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 207 of SEQ ID NO: 9.

[0082] In some embodiments, a subsequence of a given nucleotide sequence provided at the 3' end of a 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 at 3' (i.e., downstream in relation to the complete sequence of the given nucleotide sequence) relative to the midpoint of the given nucleotide sequence. For example, the sequence provided at the 3' end of SEQ ID NO: 9 may include 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 208-414 of SEQ ID NO: 9.

[0083] In some embodiments, the subsequence of a given nucleotide sequence provided at the 5' end of a given nucleotide sequence includes one or more nucleotides provided within 25 nucleotides of the 5' nucleotide of the given nucleotide sequence, for example, within one of 20, 15, 10, or 5 nucleotides. In some embodiments, the subsequence of a given nucleotide sequence provided at the 5' end of a given nucleotide sequence includes the 5' nucleotide of the given nucleotide sequence. For example, the sequence provided at the 5' end of SEQ ID NO: 9 may include one or more nucleotides provided at positions 1-25 of SEQ ID NO: 9, for example, positions 1-20, 1-15, 1-10, or 1-5. For example, the sequence provided at the 5' end of SEQ ID NO: 9 may include position 1 of SEQ ID NO: 9.

[0084] In some embodiments, the subsequence of a given nucleotide sequence provided at the 3' end of a given nucleotide sequence includes one or more nucleotides provided within 25 nucleotides of the 3' nucleotide of the given nucleotide sequence, for example, within one of 20, 15, 10, or 5 nucleotides. In some embodiments, the subsequence of a given nucleotide sequence provided at the 3' end of a given nucleotide sequence includes the 3' nucleotide of the given nucleotide sequence. For example, the sequence provided at the 3' end of SEQ ID NO: 9 may include one or more nucleotides provided at positions 389-414 of SEQ ID NO: 9, for example, positions 394-414, 399-414, 404-414, or 409-414. For example, the sequence provided at the 3' end of SEQ ID NO: 9 may include position 414 of SEQ ID NO: 9.

[0085] In some embodiments, the second nucleotide sequence includes a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 7 in a region 5' relative to its midpoint. In some embodiments, the second nucleotide sequence includes a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 8 or SEQ ID NO: 377 in a region 3' relative to its midpoint.

[0086] In some embodiments, a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 7 occupies at least 5% of the 5' region relative to the midpoint of the second nucleotide sequence, for example, 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%. In some embodiments, a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 8 or SEQ ID NO: 377 occupies at least 5% of the 3' region relative to the midpoint of the second nucleotide sequence, for example, 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%.

[0087] In some embodiments, a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 7 includes one or more nucleotides provided within 25 nucleotides of the 5' nucleotide of the second nucleotide sequence, for example, at positions 20, 15, 10, or 5 nucleotides. In some embodiments, a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 7 includes the 5' nucleotide of the second nucleotide sequence. In some embodiments, a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 8 or SEQ ID NO: 377 includes one or more nucleotides provided within 25 nucleotides of the 3' nucleotide of the second nucleotide sequence, for example, at positions 20, 15, 10, or 5 nucleotides. In some embodiments, a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 8 or SEQ ID NO: 377 includes the 3' nucleotide of the second nucleotide sequence.

[0088] An exemplary sequence that has a nucleotide sequence at its 5' end that is at least 80% sequence-identical to sequence number 7 is sequence number 9. Positions 1-102 of sequence number 9 correspond to positions 1-102 of sequence number 7.

[0089] An exemplary sequence that has a nucleotide sequence at its 3' end that is at least 80% sequence-identical to sequence number 8 is sequence number 9. Positions 253-414 of sequence number 9 correspond to positions 1-162 of sequence number 8.

[0090] In some embodiments, the second nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in SEQ ID NO: 229. Exemplary sequences that fit the consensus nucleotide sequence of SEQ ID NO: 229 include SEQ ID NOs: 10 and 228.

[0091] In some embodiments, the second nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity with sequence number 9 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%). In some embodiments, the second nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity with sequence number 10 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0092] In some embodiments, the second nucleotide sequence includes a "C" at the position corresponding to position 5766 of SEQ ID NO: 30 (see, for example, position 411 of SEQ ID NO: 9 and position 261 of SEQ ID NO: 10).

[0093] In some embodiments, the second nucleotide sequence consists of fewer than 1044 nucleotides, for example, one of <1000 nucleotides, <900 nucleotides, <800 nucleotides, <750 nucleotides, <700 nucleotides, <650 nucleotides, <600 nucleotides, <550 nucleotides, or <500 nucleotides.

[0094] In some embodiments, the second nucleotide sequence consists of fewer than 500 nucleotides, for example, one of <450 nucleotides, <400 nucleotides, <350 nucleotides, <300 nucleotides, <250 nucleotides, or <200 nucleotides. In some embodiments, the second nucleotide sequence consists of 1044 nucleotides. In some embodiments, the second nucleotide sequence consists of 414 nucleotides. In some embodiments, the second nucleotide sequence consists of 264 nucleotides. In some embodiments, the second nucleotide sequence consists of 189 nucleotides.

[0095] In some embodiments, the second nucleotide sequence includes: (i) 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 with SEQ ID NO: 7 at its 5' end; (ii) 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 with SEQ ID NO: 8 at its 3' end; (iii) containing a "C" at the position corresponding to position 5766 of SEQ ID NO: 30; and (iv) consisting of fewer than 1044 nucleotides. Examples of such a second nucleotide sequence include SEQ ID NOs: 9 and 10.

[0096] In some embodiments, the second nucleotide sequence includes: (i) 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 with SEQ ID NO: 7 at its 5' end; (ii) 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 with SEQ ID NO: 8 at its 3' end; (iii) containing a "C" at the position corresponding to position 5766 of SEQ ID NO: 30; and (iv) consisting of fewer than 500 nucleotides. An example of such a second nucleotide sequence is SEQ ID NO: 9.

[0097] In some embodiments, the second nucleotide sequence includes: (i) 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 with SEQ ID NO: 7 at its 5' end; (ii) 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 with SEQ ID NO: 8 at its 3' end; (iii) containing a "C" at the position corresponding to position 5766 of SEQ ID NO: 30; and (iv) consisting of fewer than 300 nucleotides. An example of such a second nucleotide sequence is SEQ ID NO: 10.

[0098] In some embodiments, the polynucleotides according to the present disclosure include 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 with a nucleotide sequence selected from SEQ ID NOs. 9, 10, 228, 238-255, 375, and 376, or a nucleotide sequence consisting of such a sequence (e.g., a second nucleotide sequence).

[0099] In some embodiments, the polynucleotides of the present disclosure do not include the nucleotide sequence of SEQ ID NO: 240. In some embodiments, the polynucleotides of the present disclosure include 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 with a nucleotide sequence selected from SEQ ID NOs: 9, 10, 228, 238, 239, 241-255, 375, and 376, or a nucleotide sequence consisting of such a sequence (e.g., a second nucleotide sequence).

[0100] In some embodiments, the polynucleotide according to the disclosure includes a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 9 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%), or includes a nucleotide sequence consisting thereof (e.g., a second nucleotide sequence). In some embodiments, the polynucleotide according to the disclosure includes a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 10 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%), or includes a nucleotide sequence consisting thereof (e.g., a second nucleotide sequence). In some embodiments, the polynucleotides according to the present disclosure include a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 228 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%), or a nucleotide sequence consisting thereof (e.g., a second nucleotide sequence).

[0101] Third nucleotide sequence The third nucleotide sequence provided in this disclosure comprises or consists of a nucleotide sequence that is a variant of human SMN2 exon 7.

[0102] Therefore, in some embodiments, the third nucleotide sequence is not comprised of or does not include SEQ ID NO: 12. In some embodiments, the third nucleotide sequence comprises or consists of a nucleotide sequence having <100% sequence identity with SEQ ID NO: 12.

[0103] In some embodiments, the third nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in SEQ ID NO: 11. It will be understood that "the nucleotide sequence described in SEQ ID NO: 11" refers to a nucleotide sequence that conforms to the consensus nucleotide sequence of SEQ ID NO: 11. Exemplary sequences that conform to the consensus nucleotide sequence of SEQ ID NO: 11 include SEQ ID NOs: 13 and 14.

[0104] In some embodiments, the third nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in SEQ ID NO: 26. “The nucleotide sequence described in SEQ ID NO: 26” will be understood to refer to a nucleotide sequence that conforms to the consensus nucleotide sequence of SEQ ID NO: 26. Exemplary sequences that conform to the consensus nucleotide sequence of SEQ ID NO: 26 include SEQ ID NOs: 13 and 27.

[0105] In some embodiments, the third nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in SEQ ID NO: 225. Exemplary sequences that fit the consensus nucleotide sequence of SEQ ID NO: 225 include SEQ ID NOs: 223 and 224.

[0106] In some embodiments, the third nucleotide sequence includes or comprises a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in Sequence ID No. 226.

[0107] In some embodiments, the third nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity with sequence number 13 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%). In some embodiments, the third nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity with sequence number 14 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%). In some embodiments, the third nucleotide sequence includes or consists of a nucleotide sequence having sequence identity with sequence number 27 of at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0108] In some embodiments, the third nucleotide sequence includes "A" at the position corresponding to position 2 of SEQ ID NO: 12 (see, for example, position 2 of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 26, and SEQ ID NO: 27).

[0109] In some embodiments, the third nucleotide sequence includes the insertion "GCCACC" after the position corresponding to position 6 of SEQ ID NO: 12 (see, for example, positions 7-12 of SEQ ID NO: 14).

[0110] In some embodiments, the third nucleotide sequence includes "TG" at positions 8-9 of SEQ ID NO: 12 (see, for example, positions 14 and 15 of SEQ ID NO: 14).

[0111] In some embodiments, the third nucleotide sequence includes an insertion of "A" after the position corresponding to position 48 in SEQ ID NO: 12 (see, for example, position 49 in SEQ ID NO: 13 and position 55 in SEQ ID NO: 14).

[0112] In some embodiments, the third nucleotide sequence includes "G" at the position corresponding to position 24 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes a nucleotide deletion at the position corresponding to position 20 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "T" at the position corresponding to position 27 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "T" at the position corresponding to position 28 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "G" at the position corresponding to position 21 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "A" at the position corresponding to position 29 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "T" at the position corresponding to position 21 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "A" at the position corresponding to position 31 of SEQ ID NO: 12 and "A" at the position corresponding to position 34 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes deletions at the positions corresponding to positions 9 through 20 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes a deletion at positions 33-41 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "CAC" at positions 34-36 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes an insertion of "G" after the position corresponding to position 39 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "GC" at positions 47-48 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes an insertion of "CACCATG" after the position corresponding to position 48 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "C" at the position corresponding to position 48 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes an insertion of "AA" after the position corresponding to position 48 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "CAT" at positions 49-51 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "GCC" at positions 46-48 of SEQ ID NO: 12.

[0113] In some embodiments, the third nucleotide sequence includes "CCA" at positions 49-51 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes an insertion of "T" after the position corresponding to position 51 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "GC" at positions 39-40 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes an insertion of "CA" after the position corresponding to position 40 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "ATG" at positions 43-45 of SEQ ID NO: 12 and a deletion at the position corresponding to position 46 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes an insertion of "GCCACCATG" after the position corresponding to position 9 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes an insertion of "AGCACCATG" after the position corresponding to position 15 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "G" at the position corresponding to position 16 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes the insertion of "CACCATG" after the position corresponding to position 15 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes the insertion of "ATG" after the position corresponding to position 15 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes the insertion of "ATG" after the position corresponding to position 21 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes the insertion of "GG" after the position corresponding to position 48 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes the insertion of "AT" after the position corresponding to position 30 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "G" at the position corresponding to position 31 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes the insertion of "G" after the position corresponding to position 33 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes the insertion of "A" after the position corresponding to position 43 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "GG" at positions corresponding to positions 45 and 46 of SEQ ID NO: 12.In some embodiments, the third nucleotide sequence includes an insertion of "AT" after the position corresponding to position 52 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes deletions at positions 49 and 50 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "G" at the position corresponding to position 51 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "G" at the position corresponding to position 40 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "A" at the position corresponding to position 35 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes an insertion of "A" after the position corresponding to position 39 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "G" at the position corresponding to position 41 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "C" at the position corresponding to position 43 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes an insertion of "ATG" after the position corresponding to position 42 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes an insertion of "TG" after the position corresponding to position 45 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes an insertion of "ATGGA" after the position corresponding to position 44 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "A" at the position corresponding to position 44 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "G" at the position corresponding to position 49 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes a deletion at the position corresponding to position 50 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "A" at the position corresponding to position 48 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "G" at the position corresponding to position 50 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes "G" at the position corresponding to position 44 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes a deletion at the position corresponding to position 49 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes a deletion at positions 42-44 of sequence number 12.In some embodiments, the third nucleotide sequence includes an insertion of "G" after the position corresponding to position 39 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes a deletion at the position corresponding to position 48 of SEQ ID NO: 12. In some embodiments, the third nucleotide sequence includes an insertion of "GA" after the position corresponding to position 48 of SEQ ID NO: 12.

[0114] In some embodiments, the third nucleotide sequence includes or consists of: (i) a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in SEQ ID NO: 11; and (ii) including an "A" at the position corresponding to position 2 of SEQ ID NO: 12; and (iii) including an insertion of an "A" after the position corresponding to position 48 of SEQ ID NO: 12. An example of such a third nucleotide sequence is SEQ ID NO: 13.

[0115] In some embodiments, the third nucleotide sequence includes or consists of: (i) a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in SEQ ID NO: 11; and (ii) including "A" at the position corresponding to position 2 of SEQ ID NO: 12; and (iii) including an insertion of "A" after the position corresponding to position 48 of SEQ ID NO: 12; and (iv) including an insertion of "GCCACC" after the position corresponding to position 6 of SEQ ID NO: 12; and (v) including "TG" at the positions corresponding to positions 8 and 9 of SEQ ID NO: 12. An example of such a third nucleotide sequence is SEQ ID NO: 14.

[0116] In some embodiments, the third nucleotide sequence includes or consists of: (i) a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in SEQ ID NO: 26; and (ii) including an "A" at the position corresponding to position 2 of SEQ ID NO: 12; and (iii) including a deletion at the position corresponding to position 20 of SEQ ID NO: 12; and (iv) including an insertion of an "A" after the position corresponding to position 48 of SEQ ID NO: 12. An example of such a third nucleotide sequence is SEQ ID NO: 27.

[0117] In some embodiments, the polynucleotides according to the present disclosure include, or comprise, 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 with a nucleotide sequence selected from SEQ ID NOs. 13, 14, 27, 223, 224, and 256-329, or a nucleotide sequence consisting of such a sequence (e.g., a third nucleotide sequence).

[0118] In some embodiments, the polynucleotides of the present disclosure do not include the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the polynucleotides of the present disclosure include 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 with a nucleotide sequence selected from SEQ ID NOs: 14, 27, 223, 224, and 256-329, or a nucleotide sequence consisting of such a sequence (e.g., a third nucleotide sequence).

[0119] In some embodiments, the polynucleotides according to the disclosure include a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 13 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%), or a nucleotide sequence consisting thereof (e.g., a third nucleotide sequence). In some embodiments, the polynucleotides according to the disclosure include a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 14 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%), or a nucleotide sequence consisting thereof (e.g., a third nucleotide sequence). In some embodiments, the polynucleotides according to the disclosure include a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 27 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%), or a nucleotide sequence consisting thereof (e.g., a third nucleotide sequence). In some embodiments, the polynucleotides according to the disclosure include a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 223 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%), or a nucleotide sequence consisting thereof (e.g., a third nucleotide sequence). In some embodiments, the polynucleotides according to the present disclosure include a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 224 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%), or a nucleotide sequence consisting thereof (e.g., a third nucleotide sequence).

[0120] Fourth nucleotide sequence The fourth nucleotide sequence provided in this disclosure includes, or consists of, a nucleotide sequence that is a variant of human SMN2 intron 7.

[0121] Therefore, in some embodiments, the fourth nucleotide sequence is not comprised of or does not include SEQ ID NO: 18. In some embodiments, the fourth nucleotide sequence comprises or consists of a nucleotide sequence having <100% sequence identity with SEQ ID NO: 18.

[0122] In some embodiments, the fourth nucleotide sequence includes 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 with the nucleotide sequence described in SEQ ID NO: 380 at its 5' end. It will be understood that "the nucleotide sequence described in SEQ ID NO: 380" refers to a nucleotide sequence that conforms to the consensus nucleotide sequence of SEQ ID NO: 380. Exemplary sequences that conform to the consensus nucleotide sequence of SEQ ID NO: 380 include SEQ ID NO: 15 and SEQ ID NO: 379.

[0123] In some embodiments, the fourth nucleotide sequence includes 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 with sequence number 15 at its 5' end. In some embodiments, the fourth nucleotide sequence includes 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 with sequence number 379 at its 5' end. In some embodiments, the fourth nucleotide sequence includes a nucleotide sequence at its 3' end that has at least 80% sequence identity with sequence number 16 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%). In some embodiments, the fourth nucleotide sequence includes a nucleotide sequence at its 5' end that has at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) sequence identity with sequence number 15, and includes a nucleotide sequence at its 3' end that has at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) sequence identity with sequence number 16. In some embodiments, the fourth nucleotide sequence includes a nucleotide sequence at its 5' end that has at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) sequence identity with sequence number 379, and includes a nucleotide sequence at its 3' end that has at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) sequence identity with sequence number 16.

[0124] In some embodiments, the fourth nucleotide sequence includes a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 15 or SEQ ID NO: 379 in the 5' region relative to its midpoint. In some embodiments, the fourth nucleotide sequence includes a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 16 in the 3' region relative to its midpoint.

[0125] In some embodiments, a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 15 or SEQ ID NO: 379 occupies at least 5% of the 5' region relative to the midpoint of the fourth nucleotide sequence, for example, 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%. In some embodiments, a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 16 occupies at least 5% of the 3' region relative to the midpoint of the fourth nucleotide sequence, for example, 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%.

[0126] In some embodiments, a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 15 or SEQ ID NO: 379 includes one or more nucleotides provided within 25 nucleotides of the 5' nucleotide of the fourth nucleotide sequence, for example, at the position of 20, 15, 10, or 5 nucleotides. In some embodiments, a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 15 or SEQ ID NO: 379 includes the 5' nucleotide of the fourth nucleotide sequence. In some embodiments, a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 16 includes one or more nucleotides provided within 25 nucleotides of the 3' nucleotide of the fourth nucleotide sequence, for example, at the position of 20, 15, 10, or 5 nucleotides. In some embodiments, a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 16 includes the 3' nucleotide of the fourth nucleotide sequence.

[0127] An exemplary sequence that has a nucleotide sequence at its 5' end that is at least 80% sequence-identical to sequence number 15 is sequence number 17. Positions 1-102 of sequence number 17 correspond to positions 1-102 of sequence number 15.

[0128] An exemplary sequence that has a nucleotide sequence at its 3' end that is at least 80% sequence-identical to sequence number 16 is sequence number 18. Positions 295-444 of sequence number 18 correspond to positions 1-150 of sequence number 16.

[0129] In some embodiments, the fourth nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity with sequence number 16 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%). In some embodiments, the fourth nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity with sequence number 17 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%). In some embodiments, the fourth nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity with sequence number 18 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0130] In some embodiments, the fourth nucleotide sequence consists of fewer than 444 nucleotides, for example, one of <425 nucleotides, <400 nucleotides, <350 nucleotides, <300 nucleotides, <250 nucleotides, or <200 nucleotides. In some embodiments, the fourth nucleotide sequence consists of fewer than 255 nucleotides. In some embodiments, the fourth nucleotide sequence consists of 252 nucleotides. In some embodiments, the fourth nucleotide sequence consists of 177 nucleotides.

[0131] In some embodiments, the fourth nucleotide sequence consists of more than 444 nucleotides, for example, >450 nucleotides, >475 nucleotides, or >500 nucleotides. In some embodiments, the fourth nucleotide sequence consists of 508 nucleotides.

[0132] In some embodiments, the fourth nucleotide sequence includes "C" at the position corresponding to position 441 of SEQ ID NO: 18. In some embodiments, the fourth nucleotide sequence includes "T" at the position corresponding to position 441 of SEQ ID NO: 18. In some embodiments, the fourth nucleotide sequence includes "TT" at the positions corresponding to positions 436 and 437 of SEQ ID NO: 18. In some embodiments, the fourth nucleotide sequence includes "TCCTC" at the positions corresponding to positions 11-15 of SEQ ID NO: 18. In some embodiments, the fourth nucleotide sequence includes an insertion of "TTT" after the position corresponding to position 10 of SEQ ID NO: 18. In some embodiments, the fourth nucleotide sequence includes an insertion of "CCC" after the position corresponding to position 10 of SEQ ID NO: 18.

[0133] In some embodiments, the fourth nucleotide sequence includes: (i) 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 with SEQ ID NO: 15 at its 5' end; (ii) 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 with SEQ ID NO: 16 at its 3' end; and (iii) fewer than 500 nucleotides. Examples of such a fourth nucleotide sequence include SEQ ID NOs: 17 and 18.

[0134] In some embodiments, the fourth nucleotide sequence includes: (i) 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 with SEQ ID NO: 15 at its 5' end; (ii) 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 with SEQ ID NO: 16 at its 3' end; and (iii) consisting of fewer than 255 nucleotides. An example of such a fourth nucleotide sequence is SEQ ID NO: 17.

[0135] In some embodiments, the polynucleotides according to the present disclosure include, or include, 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 with a nucleotide sequence selected from SEQ ID NOs. 18, 227, 330-370, and 381, or a nucleotide sequence consisting of such a sequence (e.g., a fourth nucleotide sequence).

[0136] In some embodiments, the polynucleotides of the present disclosure do not include the nucleotide sequence of SEQ ID NO: 337. In some embodiments, the polynucleotides of the present disclosure include 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 with a nucleotide sequence selected from SEQ ID NOs: 18, 227, 330-336, 338-370, and 381, or a nucleotide sequence consisting of such a sequence (e.g., a third nucleotide sequence).

[0137] In some embodiments, the polynucleotides according to the disclosure include a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 18 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%), or a nucleotide sequence consisting thereof (e.g., a fourth nucleotide sequence). In some embodiments, the polynucleotides according to the disclosure include a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 227 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%), or a nucleotide sequence consisting thereof (e.g., a fourth nucleotide sequence).

[0138] Fifth nucleotide sequence The fifth nucleotide sequence provided in this disclosure comprises or consists of a nucleotide sequence that is a variant of human SMN2 exon 8.

[0139] Therefore, in some embodiments, the fifth nucleotide sequence is not comprised of or does not include SEQ ID NO: 32. In some embodiments, the fifth nucleotide sequence comprises or includes a nucleotide sequence having <100% sequence identity with SEQ ID NO: 32.

[0140] In some embodiments, the fifth nucleotide sequence consists of the dinucleotide "GA", "TG", or "TT". In some embodiments, the fifth nucleotide sequence consists of the dinucleotide "GA". In some embodiments, the fifth nucleotide sequence consists of the dinucleotide "TG". In some embodiments, the fifth nucleotide sequence consists of the dinucleotide "TT".

[0141] In some embodiments, the fifth nucleotide sequence consists of a trinucleotide "GAG".

[0142] In some embodiments, the fifth nucleotide sequence consists of nucleotide "A".

[0143] In some embodiments, the fifth nucleotide sequence codes for the target polypeptide (for example, as described below). In some embodiments, the fifth nucleotide sequence codes for the target polypeptide and includes "GA", "TG", or "TT" at positions 1 and 2. In some embodiments, the fifth nucleotide sequence codes for the target polypeptide and includes "GA" at positions 1 and 2. In some embodiments, the fifth nucleotide sequence codes for the target polypeptide and includes "TG" at positions 1 and 2. In some embodiments, the fifth nucleotide sequence codes for the target polypeptide and includes "TT" at positions 1 and 2.

[0144] In some embodiments, the fifth nucleotide sequence encodes the polypeptide of interest and includes "GAG" at positions 1-3.

[0145] In some embodiments, the fifth nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in SEQ ID NO: 19. “The nucleotide sequence described in SEQ ID NO: 19” will be understood to refer to a nucleotide sequence that conforms to the consensus nucleotide sequence of SEQ ID NO: 19. Exemplary sequences that conform to the consensus nucleotide sequence of SEQ ID NO: 19 include SEQ ID NOs: 20 and 21.

[0146] In some embodiments, the fifth nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity with sequence number 20 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%). In some embodiments, the fifth nucleotide sequence includes or consists of a nucleotide sequence having at least 80% sequence identity with sequence number 21 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0147] In some embodiments, the fifth nucleotide sequence includes "GA" at positions corresponding to positions 1 and 2 of SEQ ID NO: 32 (see, for example, positions 1 and 2 of SEQ ID NO: 20).

[0148] In some embodiments, the fifth nucleotide sequence includes "TG" at positions corresponding to positions 1 and 2 of SEQ ID NO: 32 (see, for example, positions 1 and 2 of SEQ ID NO: 21).

[0149] In some embodiments, the fifth nucleotide sequence contains fewer than 577 nucleotides. In some embodiments, the fifth nucleotide sequence consists of <500 nucleotides, for example, one of <400 nucleotides, <300 nucleotides, <200 nucleotides, <100 nucleotides, or <50 nucleotides.

[0150] In some embodiments, the fifth nucleotide sequence includes fewer than 25 nucleotides, for example, one of <20 nucleotides, <15 nucleotides, <10 nucleotides, or <5 nucleotides.

[0151] In some embodiments, the fifth nucleotide sequence contains 23 nucleotides. In some embodiments, the fifth nucleotide sequence contains 2 nucleotides.

[0152] In some embodiments, the fifth nucleotide sequence contains 11 nucleotides. In some embodiments, the fifth nucleotide sequence contains 8 nucleotides. In some embodiments, the fifth nucleotide sequence contains 5 nucleotides.

[0153] In some embodiments, the fifth nucleotide sequence includes or consists of a nucleotide sequence having at least 80% (e.g., one of ≥85%, ≥90%, ≥95%, or 100%) sequence identity with positions 1 to 23 of sequence number 32.

[0154] In some embodiments, the fifth nucleotide sequence includes an insertion of "G" after the position corresponding to position 2 of SEQ ID NO: 32.

[0155] In some embodiments, the fifth nucleotide sequence includes or consists of a nucleotide sequence having at least 80% (e.g., ≥90% or 100%) sequence identity with positions 1 to 11 of SEQ ID NO: 32. In some embodiments, the fifth nucleotide sequence includes or consists of a nucleotide sequence having at least 80% (e.g., 100%) sequence identity with positions 1 to 8 of SEQ ID NO: 32. In some embodiments, the fifth nucleotide sequence includes or consists of a nucleotide sequence having at least 80% (e.g., 100%) sequence identity with positions 1 to 5 of SEQ ID NO: 32. In some embodiments, the fifth nucleotide sequence includes or consists of positions 1 and 2 of SEQ ID NO: 32.

[0156] In some embodiments, the fifth nucleotide sequence includes "A" at the position corresponding to position 1 of SEQ ID NO: 32. In some embodiments, the fifth nucleotide sequence includes an insertion of "T" after the position corresponding to position 1 of SEQ ID NO: 32. In some embodiments, the fifth nucleotide sequence includes "G" at the position corresponding to position 3 of SEQ ID NO: 32.

[0157] In some embodiments, the fifth nucleotide sequence includes or consists of: (i) a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 19 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); and (ii) containing "GA" at positions corresponding to positions 1 and 2 of SEQ ID NO: 32. An example of such a fifth nucleotide sequence is SEQ ID NO: 20.

[0158] In some embodiments, the fifth nucleotide sequence includes or consists of: (i) a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 19 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); and (ii) containing "TG" at positions corresponding to positions 1 and 2 of SEQ ID NO: 32. An example of such a fifth nucleotide sequence is SEQ ID NO: 21.

[0159] In some embodiments, the polynucleotides according to this disclosure include, or comprise 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 with a nucleotide sequence selected from sequence numbers 20, 21, and 371-374, or a nucleotide sequence consisting thereof (e.g., a fifth nucleotide sequence).

[0160] In some embodiments, the polynucleotides of the present disclosure do not include the nucleotide sequence of SEQ ID NO: 20. In some embodiments, the polynucleotides of the present disclosure include 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 with a nucleotide sequence selected from SEQ ID NOs: 21 and 371-374, or a nucleotide sequence consisting of such a nucleotide sequence (e.g., a fifth nucleotide sequence).

[0161] In some embodiments, the polynucleotides according to the Disclosure include a dinucleotide "TG" or a nucleotide sequence consisting thereof (e.g., a fifth nucleotide sequence). In some embodiments, the polynucleotides according to the Disclosure include a dinucleotide "GA" or a nucleotide sequence consisting thereof (e.g., a fifth nucleotide sequence). In some embodiments, the polynucleotides according to the Disclosure include a dinucleotide "TT" or a nucleotide sequence consisting thereof (e.g., a fifth nucleotide sequence). In some embodiments, the polynucleotides according to the Disclosure include a trinucleotide "GAG" or a nucleotide sequence consisting thereof (e.g., a fifth nucleotide sequence). In some embodiments, the polynucleotides according to the disclosure include a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 20 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%), or a nucleotide sequence consisting thereof (e.g., a fifth nucleotide sequence). In some embodiments, the polynucleotides according to the disclosure include a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 21 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%), or a nucleotide sequence consisting thereof (e.g., a fifth nucleotide sequence).

[0162] Nucleotide sequence encoding the target polypeptide In some embodiments, the fifth nucleotide sequence according to the Disclosure comprises or consists of a nucleotide sequence encoding the polypeptide of interest. If present, the sixth nucleotide sequence according to the Disclosure comprises or consists of a nucleotide sequence encoding the polypeptide of interest.

[0163] The target polypeptide can be any polypeptide.

[0164] In some embodiments, the polypeptides of interest according to this disclosure may be antigen-binding polypeptides, aptamers, antigen-binding polypeptide complexes, antibodies or their antigen-binding fragments or derivatives, Fc fusion proteins, anticoagulant factors, blood factors, bone morphogenetic proteins, ligand decoy receptors, receptor decoy ligands, enzymes, growth factors, hormones, interferons, interleukins, thrombolytic substances, transcription factors, epigenetic modifiers, constituent proteins of site-directed nuclease nucleic acid editing systems (e.g., CRISPR / Cas9 system, CRISPR / Cpf1 system, CRISPR / C2c1 system, CRISPR / C2c2 system, CRISPR / C2c3 system, ZFN system, or TALEN system), constituent proteins of ribonucleoproteins, or viral proteins (e.g., capsid proteins or viral enzymes).

[0165] In some embodiments, the polypeptide of interest according to this disclosure may be an antigen-binding polypeptide or an antigen-binding polypeptide complex. In some embodiments, the polypeptide of interest may be a chimeric antigen receptor (CAR).

[0166] In some embodiments, the target polypeptide is a polypeptide suitable for use in the treatment or prevention of a disease / symptom. In some embodiments, the target polypeptide is a detectable polypeptide or a polypeptide having detectable activity.

[0167] A polypeptide suitable for use in the treatment or prevention of a disease / symptom may be any polypeptide whose administration is useful in treating or preventing the disease / symptom. In some embodiments, a polypeptide suitable for use in the treatment or prevention of a disease / symptom may be a polypeptide whose deficiency is positively associated with the disease or symptom, or which is involved in the pathogenesis of the disease or symptom. For example, in some embodiments, the polypeptide of interest may be MeCP2. MeCP2 deficiency is associated with Rett syndrome.

[0168] In some embodiments, polypeptides suitable for use in the treatment or prevention of a disease / symptom may be polypeptides whose expression or activity is positively related to the disease or symptom, or which inhibit the expression and / or activity of factors involved in the pathogenesis of the disease or symptom.

[0169] Detectable polypeptides may be or may include fluorescent polypeptides. Fluorescent polypeptides include green fluorescent protein and its variants (e.g., enhanced green fluorescent protein), yellow fluorescent protein (e.g., citrin), red fluorescent protein and its variants (e.g., mOrange, mCherry), blue fluorescent protein and its variants (e.g., TagBFP), cyan fluorescent protein and its variants (e.g., mTurquoise, cerulean), allophycocyanin, phycocyanin, phycoerythrin, and phycoerythrocyanin.

[0170] Detectable polypeptides may be epitope tags or may contain epitope tags. Examples of epitope tags include His (e.g., 6XHis), FLAG, c-Myc, StrepTag, hemagglutinin, 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).

[0171] A polypeptide with detectable activity may be or may contain an enzyme moiety. Enzyme moieties include, for example, luciferase, glucose oxidase, galactosidase (e.g., beta-galactosidase), glucolinidase, phosphatase (e.g., alkaline phosphatase), peroxidase (e.g., horseradish peroxidase), and cholinesterase.

[0172] The target polypeptide expressed from the polynucleotides of this disclosure may further include one or more foreign amino acids added to the N-terminus of the polypeptide, i.e., immediately upstream of the amino acid sequence of the target polypeptide. Such foreign amino acids may be characterized by forming an N-terminal tag on the target polypeptide. It may be desirable to minimize or completely remove the size of such foreign amino acids / N-terminal tag on the target polypeptide.

[0173] In some embodiments, the N-terminal tag consists of one of fewer than 50 amino acids, for example, ≤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 target polypeptide lacks an N-terminal tag.

[0174] In some embodiments, the polypeptides of this disclosure include 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 those skilled in the art of molecular biology and can be used. In some embodiments, the cleavage sequence includes an autocleavage site. Autocleavage sites include a 2A cleavage sequence derived from the picornavirus "NPGP" that is cleaved at "G / P". Further autocleavage sites are described, for example, in Kim et al., PLoS ONE (2011) 6:e18556 (which is incorporated herein by reference in its entirety), and include, for example, the T2A, E2A, P2A, and F2A cleavage sites. The amino acid sequences of the T2A, E2A, P2A, and F2A cleavage sites are shown in SEQ ID NOs. 107, 108, 109, and 110, respectively.

[0175] A cleavage site may be included in the polypeptide according to this disclosure to provide the removal of a foreign amino acid attached to 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 in this specification for the removal of an N-terminal tag as described above. Accordingly, in some embodiments, the polypeptide according to this disclosure includes a cleavage site adjacent to a foreign amino acid, for example, a foreign amino acid forming an N-terminal tag (i.e., immediately downstream in the amino acid sequence of the polypeptide).

[0176] In some embodiments, the cleavage site according to the Disclosure is a 2A cleavage site, selected from, for example, T2A, E2A, P2A, and F2A cleavage sites. In some embodiments, the cleavage site is a T2A cleavage site. In some embodiments, the polypeptide according to the Disclosure comprises an amino acid sequence having at least 60%, preferably ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity with SEQ ID NOs: 107, 108, 109, or 110.

[0177] In some embodiments, the nucleotide sequence encoding the polypeptide of interest according to this disclosure comprises or consists of a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with SEQ ID NO: 213 or 217.

[0178] Further nucleotide sequences of polynucleotides The polynucleotides provided for in this disclosure may include additional nucleotide sequences and / or sequence features in addition to the first, second, third, fourth, fifth, and / or sixth nucleotide sequences described above in this specification.

[0179] The polynucleotides of this disclosure include a 5' (i.e., upstream in relation to the nucleotide sequence of the polynucleotide) start codon relative to the nucleotide sequence encoding the polypeptide of interest. The start codon is preferably a trinucleotide "ATG".

[0180] In some embodiments, the start codon is provided to the polynucleotide after splicing (e.g., splicing of a polyribonucleotide transcribed from the polynucleotide if the polynucleotide is a polydeoxyribonucleotide) so that the start codon is provided to the mature mRNA molecule to function as a start codon for translation of the polypeptide of interest (encoded by the fifth or sixth nucleotide sequence).

[0181] In some embodiments, the splicing referred to in the previous paragraph is splicing performed in the presence of a splicing modifier (e.g., as described herein). For example, splicing of a polyribonucleotide having the nucleotide sequence of SEQ ID NO: 22 in the presence of RG7800 / RG76196 results in the production of a mature mRNA molecule containing a start codon for translation of the polypeptide of interest. In some embodiments, the splicing referred to in the previous paragraph is splicing performed in the absence of a splicing modifier (e.g., as described herein). For example, splicing of a polyribonucleotide having the nucleotide sequence of SEQ ID NO: 28 in the absence of a splicing modifier results in the production of a mature mRNA molecule containing a start codon for translation of the polypeptide of interest.

[0182] 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 the polypeptide of interest. In some embodiments, the Kozak sequence consists of a nucleotide sequence matching the consensus of SEQ ID NO: 33. In some embodiments, the Kozak sequence consists of SEQ ID NO: 34.

[0183] In some embodiments, the polynucleotide comprises a nucleotide sequence consisting of 5' SEQ ID NO: 35 relative to the nucleotide sequence encoding the polypeptide of interest.

[0184] In some embodiments, SEQ ID NO: 35 is provided to a polynucleotide after splicing (e.g., splicing of a polyribonucleotide transcribed from a polynucleotide if the polynucleotide is a polydeoxyribonucleotide) so that the start codon of SEQ ID NO: 35 is provided to a mature mRNA molecule to function as a start codon for translation of the polypeptide of interest (encoded by the fifth or sixth nucleotide sequence).

[0185] In some embodiments, the splicing referred to in the previous paragraph is splicing performed in the presence of splicing modifiers (e.g., those described herein). In some embodiments, the splicing referred to in the previous paragraph is splicing performed in the absence of splicing modifiers (e.g., those described herein).

[0186] In some embodiments, the polynucleotide further comprises a promoter sequence, preferably 5' relative to the first nucleotide sequence. In some embodiments, the polynucleotide further comprises one or more enhancer sequences, preferably 5' relative to the first nucleotide sequence.

[0187] In some embodiments, the polynucleotide further comprises a stop codon. The stop codon is preferably provided immediately 3' (i.e., downstream in relation to the nucleotide sequence of the polynucleotide) to the trinucleotide encoding the terminal amino acid of the polypeptide encoded by the fifth or sixth nucleotide sequence.

[0188] In some embodiments, the polynucleotide further comprises a polyadenylation signal sequence. In preferred embodiments, the polyadenylation signal sequence is provided 3' (i.e., downstream in relation to the nucleotide sequence of the polynucleotide) relative to the nucleotide sequence encoding the polypeptide of interest. In some embodiments, the polynucleotide further comprises a terminator sequence. The terminator sequence is preferably 3' (3' relative to the polyadenylation signal sequence, if present) relative to the nucleotide sequence encoding the polypeptide of interest.

[0189] In preferred embodiments, the constituent nucleotide sequences of the polynucleotide (i.e., the first, second, third, fourth, fifth, and / or sixth nucleotide sequences as described above herein) are provided directly 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.

[0190] The linker nucleotide sequence may contain, or consist of, one of 1 to 10 nucleotides, for example, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0191] Where the polynucleotide according to this disclosure comprises one or more linker nucleotide sequences (i.e., provided between the first, second, third, fourth, fifth, and / or sixth nucleotide sequences), the linker sequences are preferably selected such that they do not substantially affect the post-transcriptional processing of the polynucleotide when the polynucleotide is a polyribonucleotide. In preferred embodiments of the polynucleotide according to this disclosure comprising one or more linker sequences, the splicing of the polynucleotide when the polynucleotide is a polyribonucleotide is substantially the same as the splicing of an equivalent polyribonucleotide lacking linker nucleotide sequences.

[0192] Similarly, if the polynucleotide according to the present disclosure comprises one or more linker nucleotide sequences (i.e., provided between the first, second, third, fourth, fifth, and / or sixth nucleotide sequences), the linker sequences are preferably selected so as not to alter the amino acid sequence of the polypeptide encoded by the polynucleotide. In preferred embodiments of the present disclosure comprising one or more linker sequences, the polynucleotide encodes the same polypeptide as an equivalent polyribonucleotide lacking the linker nucleotide sequences.

[0193] In some embodiments, the polynucleotide further comprises an inverted terminal repeat (ITR) sequence. In some embodiments, the polynucleotide comprises an ITR at 5' relative to the first nucleotide sequence (and, if present, at 5' relative to the promoter sequence and / or enhancer sequence). In some embodiments, the polynucleotide comprises an ITR at 3' relative to the nucleotide sequence encoding the polypeptide of interest (and, if present, at 3' relative to the stop codon, polyadenylation signal sequence and / or terminator sequence).

[0194] In some embodiments, the polynucleotide includes an ITR sequence at its 5' end and an ITR sequence 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 of the first nucleotide of the polynucleotide, for example, within 1 to 20, 1 to 15, 1 to 10, or 1 to 5 nucleotides (i.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 of the final nucleotide of the polynucleotide, for example, within 1 to 20, 1 to 15, 1 to 10, or 1 to 5 nucleotides (i.e., position 1 of the nucleotide sequence of the polynucleotide).

[0195] In some embodiments, the polynucleotides of this disclosure (i.e., including the first, second, third, fourth, fifth, and / or sixth nucleotide sequences described herein) have a size that enables delivery as gene therapy, i.e., in a suitable vector. In some embodiments, the polynucleotides consist of nucleotide sequences having a size within the packaging limits of a vector for delivering the polynucleotides.

[0196] In some embodiments, the polynucleotide has a size within the packaging limit of the AAV vector. In some embodiments, the polynucleotide has a size within the packaging limit of one of the following serotypes of the AAV vector: AAV9, AAV9.45, AAV-PHP.B, AAV1, AAV2, AAV2i8, AAV5, AAV6, AAV8, AAV10, or AAVrh74. In some embodiments, the polynucleotide has a size within the packaging limit of one of the following serotypes of the AAV vector: AAV9, AAV9.45, AAV-PHP.B, AAV1, AAV2, AAV2.7m8, AAV2i8, AAV5, AAV6, AAV8, AAV10, or AAVrh74.

[0197] In some embodiments, the polynucleotides of the Disclosure (i.e., including the first, second, third, fourth, fifth and / or sixth nucleotide sequences described herein) consist of fewer than 6,000 nucleotides, for example, 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.

[0198] In preferred embodiments, the polynucleotides of the present disclosure consist of fewer nucleotides than known SMN2 exon 6-exon 8 derived transgene expression systems (e.g., SMN2 exon 6-exon 8 derived transgene expression systems described in Zhang, et al., Gene Ther. (2001) 8:1532-1538, International Publication No. 2022 / 204471, Monteys et al. Nature (2021) 596:291-295, or International Publication No. 2021 / 163556).

[0199] In some embodiments, the polynucleotides of the present disclosure consist of fewer nucleotides than the polynucleotide comprising: (i) SEQ ID NO: 36 and (ii) the nucleotide sequence encoding the polypeptide of interest (i.e., adjacent to and 3' of SEQ ID NO: 36, in relation to the complete sequence of the polynucleotide). Such polynucleotides are described, for example, in International Publication No. 2021 / 163556.

[0200] Herein, for brevity, a polynucleotide comprising (i) SEQ ID NO: 36 and (ii) the nucleotide sequence encoding the polypeptide of interest (i.e., adjacent to and 3' of SEQ ID NO: 36 in relation to the complete sequence of the polynucleotide) is also referred to herein simply as the "SMN2ind minigene polynucleotide."

[0201] In some embodiments, the polynucleotides of the present disclosure consist of a number of nucleotides less than 1 times the number of nucleotides of the SMN2ind minigene polynucleotide, for example ≤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, or ≤0.25 times.

[0202] In some embodiments, the polynucleotide regions of the present disclosure formed by the first, second, third, fourth, and fifth nucleotide sequences consist of fewer nucleotides than the number of nucleotides in SEQ ID NO: 36. In some embodiments, the polynucleotide regions of the present disclosure formed by the first, second, third, fourth, and fifth nucleotide sequences consist of a number of nucleotides less than one times the number of nucleotides in SEQ ID NO: 36, for example ≤0.99, ≤0.95, ≤0.9, ≤0.85, ≤0.8, ≤0.75, ≤0.7, ≤0.65, ≤0.6, ≤0.55, ≤0.5, ≤0.45, ≤0.4, ≤0.35, ≤0.3, or ≤0.25.

[0203] The polynucleotides of this disclosure may include a 5' cap, 5' UTR, 3' UTR, and / or PolyA tail nucleotide sequence.

[0204] In some embodiments, the polynucleotide includes a 5' UTR at 5' (i.e., upstream in relation to the nucleotide sequence of the polynucleotide) relative to the start codon.

[0205] In some embodiments, the polynucleotide includes a 3' UTR at 3' (i.e., downstream in relation to the nucleotide sequence of the polynucleotide) relative to the stop codon. In some embodiments, the polynucleotide includes a 5' UTR relative to the polyadenylation signal sequence. In some embodiments, the polynucleotide includes a 3' UTR at 3' relative to the stop codon and a 5' UTR relative to the polyadenylation signal sequence.

[0206] In some embodiments, the polynucleotides of the present disclosure include one or more nucleotide sequences encoding selection markers to facilitate the identification and / or selection of cells containing / expressing the polynucleotides. Examples of selection markers include proteins that confer resistance to antibiotics or other toxins, such as blastosidine, ampicillin, neomycin, methotrexate, or tetracycline, and proteins that complement nutritional deficiencies.

[0207] In some embodiments, the polynucleotides of the Disclosure include a nucleotide sequence encoding an internal ribosome entry site (IRES). In some embodiments, the polynucleotides include a nucleotide sequence that allows two or more polypeptides to be translated separately from a single polyribonucleotide.

[0208] The polynucleotides of this disclosure may be provided in purified or isolated forms, i.e., from other nucleic acids or naturally occurring biological materials.

[0209] Specific exemplary polynucleotides In some embodiments, the polynucleotides according to this disclosure include: A first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with a nucleotide sequence selected from column A of Table A; A second nucleotide sequence containing, or consisting of, a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with a nucleotide sequence selected from column B of Table A; A third nucleotide sequence containing, or consisting of, a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with a nucleotide sequence selected from column C of Table A; A fourth nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with a nucleotide sequence selected from column D of Table A; and A fifth nucleotide sequence that includes, or consists of, a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with a nucleotide sequence selected from column E of Table A.

[0210] As per the previous paragraph, in some embodiments, the nucleotide sequences selected from column A of Table A, the nucleotide sequences selected from column B of Table A, the nucleotide sequences selected from column C of Table A, the nucleotide sequences selected from column B of Table A, the nucleotide sequences selected from column D of Table A, the nucleotide sequences selected from column B of Table A, and the nucleotide sequences selected from column E of Table A are all selected from the same row of Table A. For example, in some embodiments, the nucleotide sequences selected from columns A, B, C, D, and E of Table A may be selected from row 3 of Table 1, and thus may be sequence numbers 5, 9, 14, 18, and 20, respectively.

[0211] In some embodiments, the polynucleotides include: (i) a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in SEQ ID NO: 1; and containing "GGC" at positions corresponding to positions 82-84 of SEQ ID NO: 2; and (iii) a first nucleotide sequence consisting of ≤45 nucleotides; (i) 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 with sequence number 7 at its 5' end; and (ii) 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 with sequence number 8 at its 3' end; and (iii) a "C" at the position corresponding to position 5766 of sequence number 30; and (iv) a second nucleotide sequence consisting of fewer than 300 nucleotides; (i) a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in SEQ ID NO: 11; and (ii) a third nucleotide sequence containing "A" at the position corresponding to position 2 of SEQ ID NO: 12; and (iii) an insertion of "A" after the position corresponding to position 48 of SEQ ID NO: 12; (i) 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 with SEQ ID NO: 15 at its 5' end; and (ii) 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 with SEQ ID NO: 16 at its 3' end; and (iii) a fourth nucleotide sequence consisting of fewer than 255 nucleotides; and The fifth nucleotide sequence, consisting of the dinucleotide "GA".

[0212] In some embodiments, the polynucleotides include: A first nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% sequence identity with sequence number 106 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A second nucleotide sequence containing, or consisting of, a nucleotide sequence having at least 80% sequence identity with sequence number 10 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A third nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having at least 80% sequence identity with sequence number 13 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A fourth nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having at least 80% sequence identity with sequence number 17 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); and The fifth nucleotide sequence, consisting of the dinucleotide "GA".

[0213] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 22 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0214] In some embodiments, the polynucleotides include: A first nucleotide sequence comprising, or consisting of, a nucleotide sequence having sequence identity with sequence number 3 by at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A second nucleotide sequence containing, or consisting of, a nucleotide sequence having at least 80% sequence identity with sequence number 10 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A third nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having at least 80% sequence identity with sequence number 13 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A fourth nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having at least 80% sequence identity with sequence number 17 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); and The fifth nucleotide sequence, consisting of the dinucleotide "GA".

[0215] In some embodiments, the polynucleotide comprises a nucleotide sequence having sequence identity with SEQ ID NO: 23 of at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0216] In some embodiments, the polynucleotides include: (i) A first nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in Sequence ID No. 1; and containing "GGC" at positions 82-84 of Sequence ID No. 2; and (iii) containing "CTG" at positions 109-111 of Sequence ID No. 2; (i) 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 with sequence number 7 at its 5' end; and (ii) 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 with sequence number 8 at its 3' end; and (iii) a "C" at the position corresponding to position 5766 of sequence number 30; and (iv) a second nucleotide sequence consisting of fewer than 500 nucleotides; (i) A third nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in SEQ ID NO: 11; and (ii) A third nucleotide sequence containing "A" at the position corresponding to position 2 of SEQ ID NO: 12; and (iii) An insertion of "A" after the position corresponding to position 48 of SEQ ID NO: 12; and (iv) An insertion of "GCCACC" after the position corresponding to position 6 of SEQ ID NO: 12; and (v) A third nucleotide sequence containing "TG" at the positions corresponding to positions 8 and 9 of SEQ ID NO: 12; (i) 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 with SEQ ID NO: 15 at its 5' end; and (ii) 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 with SEQ ID NO: 16 at its 3' end; and (iii) a fourth nucleotide sequence consisting of fewer than 500 nucleotides; and A fifth nucleotide sequence containing, or consisting of, a nucleotide sequence having at least 80% sequence identity with sequence number 20 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0217] In some embodiments, the polynucleotides include: A first nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having at least 80% sequence identity with sequence number 5 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A second nucleotide sequence containing, or consisting of, a nucleotide sequence having at least 80% sequence identity with sequence number 9 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A third nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having at least 80% sequence identity with sequence number 14 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A fourth nucleotide sequence comprising, or consisting of, a nucleotide sequence having sequence identity with sequence number 18 of at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); and A fifth nucleotide sequence containing, or consisting of, a nucleotide sequence having at least 80% sequence identity with sequence number 20 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0218] In some embodiments, the polynucleotide comprises a nucleotide sequence having sequence identity with SEQ ID NO: 24 by at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0219] In some embodiments, the polynucleotides include: (i) A first nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in SEQ ID NO: 1; and containing "GGC" at positions 82-84 of SEQ ID NO: 2; and (iii) containing "CAG" at positions 109-111 of SEQ ID NO: 2; (i) 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 with sequence number 7 at its 5' end; and (ii) 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 with sequence number 8 at its 3' end; and (iii) a "C" at the position corresponding to position 5766 of sequence number 30; and (iv) a second nucleotide sequence consisting of fewer than 500 nucleotides; (i) a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in SEQ ID NO: 11; and (ii) a third nucleotide sequence containing "A" at the position corresponding to position 2 of SEQ ID NO: 12; and (iii) an insertion of "A" after the position corresponding to position 48 of SEQ ID NO: 12; (i) 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 with SEQ ID NO: 15 at its 5' end; and (ii) 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 with SEQ ID NO: 16 at its 3' end; and (iii) a fourth nucleotide sequence consisting of fewer than 500 nucleotides; and A fifth nucleotide sequence containing, or consisting of, a nucleotide sequence having at least 80% sequence identity with sequence number 21 (for example, one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0220] In some embodiments, the polynucleotides include: A first nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having sequence identity with sequence number 6 by at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A second nucleotide sequence containing, or consisting of, a nucleotide sequence having at least 80% sequence identity with sequence number 9 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A third nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having at least 80% sequence identity with sequence number 13 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A fourth nucleotide sequence comprising, or consisting of, a nucleotide sequence having sequence identity with sequence number 18 of at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); and A fifth nucleotide sequence containing, or consisting of, a nucleotide sequence having at least 80% sequence identity with sequence number 20 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0221] In some embodiments, the polynucleotide comprises a nucleotide sequence having sequence identity with SEQ ID NO: 25 of at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0222] In some embodiments, the polynucleotides include: (i) A nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in Sequence ID No. 1; and containing "GGC" at positions corresponding to positions 82-84 of Sequence ID No. 2; and (iii) A first nucleotide sequence consisting of ≤45 nucleotides.

[0223] In some embodiments, the polynucleotides include: (i) 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 with sequence number 7 at its 5' end; and (ii) 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 with sequence number 8 at its 3' end; and (iii) a "C" at the position corresponding to position 5766 of sequence number 30; and (iv) a second nucleotide sequence consisting of fewer than 300 nucleotides; (i) a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in SEQ ID NO: 11; and (ii) a third nucleotide sequence containing "A" at the position corresponding to position 2 of SEQ ID NO: 12; and (iii) an insertion of "A" after the position corresponding to position 48 of SEQ ID NO: 12; (i) 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 with SEQ ID NO: 15 at its 5' end; and (ii) 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 with SEQ ID NO: 16 at its 3' end; and (iii) a fourth nucleotide sequence consisting of fewer than 255 nucleotides; and A fifth nucleotide sequence consisting of the trinucleotide "GAG".

[0224] In some embodiments, the polynucleotides include: A first nucleotide sequence comprising, or consisting of, a nucleotide sequence having sequence identity with sequence number 3 by at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A second nucleotide sequence containing, or consisting of, a nucleotide sequence having at least 80% sequence identity with sequence number 10 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A third nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having at least 80% sequence identity with sequence number 13 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A fourth nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having at least 80% sequence identity with sequence number 17 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); and A fifth nucleotide sequence consisting of the trinucleotide "GAG".

[0225] In some embodiments, the polynucleotide comprises a nucleotide sequence having sequence identity with SEQ ID NO: 28 of at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0226] In some embodiments, the polynucleotides include: (i) A nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in Sequence ID No. 1; and containing "GCC" at positions corresponding to positions 82-84 of Sequence ID No. 2; and (iii) A first nucleotide sequence consisting of ≤45 nucleotides.

[0227] In some embodiments, the polynucleotides include: (i) 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 with sequence number 7 at its 5' end; and (ii) 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 with sequence number 8 at its 3' end; and (iii) a "C" at the position corresponding to position 5766 of sequence number 30; and (iv) a second nucleotide sequence consisting of fewer than 300 nucleotides; (i) a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in SEQ ID NO: 11; and (ii) a third nucleotide sequence containing "A" at the position corresponding to position 2 of SEQ ID NO: 12; and (iii) a deletion at the position corresponding to position 20 of SEQ ID NO: 12; and (iv) an insertion of "A" after the position corresponding to position 48 of SEQ ID NO: 12; (i) 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 with SEQ ID NO: 15 at its 5' end; and (ii) 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 with SEQ ID NO: 16 at its 3' end; and (iii) a fourth nucleotide sequence consisting of fewer than 255 nucleotides; and A fifth nucleotide sequence consisting of the trinucleotide "GAG".

[0228] In some embodiments, the polynucleotides include: A first nucleotide sequence comprising, or consisting of, a nucleotide sequence having sequence identity with sequence number 3 by at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A second nucleotide sequence containing, or consisting of, a nucleotide sequence having at least 80% sequence identity with sequence number 10 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A third nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having at least 80% sequence identity with sequence number 27 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A fourth nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having at least 80% sequence identity with sequence number 17 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); and A fifth nucleotide sequence consisting of the trinucleotide "GAG".

[0229] In some embodiments, the polynucleotide comprises a nucleotide sequence having sequence identity with SEQ ID NO: 29 of at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0230] In some embodiments, the polynucleotide is not comprised of or does not contain SEQ ID NO: 36.

[0231] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with one of sequence numbers 37 to 105.

[0232] In some embodiments, the polynucleotides include: (i) a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in Sequence ID No. 221; and (ii) a first nucleotide sequence consisting of ≤45 nucleotides; (i) 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 with sequence number 7 at its 5' end; and (ii) 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 with sequence number 8 at its 3' end; and (iii) a "C" at the position corresponding to position 5766 of sequence number 30; and (iv) a second nucleotide sequence consisting of fewer than 300 nucleotides; (i) a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in SEQ ID NO: 11; and (ii) a third nucleotide sequence containing "A" at the position corresponding to position 2 of SEQ ID NO: 12; and (iii) an insertion of "A" after the position corresponding to position 48 of SEQ ID NO: 12; (i) 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 with SEQ ID NO: 15 at its 5' end; and (ii) 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 with SEQ ID NO: 16 at its 3' end; and (iii) a fourth nucleotide sequence consisting of fewer than 255 nucleotides; and The fifth nucleotide sequence, consisting of the dinucleotide "TG".

[0233] In some embodiments, the polynucleotides include: A first nucleotide sequence comprising, or consisting of, a nucleotide sequence having sequence identity with sequence number 219 by at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A second nucleotide sequence containing, or consisting of, a nucleotide sequence having at least 80% sequence identity with sequence number 10 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A third nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having at least 80% sequence identity with sequence number 13 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A fourth nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having at least 80% sequence identity with sequence number 17 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); and The fifth nucleotide sequence, consisting of the dinucleotide "TG".

[0234] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity with sequence number 111 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0235] In some embodiments, the polynucleotides include: (i) a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in Sequence ID No. 221; and (ii) a first nucleotide sequence consisting of ≤45 nucleotides; (i) 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 with sequence number 7 at its 5' end; and (ii) 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 with sequence number 8 at its 3' end; and (iii) a "C" at the position corresponding to position 5766 of sequence number 30; and (iv) a second nucleotide sequence consisting of fewer than 300 nucleotides; (i) A third nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in Sequence ID No. 225; (i) 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 with SEQ ID NO: 15 at its 5' end; and (ii) 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 with SEQ ID NO: 16 at its 3' end; and (iii) a fourth nucleotide sequence consisting of fewer than 255 nucleotides; and The fifth nucleotide sequence, consisting of the dinucleotide "TG".

[0236] In some embodiments, the polynucleotides include: A first nucleotide sequence comprising, or consisting of, a nucleotide sequence having sequence identity with sequence number 219 by at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A second nucleotide sequence containing, or consisting of, a nucleotide sequence having at least 80% sequence identity with sequence number 10 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A third nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having at least 80% sequence identity with sequence number 223 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A fourth nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having at least 80% sequence identity with sequence number 17 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); and The fifth nucleotide sequence, consisting of the dinucleotide "TG".

[0237] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 116 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0238] In some embodiments, the polynucleotides include: (i) a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in Sequence ID No. 221; and (ii) a first nucleotide sequence consisting of ≤45 nucleotides; (i) 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 with sequence number 7 at its 5' end; and (ii) 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 with sequence number 8 at its 3' end; and (iii) a "C" at the position corresponding to position 5766 of sequence number 30; and (iv) a second nucleotide sequence consisting of fewer than 300 nucleotides; (i) A third nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in Sequence ID No. 225; (i) a nucleotide sequence having sequence identity with sequence number 227 by at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); and (iii) a fourth nucleotide sequence consisting of fewer than 280 nucleotides; and The fifth nucleotide sequence, consisting of the dinucleotide "TG".

[0239] In some embodiments, the polynucleotides include: A first nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having sequence identity with sequence number 220 by at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A second nucleotide sequence containing, or consisting of, a nucleotide sequence having at least 80% sequence identity with sequence number 10 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A third nucleotide sequence containing, or consisting of, a nucleotide sequence having at least 80% sequence identity with sequence number 224 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A fourth nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having sequence identity with sequence number 227 by at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); and The fifth nucleotide sequence, consisting of the dinucleotide "TG".

[0240] In some embodiments, the polynucleotide comprises a nucleotide sequence having sequence identity with sequence number 164 of at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0241] In some embodiments, the polynucleotides include: (i) a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in Sequence ID No. 221; and (ii) a first nucleotide sequence consisting of ≤45 nucleotides; (i) A second nucleotide sequence comprising, or consisting of, a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in Sequence ID No. 229; (i) A third nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with the nucleotide sequence described in Sequence ID No. 225; (i) a nucleotide sequence having sequence identity with sequence number 227 by at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); and (iii) a fourth nucleotide sequence consisting of fewer than 280 nucleotides; and The fifth nucleotide sequence, consisting of the dinucleotide "TG".

[0242] In some embodiments, the polynucleotides include: A first nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having sequence identity with sequence number 220 by at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A second nucleotide sequence containing, or consisting of, a nucleotide sequence having at least 80% sequence identity with sequence number 228 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A third nucleotide sequence containing, or consisting of, a nucleotide sequence having at least 80% sequence identity with sequence number 224 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); A fourth nucleotide sequence comprising, or consisting thereof, a nucleotide sequence having sequence identity with sequence number 227 by at least 80% (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%); and The fifth nucleotide sequence, consisting of the dinucleotide "TG".

[0243] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 171 (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%).

[0244] In some embodiments, the polynucleotide includes a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with one of sequence numbers 111-200. In some embodiments, the polynucleotide includes a nucleotide sequence having at least 80% sequence identity (e.g., one of ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) with one of sequence numbers 37-105 or 111-200.

[0245] Splicing modifying factor Aspects and embodiments of the present disclosure relate to splicing modifying factors. A splicing modifying factor is a molecule that affects the splicing of a polynucleotide. Such molecules are reviewed, for example, 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 incorporated herein by reference in their entirety.

[0246] Splicing modifying factors generally reduce / prevent the association of factors required for normal post-transcriptional processing (e.g., components of the spliceosome) with RNA. Splicing modifying factors typically bind to the nucleotide sequence of a polynucleotide and inhibit or promote the association of RNA-binding proteins and / or non-coding RNAs that function as splicing activating or inhibitory factors. By competitively inhibiting the recruitment of splicing activating and splicing inhibitory factors to the polynucleotide, splicing modifying factors can alter the balance of splicing regulatory RNA structures and thus promote an increase or decrease in exon skipping or inclusion and / or intron retention or excision in the mature RNA molecules produced by splicing.

[0247] In some embodiments, the splicing modifying factor is a small molecule or a splice-switching nucleic acid (e.g., a splice-switching oligonucleotide).

[0248] "Small molecule" refers to an organic compound with a low molecular weight (<1000 daltons, typically about 300 to about 700 daltons).

[0249] Splice-switching nucleic acids are outlined, for example, in Haves and Hastings, Nucleic Acids Res. (2016) 44(14):6549-6563, which is incorporated herein by reference in its entirety. Splice-switching nucleic acids include, for example, splice-switching oligonucleotides (SSOs). They disrupt the normal splicing of a target RNA transcript by blocking RNA:RNA base pairing and / or protein:RNA binding interactions that occur between the components of the splicing mechanism and pre-mRNA. Splice-switching nucleic acids may be designed to target specific regions of a target transcript, for example, to promote the skipping of a desired exon, and / or the inclusion of a desired exon, and / or the retention of a desired intron, and / or the excision of a desired intron. SSOs often involve modifications to oligonucleotide sugar-phosphate backbones to reduce / prevent RNA degradation, such as phosphorothioate bonds, phosphorodiamidate bonds such as phosphorodiamidate morpholino (PMO), and may include peptide nucleic acids (PNA), locked nucleic acids (LNA), methoxyethyl nucleotide modifications, such as 2'O-methyl (2'OMe) and 2'-O-methoxyethyl (MOE) ribose modifications and / or 5'-methylcytosine modifications.

[0250] Small molecule splicing modifiers intended in accordance with this disclosure include RG-7800 and RG-7916 (also known as risdiplam), and their analogues. RG-7800 and RG-7916 bind to the 5' splice site of intron 7 and exon splicing enhancer 2 of exon 7 of human SMN2, thereby stabilizing the transient double-stranded RNA structure formed by the SMN2 pre-mRNA and U1 snRNP complex, and promoting the inclusion of exon 7 in the mature RNA molecule obtained after splicing of pre-mRNA transcribed from SMN2. RG-7800 and RG-7916 are useful for restoring functional SMN2 protein expression from SMN2 alleles containing the spinal muscular atrophy (SMA)-associated polymorphism c.840C>T (which enhances exon 7 skipping). RG-7916 (DrugBank Acc. number DB15305) is a close structural analog of RG-7800 with improved potency, pharmacokinetics, and safety profiles, and is FDA approved for the treatment of SMA. Further analogues of RG-7800 and RG-7916 with similar splicing modifier activity include SMN-C2, SMN-C3, SMN-C5, and TEC-1.

[0251] Branapram (DrugBank Acc. number DB14918; also known as LMI-070) is another small molecule splicing modifier that promotes the inclusion of exon 7 in mature RNA expressed from SMN2. Similar to RG-7800 and RG-7916, branapram binds to the 5' splice site of intron 7 in human SMN2.

[0252] PK4C9 (also known as homocarbonyltopsentin) is another small molecule splicing modifier that promotes the inclusion of exon 7 in mature RNA expressed from SMN2. It is thought to improve the accessibility of the 5' splice site by binding to exon 7 and the 5' splice site of TSL2 and stabilizing the triloop structure of TSL2.

[0253] Small molecule splicing modifiers that promote the inclusion of exon 7 in mature RNA expressed from SMN2 are described, for example, in International Publication No. 2015 / 173181 and International Publication No. 2009 / 151546, both of which are incorporated herein by reference. Further small molecule splicing modifiers that promote the inclusion of exon 7 in mature RNA expressed from SMN2 are described, for example, in International Publication No. 2022 / 204471, both of which are incorporated herein by reference.

[0254] Nusinersen (DrugBank Acc. number 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 hybridizes to and occupies the intron splicing silencer site 1 of intron 7, thereby inhibiting the association of splicing suppressor ribonucleoproteins hnRNP A1 / A2 and thus promoting the inclusion of exon 7 in mature RNA molecules.

[0255] The splicing modifiers relating to this disclosure preferably promote the inclusion of SMN2 exon 7 in RNA obtained after splicing of pre-mRNA transcribed from human SMN2. Such molecules increase the proportion of RNA molecules containing SMN2 exon 7 among the RNA molecules obtained after splicing of pre-mRNA transcribed from human SMN2 (i.e., compared to the proportion obtained in the absence of the splicing modifier). For brevity, “splicing modifiers that promote the inclusion of SMN2 exon 7 in RNA obtained after splicing of pre-mRNA transcribed from human SMN2” may be referred to herein simply as “splicing modifiers that promote SMN2 exon 7 inclusion.”

[0256] In some embodiments, splicing modifiers promote the inclusion of exon 7 in RNA obtained after splicing of pre-mRNA transcribed from human SMN2 alleles containing c.840C>T. That is, in some embodiments, splicing modifiers increase the proportion of RNA molecules containing SMN2 exon 7 among the RNA molecules obtained after splicing of pre-mRNA transcribed from human SMN2 alleles containing c.840C>T (i.e., compared to the proportion obtained in the absence of splicing modifiers).

[0257] In some embodiments, the splicing modifier increases the level of human SMN2 protein containing the amino acid encoded by exon 7 of human SMN2 (i.e., compared to the level detected in the absence of the splicing modifier). In some embodiments, the splicing modifier increases the proportion of polypeptides expressed from human SMN2 that contain the amino acid encoded by exon 7 of human SMN2 (i.e., compared to the proportion obtained in the absence of the splicing modifier). In some embodiments, the splicing modifier increases the proportion of polypeptides expressed from human SMN2 alleles containing c.840C>T that contain the amino acid encoded by exon 7 of human SMN2 (i.e., compared to the proportion obtained in the absence of the splicing modifier).

[0258] In some embodiments, the splicing modifier facilitates the inclusion of a third nucleotide sequence in the product of the splicing of a polynucleotide according to the Disclosure (i.e., when the polynucleotide is a polyribonucleotide). In some embodiments, the splicing modifier increases the proportion of molecules containing a third nucleotide sequence among the molecules obtained after the splicing of a polynucleotide according to the Disclosure (i.e., when the polynucleotide is a polyribonucleotide).

[0259] In some embodiments, the splicing modifier promotes the inclusion of the nucleotide sequence of SEQ ID NO: 13 in the molecule obtained after splicing the polyribonucleotide sequence of SEQ ID NO: 22. In some embodiments, the splicing modifier increases the proportion of molecules containing the nucleotide sequence of SEQ ID NO: 13 in the molecule obtained after splicing the polyribonucleotide sequence of SEQ ID NO: 22.

[0260] In some embodiments, the splicing modifier promotes the inclusion of the nucleotide sequence of SEQ ID NO: 13 in the molecule obtained after splicing the polyribonucleotide sequence of SEQ ID NO: 23. In some embodiments, the splicing modifier increases the proportion of molecules containing the nucleotide sequence of SEQ ID NO: 13 in the molecule obtained after splicing the polyribonucleotide sequence of SEQ ID NO: 23.

[0261] In some embodiments, the splicing modifier promotes the inclusion of the nucleotide sequence of SEQ ID NO: 14 in the molecule obtained after splicing the polyribonucleotide sequence of SEQ ID NO: 24. In some embodiments, the splicing modifier increases the proportion of molecules containing the nucleotide sequence of SEQ ID NO: 14 in the molecule obtained after splicing the polyribonucleotide sequence of SEQ ID NO: 24.

[0262] In some embodiments, the splicing modifier promotes the inclusion of the nucleotide sequence of SEQ ID NO: 13 in the molecule obtained after splicing the polyribonucleotide sequence of SEQ ID NO: 25. In some embodiments, the splicing modifier increases the proportion of molecules containing the nucleotide sequence of SEQ ID NO: 13 in the molecule obtained after splicing the polyribonucleotide sequence of SEQ ID NO: 25.

[0263] In some embodiments, the splicing modifier promotes the inclusion of the nucleotide sequence of SEQ ID NO: 13 in the molecule obtained after splicing the polyribonucleotide sequence of SEQ ID NO: 28. In some embodiments, the splicing modifier increases the proportion of molecules containing the nucleotide sequence of SEQ ID NO: 13 in the molecule obtained after splicing the polyribonucleotide sequence of SEQ ID NO: 28.

[0264] In some embodiments, the splicing modifier promotes the inclusion of the nucleotide sequence of SEQ ID NO: 27 in the molecule obtained after splicing the polyribonucleotide sequence of SEQ ID NO: 29. In some embodiments, the splicing modifier increases the proportion of molecules containing the nucleotide sequence of SEQ ID NO: 27 in the molecule obtained after splicing the polyribonucleotide sequence of SEQ ID NO: 29.

[0265] Splicing modifiers possessing such functional properties can be identified, for example, by analysis in a suitable in vitro assay. Such assays may involve culturing cells in vitro in or out of the presence of candidate splicing modifiers and analyzing the RNA and / or proteins produced after a period of time appropriate for the effects of the candidate splicing modifiers to be observed. For example, cells can be transfected with a vector containing DNA having the sequence of SEQ ID NO: 22 and cultured for an appropriate period (e.g., 24, 48, 72 hours) in or out of the presence of candidate splicing modifiers. Subsequently, RNA can be isolated from the cells and analyzed (e.g., by qRT-PCR) to determine the level / percentage of mature RNA molecules containing the nucleotide sequence of SEQ ID NO: 13.

[0266] In some embodiments, the splicing modifier that promotes SMN2 exon 7 inclusion according to the present disclosure is a compound of formula (I) of International Publication No. 2015 / 173181. In some embodiments, the splicing modifier that promotes SMN2 exon 7 inclusion according to the present disclosure is a compound selected from those listed in claim 39 of International Publication No. 2015 / 173181. In some embodiments, the splicing modifier that promotes SMN2 exon 7 inclusion according to the present disclosure is a compound selected from those listed in claim 40 of International Publication No. 2015 / 173181.

[0267] In some embodiments, the splicing modifier according to the present disclosure is selected from the following: 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 the following: 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 a preferred embodiment, the splicing modifier is risdiplam (RG-7916).

[0268] Functional characteristics of polynucleotides In aspects and embodiments of the present disclosure, the polynucleotides of the present disclosure may be characterized by reference to one or more functional characteristics.

[0269] In some embodiments, when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion results in a polyribonucleotide that substantially lacks a third nucleotide sequence. In some embodiments, when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion results in a polyribonucleotide that contains a third nucleotide sequence.

[0270] In some embodiments, in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion, the inclusion of a third nucleotide sequence is facilitated in the splicing of the polyribonucleotide, which is favorable for the production of a mature RNA molecule encoding the polypeptide of interest, and thus for the expression of the polypeptide of interest at the protein level. Such polynucleotides may be referred to herein as "ON-switch" polynucleotides.

[0271] In some embodiments, cells containing the ON-switched polynucleotide of this disclosure substantially do not express the polypeptide of interest in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, cells containing the ON-switched polynucleotide express the polypeptide of interest in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion.

[0272] Exemplary ON-switched polynucleotides in this disclosure include polynucleotides containing SEQ ID NOs. 22, 23, 24, or 25. Further exemplary ON-switched polynucleotides in this disclosure include polynucleotides containing any one of SEQ ID NOs. 37-105.

[0273] The inclusion of the third nucleotide sequence described herein in the polyribonucleotide splicing product according to this disclosure can be evaluated using methods well known to those skilled in the art. Such methods include qRT-PCR-based methods for the detection and / or quantification of RNA molecules containing the nucleotide sequence of interest.

[0274] For example, cells may be transduced in vitro with a vector containing the polyribonucleotide according to the present disclosure, and subsequently cultured in vitro for a period appropriate to observe the effect of the present disclosure on polyribonucleotide splicing, with or without a splicing modifier that promotes SMN2 exon 7 inclusion. After such a period, total RNA may be isolated from the cells, cDNA may be prepared from the total RNA, and the number / percentage of mature RNA molecules containing the third nucleotide sequence may be assessed by qPCR using oligonucleotides (e.g., oligonucleotides that hybridize to nucleotide sequences spanning exon:exon boundaries) that provide specific amplification and / or detection of polyribonucleotide splicing products. qPCR analysis may use oligonucleotides that enable distinction between RNA molecules containing the third nucleotide sequence and RNA molecules lacking the third nucleotide sequence. Such qRT-PCR-based methods for analyzing polyribonucleotide splicing products are described in the experimental examples of the present disclosure.

[0275] The expression of the target polypeptide can be evaluated using any suitable technique for the detection and / or quantification of the related polypeptide. Such techniques include, for example, antibody-based methods (e.g., flow cytometry, immunocytochemistry, Western blotting, ELISA), fluorescence microscopy, and flow cytometry. In some embodiments, the expression of the target polypeptide can be evaluated as described in the experimental examples of this disclosure. In preferred embodiments, the expression of the target polypeptide may be evaluated by flow cytometry.

[0276] In this specification, in multiple / populations of polyribonucleotides obtained after splicing that "substantially lacks" the third nucleotide sequence, the third nucleotide sequence may be present in less than 15% of the polyribonucleotides, e.g., ≤10%, ≤5%, ≤4%, ≤3%, ≤2%, or ≤1%. Conversely, in multiple / populations of polyribonucleotides obtained after splicing that "contains" the third nucleotide sequence, the third nucleotide sequence may be present in more than 80% of the polyribonucleotides, e.g., ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%.

[0277] In this specification, within a group / population of cells that "substantially do not express" the polypeptide of interest, the polypeptide may be expressed by less than 15% of the cells, e.g., ≤10%, ≤5%, ≤4%, ≤3%, ≤2%, or ≤1%. Conversely, within a group / population of cells that "express" the polypeptide of interest, the polypeptide may be expressed by more than 80% of the cells, e.g., ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%.

[0278] In some embodiments, cells that “substantially do not express” the polypeptide of interest may exhibit an expression level of the polypeptide of interest that is less than 0.2 times the expression level of cells that express the polypeptide of interest, for example ≤0.1, ≤0.09, ≤0.08, ≤0.07, ≤0.06, ≤0.05, ≤0.04, ≤0.03, ≤0.02, or ≤0.01. In some embodiments, cells that “express” the polypeptide of interest may exhibit an expression level of the polypeptide of interest that is more than 5 times the expression level of cells that “substantially do not express” the polypeptide of interest, for example ≥10, ≥20, ≥50, ≥100, ≥1000, ≥5000, or ≥10000.

[0279] In some embodiments, the level of RNA containing a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence according to SEQ ID NO: 11 in cells containing polynucleotides according to this disclosure after culture in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion is more than 1 times the level in equivalent cells cultured in the absence of the splicing modifier that promotes SMN2 exon 7 inclusion, for example, ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥10 times, ≥20 times, ≥50 times, ≥100 times, ≥1000 times, ≥5000 times, or ≥10000 times.

[0280] In some embodiments, the level of RNA containing a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence according to SEQ ID NO: 11 in cells containing the polynucleotide according to this disclosure, after culture in the absence of splicing modifiers that promote SMN2 exon 7 inclusion, is less than 100 times the level of RNA containing a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence according to SEQ ID NO: 11 in equivalent cells that do not contain the polynucleotide, for example, ≤50 times, ≤20 times, ≤10 times, ≤5 times, ≤4 times, ≤3 times, ≤2 times, or ≤1 time.

[0281] In some embodiments, the level of the target polypeptide in cells containing the polynucleotide (e.g., ON-switched polynucleotide) according to this disclosure in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion is more than 1 times the level in equivalent cells cultured in the absence of the splicing modifier that promotes SMN2 exon 7 inclusion, for example, ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥10 times, ≥20 times, ≥50 times, ≥100 times, ≥1000 times, ≥5000 times, or ≥10000 times.

[0282] In some embodiments, in the absence of splicing modifiers that promote SMN2 exon 7 inclusion, the expression level of the target polypeptide in cells containing the polynucleotide according to this disclosure (e.g., ON-switched polynucleotide) is less than 100 times the level of the target polypeptide in equivalent cells that do not contain the polynucleotide, for example, ≤50 times, ≤20 times, ≤10 times, ≤5 times, ≤4 times, ≤3 times, ≤2 times, or ≤1 time.

[0283] In some embodiments, the proportion of cells expressing the polypeptide of interest within a population of cells containing the polynucleotide according to this disclosure (e.g., ON-switched polynucleotide) cultured in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion is greater than 1, for example, ≥2, ≥3, ≥4, ≥5, ≥10, ≥20, ≥50, ≥100, ≥1000, ≥5000 or ≥10000, compared to the proportion of such cells expressing the polypeptide of interest cultured in the absence of the splicing modifier that promotes SMN2 exon 7 inclusion.

[0284] In some embodiments, the proportion of cells expressing the polypeptide of interest within a population of cells containing the polynucleotide according to this disclosure (e.g., ON-switched polynucleotide) cultured in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion is less than 100 times, e.g., ≤50 times, ≤20 times, ≤10 times, ≤5 times, ≤4 times, ≤3 times, ≤2 times, or ≤1 time, compared to the proportion of such cells expressing the polypeptide of interest cultured in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion.

[0285] In some embodiments, in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion, the inclusion of a third nucleotide sequence is facilitated in the splicing of the polyribonucleotide, which is advantageous for the production of a mature RNA molecule encoding an early stop codon, and therefore for the prevention of the expression of the target polypeptide at the protein level. Such polynucleotides may be referred to herein as “OFF-switch” polynucleotides.

[0286] In some embodiments, cells containing the OFF-switch polynucleotide of this disclosure express the polypeptide of interest in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion. In some embodiments, cells containing the OFF-switch polynucleotide do not express the polypeptide of interest in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion.

[0287] The exemplary OFF-switched polynucleotides described herein include polynucleotides containing SEQ ID NO: 28 or 29.

[0288] In some embodiments, the level of RNA containing a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence according to SEQ ID NO: 26 in cells containing polynucleotides according to this disclosure after culture in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion is more than 1 times the level in equivalent cells cultured in the absence of the splicing modifier that promotes SMN2 exon 7 inclusion, for example, ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥10 times, ≥20 times, ≥50 times, ≥100 times, ≥1000 times, ≥5000 times, or ≥10000 times.

[0289] In some embodiments, after culturing in the absence of splicing modifiers that promote SMN2 exon 7 inclusion, the level of RNA containing a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence according to SEQ ID NO: 26 in cells containing the polynucleotide according to this disclosure is less than 100 times the level of the target polypeptide in equivalent cells that do not contain the polynucleotide, for example, ≤50 times, ≤20 times, ≤10 times, ≤5 times, ≤4 times, ≤3 times, ≤2 times, or ≤1 times.

[0290] In some embodiments, the level of the target polypeptide in cells containing the polynucleotide according to this disclosure (e.g., OFF-switched polynucleotide) in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion is more than 1 times the level in equivalent cells cultured in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion, for example, ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥10 times, ≥20 times, ≥50 times, ≥100 times, ≥1000 times, ≥5000 times, or ≥10000 times.

[0291] In some embodiments, the expression level of the target polypeptide in cells containing the polynucleotide according to this disclosure (e.g., an OFF-switched polynucleotide) in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion is less than 100 times the level of the target polypeptide in equivalent cells without the polynucleotide, for example, ≤50 times, ≤20 times, ≤10 times, ≤5 times, ≤4 times, ≤3 times, ≤2 times, or ≤1 time.

[0292] In some embodiments, the proportion of cells expressing the polypeptide of interest in a population of cells containing the polynucleotide according to this disclosure (e.g., an OFF-switched polynucleotide) cultured in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion is greater than 1, for example, ≥2, ≥3, ≥4, ≥5, ≥10, ≥20, ≥50, ≥100, ≥1000, ≥5000 or ≥10000, compared to the proportion of such cells expressing the polypeptide of interest cultured in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion.

[0293] In some embodiments, the proportion of cells expressing the polypeptide of interest within a population of cells containing the polynucleotide according to this disclosure (e.g., an OFF-switched polynucleotide) cultured in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion is less than 100 times, for example, ≤50 times, ≤20 times, ≤10 times, ≤5 times, ≤4 times, ≤3 times, ≤2 times, or ≤1 time, compared to the proportion of such cells expressing the polypeptide of interest cultured in the absence of the splicing modifier that promotes SMN2 exon 7 inclusion.

[0294] The polynucleotides of this disclosure have novel and / or improved properties compared to known transgene expression systems, such as known transgene expression systems that include an SMSM-mediated switch derived from SMN2 exons 6-8 (e.g., transgene expression systems derived from SMN2 exons 6-8 described in Zhang, et al., Gene Ther. (2001) 8:1532-1538, International Publication No. 2022 / 204471, Monteys et al. Nature (2021) 596:291-295, or International Publication No. 2021 / 163556).

[0295] In some embodiments, the polynucleotides of this disclosure have novel and / or improved properties compared to the SMN2ind minigene polynucleotide (as defined above herein).

[0296] In some embodiments, cells containing the polynucleotide of the Disclosure cultured in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion produce a population of RNA molecules having an increased proportion of RNA molecules containing nucleotide sequences having at least 80% sequence identity to the nucleotide sequence described in SEQ ID NO: 11 or 26, compared to a population of RNA molecules cultured under the same conditions. That is, in some embodiments, the polynucleotide of the Disclosure is more effective than the SMN2ind minigene polynucleotide in promoting the inclusion of nucleotide sequences having at least 80% sequence identity to the nucleotide sequence described in SEQ ID NO: 11 or 26 in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion. In other words, the polynucleotide of the Disclosure is more responsive than the SMN2ind minigene polynucleotide to the induction of SMN2 exon 7 variant inclusion in splicing products in response to a splicing modifier that promotes SMN2 exon 7 inclusion.

[0297] In some embodiments, the proportion of RNA molecules containing nucleotide sequences having at least 80% sequence identity with the nucleotide sequences described in SEQ ID NO: 11 or 26 among RNA molecules obtained from cells containing the polynucleotide of the present disclosure cultured in the presence of splicing modifiers that promote SMN2 exon 7 inclusion is greater than 1, for example, ≥1.01, ≥1.02, ≥1.03, ≥1.04, ≥1.05, ≥1.1, ≥1.2, ≥1.3, ≥1.4, ≥1.5, ≥1.6, ≥1.7, ≥1.8, ≥1.9, ≥2, ≥3, ≥4, ≥5, ≥6, ≥7, ≥8, ≥9, or ≥10 times the proportion of RNA molecules containing nucleotide sequences having at least 80% sequence identity with the nucleotide sequences described in SEQ ID NO: 11 or 26 among RNA molecules obtained from cells containing the SMN2ind minigene polynucleotide and cultured under the same conditions.

[0298] It will be understood that the functional properties described herein are evaluated using the same experimental conditions for the evaluation of cells containing different polynucleotides (i.e., the polynucleotides of this disclosure and the SMN2ind minigene polynucleotide). For example, the same cell type, the same splicing modifiers that promote SMN2 exon 7 inclusion, the same concentrations of splicing modifiers that promote SMN2 exon 7 inclusion are used, the same culture period is provided, and the cells are analyzed in the same manner to determine the percentage of RNA molecules containing the relevant nucleotide sequence and / or the level of the polypeptide of interest.

[0299] In some embodiments, cells containing the polynucleotide of the present disclosure, cultured in the absence of splicing modifiers that promote SMN2 exon 7 inclusion, are produced by cells containing the SMN2ind minigene polynucleotide and produce a population of RNA molecules having a reduced proportion of RNA molecules containing nucleotide sequences having at least 80% sequence identity to the nucleotide sequences described in SEQ ID NO: 11 or 26, compared to a population of RNA molecules cultured under the same conditions.

[0300] In other words, in some embodiments, the polynucleotides of the present disclosure are more effective than the SMN2ind minigene polynucleotide in excluding nucleotide sequences having at least 80% sequence identity with the nucleotide sequence described in SEQ ID NO: 11 or 26 in the absence of splicing modifiers that promote SMN2 exon 7 inclusion. That is, the polynucleotides of the present disclosure are less "leaky" than the SMN2ind minigene polynucleotide with respect to SMN2 exon 7 variant inclusion in splicing products in the absence of splicing modifiers that promote SMN2 exon 7 inclusion. The leaky nature of the production of mature RNA molecules containing SMN2 exon 7 variants (i.e., in the absence of splicing modifiers that promote SMN2 exon 7 inclusion) may also be referred to herein as "background" production of such RNA molecules.

[0301] In some embodiments, the proportion of RNA molecules containing nucleotide sequences having at least 80% sequence identity to the nucleotide sequence described in SEQ ID NO: 11 or 26 among RNA molecules obtained from cells containing the polynucleotide of this disclosure cultured in the absence of splicing modifiers that promote SMN2 exon 7 inclusion is the proportion of RNA molecules obtained from cells containing the SMN2ind minigene polynucleotide and cultured under the same conditions as SEQ ID NO: 11 or 26. The ratio of RNA molecules containing a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in 26 is less than 1, for example ≤0.99, ≤0.95, ≤0.9, ≤0.85, ≤0.8, ≤0.75, ≤0.7, ≤0.65, ≤0.6, ≤0.55, ≤0.5, ≤0.45, ≤0.4, ≤0.35, ≤0.3, ≤0.25, ≤0.2, ≤0.15, ≤0.1, ≤0.05, or ≤0.01.

[0302] In some embodiments, cells containing the polynucleotide of the Disclosure (e.g., ON-switched polynucleotide) cultured in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion exhibit increased levels of protein expression of the target polypeptide compared to cells containing the SMN2ind minigene polynucleotide cultured under the same conditions. That is, in some embodiments, the polynucleotide of the Disclosure is more effective than the SMN2ind minigene polynucleotide in promoting protein expression of the target polypeptide in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion. In other words, the polynucleotide of the Disclosure is more responsive than the SMN2ind minigene polynucleotide to inducing the expression of the target polypeptide in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion.

[0303] For the purpose of such comparison, it will be understood that different polynucleotides (i.e., the polynucleotide of this disclosure and the SMN2ind minigene polynucleotide) preferably encode the same polypeptide of interest.

[0304] In some embodiments, the protein expression level of the polypeptide of interest from cells containing the polynucleotide of this disclosure (e.g., ON-switched polynucleotide) cultured in the presence of a splicing modifier that promotes SMN2 exon 7 inclusion is greater than 1 times, for example, ≥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.

[0305] In some embodiments, cells containing the polynucleotide of the Disclosure (e.g., ON-switched polynucleotide) cultured in the absence of splicing modifiers that promote SMN2 exon 7 inclusion exhibit reduced levels of protein expression of the target polypeptide compared to cells containing the SMN2ind minigene polynucleotide cultured under the same conditions. That is, in some embodiments, the polynucleotide of the Disclosure is more effective than the SMN2ind minigene polynucleotide in preventing protein expression of the target polypeptide in the absence of splicing modifiers that promote SMN2 exon 7 inclusion. In other words, the polynucleotide of the Disclosure is less "leaky" than the SMN2ind minigene polynucleotide with respect to the expression of the target polypeptide in the absence of splicing modifiers that promote SMN2 exon 7 inclusion. The leaching of the expression of the target polypeptide (i.e., in the absence of splicing modifiers that promote SMN2 exon 7 inclusion) may also be referred to herein as "background" expression of the polypeptide.

[0306] In some embodiments, the protein expression level of the polypeptide of interest from cells containing the polynucleotide of this disclosure (e.g., an ON-switched polynucleotide) cultured in the absence of splicing modifiers that promote SMN2 exon 7 inclusion is less than 1x the protein expression level of the polypeptide of interest from cells containing the SMN2ind minigene polynucleotide cultured under the same conditions, e.g., ≤0.99x, ≤0.95x, ≤0.9x, ≤0.85x, ≤0.8x, ≤0.75x, ≤0.7x, ≤0.65x, ≤0.6x, ≤0.55x, ≤0.5x, ≤0.45x, ≤0.4x, ≤0.35x, ≤0.3x, ≤0.25x, ≤0.2x, ≤0.15x, ≤0.1x, ≤0.05x, or ≤0.01x.

[0307] In some embodiments, the polypeptide of interest expressed from the polynucleotide of this disclosure contains fewer foreign additional amino acids (i.e., amino acids additional to the amino acid sequence of the polypeptide of interest) compared to the polypeptide of interest expressed from the SMN2ind minigene polynucleotide. In some embodiments, the polypeptide of interest expressed from the polynucleotide of this disclosure contains a smaller N-terminal tag (i.e., formed from foreign additional amino acids in addition to the amino acid sequence of the polypeptide of interest) compared to the polypeptide of interest expressed from the SMN2ind minigene polynucleotide. In other words, in some embodiments, the polynucleotide of this disclosure provides inducible expression of the polypeptide of interest containing fewer foreign additional amino acids compared to the polypeptide of interest expressed from the SMN2ind minigene polynucleotide.

[0308] vector This disclosure provides vectors comprising polynucleotides according to this disclosure. Since a vector is also a polynucleotide, it will be understood that in some embodiments, the polynucleotides according to this disclosure can be vectors.

[0309] As used herein, “vector” refers to a polynucleotide used as a vehicle for transferring exogenous nucleic acids into cells. A vector may be a vector for the expression of nucleic acids in cells (i.e., a vector may be an expression vector). Such a vector may include a promoter sequence operably ligated to the nucleotide sequence to be expressed. A vector may also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art may be used in the vector according to this disclosure.

[0310] The term "operably linked" may include situations in which the nucleic acid encoding the polypeptide of interest according to this disclosure and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked in such a manner that the expression of the nucleic acid encoding the polypeptide is under the influence or control of the regulatory nucleic acid sequence (thereby forming an expression cassette). Thus, if the regulatory sequence is capable of transcribing the nucleic acid sequence, the regulatory sequence is operably linked to the selected nucleic acid sequence. The resulting transcript can then be translated into the desired polypeptide.

[0311] Vectors intended in connection with this disclosure include DNA vectors, RNA vectors, plasmids (e.g., conjugated plasmids (e.g., F plasmids), non-conjugated plasmids, R plasmids, col plasmids, episomes), viral vectors (e.g., retroviral vectors, e.g., gamma retroviral vectors (e.g., mouse leukemia virus (MLV)-derived vectors, e.g., SFG vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, baculovirus vectors, and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), for example, as described in Maus et al., Annu Rev Immunol (2014) 32:189-225 and Morgan and Boyerinas, Biomedicines (2016) 4:9, both of which are incorporated herein by reference in their entirety.

[0312] In some embodiments, the vector may be a eukaryotic vector, i.e., a vector containing elements necessary for protein expression from the vector in eukaryotic cells. In some embodiments, the vector may be a mammalian vector containing, for example, a cytomegalovirus (CMV) or SV40 promoter that drives protein expression. In some embodiments, the vector may contain CMV (e.g., mCMV), SV40, RSV, or PGK promoter.

[0313] In some embodiments, the polynucleotides according to the Disclosure (e.g., the vectors according to the Disclosure) include a CMV promoter, a CAG promoter, an hEF1a promoter, an hUbiC promoter, an RSV promoter, a TK promoter, a PGK promoter, or a CAG minimal promoter.

[0314] In some embodiments, the polynucleotides of the present disclosure (e.g., the vectors of the present disclosure) include 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 with SEQ ID NOs. 201, 202, 203, 204, 205, 206, 207, or 208, or a promoter having a nucleotide sequence comprising such a nucleotide sequence.

[0315] In some embodiments, the vector is selected based on its directivity to the cell type / tissue / organ to which the polynucleotides according to this disclosure are desired. In some embodiments, the vector is selected based on its directivity to the cell type / tissue / organ to which the polypeptide of interest is desired to express. For example, it may be desirable to deliver the polynucleotides to the cell type / tissue / organ affected by the disease / symptoms treated / prevented according to this disclosure (e.g., the cell type / tissue / organ in which the symptoms of the disease / symptoms appear) and / or to express the polypeptide of interest.

[0316] For example, it may be desirable to deliver the polynucleotide of the present disclosure encoding the polypeptide of interest to nerve cells / tissues, and a vector having directivity to such cells / tissues may be used in such cases (i.e., a nerve-directed vector).

[0317] In preferred embodiments, the vector is an adeno-associated virus (AAV) vector. Adeno-associated virus vectors and their use in vector gene therapy are outlined, for example, 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 incorporated herein by reference in their entirety. In some embodiments, the vector may be an adeno-associated virus vector as described in Wang et al., Nat. Rev. Drug Discov. (2019) 18:358-378. In some embodiments, the vector may be an adeno-associated virus vector as described in Li and Samulski, Nat. Rev. Genet. (2020) 12:255-272.

[0318] In some embodiments, the vector is a self-complementary adeno-associated virus (scAAV) vector. Self-complementary adeno-associated virus vectors are described, for example, in McCarty, Mol Ther. (2008) 16(10):1648-56, which is incorporated herein by reference in its entirety. Conventional AAVs have a single-stranded DNA genome and rely on the DNA replication mechanism of the transduced cell to synthesize the complementary strand, delaying transgene expression. In contrast, scAAVs contain a complementary sequence that spontaneously anneals upon infection, eliminating the need for DNA synthesis in the transduced host cell. Compared to classical single-stranded AAV vectors, scAAV vectors have been shown to accelerate the initiation of transgene expression and increase the level of transgene expression.

[0319] In some embodiments, the vector may be an adeno-associated virus 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 may be an adeno-associated virus vector of one of the following serotypes: AAV9 (including AAV9 variants AAV-PHP.B and AAV9.45), AAV1, AAV2 (including AAV2 variants AAV2.7m8 and AAV2i8), AAV5, AAV6, AAV8, AAV10, or AAVrh74. In some embodiments, the vector is an AAV9 vector.

[0320] In some embodiments, the vector includes modifications that increase binding to and / or transduction to the target cell type (i.e., compared to the level of binding / transduction with the unmodified vector). In some embodiments, the modifications are to the capsid protein.

[0321] In some embodiments, the vector comprises a capsid protein containing 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 incorporated herein by reference in its entirety), for example, the cell-targeting peptides shown in Tables 1, 2, 3, or 4.

[0322] In some embodiments, the vector comprises a capsid protein in which one or more tyrosine residues, for example, one or more surface-exposed tyrosine residues, are substituted. In some embodiments, one or more tyrosine residues of the capsid protein are substituted with phenylalanine. In some embodiments, the vector comprises a capsid protein in which one or more tyrosine residues are substituted with another amino acid, as described in Iida et al., Biomed Res Int. (2013) 2013:974819 (which is incorporated herein by reference in its entirety).

[0323] In some embodiments, the vector may be the adeno-associated virus vector described by Burning and Srivastava (above). In some embodiments, the vector may be the adeno-associated virus vector described by Iida et al. (above).

[0324] In some embodiments, the vector includes a regulatory element for the inducible expression of the polynucleotides of this disclosure.

[0325] Sequences that control polynucleotide expression may provide polynucleotide expression by specific cell types or tissues. For example, expression may be under the control of cell type or tissue-specific promoters.

[0326] The promoters for cell-type or tissue-specific expression of polynucleotides according to this disclosure can be selected according to the disease / symptom being treated / prevented. For example, a promoter may drive expression in cell types / tissues / organs affected by the disease / symptom (e.g., cell types / tissues / organs where the symptoms of the disease / symptom appear).

[0327] In some embodiments, the promoter may provide polynucleotide expression in neuronal cells / tissues. In some embodiments, the promoter may be a neuron-specific promoter (e.g., CaMKII, NSE, or SynI-miniCMV promoter). In some embodiments, the promoter may provide polynucleotide expression in muscle cells / tissues (e.g., cardiomyocytes and / or skeletal muscle cells / tissues). In some embodiments, the promoter may be a cardiac or cardiomyocyte-specific promoter (e.g., cTNT, α-MHC, or MLC2v promoter). In some embodiments, the promoter may be a skeletal muscle / striated muscle cell-specific promoter (e.g., MCK, MHCK7, or desmin promoter). In some embodiments, the promoter may be a vascular endothelial cell-specific promoter (e.g., Tie2 promoter). In some embodiments, the promoter may be a vascular smooth muscle cell-specific promoter (e.g., SM22a promoter). In some embodiments, the promoter may be a monocyte / macrophage-specific promoter (e.g., LysM promoter).

[0328] Sequences for controlling polynucleotide expression may provide polynucleotide expression in response to a given drug / signal, for example. For example, expression may be under the control of an inducible promoter. Drugs may provide inducible polynucleotide expression in vivo by administering the drug to subjects to whom the modified cells according to this disclosure have been administered, or ex vivo / in vitro by administering the drug to cells in culture ex vivo or in vitro.

[0329] In some embodiments, the polynucleotides or vectors of this disclosure may utilize a conditional expression system to control the expression of the polynucleotide by cells containing the polynucleotide / vector. "Conditional expression" may also be referred to herein as "inducible expression" and refers to expression that depends on specific conditions, such as the presence of a specific drug. Conditional expression systems are well known in the art and are outlined, for example, in Ryding et al. Journal of Endocrinology (2001) 171, 1-14, which are incorporated herein by reference in their entirety.

[0330] cell The disclosure also provides cells containing or expressing polynucleotides according to the disclosure. Cells containing or expressing vectors according to the disclosure are also provided.

[0331] The polynucleotides (e.g., polyribonucleotides) according to this disclosure can be produced in cells by transcription from polynucleotides (e.g., polydeoxyribonucleotides) that encode polynucleotides.

[0332] The cells may be eukaryotic cells, such as mammalian cells. Mammals may be primates (rhesus monkeys, crab-eating macaques, non-human primates, or humans) or non-human mammals (e.g., rabbits, guinea pigs, rats, mice or other rodents (including any animals of the order Rodentia), cats, dogs, pigs, sheep, goats, cattle (including cows, such as dairy cows, or any animals of the order Artiodactyla), horses (including any animals of the order Perissodactyla), donkeys, and non-human primates). In preferred embodiments, the cells may be human cells.

[0333] The cells may be immune cells. The cells may be hematopoietic cells, such as neutrophils, eosinophils, basophils, dendritic cells, lymphocytes, or monocytes. Lymphocytes may be, for example, T cells, B cells, NK cells, NKT cells, or innate lymphoid cells (ILCs), or their precursors. The cells may express, for example, CD3 polypeptides (e.g., CD3γ, CD3ε, CD3ζ, or CD3δ), TCR polypeptides (TCRα or TCRβ), CD27, CD28, CD4, or CD8. In some embodiments, the cells are T cells. In some embodiments, the T cells are CD3+ T cells. In some embodiments, the T cells are CD3+, CD8+ T cells. In some embodiments, the T cells are cytotoxic T cells (e.g., cytotoxic T lymphocytes (CTLs)).

[0334] The Disclosure also provides a method for producing cells containing or expressing polynucleotides / vectors according to the Disclosure, the method comprising introducing the polynucleotides / vectors according to the Disclosure into cells. In some embodiments, introducing the polynucleotides / vectors according to the Disclosure into cells includes transformation, transfection, electroporation, or transduction (e.g., adeno-associated virus transduction). In some embodiments, the polynucleotides / vectors are introduced into cells in vivo by targeting, for example, a vector according to the Disclosure (e.g., a viral vector, e.g., an adeno-associated virus vector). In some embodiments, the polynucleotides / vectors are introduced into cells in culture ex vivo or in vitro.

[0335] Cells according to the present disclosure can be produced using any suitable method. Such methods may include nucleic acid transfer for permanent (i.e., stable) or transient expression of the polynucleotides of the present disclosure. In some embodiments, after introduction into cells, the polynucleotides may be incorporated into or form part of the cell's genomic DNA. In some embodiments, after introduction into cells, the polynucleotides may be maintained outside the chromosome.

[0336] Any suitable genetic engineering platform may be used, including gamma retroviral vectors, lentiviral vectors, adenoviral vectors, DNA transfection, transposon-based gene delivery, and RNA transfection, for example, described in Maus et al., Annu Rev Immunol. (2014) 32:189-225, which is incorporated herein by reference in its entirety. Methods also include, for example, those described in Wang and Riviere Mol Ther Oncolytics. (2016) 3:16015 (which is incorporated herein by reference in its entirety). Suitable methods for introducing nucleic acids / vectors into cells include transduction, transfection, and electroporation.

[0337] In some embodiments, the method further includes maintaining the cells under conditions suitable for the expression of polynucleotides / vectors by the cells.

[0338] This disclosure also provides cells that can be obtained or obtained by the methods relating to this disclosure.

[0339] composition This disclosure also provides compositions comprising the polynucleotides, vectors, and cells described herein. In particular, this disclosure provides pharmaceutical compositions and pharmaceuticals comprising the polynucleotides, vectors, and cells described herein.

[0340] Such compositions may include relevant articles (i.e., polynucleotides / vectors / cells) in formulations suitable for clinical use. This disclosure relates, in particular, to pharmaceutical compositions / pharmaceuticals comprising polynucleotides and vectors according to this disclosure.

[0341] The compositions disclosed herein include one or more pharmaceutically acceptable carriers (e.g., liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g., starch, cellulose, cellulose derivatives, polyols, 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, methylparaben, propylparaben), and antioxidants. It may contain (for example, vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (for example, magnesium stearate, talc, silica, stearic acid, vegetable stearin), binders (for example, sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilizers, solubilizers, surfactants (for example, wetting agents), masking agents or colorants (for example, titanium dioxide).

[0342] As used herein, the term “pharmaceutically acceptable” means that, within the bounds of sound medical judgment, a compound, component, material, composition, dosage form, etc., is suitable for use in contact with the tissue of the subject of concern (e.g., a human subject) without excessive toxicity, irritation, allergic reaction, or other problems or complications, and is commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, colorant, flavoring agent, or sweetener of a composition according to this disclosure must also be “acceptable” in the sense that it is compatible with the other components of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking agents, colorants, flavoring agents, or sweeteners can be found in standard pharmaceutical texts, such as Remington's "The Science and Practice of Pharmacy" (Ed. (A) Adejare), 23rd Edition (2020), Academic Press.

[0343] The pharmaceutical compositions / pharmaceuticals described herein may be formulated for administration to a subject, for example, through a route of administration appropriate to the nature of the therapeutic agent and the disease being treated / prevented. In some embodiments, the pharmaceutical compositions / pharmaceuticals may be formulated for parenteral administration, systemic administration, topical administration, intracavitary administration, intravascular administration, intravenous administration, intra-arterial administration, intramuscular administration, intrathecal administration, intraocular administration, intraconjunctival administration, intratumoral administration, subcutaneous administration, intradermal administration, oral administration, or transdermal administration. In some embodiments, the pharmaceutical compositions / pharmaceuticals may be formulated for administration by injection or infusion, or by ingestion.

[0344] Pharmaceuticals and pharmaceutical compositions may be formulated for administration to blood vessels or to target tissues / organs (e.g., tissues / organs affected by a disease / symptom, e.g., tissues / organs where symptoms of the disease / symptom appear).

[0345] Pharmaceutical compositions / pharmaceuticals may contain polynucleotides / vectors / cells in sterile or isotonic media. Pharmaceutical compositions / pharmaceuticals may be provided in a fluid form, including a gel. Fluid formulations may be formulated for administration by injection or infusion (e.g., via a cannula) into blood vessels or selected areas of the human or animal body. Pharmaceutical compositions / pharmaceuticals may be provided in a solid form, such as a lyophilized form.

[0346] This disclosure also provides methods for producing pharmaceutical compositions / pharmaceuticals according to this disclosure. Such methods may involve mixing polynucleotides / vectors / cells described herein with pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking agents, colorants, flavoring agents, or sweeteners. Such methods generally involve associating polynucleotides / vectors / cells with carriers constituting one or more auxiliary components. Generally, compositions are prepared by homogeneously and closely associating an active compound with a carrier (e.g., a liquid carrier, a finely divided solid carrier, etc.) and then, if necessary, shaping the product.

[0347] The polynucleotides, vectors, cells, and compositions relating to this disclosure may be modified and / or formulated to facilitate delivery to and / or uptake by a target cell type / tissue / organ (e.g., a cell type / tissue / organ on which symptoms of a disease / symptom appear).

[0348] Strategies for targeted delivery of polynucleotides are outlined, for example, 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 incorporated herein by reference in their entirety.

[0349] In some embodiments, the articles of the Disclosure may be encapsulated in nanoparticles or liposomes. In some embodiments, the articles of the Disclosure may associate (covalently or noncovalently) with cell-permeable peptides (e.g., protein transduction domains, Trojan peptides, arginine-rich peptides, Vectocell peptides), cationic polymers, cationic lipids, or viral carriers.

[0350] Nanoparticles can be organic, such as micelles, liposomes, proteins, solid lipid particles, solid polymer particles, dendrimers, and polymer therapeutics. Nanoparticles can also be inorganic, such as nanotubes or metal particles to which organic molecules are optionally added. In some embodiments, the nanoparticles are those described in Chen et al., Mol Ther Methods Clin Dev. (2016) 3:16023 (which is incorporated herein by reference in its entirety). In some embodiments, the nanoparticles are PLGA, polypeptides, poly(β-aminoesters), DOPE, β-cyclodextrin-containing polycations, linear PEI, PAMAM dendrimers, branched PEI, chitosan, or polyphosphoester nanoparticles.

[0351] In some embodiments, the polynucleotides and vectors according to the Disclosure include modifications to incorporate one or more moieties that facilitate delivery to and / or uptake by a target cell type, organ, or tissue (e.g., a cell type, tissue, or organ on which symptoms of a disease / symptom appear). In some embodiments, the polynucleotides or vectors according to the Disclosure are ligated (e.g., chemically conjugated) to one or more moieties that facilitate delivery to and / or uptake by a target cell type, tissue, or organ.

[0352] The portions that facilitate delivery to and / or uptake by target cell types, tissues, or organs are described, for example, in Benizri et al., Bioconjug Chem. (2019) 30(2):366-383 (the whole of which is incorporated herein by reference). Such portions include, for example, N-acetylgalactosamine (GalNAc), α-tocopherol, cell-permeable peptides, nucleic acid aptamers, antibodies and their antigen-binding fragments / derivatives, cholesterol, squalene, polyethylene glycol (PEG), fatty acids (e.g., palmitic acid), and nuclear lipid portions.

[0353] The articles of this disclosure may be formulated in a sustained-release delivery system to release polynucleotides, vectors, cells, or compositions at a predetermined rate. The sustained-release delivery system may maintain a constant drug / therapeutic / prophylactic concentration over a specific period of time. In some embodiments, the articles of this disclosure may be formulated in liposomes, gels, implants, devices, or drug-polymer conjugates, such as hydrogels.

[0354] In some embodiments, the compositions according to this disclosure may further include splicing modifiers (e.g., those described herein) that promote SMN2 exon 7 inclusion.

[0355] Therapeutic / preventive applications The polynucleotides, vectors, cells, and compositions of this disclosure are used for therapeutic and prophylactic purposes.

[0356] Accordingly, this disclosure provides polynucleotides, vectors, cells, or compositions described herein for use in methods of medical treatment or prevention. Also provided are polynucleotides, vectors, cells, or compositions described herein for use in methods of treating or preventing the diseases / conditions described herein. Also provided are the use of polynucleotides, vectors, cells, or compositions described herein in the manufacture of pharmaceuticals for treating or preventing the diseases or conditions described herein. Also provided are methods of treating or preventing the diseases or conditions described herein, comprising administering a therapeutically effective or preventively effective amount of the polynucleotides, vectors, cells, or compositions described herein to a subject.

[0357] The interventions described in the previous paragraph may be effective in reducing the onset or progression of the disease / symptom, alleviating the symptoms of the disease / symptom, or reducing the pathogenesis of the disease / symptom. Interventions may be effective in preventing the progression of the disease / symptom, for example, in preventing the worsening of the progression of the disease / symptom, or in slowing its rate of onset. In some embodiments, interventions may result in improvement of the disease / symptom, for example, a reduction in the symptoms of the disease / symptom or a reduction in any other correlation of the severity / activity of the disease / symptom. In some embodiments, interventions may prevent the progression / onset of the disease / symptom at a later stage (e.g., the chronic stage).

[0358] It will be understood that the polynucleotides, vectors, cells, and compositions described herein may be used to treat / prevent any disease / condition for which a therapeutic or preventive benefit is to be obtained from an increase in the level of the target polypeptide (i.e., the target polypeptide encoded by the polynucleotide).

[0359] For example, a disease / symptom may be a disease / symptom associated with and / or characterized by a deficiency / insufficiency of the polypeptide of interest. A deficiency / insufficiency of the polypeptide of interest may be positively associated with the onset, development, or progression of the disease / symptom, and / or with the severity of one or more symptoms of the disease / symptom. A deficiency / insufficiency of the polypeptide of interest may be a risk factor for the onset, development, or progression of the disease / symptom. A disease / symptom may be characterized, for example, by a reduced level of expression or activity of the polypeptide of interest compared to the level of expression / activity in the absence of the disease / symptom. In some embodiments, a disease / symptom may be characterized by a reduced number / percentage / activity of cells expressing the polypeptide of interest compared to the level / number / percentage / activity in the absence of the disease / symptom (e.g., in a healthy subject or in equivalent non-disease tissue).

[0360] For example, in some embodiments, the polypeptide of interest may be MeCP2, and the disease / symptom treated / prevented in accordance with this disclosure may be a disease / symptom caused by a deficiency / insufficiency of MeCP2, such as Rett syndrome.

[0361] As a further example, in embodiments where the polypeptide of interest is a polypeptide capable of inhibiting the expression and / or activity of a target antigen of interest, the disease / symptom may be a disease / symptom in which the target antigen, or cells containing / expressing the target antigen, is pathologically involved, for example, an increase in the level / activity of the target antigen, or an increase in the number / percentage / activity of cells containing / expressing the target antigen, is positively associated with the onset, development, or progression of the disease / symptom, and / or the severity of one or more symptoms of the disease / symptom. In some embodiments, an increase in the level / activity of the target antigen, or an increase in the number / percentage / activity of cells containing / expressing the target antigen, may be a risk factor for the onset, development, or progression of the disease / symptom. The disease / symptom may be characterized, for example, by an increase in the level of expression or activity of the target antigen compared to the level of expression / activity in the absence of the disease / symptom. In some embodiments, the disease / symptom may be characterized by an increase in the number / percentage / activity of cells expressing the target antigen compared to the level / number / percentage / activity in the absence of the disease / symptom (e.g., in a healthy subject or in equivalent non-disease tissue). The therapeutic / prophylactic interventions provided in this disclosure may achieve one or more of the following in subjects (compared to equivalent untreated subjects or subjects treated with appropriate controls): reduction of the level of the target antigen; reduction of the activity of the target antigen; and / or reduction of the number / percentage / activity of cells containing / expressing the target antigen.

[0362] As a further example, a disease / symptom may be a disease / symptom treated by nucleic acid editing, and the polypeptide of interest may be a constituent protein of the appropriate site-specific nuclease nucleic acid editing system.

[0363] This disclosure provides articles of the disclosure for use, methods of using articles of the disclosure, and methods of administering polynucleotides, vectors, cells, and compositions of the disclosure to a subject (e.g., a subject requiring treatment). In some embodiments, the methods include administering to a subject a splicing modifier that promotes SMN2 exon 7 inclusion (e.g., a splicing modifier that promotes SMN2 exon 7 inclusion as described herein).

[0364] The administration of the articles of this disclosure is preferably a “therapeutic effective” or “preventive effective” dose, which is sufficient to demonstrate a therapeutic or preventive benefit to the subject. The actual amount administered, as well as the rate and time course of administration, depends on the nature and severity of the disease / symptom and the specific article being administered. The prescription of the treatment, such as the determination of the dosage, is the responsibility of the general practitioner and other physicians, and typically takes into account the disease / disorder being treated, the symptoms of the individual subject, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the techniques and protocols described above can be found in Remington's “The Science and Practice of Pharmacy” (ed. (A) Adejare), 23rd Edition (2020), Academic Press.

[0365] The articles of this disclosure may be administered parenterally, systemically, intravenously, intra-arterially, intramuscularly, intracavitally, intrathecally, intraocularly, intravitreously, intraconjunctivally, subretinally, suprachoroidally, subcutaneously, intradermally, intrathecally, orally, nasally, topically, or percutaneously. Administration may be by injection or infusion. Administration of the articles of this disclosure may be intratumoral. In some cases, the articles of this disclosure may be formulated for targeted delivery to specific cells, tissues, organs, and / or tumors.

[0366] Multiple doses of the articles of this disclosure may be provided. The multiple doses may be separated by a predetermined time interval, which may be 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 one of 1, 2, 3, 4, 5 or 6 months.

[0367] In some embodiments, the Disclosure provides articles of the Disclosure for use, uses of articles of the Disclosure, and methods comprising administering (i) polynucleotides, vectors, cells, or compositions according to the Disclosure, and (ii) splicing modifiers that promote SMN2 exon 7 inclusion, to a subject (e.g., a subject requiring treatment).

[0368] In embodiments according to the aspects of the preceding paragraph, the provision of (i) and (ii) may be as a combination therapy. In some embodiments, (i) and (ii) may be provided simultaneously or sequentially.

[0369] Co-administration refers to administering two or more drugs (e.g., polynucleotides, vectors, cells or compositions, and splicing modifiers that promote SMN2 exon 7 inclusion according to this disclosure) together, for example, as a pharmaceutical composition containing both drugs (i.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, for example, into the same artery, vein, or other blood vessel. Sequential administration refers to administering one drug, and then administering another drug separately after a given time interval. As is the case in some embodiments, the drugs do not need to be administered via the same route. The time interval may be any time interval.

[0370] In some embodiments, the splicing modifier that promotes SMN2 exon 7 inclusion is administered to the subject after administration of the polynucleotide, vector, cell, or composition according to the Disclosure. In some embodiments, the polynucleotide, vector, cell, or composition according to the Disclosure is administered to the subject, and then the splicing modifier that promotes SMN2 exon 7 inclusion is administered to the subject sequentially thereafter.

[0371] In some embodiments, the splicing modifier that promotes SMN2 exon 7 inclusion is administered in an amount and / or with periodicity selected to achieve a desired expression level of the polypeptide of interest. That is, in some embodiments, the amount and / or periodicity of the SMN2 exon 7 inclusion promoting splicing modifier administered to a subject according to this disclosure is selected to achieve a desired expression level of the polypeptide of interest. In some embodiments, the amount and / or periodicity of the SMN2 exon 7 inclusion promoting splicing modifier administered to a subject is adjusted to alter (i.e., decrease or increase) the expression level of the polypeptide of interest in the subject throughout the course of administration.

[0372] In embodiments where the polynucleotide of this disclosure is an ON-switch polynucleotide, the dose and / or periodicity of the administration of the splicing modifier that promotes SMN2 exon 7 inclusion may be increased to increase the expression level of the polypeptide of interest. Conversely, the dose and / or periodicity of the administration of the splicing modifier that promotes SMN2 exon 7 inclusion may be decreased to decrease the expression level of the polypeptide of interest.

[0373] In embodiments where the polynucleotide of this disclosure is an OFF-switch polynucleotide, the dose and / or periodicity of the splicing modifier that promotes SMN2 exon 7 inclusion may be reduced to increase the expression level of the polypeptide of interest. Conversely, the dose and / or periodicity of the splicing modifier that promotes SMN2 exon 7 inclusion may be increased to decrease the expression level of the polypeptide of interest.

[0374] Further methods The disclosure also provides methods for modifying cells to contain or express polynucleotides according to the disclosure, including introducing polynucleotides or vectors according to the disclosure into cells.

[0375] In some embodiments, introducing polynucleotides or vectors according to this disclosure into cells includes transformation, transfection, electroporation, or transduction (e.g., retroviral transduction).

[0376] Transfection refers to the process of introducing nucleic acids into cells using means other than viral infection, and is therefore a non-viral method. Transfection can be carried out by physical / mechanical methods (including electroporation, sonoporation, magnetofection, gene microinjection, and laser irradiation) or chemical methods (liposome-based or non-liposome-based). Liposome-based transfection reagents are chemicals that enable the formation of positively charged lipid aggregates, which can then fuse with the phospholipid bilayer of cells to facilitate the entry of foreign genetic material. Examples of liposome-based transfection reagents include, but are not limited to, Oligofectamine®, Lipofectamine®, and DharmaFECT®. Examples of non-liposome transfection reagents include, but are not limited to, calcium phosphate, nanoparticles, polymers, dendrimers, and non-liposomal lipids. An example of a non-liposome transfection reagent is polyethyleneimine (PEI).

[0377] Electroporation may be carried out, for example, as described in Koh et al., Molecular Therapy-Nucleic Acids (2013) 2, e114, which is incorporated in whole herein by reference.

[0378] Transduction is the process by which nucleic acids can be introduced into cells by a virus or viral vector. Therefore, in some embodiments, the polynucleotide is or is contained within a viral vector, or the vector is a viral vector. Transduction of immune cells by viral vectors is described, for example, in Simmons and Alberola-Ila, Methods Mol Biol. (2016) 1323:99-108, which is incorporated herein by reference in its entirety. In the methods of this disclosure, agents may be used to enhance the efficiency of transduction. Hexadimethrin bromide (Polybrene) is a cationic polymer commonly used to improve transduction by neutralizing the charge repulsion between virions expressed on the cell surface and sialic acid residues. Other drugs commonly used to enhance transduction include, for example, LentiBOOST (Sirion Biotech), Retronectin (Takara), Vectofusin (Miltenyi Biotech), and poloxamers such as SureENTRY (Qiagen) and ViraDuctin (Cell Biolabs).

[0379] In some embodiments, the method comprises centrifuging cells into which the polynucleotides or vectors according to the Disclosure are to be introduced in the presence of a cell culture medium containing a polynucleotide-containing viral vector (referred to in the art as "spinfection").

[0380] In some embodiments, the method includes culturing cells under conditions suitable for the expression of polynucleotides or vectors by cells. In some embodiments, the method includes culturing cells under conditions suitable for the transcription of polydeoxyribonucleotides. In some embodiments, the method includes culturing cells under conditions suitable for post-transcriptional processing (e.g., splicing) of polyribonucleotides. In some embodiments, the method includes culturing cells under conditions suitable for the translation of polypeptides from polyribonucleotides.

[0381] Methods for culturing (including generating and / or growing) cell populations in vitro / ex vivo, including appropriate culture conditions (i.e., cell culture medium, additives, stimuli, temperature, gaseous atmosphere), cell number, and culture period, are well known to those skilled in the art. Conveniently, cell cultures according to this disclosure can be maintained at 37°C in a humidified atmosphere containing 5% CO2.

[0382] This disclosure also provides a method for modifying cells to express a polypeptide of interest, comprising introducing a polynucleotide or vector according to this disclosure into the cells. Where the polynucleotide is an OFF switch or the vector contains / encodes it, the cells may express the polypeptide of interest after the polynucleotide / vector has been introduced into the cells.

[0383] Where the polynucleotide is an ON switch or the vector contains / encodes it, the method may further include contacting the cells with a splicing modifier that promotes SMN2 exon 7 inclusion as described herein. Thus, in some embodiments, the method includes (i) introducing the polynucleotide or vector according to the disclosure into cells; and (ii) subsequently contacting the cells with a splicing modifier that promotes SMN2 exon 7 inclusion.

[0384] "Bringing cells into contact with splicing modifiers" may include bringing cells into contact with splicing modifiers in cell culture, and this can be achieved by applying splicing modifiers to cells in culture.

[0385] This disclosure also provides a method for inhibiting the expression of a target polypeptide in cells. The method comprises contacting cells containing a polynucleotide or vector according to this disclosure that includes / encodes an OFF switch with a splicing modifier that promotes SMN2 exon 7 inclusion, as described herein.

[0386] Any suitable amount / concentration of splicing modifier may be used in the method of the present disclosure. It will be understood that the amount / concentration of splicing modifier is preferably selected to achieve the desired effect, namely increased inclusion of the SMN2 exon 7 variant nucleotide sequence in the splicing product of the relevant polyribonucleotide.

[0387] subject The subjects of various aspects of this disclosure may be any animal or human. Therapeutic and prophylactic uses may be in humans or animals (veterinary use).

[0388] Subjects administered with the articles of this disclosure (e.g., in accordance with therapeutic or prophylactic interventions) may be subjects requiring such interventions. Subjects are preferably mammals, more preferably humans. Subjects may be non-human mammals, but more preferably humans. Subjects may be male or female. Subjects may be patients.

[0389] Subjects may have (for example, have been diagnosed with) any of the diseases or conditions described herein, may be suspected of having such a disease / condition, or may be at risk of developing / experiencing such a disease / condition. In embodiments of this disclosure, subjects may be selected for treatment by a method based on the characterization of one or more markers of such disease / condition.

[0390] kit This disclosure also provides a kit of parts.

[0391] In some aspects and embodiments, the kit of parts according to the Disclosure comprises (i) a polynucleotide, vector, or pharmaceutical composition according to the Disclosure, and (ii) a splicing modifier that promotes SMN2 exon 7 inclusion.

[0392] The kit of parts according to this disclosure may include predetermined amounts of the articles described in (i) and / or (ii), as described in the preceding paragraph. In some embodiments, the articles according to (i) and / or (ii) are provided in a container (e.g., in a vial or bottle). The kit may provide the articles according to (i) and / or (ii) together with instructions (e.g., a protocol) on how to use them in accordance with the therapeutic or prophylactic intervention described herein.

[0393] In some embodiments, the kit of parts may include polynucleotides or vectors according to the Disclosure, and optionally, materials for introducing the polynucleotides / vectors into cells. In some embodiments, the kit of parts may include a system for producing cells according to the Disclosure. In some embodiments, the kit of parts may include a (closed) bag cell incubation system into which polynucleotides or vectors can be introduced into cells.

[0394] In some embodiments, the kit of parts may include materials for formulating the polynucleotide or vector according to the Disclosure into a pharmaceutical composition, such as a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0395] In some embodiments, the kit of parts further includes reagents, buffers, and / or standards necessary for carrying out the methods according to the Disclosure. The kit according to the Disclosure may include instructions for use, for example, in the form of an instruction booklet or leaflet. The instructions may include protocols for carrying out any one or more of the methods described herein.

[0396] The manufacture of the kit of parts according to this disclosure preferably follows standard procedures known to those skilled in the art.

[0397] Sequence identity The "sequence identity" between a given nucleotide sequence (for example, a polynucleotide) and a reference nucleotide sequence is calculated by aligning the sequences, introducing gaps as needed to achieve maximum percentage sequence identity between the two sequences, and then determining the percentage of nucleotides in the given nucleotide sequence that are identical to those in the reference nucleotide sequence. Similarly, the "sequence identity" between a given amino acid sequence (for example, a polypeptide) and a reference amino acid sequence is calculated by aligning the sequences, introducing gaps as needed to achieve maximum percentage sequence identity between the two sequences, and then determining the percentage of amino acids in the given amino acid sequence that are identical to those in the reference amino acid sequence.

[0398] Pairwise and multiple sequence alignments for the purpose of evaluating sequence identity between two or more nucleotide or amino acid sequences can be achieved using various methods known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega (Soding, J.2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al. 2000, J.Mol.Biol.(2000)302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)), and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4)772-780). When using such software, it is preferable to use default parameters, for example, gap penalty and expansion penalty.

[0399] [Table 1] TIFF2026522319000003.tif255170 TIFF2026522319000004.tif255170 TIFF2026522319000005.tif255170 TIFF2026522319000006.tif255170 TIFF2026522319000007.tif255170 TIFF2026522319000008.tif255170 TIFF2026522319000009.tif255170 TIFF2026522319000010.tif255170 TIFF2026522319000011.tif255170 TIFF2026522319000012.tif255170 TIFF2026522319000013.tif255170 TIFF2026522319000014.tif255170 TIFF2026522319000015.tif255170 TIFF2026522319000016.tif255170 TIFF2026522319000017.tif255170 TIFF2026522319000018.tif255170 TIFF2026522319000019.tif255170 TIFF2026522319000020.tif255170 TIFF2026522319000021.tif255170 TIFF2026522319000022.tif255170 TIFF2026522319000023.tif255170 TIFF2026522319000024.tif255170 TIFF2026522319000025.tif255170 TIFF2026522319000026.tif255170 TIFF2026522319000027.tif255170 TIFF2026522319000028.tif255170 TIFF2026522319000029.tif255170 TIFF2026522319000030.tif255170 TIFF2026522319000031.tif255170 TIFF2026522319000032.tif255170 TIFF2026522319000033.tif255170 TIFF2026522319000034.tif255170 TIFF2026522319000035.tif255170 TIFF2026522319000036.tif255170 TIFF2026522319000037.tif255170 TIFF2026522319000038.tif255170 TIFF2026522319000039.tif255170 TIFF2026522319000040.tif255170 TIFF2026522319000041.tif255170 TIFF2026522319000042.tif255170 TIFF2026522319000043.tif255170 TIFF2026522319000044.tif255170 TIFF2026522319000045.tif229170

[0400] Numbered descriptions The following numbered paragraphs describe specific aspects and embodiments of this disclosure.

[0401] A polynucleotide containing the following from 1.5' to 3', (i) A first nucleotide sequence comprising a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in Sequence ID No. 222, but not comprising Sequence ID No. 2; (ii) A second nucleotide sequence comprising fewer than 1044 nucleotides, comprising a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 7 at its 5' end and a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in SEQ ID NO: 378 at its 3' end; (iii) A third nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in Sequence ID No. 226; (iv) A fourth nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in SEQ ID NO: 380 at its 5' end, and having at least 80% sequence identity with the nucleotide sequence described in SEQ ID NO: 16 at its 3' end; (v)(a) comprising a dinucleotide "GA", "TG", or "TT", or (b) comprising a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in SEQ ID NO: 19, wherein the nucleotide sequence contains "GA", "TG", or "TT" at positions corresponding to positions 1 and 2 of SEQ ID NO: 19, or (c) a fifth nucleotide sequence encoding the target polypeptide and containing "GA", "TG", or "TT" at positions 1 and 2; and (vi) If the fifth nucleotide sequence is the nucleotide sequence described in (v)(a) or (v)(b), then the sixth nucleotide sequence encoding the polypeptide of interest; Includes, A polynucleotide containing a start codon at the 5' end of the nucleotide sequence encoding the target polypeptide.

[0402] 2. The polynucleotide according to para1, wherein, if the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes the inclusion of SMN2 exon 7 results in a polyribonucleotide substantially lacking a third nucleotide sequence.

[0403] 3. A polynucleotide according to para1 or para2, wherein the first nucleotide sequence includes the nucleotide sequence described in SEQ ID NO: 222.

[0404] 4. A polynucleotide according to any one of paras1 to 3, wherein the first nucleotide sequence includes or consists of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 106, SEQ ID NO: 219, or SEQ ID NO: 220.

[0405] 5. A polynucleotide as described in any one of paras1 to 4, wherein the second nucleotide sequence contains SEQ ID NO: 7 at its 5' end and SEQ ID NO: 8 or SEQ ID NO: 377 at its 3' end.

[0406] 6. A polynucleotide described in any one of paras1 to 5, wherein the second nucleotide sequence consists of fewer than 500 nucleotides.

[0407] 7. A polynucleotide as described in any one of paras1 to 6, wherein the second nucleotide sequence includes or consists of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 228.

[0408] 8. A polynucleotide described in any one of paras1 to 7, wherein the third nucleotide sequence consists of the nucleotide sequence described in Sequence ID No. 226.

[0409] 9. A polynucleotide described in any one of paras1 to 8, wherein the third nucleotide sequence consists of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 223, or SEQ ID NO: 224.

[0410] 10. A polynucleotide as described in any one of paras1 to 9, wherein the fourth nucleotide sequence contains SEQ ID NO: 15 or SEQ ID NO: 379 at its 5' end and SEQ ID NO: 16 at its 3' end.

[0411] 11. A polynucleotide described in any one of paras1 to 10, wherein the fourth nucleotide sequence consists of fewer than 500 nucleotides.

[0412] 12. A polynucleotide as described in any one of paras1 to 11, wherein the fourth nucleotide sequence includes or consists of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 227, or SEQ ID NO: 340.

[0413] 13. A polynucleotide according to any one of paras1 to 12, wherein the fifth nucleotide sequence consists of (a) a dinucleotide of "GA" or "TG", or (b) a nucleotide sequence described in Sequence ID No. 19, or consists of such a sequence.

[0414] 14. A polynucleotide as described in any one of paras1 to 13, wherein the fifth nucleotide sequence consists of (a) a dinucleotide of "GA" or "TG", or (b) includes or consists of SEQ ID NO: 20 or SEQ ID NO: 21.

[0415] 15. A polynucleotide according to any one of paras1 to 14, comprising a nucleotide sequence having at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs. 22, 23, 24, 25, 111, 114, 116, 164, and 171.

[0416] 16. A polynucleotide described in any one of paras1 to 15, further comprising a promoter sequence at the 5' end relative to the start codon.

[0417] 17. A polynucleotide as described in any one of paras1 to 16, further comprising a polyadenylation sequence at the 3' end of the nucleotide sequence encoding the target polypeptide.

[0418] 18. A polynucleotide described in any one of paras1 to 17, having an inverted terminal repeat (ITR) sequence at its 5' end and an ITR sequence at its 3' end.

[0419] A vector containing one of the polynucleotides described in 19.paras1-18.

[0420] 20. The adeno-associated virus (AAV) vector described in para19.

[0421] 21. A pharmaceutical composition comprising a polynucleotide described in any one of paras1 to 18, or a vector described in para19 or para20, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0422] 22. A cell containing a polynucleotide described in any one of paras1 to 18, or a vector described in para19 or para20.

[0423] 23. The cells described in para22, further comprising a splicing modifier that promotes SMN2 exon 7 inclusion, optionally, the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

[0424] 24. A method for modifying cells to express a target polypeptide, (i) introducing a polynucleotide described in any one of paras1-18, or a vector described in para19 or para20, into cells; and (ii) A method comprising subsequently contacting cells with a splicing modifier that promotes SMN2 exon 7 inclusion, wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

[0425] 25. A method for expressing a target polypeptide in cells, comprising contacting the cells described in para22 with a splicing modifier that promotes SMN2 exon 7 inclusion, wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

[0426] 26. A polynucleotide according to any one of paras1 to 18, a vector according to para19 or para20, or a pharmaceutical composition according to para21, for use in a method of medical treatment or prevention.

[0427] 27. A polynucleotide according to any one of paras1 to 18, a vector according to para19 or para20, or a pharmaceutical composition according to para21, for use in treating or preventing a disease or condition in which a therapeutic or preventive benefit is obtained from an increase in the expression level of the target polypeptide.

[0428] 28. Use of a polynucleotide described in any one of paras1 to 18, a vector described in para19 or para20, or a pharmaceutical composition described in para21 in the manufacture of a pharmaceutical for treating or preventing a disease or condition in which a therapeutic or preventive benefit is obtained from an increase in the expression level of a target polypeptide.

[0429] 29. A method for treating or preventing a disease or condition for which a therapeutic or preventive benefit is obtained from an increase in the expression level of a target polypeptide, comprising administering a polynucleotide described in any one of paras1 to 18, a vector described in para19 or para20, or a pharmaceutical composition described in para21 to a target.

[0430] 30. A polynucleotide, vector, or pharmaceutical composition for use as described in para27, or as described in para28, or as described in para29, for use as described in para29, further comprising administering a splicing modifier that promotes SMN2 exon 7 inclusion to treat or prevent a disease or symptom, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

[0431] 31. A polynucleotide, vector, or pharmaceutical composition for use as described in para.27 or para.30, for use as described in para.28 or para.30, or the method as described in para.29 or para.30, for use in which the disease or symptom is characterized by a deficiency of the polypeptide of interest.

[0432] 32. It is a kit, (i) a polynucleotide described in any one of paras1 to 18, a vector described in para19 or para20, or a pharmaceutical composition described in para21, (ii) A kit comprising a splicing modifier that promotes SMN2 exon 7 inclusion, wherein optionally the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

[0433] A polynucleotide containing the following from 33.5' to 3', (i) A first nucleotide sequence comprising a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in SEQ ID NO: 1, but not comprising SEQ ID NO: 2; (ii) A second nucleotide sequence comprising fewer than 1044 nucleotides, comprising a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 7 at its 5' end and a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 8 at its 3' end; (iii) A third nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in Sequence ID No. 26, wherein the third nucleotide sequence contains "A" at the position corresponding to position 2 of Sequence ID No. 12; (iv) A fourth nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 15 at its 5' end and at least 80% sequence identity with SEQ ID NO: 16 at its 3' end; (v) a fifth nucleotide sequence consisting of (a) a trinucleotide "GAG" or (b) encoding the polypeptide of interest and containing "GAG" at positions 1-3; and (vi) If the fifth nucleotide sequence is the nucleotide sequence described in (v)(a), then the sixth nucleotide sequence encoding the target polypeptide. Includes, A polynucleotide containing a start codon at the 5' end of the nucleotide sequence encoding the target polypeptide.

[0434] 34. The polynucleotide described in para33, wherein, if the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes the inclusion of SMN2 exon 7 results in a polyribonucleotide substantially lacking a third nucleotide sequence.

[0435] 35. A polynucleotide according to para33 or para34, wherein the first nucleotide sequence includes the nucleotide sequence described in SEQ ID NO: 1.

[0436] 36. A polynucleotide as described in any one of paras33-35, wherein the first nucleotide sequence includes or consists of SEQ ID NO: 3.

[0437] 37. A polynucleotide as described in any one of paras33-36, wherein the second nucleotide sequence contains SEQ ID NO: 7 at its 5' end and SEQ ID NO: 8 at its 3' end.

[0438] 38. A polynucleotide described in any one of paras33-37, wherein the second nucleotide sequence consists of fewer than 500 nucleotides.

[0439] 39. A polynucleotide as described in any one of paras33-38, wherein the second nucleotide sequence includes or consists of sequence number 10.

[0440] 40. A polynucleotide described in any one of paras33-39, wherein the third nucleotide sequence consists of the nucleotide sequence described in SEQ ID NO: 26.

[0441] 41. A polynucleotide described in any one of paras33-40, wherein the third nucleotide sequence consists of either SEQ ID NO: 13 or SEQ ID NO: 27.

[0442] 42. A polynucleotide as described in any one of paras33 to 41, wherein the fourth nucleotide sequence contains SEQ ID NO: 15 at its 5' end and SEQ ID NO: 16 at its 3' end.

[0443] 43. A polynucleotide described in any one of paras33-42, wherein the fourth nucleotide sequence consists of fewer than 500 nucleotides.

[0444] 44. A polynucleotide as described in any one of paras33-43, wherein the fourth nucleotide sequence includes or consists of sequence number 17.

[0445] 45. A polynucleotide described in any one of paras33-44, having at least 80% sequence identity with SEQ ID NO: 28 or SEQ ID NO: 29.

[0446] 46. ​​A polynucleotide described in any one of paras33-45, further comprising a promoter sequence at the 5' end relative to the start codon.

[0447] 47. A polynucleotide as described in any one of paras33 to 46, further comprising a polyadenylation sequence at the 3' end of the nucleotide sequence encoding the target polypeptide.

[0448] 48. A polynucleotide described in any one of paras33 to 47, having an inverted terminal repeat (ITR) sequence at its 5' end and an ITR sequence at its 3' end.

[0449] A vector containing one of the polynucleotides described in 49.paras33~48.

[0450] 50. The adeno-associated virus (AAV) vector, as described in para49.

[0451] A pharmaceutical composition comprising a polynucleotide described in any one of paragraphs 33 to 48, or a vector described in paragraph 49 or paragraph 50, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0452] 52. A cell containing a polynucleotide described in any one of paras33-48, or a vector described in para49 or para50.

[0453] 53. Cells as described in para52, further comprising a splicing modifier that promotes SMN2 exon 7 inclusion, wherein optionally the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

[0454] 54. A method for modifying cells to express a target polypeptide, comprising introducing a polynucleotide described in any one of paras33-48, or a vector described in para49 or para50, into cells.

[0455] 55. A method for inhibiting the expression of a target polypeptide in cells, comprising contacting the cells described in para. 52 with a splicing modifier that promotes SMN2 exon 7 inclusion, wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

[0456] 56. A polynucleotide according to any one of paras33-48, a vector according to para49 or para50, or a pharmaceutical composition according to para51, for use in a method of medical treatment or prevention.

[0457] 57. A polynucleotide according to any one of paras33-48, a vector according to para49 or para50, or a pharmaceutical composition according to para51, for use in treating or preventing a disease or condition for which a therapeutic or preventive benefit is obtained from an increase in the expression level of a target polypeptide.

[0458] 58. Use of a polynucleotide described in any one of paras33-48, a vector described in para49 or para50, or a pharmaceutical composition described in para51 in the manufacture of a pharmaceutical for treating or preventing a disease or condition in which a therapeutic or preventive benefit is obtained from an increase in the expression level of a target polypeptide.

[0459] 59. A method for treating or preventing a disease or condition for which a therapeutic or preventive benefit is obtained from an increase in the expression level of a target polypeptide, comprising administering to a target polynucleotide described in any one of paras33 to 48, a vector described in para49 or para50, or a pharmaceutical composition described in para51.

[0460] 60. A polynucleotide, vector, or pharmaceutical composition for use as described in para. 57, for use as described in para. 58, or by method as described in para. 59, for a disease or condition characterized by a deficiency of the polypeptide of interest.

[0461] 61. It is a kit, (i) A polynucleotide described in any one of paras33 to 48, a vector described in para49 or para50, or a pharmaceutical composition described in para51, (ii) A kit comprising a splicing modifier that promotes SMN2 exon 7 inclusion, wherein optionally the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

[0462] 1A. A polynucleotide containing the following from 5' to 3', (i) A first nucleotide sequence comprising a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in SEQ ID NO: 1, but not comprising SEQ ID NO: 2; (ii) A second nucleotide sequence comprising fewer than 1044 nucleotides, comprising a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 7 at its 5' end and a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 8 at its 3' end; (iii) A third nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in SEQ ID NO: 11, wherein the third nucleotide sequence includes "A" at the position corresponding to position 2 of SEQ ID NO: 12 and includes an insertion of "A" immediately after the position corresponding to position 48 of SEQ ID NO: 12; (iv) A fourth nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 15 at its 5' end and at least 80% sequence identity with SEQ ID NO: 16 at its 3' end; (v)(a) comprising a dinucleotide "GA", "TG", or "TT", or (b) comprising a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in SEQ ID NO: 19, wherein the nucleotide sequence contains "GA", "TG", or "TT" at positions corresponding to positions 1 and 2 of SEQ ID NO: 19, or (c) a fifth nucleotide sequence encoding the target polypeptide and containing "GA", "TG", or "TT" at positions 1 and 2; and (vi) If the fifth nucleotide sequence is the nucleotide sequence described in (v)(a) or (v)(b), then the sixth nucleotide sequence encoding the polypeptide of interest; Includes, A polynucleotide containing a start codon at the 5' end of the nucleotide sequence encoding the target polypeptide.

[0463] 2A. The polynucleotide described in para1A, wherein, if the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes the inclusion of SMN2 exon 7 results in a polyribonucleotide substantially lacking a third nucleotide sequence.

[0464] 3A. A polynucleotide according to para1A or para2A, wherein the first nucleotide sequence includes the nucleotide sequence described in SEQ ID NO: 1.

[0465] 4A. A polynucleotide as described in any one of paras1A to 3A, wherein the first nucleotide sequence includes or consists of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0466] 5A. A polynucleotide described in any one of paras1A to 4A, wherein the second nucleotide sequence contains SEQ ID NO: 7 at its 5' end and SEQ ID NO: 8 at its 3' end.

[0467] 6A. A polynucleotide described in any one of paras1A to 5A, in which the second nucleotide sequence consists of fewer than 500 nucleotides.

[0468] 7A. A polynucleotide as described in any one of paras1A to 6A, wherein the second nucleotide sequence includes or consists of SEQ ID NO: 9 or SEQ ID NO: 10.

[0469] 8A. A polynucleotide described in any one of paras1A to 7A, wherein the third nucleotide sequence consists of the nucleotide sequence described in Sequence ID No. 11.

[0470] 9A. A polynucleotide described in any one of paras1A to 8A, wherein the third nucleotide sequence consists of SEQ ID NO: 13 or SEQ ID NO: 14.

[0471] 10A. A polynucleotide as described in any one of paras1A to 9A, wherein the fourth nucleotide sequence contains SEQ ID NO: 15 at its 5' end and SEQ ID NO: 16 at its 3' end.

[0472] 11A. A polynucleotide described in any one of paras1A to 10A, wherein the fourth nucleotide sequence consists of fewer than 500 nucleotides.

[0473] 12A. A polynucleotide as described in any one of paras1A to 11A, wherein the fourth nucleotide sequence includes or consists of SEQ ID NO: 17 or SEQ ID NO: 18.

[0474] 13A. A polynucleotide as described in any one of paras1A to 12A, wherein the fifth nucleotide sequence consists of (a) the dinucleotide "GA" or (b) the nucleotide sequence described in Sequence ID No. 19, or consists of the same.

[0475] 14A. A polynucleotide as described in any one of paras1A to 13A, wherein the fifth nucleotide sequence consists of (a) the dinucleotide "GA" or (b) sequence number 20 or sequence number 21, or consists of either.

[0476] 15A. A polynucleotide according to any one of paras1A to 14A, comprising a nucleotide sequence having at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs. 22, SEQ ID NOs. 23, SEQ ID NOs. 24, and SEQ ID NOs.

[0477] 16A. A polynucleotide described in any one of paras1A to 15A, further comprising a promoter sequence 5' relative to the start codon.

[0478] 17A. A polynucleotide as described in any one of paras1A to 16A, further comprising a polyadenylation sequence at the 3' end of the nucleotide sequence encoding the target polypeptide.

[0479] 18A. A polynucleotide described in any one of paras1A to 17A, which contains an inverted terminal repeat (ITR) sequence at its 5' end and an ITR sequence at its 3' end.

[0480] A vector containing one of the polynucleotides described in 19A.paras1A to 18A.

[0481] 20A. An adeno-associated virus (AAV) vector, as described in para19A.

[0482] A pharmaceutical composition comprising a polynucleotide described in any one of 21A.paras1A to 18A, or a vector described in para19A or para20A, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0483] Cells containing a polynucleotide described in any one of 22A.paras1A~18A, or a vector described in para19A or para20A.

[0484] 23A. The cells described in para22A, further comprising a splicing modifier that promotes SMN2 exon 7 inclusion, wherein optionally the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

[0485] 24A. A method for modifying cells to express a target polypeptide, (i) introducing a polynucleotide described in any one of paras1A to 18A, or a vector described in para19A or para20A, into cells; and (ii) A method comprising subsequently contacting cells with a splicing modifier that promotes SMN2 exon 7 inclusion, wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

[0486] 25A. A method for expressing a target polypeptide in cells, comprising contacting the cells described in para22A with a splicing modifier that promotes SMN2 exon 7 inclusion, wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

[0487] 26A. A polynucleotide according to any one of paras1A to 18A, a vector according to para19A or para20A, or a pharmaceutical composition according to para21A, for use in a method of medical treatment or prevention.

[0488] 27A. A polynucleotide according to any one of paras1A to 18A, a vector according to para19A or para20A, or a pharmaceutical composition according to para21A, for use in treating or preventing a disease or condition in which a therapeutic or preventive benefit is obtained from an increase in the expression level of a target polypeptide.

[0489] 28A. Use of a polynucleotide described in any one of paras1A to 18A, a vector described in para19A or para20A, or a pharmaceutical composition described in para21A in the manufacture of a pharmaceutical for treating or preventing a disease or condition in which a therapeutic or preventive benefit is obtained from an increase in the expression level of a target polypeptide.

[0490] 29A. A method for treating or preventing a disease or condition for which a therapeutic or preventive benefit is obtained from an increase in the expression level of a target polypeptide, comprising administering a polynucleotide described in any one of paras1A to 18A, a vector described in para19A or para20A, or a pharmaceutical composition described in para21A to para21A to a target.

[0491] 30A. A polynucleotide, vector, or pharmaceutical composition for use as described in para27A, for use as described in para28A, or as described in para29A, further comprising administering a splicing modifier that promotes SMN2 exon 7 inclusion to a target, optionally wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

[0492] 31A. A polynucleotide, vector, or pharmaceutical composition for use as described in para27A or para30A, for use as described in para28A or para30A, or as described in para29A or para30A, for use in which the disease or condition is characterized by a deficiency of the polypeptide of interest.

[0493] 32A. Kit, (i) a polynucleotide described in any one of paras1A to 18A, a vector described in para19A or para20A, or a pharmaceutical composition described in para21A, (ii) A kit comprising a splicing modifier that promotes SMN2 exon 7 inclusion, wherein optionally the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

[0494] 33A. A polynucleotide containing the following from 5' to 3', (i) A first nucleotide sequence comprising a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in SEQ ID NO: 1, but not comprising SEQ ID NO: 2; (ii) A second nucleotide sequence comprising fewer than 1044 nucleotides, comprising a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 7 at its 5' end and a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 8 at its 3' end; (iii) A third nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in Sequence ID No. 26, wherein the third nucleotide sequence contains "A" at the position corresponding to position 2 of Sequence ID No. 12; (iv) A fourth nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 15 at its 5' end and at least 80% sequence identity with SEQ ID NO: 16 at its 3' end; (v) a fifth nucleotide sequence consisting of (a) a trinucleotide "GAG" or (b) encoding the polypeptide of interest and containing "GAG" at positions 1-3; and (vi) If the fifth nucleotide sequence is the nucleotide sequence described in (v)(a), then the sixth nucleotide sequence encoding the target polypeptide. Includes, A polynucleotide containing a start codon at the 5' end of the nucleotide sequence encoding the target polypeptide.

[0495] 34A. The polynucleotide described in para33A, wherein, if the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes the inclusion of SMN2 exon 7 results in a polyribonucleotide substantially lacking a third nucleotide sequence.

[0496] 35A. A polynucleotide according to para33A or para34A, wherein the first nucleotide sequence includes the nucleotide sequence described in SEQ ID NO: 1.

[0497] 36A. A polynucleotide as described in any one of paras33A to 35A, wherein the first nucleotide sequence contains or consists of SEQ ID NO: 3.

[0498] 37A. A polynucleotide as described in any one of paras33A to 36A, wherein the second nucleotide sequence contains SEQ ID NO: 7 at its 5' end and SEQ ID NO: 8 at its 3' end.

[0499] 38A. A polynucleotide described in any one of paras33A to 37A, wherein the second nucleotide sequence consists of fewer than 500 nucleotides.

[0500] 39A. A polynucleotide as described in any one of paras33A to 38A, wherein the second nucleotide sequence contains or consists of sequence number 10.

[0501] 40A. A polynucleotide described in any one of paras33A to 39A, wherein the third nucleotide sequence consists of the nucleotide sequence described in Sequence ID No. 26.

[0502] 41A. A polynucleotide described in any one of paras33A to 40A, wherein the third nucleotide sequence consists of either SEQ ID NO: 13 or SEQ ID NO: 27.

[0503] 42A. A polynucleotide as described in any one of paras33A to 41A, wherein the fourth nucleotide sequence contains SEQ ID NO: 15 at its 5' end and SEQ ID NO: 16 at its 3' end.

[0504] 43A. A polynucleotide described in any one of paras33A to 42A, wherein the fourth nucleotide sequence consists of fewer than 500 nucleotides.

[0505] 44A. A polynucleotide as described in any one of paras33A to 43A, wherein the fourth nucleotide sequence contains or consists of SEQ ID NO: 17.

[0506] 45A. A polynucleotide described in any one of paras33A to 44A, having at least 80% sequence identity with SEQ ID NO: 28 or SEQ ID NO: 29.

[0507] 46A. A polynucleotide described in any one of paras33A to 45A, further comprising a promoter sequence 5' relative to the start codon.

[0508] 47A. A polynucleotide as described in any one of paras33A to 46A, further comprising a polyadenylation sequence at the 3' end of the nucleotide sequence encoding the target polypeptide.

[0509] 48A. A polynucleotide described in any one of paras33A to 47A, which contains an inverted terminal repeat (ITR) sequence at its 5' end and an ITR sequence at its 3' end.

[0510] A vector containing one of the polynucleotides described in 49A.paras33A~48A.

[0511] 50A. An adeno-associated virus (AAV) vector, as described in para49A.

[0512] A pharmaceutical composition comprising a polynucleotide described in any one of 51A.paras33A~48A, or a vector described in para49A or para50A, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0513] A cell containing a polynucleotide described in any one of 52A.paras33A~48A, or a vector described in para49A or para50A.

[0514] 53A. Cells as described in para52A, further comprising a splicing modifier that promotes SMN2 exon 7 inclusion, wherein optionally the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

[0515] 54A. A method for modifying cells to express a target polypeptide, comprising introducing a polynucleotide described in any one of paras33A to 48A, or a vector described in para49A or para50A, into cells.

[0516] 55A. A method for inhibiting the expression of a target polypeptide in cells, comprising contacting the cells described in para52A with a splicing modifier that promotes SMN2 exon 7 inclusion, wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

[0517] 56A. A polynucleotide according to any one of paras33A to 48A, a vector according to para49A or para50A, or a pharmaceutical composition according to para51A, for use in a method of medical treatment or prevention.

[0518] 57A. A polynucleotide according to any one of paras33A to 48A, a vector according to para49A or para50A, or a pharmaceutical composition according to para51A, for use in treating or preventing a disease or condition in which a therapeutic or preventive benefit is obtained from an increase in the expression level of a target polypeptide.

[0519] 58A. Use of a polynucleotide described in any one of paras33A to 48A, a vector described in para49A or para50A, or a pharmaceutical composition described in para51A in the manufacture of a pharmaceutical for treating or preventing a disease or condition in which a therapeutic or preventive benefit is obtained from an increase in the expression level of a target polypeptide.

[0520] 59A. A method for treating or preventing a disease or condition for which a therapeutic or preventive benefit is obtained from an increase in the expression level of a target polypeptide, comprising administering a polynucleotide described in any one of paras33A to 48A, a vector described in para49A or para50A, or a pharmaceutical composition described in para51A to a target.

[0521] 60A. A polynucleotide, vector, or pharmaceutical composition for use as described in para57A, for use as described in para58A, or as described in para59A, for use in which the disease or condition is characterized by a deficiency of the polypeptide of interest.

[0522] 61A. Kit, (i) a polynucleotide described in any one of paras33A to 48A, a vector described in para49A or para50A, or a pharmaceutical composition described in para51A, (ii) A kit comprising a splicing modifier that promotes SMN2 exon 7 inclusion, wherein optionally the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

[0523] [Table 2] TIFF2026522319000047.tif255170 TIFF2026522319000048.tif255170 TIFF2026522319000049.tif255170 TIFF2026522319000050.tif255170 TIFF2026522319000051.tif198170

[0524] This disclosure includes combinations of the described embodiments and preferred features, unless such combinations are clearly unacceptable or expressly avoided.

[0525] Section headings used in this specification are for structural purposes only and should not be construed as limiting the subjects described herein.

[0526] Herein, aspects and embodiments of the present disclosure will be described by reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents referenced herein are incorporated herein by reference.

[0527] Throughout this specification, including the following claims, it will be understood that, unless context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” mean the inclusion of the described integer or process, or group of integers or processes, but not the exclusion of any other integer or process, or group of integers or processes.

[0528] As used herein, “peptide” refers to a chain of two or more amino acid monomers linked by peptide bonds. Peptides typically have a length of about 2 to 50 amino acids. “Polypeptide” is a polymer chain of two or more peptides. Polypeptides typically have a length of more than about 50 amino acids. References to peptides, polypeptides, and proteins herein also include glycopeptides / glycopeptides / glycoproteins, lipopeptides / lipopeptides / lipoproteins, nucleopeptides / nucleopolypeptides / nucleoproteins, etc.

[0529] As used herein, an amino acid sequence or polypeptide region that "corresponds" to a particular reference amino acid sequence or polypeptide region has at least 60% sequence identity with the amino acid sequence of the amino acid sequence / polypeptide / region, for example, at least ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%. The amino acid sequence / region / position of a polypeptide / amino acid sequence that "corresponds" to a particular reference amino acid sequence / region / position of the polypeptide / amino acid sequence can be identified by sequence alignment of the target sequence to the reference sequence using sequence alignment software such as ClustalOmega (Soding, J.2005, Bioinformatics 21, 951-960).

[0530] Similarly, a nucleotide sequence or polynucleotide region that "corresponds" to a particular reference nucleotide sequence or polynucleotide region has at least 60% sequence identity with the amino acid sequence of the nucleotide sequence / polynucleotide / region, for example, at least ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%. The polynucleotide / region / position of a polynucleotide / nucleotide sequence that "corresponds" to a specified reference nucleotide sequence / region / position of a polynucleotide / nucleotide sequence can be identified by sequence alignment of the target sequence relative to the reference sequence using sequence alignment software such as ClustalOmega (Soding, J.2005, Bioinformatics 21, 951-960).

[0531] As used herein, an amino acid sequence "derived" from a reference amino acid sequence (e.g., an amino acid sequence of a peptide / polypeptide / domain / region) consists of, or comprises, an amino acid sequence having at least 60% sequence identity with respect to the reference amino acid sequence, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Similarly, a nucleotide sequence "derived" from a reference nucleotide sequence (e.g., a nucleotide sequence of a polynucleotide) contains or consists of a nucleotide sequence having at least 60% sequence identity with respect to the reference nucleotide sequence, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0532] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless otherwise explicitly indicated by the context. Ranges may be expressed herein as “about” one particular value and / or “about” another particular value. Where such ranges are expressed, alternative embodiments include one particular value and / or other particular values. Similarly, the use of the antecedent “about” will be understood to mean that when a value is expressed as an approximation, a particular value forms an alternative embodiment.

[0533] Where nucleic acid sequences are disclosed herein, their reverse complements are also expressly intended.

[0534] The methods described herein may preferably be carried out in vitro. The term “in vitro” is intended to encompass procedures performed using cells in culture, while the term “in vivo” is intended to encompass procedures using / on intact multicellular organisms. [Brief explanation of the drawing]

[0535] Herein, embodiments and experiments demonstrating the principles of this disclosure will be described with reference to the accompanying drawings. [Figure 1]Schematic diagram of the in-frame ON switch design. The cassette containing the in-frame switch is constructed using the exon 6-intron 6-exon 7-intron 7-exon 8 region of human SMN2 and the coding sequence of the target gene (GOI) downstream of exon 8. The translation start site is at the 5' end of exon 6. Intron 6 has been modified to reduce its length, and exon 7 contains an insertion of nucleotide A after position 48. The sequence of exon 8 is only the first 23 nucleotides. In the absence of the ris diplam splicing modifier, exon 7 is excluded, resulting in a frameshift in the coding sequence of the GOI. Conversely, ris diplam treatment results in the inclusion of exon 7 and the correct translation of the GOI open reading frame and its protein production. The asterisk indicates the insertion of nucleotide "A" that causes the frameshift. [Figure 2] Small molecule-mediated expression of GFP from an in-frame ON switch 48 hours after treatment with a risdiplam analog. FACS-based measurement of GFP expression induced by risdiplam at increasing concentrations in HEK293 cells stably expressing the in-frame ON switch pLS41. The graph shows the median GFP expression at each risdiplam concentration 48 hours after risdiplam treatment, normalized to the median GFP expression in DMSO-treated control cells. [Figure 3]Analysis of risdiplam-induced reporter expression and background reporter expression (i.e., in the absence of risdiplam) for the ON-switching of pLS41 and pLS76. (A) Schematic diagram showing the changes in the modified (Mod) in-frame switch (i.e., pLS76) relative to the wild-type (WT) sequence of SMN2 intron 6 and exon 7. (BD) FACS-based measurement of GFP expression upon induction by risdiplam at increasing concentrations in HEK293 cells stably expressing pLS41 or pLS76 constructs. (B) Median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Data from Figure 3B shown as the median total GFP signal unnormalized to DMSO control. (D) Percentage of GFP-positive (%GFP+) cells divided by %GFP+ cells at the highest concentration of risdiplam treatment (1 μM). (E) Background level of median total GFP expression in wild-type HEK293 cells (WT) or cells expressing the indicated construct in the absence of risdiplam. (F) Results of RT-PCR analysis of splicing products performed on HEK293 cells stably expressing pLS76 and treated with low (100 nM) or high (1 μM) concentrations of risdiplam or DMSO. [Figure 4]Analysis of risdiplam-induced reporter expression and background reporter expression (i.e., in the absence of risdiplam) for the ON switches of pLS159 and pLS160, which have reduced intron 6 and intron 7 lengths compared to the ON switch of pLS76. (A) Schematic diagram of the differences between the ON switches of pLS76, pLS159, and pLS160. (BD) FACS-based measurement of GFP expression induced by escalating concentrations of risdiplam in HEK293 cells stably expressing pLS76, pLS159, or pLS160 constructs. (B) Median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Data from Figure 4B shown as the median total GFP signal unnormalized to the DMSO control. (D) The percentage of GFP-positive (%GFP+) cells is shown divided by the percentage of %GFP+ cells treated with the highest concentration of risdiplam (1 μM). (E) The background level of median total GFP expression in wild-type HEK293 cells (WT) or cells expressing the indicated construct is shown in the absence of risdiplam. [Figure 5]Analysis of risdiplam-induced reporter expression and background reporter expression (i.e., in the absence of risdiplam) for the ON switches of pLS167 and pLS168, which have reduced exon 6 length compared to the ON switch of pLS76. (A) Schematic diagram of the differences between the ON switches of pLS76, pLS167 and pLS168. Modified regions are marked with dashed boxes. (BD) FACS-based measurements of GFP expression upon induction with risdiplam at increasing concentrations in HEK293 cells stably expressing pLS76, pLS167, or pLS168 constructs. (B) Median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Data from Figure 5B shown as the median total GFP signal unnormalized to the DMSO control. (D) The percentage of GFP-positive (%GFP+) cells is shown divided by the percentage of %GFP+ cells treated with the highest concentration of risdiplam (1 μM). (E) The background level of median total GFP expression in wild-type HEK293 cells (WT) or cells expressing the indicated construct is shown in the absence of risdiplam. [Figure 6]Analysis of risdiplam-inducible reporter expression and background reporter expression (i.e., in the absence of risdiplam) for the ON switch of pLS179, which combines intron shortening of pLS159 and exon 6 shortening of pLS168. (A) Schematic diagram showing the changes of the modified (Mod) in-frame switch (i.e., pLS76) relative to the wild-type (WT) sequence of SMN2 intron 6 and exon 7. (BD) FACS-based measurement of GFP expression upon induction by risdiplam at increasing concentrations in HEK293 cells stably expressing pLS76, pLS159, pLS168, or pLS179 constructs. (B) Median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Data from Figure 6B shown as the median total GFP signal unnormalized to the DMSO control. (D) The percentage of GFP-positive (%GFP+) cells is shown divided by the percentage of %GFP+ cells treated with the highest concentration of risdiplam (1 μM). (E) The background level of median total GFP expression in wild-type HEK293 cells (WT) or cells expressing the indicated construct is shown in the absence of risdiplam. (F) Results of RT-PCR analysis of splicing products performed on HEK293 cells stably expressing pLS179 and treated with low (100 nM) or high (1 μM) concentrations of risdiplam or DMSO are shown. [Figure 7]Analysis of risdiplam-induced reporter expression and background reporter expression (i.e., in the absence of risdiplam) for ON switches of pMM70, pMM71, pMM72, pMM73, and pMM112, which have reduced exon 8 length compared to the ON switch of pLS179. (A) Schematic diagram of the differences between the ON switches of pLS179, pMM70, pMM71, pMM72, pMM73, and pMM112. (BD) FACS-based measurement of GFP expression induced by escalating concentrations of risdiplam in HEK293 cells stably expressing pLS179, pMM70, pMM71, pMM72, pMM73, or pMM112 constructs. (B) Median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) The data in Figure 7B is shown as the median total GFP signal without normalization relative to the DMSO control. (D) The percentage of GFP-positive (%GFP+) cells is shown divided by the percentage of %GFP+ cells treated with the highest concentration of risdiplam (1 μM). (E) Background levels of median total GFP expression in wild-type HEK293 cells (WT) or cells expressing the indicated construct are shown in the absence of risdiplam. [Figure 8]Analysis of risdiplam-induced reporter expression and background reporter expression (i.e., in the absence of risdiplam) for the ON switch of pMM130, which has the Kozak sequence and start codon present in pMM112 removed. (A) Schematic diagram of the differences in the ON switches of pMM112 and pMM130. (BD) FACS-based measurement of GFP expression induced by escalating concentrations of risdiplam in HEK293 cells stably expressing pLS179, pMM112, or pMM130 constructs. (B) Median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Data from Figure 8B shown as the median total GFP signal unnormalized to DMSO control. (D) Percentage of GFP-positive (%GFP+) cells divided by %GFP+ cells at the highest concentration of risdiplam treatment (1 μM). (E) Background level of median total GFP expression in wild-type HEK293 cells (WT) or cells expressing the indicated construct in the absence of risdiplam. [Figure 9]Analysis of risdiplam-inducible reporter expression and background reporter expression (i.e., in the absence of risdiplam) for pMM143, pMM144, pMM145, pMM146, and pMM147 ON switches, in which the ISS-N1 region of intron 7 is mutated to reduce leakability. (A) Schematic diagram of the differences between the ON switches of pLS179, pMM143, pMM144, pMM145, pMM146, and pMM147. (BD) FACS-based measurement of GFP expression induced by risdiplam at increasing concentrations in HEK293 cells stably expressing pLS179, pMM143, pMM144, pMM145, pMM146, or pMM147 constructs. (B) Median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) The data in Figure 9B is shown as the median total GFP signal without normalization relative to the DMSO control. (D) The percentage of GFP-positive (%GFP+) cells is shown divided by the percentage of %GFP+ cells treated with the highest concentration of risdiplam (1 μM). (E) Background levels of median total GFP expression in wild-type HEK293 cells (WT) or cells expressing the indicated construct are shown in the absence of risdiplam. [Figure 10]Analysis of risdiplam-inducible reporter expression and background reporter expression (i.e., in the absence of risdiplam) for the ON switches of pMM136, pMM137, pMM138, pMM139, pMM140, pMM141, and pMM142, in which the ESE2 region of exon 7 (marked in gray based on Sivaramakrishnan et al., Nat Commun 8, 1476 (2017)) is mutated to improve switch performance. (A) Schematic diagram of the differences in the ON switches of pLS179, pMM136, pMM137, pMM138, pMM139, pMM140, pMM141, and pMM142. (BD) FACS-based measurement of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pLS179, pMM136, pMM137, pMM138, pMM139, pMM140, pMM141, or pMM142 constructs. (B) Median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Data from Figure 10B shown as the median total GFP signal unnormalized to DMSO control. (D) Percentage of GFP-positive (%GFP+) cells divided by %GFP+ cells at the highest concentration of risdiplam treatment (1 μM). (E) Background level of median total GFP expression in the absence of risdiplam in wild-type HEK293 cells (WT) or cells expressing the indicated constructs. [Figure 11]Analysis of risdiplam-induced reporter expression and background reporter expression (i.e., in the absence of risdiplam) for the ON-switching of pMM151 and pMM152, including deletions in exon 7. (A) Schematic diagram of the differences in the ON-switching of pMM112, pMM151, and pMM152. (BD) FACS-based measurement of GFP expression induced by escalating concentrations of risdiplam in HEK293 cells stably expressing pMM112, pMM151, or pMM152 constructs. (B) Median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Data from Figure 11B shown as the median total GFP signal unnormalized to DMSO control. (D) Percentage of GFP-positive (%GFP+) cells divided by %GFP+ cells at the highest concentration of risdiplam treatment (1 μM). (E) Background level of median total GFP expression in wild-type HEK293 cells (WT) or cells expressing the indicated construct in the absence of risdiplam. [Figure 12]Analysis of risdiplam-inducible reporter expression and background reporter expression (i.e., in the absence of risdiplam) for pMM56, pMM59, pMM60, pMM61, pMM62, and pMM63 ON switches, in which the ATG codon in exon 6 is removed and modifications are included in exon 7. (A)(Ai)Schematic diagram of the differences between the ON switches of pLS76 and pLS174, and (Aii)pLS174, pMM56, pMM59, pMM60, pMM61, pMM62, and pMM63. (BD)FACS-based measurement of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pLS76, pMM56, pMM59, pMM60, pMM61, pMM62, and pMM63 constructs. (B) Median GFP expression at each risdiplam concentration, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Data from Figure 12B are shown as the median total GFP signal without normalization to DMSO control. (D) Percentage of GFP-positive (%GFP+) cells divided by the percentage of %GFP+ cells at the highest risdiplam concentration (1 μM). (E) Background level of median total GFP expression in wild-type HEK293 cells (WT) or cells expressing the indicated construct in the absence of risdiplam. [Figure 13]Analysis of risdiplam-inducible reporter expression and background reporter expression (i.e., in the absence of risdiplam) for the ON switches of pMM108, pMM110, and pMM111 with the ATG codon in exon 6 removed. (A) Schematic diagram of the differences in the ON switches of pLS76, pLS176, pMM59, pMM108, pMM110, and pMM111. Insertions into the sequence of pLS174 are shown in black and bold, and mutations into the sequence of pLS174 are shown in black, italics, and bold. (BD) FACS-based measurement of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing pLS176, pMM59, pMM108, pMM110, or pMM111 constructs. (B) Median GFP expression at each risdiplam concentration, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Data from Figure 13B are shown as the median total GFP signal without normalization to DMSO control. (D) Percentage of GFP-positive (%GFP+) cells divided by the percentage of %GFP+ cells at the highest concentration of risdiplam treatment (1 μM). (E) Background levels of median total GFP expression in the absence of risdiplam in wild-type HEK293 cells (WT) or cells expressing the indicated constructs. [Figure 14]Analysis of risdiplam-inducible reporter expression and background reporter expression (i.e., in the absence of risdiplam) for the ON switches of pMM242, pMM243, pMM244, pMM245, pMM246, and pMM247, in which the ATG codon is introduced into exon 7. Insertions into the pLS174 sequence are shown in black and bold, and mutations into the pLS174 sequence are shown in black, italics, and bold. (A) Schematic diagram of the differences in the ON switches of pLS174, pMM59, pMM242, pMM243, pMM244, pMM245, pMM246, and pMM247. (BD) FACS-based measurement of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM242, pMM243, pMM244, pMM245, pMM246, or pMM247 constructs. (B) Median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Data from Figure 14B shown as the median total GFP signal unnormalized to DMSO control. (D) Percentage of GFP-positive (%GFP+) cells divided by %GFP+ cells at the highest concentration of risdiplam treatment (1 μM). (E) Background level of median total GFP expression in the absence of risdiplam in wild-type HEK293 cells (WT) or cells expressing the indicated constructs. [Figure 15]Analysis of risdiplam-inducible reporter expression and background reporter expression (i.e., in the absence of risdiplam) for the ON switches of pMM123, pMM124, pMM125, pMM126, pMM127, pMM128, and pMM129, in which the ATG codon is introduced into exon 7 downstream of ESE2. (A) Schematic diagram of the differences in the ON switches of pLS175, pMM123, pMM124, pMM125, pMM126, pMM127, pMM128, and pMM129. Insertions into the sequence of pLS175 are shown in black and bold, and mutations into the sequence of pLS175 are shown in black, italics, and bold. (BD) FACS-based measurement of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM123, pMM124, pMM125, pMM126, pMM127, pMM128, or pMM129 constructs. (B) Median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Data from Figure 15B shown as the median total GFP signal unnormalized to DMSO control. (D) Percentage of GFP-positive (%GFP+) cells divided by %GFP+ cells at the highest concentration of risdiplam treatment (1 μM). (E) Background level of median total GFP expression in the absence of risdiplam in wild-type HEK293 cells (WT) or cells expressing the indicated constructs. [Figure 16]Analysis of risdiplam-inducible reporter expression and background reporter expression (i.e., in the absence of risdiplam) for the ON switches of pMM201, pMM202, pMM203, pMM204, pMM205, and pMM206, in which the ATG codon is introduced into exon 7 downstream of ESE2. (A) Schematic diagram of the differences in the ON switches of pLS175, pMM126, pMM201, pMM202, pMM203, pMM204, pMM205, and pMM206. Insertions relative to the comparison sequence are shown in black and bold, and mutations relative to the comparison sequence are shown in black, italics, and bold. (BD) FACS-based measurement of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing pMM126, pMM201, pMM202, pMM203, pMM204, pMM205, or pMM206 constructs. (B) Median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Data from Figure 16B shown as the median total GFP signal unnormalized to DMSO control. (D) Percentage of GFP-positive (%GFP+) cells divided by %GFP+ cells at the highest concentration of risdiplam treatment (1 μM). (E) Background level of median total GFP expression in the absence of risdiplam in wild-type HEK293 cells (WT) or cells expressing the indicated constructs. [Figure 17]Analysis of risdiplam-inducible reporter expression and background reporter expression (i.e., in the absence of risdiplam) for the ON switches of pMM248, pMM249, pMM250, and pMM251, in which the ATG codon is introduced into exon 7 downstream of ESE2. (A) Schematic diagram of the differences in the ON switches of pLS175, pMM129, pMM248, pMM249, pMM250, and pMM251. Insertions into the comparison sequence are shown in black and bold, and mutations into the comparison sequence are shown in black, italics, and bold. (BD) FACS-based measurement of GFP expression upon induction by risdiplam at increasing concentrations in HEK293 cells stably expressing the pMM129, pMM248, pMM249, pMM250, and pMM251 constructs. (B) Median GFP expression at each risdiplam concentration, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Data from Figure 17B are shown as the median total GFP signal without normalization to DMSO control. (D) Percentage of GFP-positive (%GFP+) cells divided by the percentage of %GFP+ cells at the highest concentration of risdiplam treatment (1 μM). (E) Background levels of median total GFP expression in wild-type HEK293 cells (WT) or cells expressing the indicated constructs in the absence of risdiplam. [Figure 18]Analysis of risdiplam-inducible reporter expression and background reporter expression (i.e., in the absence of risdiplam) for the ON-switching of pMM252, pMM254, pMM255, pMM256, and pMM257, in which the ATG codon is introduced into exon 7 downstream of ESE2. (A) Schematic diagram of the differences in ON-switching for pMM130, pMM252, pMM254, pMM255, pMM256, and pMM257. (BD) FACS-based measurement of GFP expression upon induction by risdiplam at increasing concentrations in HEK293 cells stably expressing pMM126, pMM129, pMM130, pMM252, pMM254, pMM255, pMM256, or pMM257 constructs. (B) Median GFP expression at each risdiplam concentration, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Data from Figure 18B are shown as the median total GFP signal without normalization to DMSO control. (D) Percentage of GFP-positive (%GFP+) cells divided by the percentage of %GFP+ cells at the highest concentration of risdiplam treatment (1 μM). (E) Background levels of median total GFP expression in wild-type HEK293 cells (WT) or cells expressing the indicated constructs in the absence of risdiplam. [Figure 19]Analysis of risdiplam-inducible reporter expression and background reporter expression (i.e., in the absence of risdiplam) for the ON-switch of pMM198, pMM199, and pMM200 with ATG codons introduced across exons 7 and 8. (A) Schematic diagram of the differences in the ON-switch of pLS76, pLS175, pMM198, pMM199, and pMM200. Start codons spanning exons 7 and 8 are shown in bold and italics. (B) Schematic diagram of the predicted splicing of split-start-codon ON-switch in the absence of risdiplam (DMSO control) or in the presence of risdiplam. (CE) FACS-based measurement of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing pMM112, pMM198, pMM199, or pMM200 constructs. (C) Median GFP expression at each risdiplam concentration, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (D) Data from Figure 19C are shown as the median total GFP signal without normalization to DMSO control. (E) Percentage of GFP-positive (%GFP+) cells divided by the percentage of %GFP+ cells at the highest concentration of risdiplam treatment (1 μM). (F) Background level of median total GFP expression in wild-type HEK293 cells (WT) or cells expressing the indicated construct in the absence of risdiplam. [Figure 20]Analysis of risdiplam-inducible reporter expression and background reporter expression (i.e., in the absence of risdiplam) for the ON-switching of pMM236, pMM237, and pMM238, in which the ATG codon in exon 6 is removed, a weak Kozak sequence is introduced in exon 7, and the ATG codon is introduced in exon 8. (A) Schematic diagram of the differences in ON-switching of pLS76, pMM236, pMM237, and pMM238. (B) Schematic diagram of the predicted splicing of ON-switching of pMM236, pMM237, and pMM238 in the absence of risdiplam (DMSO control) or in the presence of risdiplam. (CE) FACS-based measurement of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing pMM112, pMM236, pMM237, or pMM238 constructs. (C) Median GFP expression at each risdiplam concentration, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (D) Data from Figure 20C are shown as the median total GFP signal without normalization to DMSO control. (E) Percentage of GFP-positive (%GFP+) cells divided by the percentage of %GFP+ cells at the highest concentration of risdiplam treatment (1 μM). (F) Background level of median total GFP expression in wild-type HEK293 cells (WT) or cells expressing the indicated construct in the absence of risdiplam. [Figure 21]Analysis of constitutive reporter expression (i.e., in the absence of risdiplam) for the OFF switches of pMM193 and pMM194, and risdiplam-inducible repression of reporter expression. (A) Schematic diagram of splicing of ON and OFF switches derived from SMN2 exons 6-8. (B) Schematic diagram of the difference between the pMM130 ON switch and the pMM193 OFF switch. (C) Schematic diagram of the difference between the OFF switches of pMM193 and pMM194. (DF) FACS-based measurement of GFP expression when HEK293 cells stably expressing pMM130, pMM193, or pMM194 constructs were treated with escalating concentrations of risdiplam. (D) Median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (E) Data from Figure 21D are shown as the median total GFP signal without normalization to the DMSO control. (F) The percentage of GFP-positive (%GFP+) cells is shown divided by the percentage of %GFP+ cells treated with the highest concentration of risdiplam (1 μM). (G) The median level of total GFP expression in wild-type HEK293 cells (WT) or in cells expressing the indicated construct is shown in the absence of risdiplam. [Figure 22]Analysis of risdiplam-inducible reporter expression and background reporter expression (i.e., in the absence of risdiplam) after replacing the IRES-blastosidine sequence with a BGH polyA signal for in-frame switch pMM259 (pMM112), pMM260 (pMM130), pMM262 (pLS168), pMM263 (pLS76), pMM272 (pMM198), and the Monteys et al. Nature (2021) 596:291-295 construct (pMM274). (A) Schematic diagram of plasmid skeleton modification. The IRES-blastosidine-SV40 sequence was replaced with a BGH polyA signal. The in-frame switch sequence remained unchanged. (B) A list of in-frame switches used in the experiment and their original numbers (CE). FACS-based measurements of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing constructs (pMM274) and Monteys et al. Nature (2021) 596:291-295. (C) The induction rate is calculated as the median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells and GFP-positive cells 48 hours after risdiplam treatment. (D) Figure 19C data is shown as the median total GFP signal without normalization relative to the DMSO control. (E) The percentage of GFP-positive (%GFP+) cells is shown divided by the percentage of %GFP+ cells at the highest concentration of risdiplam treatment (1 μM). (F) Background level of median total GFP expression in wild-type HEK293 cells (WT) or cells expressing the indicated construct in the absence of risdiplam. (G) Fragment of Figure 22C is shown, where the induction rate at a risdiplam concentration of 125 nM is calculated as the median GFP expression, normalized to the median GFP expression in DMSO-treated control cells and GFP-positive cells 48 hours after risdiplam treatment. [Figure 23]Analysis of risdiplam-induced GFP reporter expression and background GFP reporter expression (i.e., in the absence of risdiplam) for pMM259, pMM263, pMM273, and pMM274. (A) Schematic diagram of the differences in ON-switching between pMM259 and pMM273. (B) Schematic diagram of predicted splicing of split start codon ON-switching in the absence of risdiplam (DMSO control) or in the presence of risdiplam. (CE) FACS-based measurement of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing pMM259, pMM263, pMM273, and pMM274 constructs. (C) Shows the induction of GFP expression calculated as the median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (D) Shows the median total GFP signal of GFP-positive cells that are not normalized to DMSO control. (E) The percentage of GFP-positive (%GFP+) cells at each risdiplam concentration is shown divided by the percentage of %GFP+ cells at the highest risdiplam concentration (1 μM). (F) The background level of median total GFP expression in wild-type HEK293 cells (WT) or cells expressing the indicated construct is shown in the absence of risdiplam. [Figure 24]Analysis of risdiplam-induced GFP reporter expression and background GFP reporter expression (i.e., in the absence of risdiplam) for pMM273, pMM362, pMM363, and pMM364. (A) Schematic diagram of the ON-switch differences for pMM273, pMM362, pMM363, and pMM364. (BF) FACS-based measurement of GFP expression upon induction by risdiplam at increasing concentrations in HEK293 cells stably expressing the pMM273, pMM362, pMM363, and pMM364 constructs. (B) Shows the induction of GFP expression calculated as the median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Shows the median total GFP signal of GFP-positive cells that are not normalized to DMSO control. (D) The percentage of GFP-positive (%GFP+) cells at each risdiplam concentration is shown divided by the percentage of %GFP+ cells at the highest risdiplam concentration (1 μM). (E) The background level of median total GFP expression in the absence of risdiplam in wild-type HEK293 cells (WT) or in cells expressing the indicated construct. (F) The induction of GFP expression calculated as the median GFP expression at a 250 nM concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. [Figure 25]Analysis of risdiplam-induced GFP reporter expression and background GFP reporter expression (i.e., in the absence of risdiplam) for pMM273, pMM358, pMM359, pMM360, and pMM361. (A) Schematic diagram of the differences in ON-switching for pMM273, pMM358, pMM359, pMM360, and pMM361. (BF) FACS-based measurement of GFP expression upon induction by risdiplam at increasing concentrations in HEK293 cells stably expressing the pMM273, pMM358, pMM359, pMM360, and pMM361 constructs. (B) Shows the induction of GFP expression calculated as the median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Shows the median total GFP signal of GFP-positive cells that are not normalized to DMSO control. (D) The percentage of GFP-positive (%GFP+) cells at each risdiplam concentration is shown divided by the percentage of %GFP+ cells at the highest risdiplam concentration (1 μM). (E) The background level of median total GFP expression in wild-type HEK293 cells (WT) or cells expressing the indicated construct is shown in the absence of risdiplam. (F) The induction of GFP expression is shown, calculated as the median GFP expression at a 250 nM concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (G) The results of PCR splicing product analysis performed on HEK293 cells stably expressing pMM273, pMM358, pMM359, and pMM360 and treated with gradually increasing concentrations of risdiplam or DMSO are shown. [Figure 26]Analysis of risdiplam-induced GFP reporter expression and background GFP reporter expression (i.e., in the absence of risdiplam) for pMM273 and pMM365-pMM375. (A) Schematic diagram (BG) showing the differences between pMM273 and pMM365-pMM375. FACS-based measurement of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing pMM273 and pMM365-pMM375 constructs. (B) Shows the induction of GFP expression calculated as the median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Shows the median total GFP signal of GFP-positive cells that are not normalized to DMSO control. (D) Shows the percentage of GFP-positive (%GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 μM). (E) Background level of median total GFP expression in wild-type HEK293 cells (WT) or cells expressing the indicated construct in the absence of risdiplam. (F) Induction of GFP expression calculated as median GFP expression at a 250 nM concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (G) Percentage of GFP-positive (%GFP+) cells in DMSO divided by %GFP+ cells in the highest concentration of risdiplam treatment (1 μM). [Figure 27]Analysis of risdiplam-induced GFP reporter expression and background GFP reporter expression (i.e., in the absence of risdiplam) for pMM273 and pMM376-pMM387. (A) Schematic diagram (BG) showing the differences between pMM273 and pMM376-pMM387. FACS-based measurement of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing pMM273 and pMM376-pMM387 constructs. (B) Shows the induction of GFP expression calculated as the median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Shows the median total GFP signal of GFP-positive cells that are not normalized to DMSO control. (D) Shows the percentage of GFP-positive (%GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 μM). (E) Background level of median total GFP expression in wild-type HEK293 cells (WT) or cells expressing the indicated construct in the absence of risdiplam. (F) Induction of GFP expression calculated as median GFP expression at a 250 nM concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (G) Percentage of GFP-positive (%GFP+) cells in DMSO divided by %GFP+ cells in the highest concentration of risdiplam treatment (1 μM). [Figure 28]Analysis of risdiplam-induced GFP reporter expression and background GFP reporter expression (i.e., in the absence of risdiplam) for pMM273, pMM436, pMM437, and pMM438. (A) Schematic diagram (BF) showing the differences between pMM273, pMM436, pMM437, and pMM438. FACS-based measurement of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM273, pMM436, pMM437, and pMM438 constructs. (B) Shows the induction of GFP expression calculated as the median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Shows the median total GFP signal of GFP-positive cells that are not normalized to DMSO control. (D) The percentage of GFP-positive (%GFP+) cells at each risdiplam concentration is shown divided by the percentage of %GFP+ cells at the highest risdiplam concentration (1 μM). (E) The background level of median total GFP expression in the absence of risdiplam in wild-type HEK293 cells (WT) or in cells expressing the indicated construct. (F) The induction of GFP expression calculated as the median GFP expression at a 250 nM concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. [Figure 29]Analysis of risdiplam-induced GFP reporter expression and background GFP reporter expression (i.e., in the absence of risdiplam) for pMM273 and pMM477-pMM485. (A) Schematic diagram of the differences between pMM273 and pMM477-pMM485. (BF) FACS-based measurement of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing pMM273 and pMM477-pMM485 constructs. (B) Shows the induction of GFP expression calculated as the median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Shows the median total GFP signal of GFP-positive cells that are not normalized to DMSO control. (D) Shows the percentage of GFP-positive (%GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (1 μM). (E) Background level of median total GFP expression in wild-type HEK293 cells (WT) or cells expressing the indicated construct in the absence of risdiplam. (F) Induction of GFP expression calculated as median GFP expression at a concentration of 250 nM of risdiplam, normalized to median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. [Figure 30]Analysis of risdiplam-inducible GFP reporter expression and background GFP reporter expression (i.e., in the absence of risdiplam) for pMM273 and pMM464-pMM474. (A) Schematic diagram of the differences between pMM273 and pMM464-pMM474, and a table listing intron modifications in all constructs. Intron sequences added to the constructs were extracted from the SMN2 transcript. (BF) FACS-based measurement of GFP expression upon induction with increasing concentrations of risdiplam in HEK293 cells stably expressing the pMM273 and pMM464-pMM474 constructs. (B) Shows the induction of GFP expression calculated as the median GFP expression at each concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Shows the median total GFP signal of GFP-positive cells that are not normalized to the DMSO control. (D) The percentage of GFP-positive (%GFP+) cells at each risdiplam concentration is shown divided by the percentage of %GFP+ cells at the highest risdiplam concentration (1 μM). (E) The background level of median total GFP expression in the absence of risdiplam in wild-type HEK293 cells (WT) or in cells expressing the indicated construct. (F) The induction of GFP expression calculated as the median GFP expression at a 250 nM concentration of risdiplam, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. [Figure 31](A) Analysis of risdiplam-inducible GFP reporter expression and background GFP reporter expression (i.e., in the absence of risdiplam) for pMM466, pMM470, pMM472, pMM474 and pMM567-pMM570. Schematic diagram of the differences between pMM466, pMM470, pMM472, pMM474 and pMM567-pMM570. Two modifications were made to pMM567-pMM570 by introducing a single A>C mutation in exon 6 to remove the ATG start site and TAA deletion in exon 7. (BF) FACS-based measurement of GFP expression induced by risdiplam at increasing concentrations in HEK293 cells stably expressing the pMM466, pMM470, pMM472, pMM474 and pMM567-pMM570 constructs. (B) Shows the induction of GFP expression at each concentration of risdiplam, calculated as the median GFP expression at each concentration, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. (C) Shows the median total GFP signal of GFP-positive cells that are not normalized to DMSO control. (D) Shows the percentage of GFP-positive (%GFP+) cells at each concentration of risdiplam divided by the %GFP+ cells at the highest concentration of risdiplam treatment (500 nM). (E) Shows the background level of median total GFP expression in the absence of risdiplam in wild-type HEK293 cells (WT) or cells expressing the indicated construct. (F) Shows the induction of GFP expression at a concentration of 62.5 nM of risdiplam, calculated as the median GFP expression at each concentration, normalized to the median GFP expression in DMSO-treated control cells 48 hours after risdiplam treatment. [Figure 32]Analysis of risdiplam-induced GFP reporter expression and background GFP reporter expr...

Claims

1. A polynucleotide containing the following from 5' to 3', (i) A first nucleotide sequence comprising a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in Sequence ID No. 222, but not comprising Sequence ID No. 2; (ii) A second nucleotide sequence comprising fewer than 1044 nucleotides, comprising a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 7 at its 5' end, and a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in SEQ ID NO: 378 at its 3' end; (iii) A third nucleotide sequence comprising a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in Sequence ID No. 226; (iv) A fourth nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in SEQ ID NO: 380 at its 5' end, and having at least 80% sequence identity with the nucleotide sequence described in SEQ ID NO: 16 at its 3' end; (v) a fifth nucleotide sequence comprising (a) a dinucleotide "GA", "TG", or "TT", or (b) a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in SEQ ID NO: 19, wherein the nucleotide sequence contains "GA", "TG", or "TT" at positions corresponding to positions 1 and 2 of SEQ ID NO: 19, or (c) a target polypeptide containing "GA", "TG", or "TT" at positions 1 and 2; and (vi) If the fifth nucleotide sequence is the nucleotide sequence described in (v)(a) or (v)(b), a sixth nucleotide sequence encoding the target polypeptide; Includes, A polynucleotide having a start codon at the 5' end of the nucleotide sequence encoding the target polypeptide.

2. The polynucleotide according to claim 1, wherein, if the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion results in a polyribonucleotide substantially lacking the third nucleotide sequence.

3. The polynucleotide according to claim 1 or claim 2, wherein the first nucleotide sequence includes the nucleotide sequence described in SEQ ID NO:

222.

4. The polynucleotide according to any one of claims 1 to 3, wherein the first nucleotide sequence includes or consists of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 106, SEQ ID NO: 219, or SEQ ID NO:

220.

5. The polynucleotide according to any one of claims 1 to 4, wherein the second nucleotide sequence includes SEQ ID NO: 7 at its 5' end and SEQ ID NO: 8 or SEQ ID NO: 377 at its 3' end.

6. The polynucleotide according to any one of claims 1 to 5, wherein the second nucleotide sequence consists of fewer than 500 nucleotides.

7. The polynucleotide according to any one of claims 1 to 6, wherein the second nucleotide sequence includes or consists of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:

228.

8. The polynucleotide according to any one of claims 1 to 7, wherein the third nucleotide sequence comprises the nucleotide sequence described in Sequence ID No.

226.

9. The polynucleotide according to any one of claims 1 to 8, wherein the third nucleotide sequence consists of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 223, or SEQ ID NO:

224.

10. The polynucleotide according to any one of claims 1 to 9, wherein the fourth nucleotide sequence includes SEQ ID NO: 15 or SEQ ID NO: 379 at its 5' end and SEQ ID NO: 16 at its 3' end.

11. The polynucleotide according to any one of claims 1 to 10, wherein the fourth nucleotide sequence consists of fewer than 500 nucleotides.

12. The polynucleotide according to any one of claims 1 to 11, wherein the fourth nucleotide sequence includes or consists of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 227, or SEQ ID NO:

340.

13. The polynucleotide according to any one of claims 1 to 12, wherein the fifth nucleotide sequence consists of (a) a "GA" or "TG" dinucleotide, or (b) the nucleotide sequence described in Sequence ID No. 19, or consists of the same.

14. The polynucleotide according to any one of claims 1 to 13, wherein the fifth nucleotide sequence consists of (a) a dinucleotide of "GA" or "TG", or (b) includes or consists of SEQ ID NO: 20 or SEQ ID NO:

21.

15. A polynucleotide according to any one of claims 1 to 14, comprising a nucleotide sequence having at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NO: 164, SEQ ID NO: 171, SEQ ID NO: 116, SEQ ID NO: 114, SEQ ID NO: 111, SEQ ID NO: 25, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO:

22.

16. The polynucleotide according to any one of claims 1 to 15, further comprising a promoter sequence at the 5' end relative to the start codon.

17. The polynucleotide according to any one of claims 1 to 16, further comprising a polyadenylation sequence at the 3' end of the nucleotide sequence encoding the target polypeptide.

18. The polynucleotide according to any one of claims 1 to 17, comprising an inverted terminal repeat (ITR) sequence at its 5' end and an ITR sequence at its 3' end.

19. A vector comprising a polynucleotide according to any one of claims 1 to 18.

20. The vector according to claim 19, which is an adeno-associated virus (AAV) vector.

21. A pharmaceutical composition comprising a polynucleotide according to any one of claims 1 to 18, or a vector according to claim 19 or claim 20, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

22. A cell comprising a polynucleotide according to any one of claims 1 to 18, or a vector according to claim 19 or claim 20.

23. The cell according to claim 22, further comprising a splicing modifier that promotes SMN2 exon 7 inclusion, wherein the splicing modifier that promotes SMN2 exon 7 inclusion is optionally risdiplam.

24. A method for modifying cells to express a target polypeptide, (i) introducing a polynucleotide according to any one of claims 1 to 18, or a vector according to claim 19 or claim 20, into a cell; and (ii) A method comprising subsequently contacting the cells with a splicing modifier that promotes SMN2 exon 7 inclusion, wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

25. A method for expressing a target polypeptide in cells, comprising contacting the cells according to claim 22 with a splicing modifier that promotes SMN2 exon 7 inclusion, wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

26. A polynucleotide according to any one of claims 1 to 18, a vector according to claim 19 or claim 20, or a pharmaceutical composition according to claim 21, for use in a method of medical treatment or prevention.

27. A polynucleotide according to any one of claims 1 to 18, a vector according to claim 19 or 20, or a pharmaceutical composition according to claim 21, for use in treating or preventing a disease or condition for which a therapeutic or preventive benefit is obtained from an increase in the expression level of the target polypeptide.

28. Use of a polynucleotide according to any one of claims 1 to 18, a vector according to claim 19 or 20, or a pharmaceutical composition according to claim 21 in the manufacture of a pharmaceutical for treating or preventing a disease or condition for which a therapeutic or preventive benefit is obtained from an increase in the expression level of the target polypeptide.

29. A method for treating or preventing a disease or condition for which a therapeutic or preventive benefit is obtained from an increase in the expression level of the target polypeptide, comprising administering to a target polynucleotide according to any one of claims 1 to 18, a vector according to claim 19 or claim 20, or a pharmaceutical composition according to claim 21.

30. A polynucleotide, vector, or pharmaceutical composition for use according to claim 27, the use according to claim 28, or the method according to claim 29, wherein treating or preventing the disease or symptom further comprises administering to the subject a splicing modifier that promotes SMN2 exon 7 inclusion, optionally the splicing modifier that promotes SMN2 exon 7 inclusion being risdiplam.

31. A polynucleotide, vector, or pharmaceutical composition for use according to claim 27 or 30, the use according to claim 28 or 30, or the method according to claim 29 or 30, wherein the disease or symptom is characterized by a deficiency of the target polypeptide.

32. It's a kit, (i) a polynucleotide according to any one of claims 1 to 18, a vector according to claim 19 or claim 20, or a pharmaceutical composition according to claim 21, (ii) A kit comprising a splicing modifier that promotes SMN2 exon 7 inclusion, wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplum.

33. A polynucleotide containing the following from 5' to 3', (i) A first nucleotide sequence comprising a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in Sequence ID No. 1, but not comprising Sequence ID No. 2; (ii) A second nucleotide sequence comprising less than 1044 nucleotides, comprising a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 7 at its 5' end and a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 8 at its 3' end; (iii) A third nucleotide sequence having at least 80% sequence identity with the nucleotide sequence described in Sequence ID No. 26, wherein the third nucleotide sequence contains "A" at the position corresponding to position 2 of Sequence ID No. 12; (iv) A fourth nucleotide sequence having at least 80% sequence identity with sequence number 15 at its 5' end and at least 80% sequence identity with sequence number 16 at its 3' end; (v) a fifth nucleotide sequence comprising (a) a trinucleotide "GAG" or (b) encoding the polypeptide of interest and containing "GAG" at positions 1-3; and (vi) If the fifth nucleotide sequence is the nucleotide sequence described in (v)(a), a sixth nucleotide sequence encoding the target polypeptide. Includes, A polynucleotide having a start codon at the 5' end of the nucleotide sequence encoding the target polypeptide.

34. The polynucleotide according to claim 33, wherein, if the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes SMN2 exon 7 inclusion results in a polyribonucleotide substantially lacking the third nucleotide sequence.

35. The polynucleotide according to claim 33 or claim 34, wherein the first nucleotide sequence includes the nucleotide sequence described in SEQ ID NO:

1.

36. The polynucleotide according to any one of claims 33 to 35, wherein the first nucleotide sequence includes or consists of sequence number 3.

37. The polynucleotide according to any one of claims 33 to 36, wherein the second nucleotide sequence includes SEQ ID NO: 7 at its 5' end and SEQ ID NO: 8 at its 3' end.

38. The polynucleotide according to any one of claims 33 to 37, wherein the second nucleotide sequence consists of fewer than 500 nucleotides.

39. The polynucleotide according to any one of claims 33 to 38, wherein the second nucleotide sequence includes or consists of sequence number 10.

40. The polynucleotide according to any one of claims 33 to 39, wherein the third nucleotide sequence comprises the nucleotide sequence described in Sequence ID No.

26.

41. The polynucleotide according to any one of claims 33 to 40, wherein the third nucleotide sequence consists of SEQ ID NO: 13 or SEQ ID NO:

27.

42. The polynucleotide according to any one of claims 33 to 41, wherein the fourth nucleotide sequence includes SEQ ID NO: 15 at its 5' end and SEQ ID NO: 16 at its 3' end.

43. The polynucleotide according to any one of claims 33 to 42, wherein the fourth nucleotide sequence consists of fewer than 500 nucleotides.

44. The polynucleotide according to any one of claims 33 to 43, wherein the fourth nucleotide sequence includes or consists of sequence number 17.

45. A polynucleotide according to any one of claims 33 to 44, comprising at least 80% sequence identity with SEQ ID NO: 28 or SEQ ID NO:

29.

46. The polynucleotide according to any one of claims 33 to 45, further comprising a promoter sequence at the 5' end relative to the start codon.

47. The polynucleotide according to any one of claims 33 to 46, further comprising a polyadenylation sequence at the 3' end of the nucleotide sequence encoding the target polypeptide.

48. The polynucleotide according to any one of claims 33 to 47, comprising an inverted terminal repeat (ITR) sequence at its 5' end and an ITR sequence at its 3' end.

49. A vector comprising a polynucleotide according to any one of claims 33 to 48.

50. The vector according to claim 49, which is an adeno-associated virus (AAV) vector.

51. A pharmaceutical composition comprising a polynucleotide according to any one of claims 33 to 48, or a vector according to claim 49 or claim 50, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

52. A cell comprising a polynucleotide according to any one of claims 33 to 48, or a vector according to claim 49 or claim 50.

53. The cell according to claim 52, further comprising a splicing modifier that promotes SMN2 exon 7 inclusion, wherein the splicing modifier that promotes SMN2 exon 7 inclusion is optionally risdiplam.

54. A method for modifying cells to express a target polypeptide, comprising introducing a polynucleotide according to any one of claims 33 to 48, or a vector according to claim 49 or claim 50, into the cells.

55. A method for inhibiting the expression of a target polypeptide in cells, comprising contacting the cells according to claim 52 with a splicing modifier that promotes SMN2 exon 7 inclusion, wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplam.

56. A polynucleotide according to any one of claims 33 to 48, a vector according to claim 49 or claim 50, or a pharmaceutical composition according to claim 51, for use in a method of medical treatment or prevention.

57. A polynucleotide according to any one of claims 33 to 48, a vector according to claim 49 or 50, or a pharmaceutical composition according to claim 51, for use in treating or preventing a disease or condition for which a therapeutic or preventive benefit is obtained from an increase in the expression level of the target polypeptide.

58. Use of a polynucleotide according to any one of claims 33 to 48, a vector according to claim 49 or claim 50, or a pharmaceutical composition according to claim 51 in the manufacture of a pharmacopoeia for treating or preventing a disease or condition for which a therapeutic or preventive benefit is obtained from an increase in the expression level of the target polypeptide.

59. A method for treating or preventing a disease or condition for which a therapeutic or preventive benefit is to be obtained from an increase in the expression level of the target polypeptide, comprising administering to a subject a polynucleotide according to any one of claims 33 to 48, a vector according to claim 49 or claim 50, or a pharmaceutical composition according to claim 51.

60. A polynucleotide, vector, or pharmaceutical composition for use according to claim 57, the use according to claim 58, or the method according to claim 59, wherein the disease or symptom is characterized by a deficiency of the target polypeptide.

61. It's a kit, (i) a polynucleotide according to any one of claims 33 to 48, a vector according to claim 49 or claim 50, or a pharmaceutical composition according to claim 51, (ii) A kit comprising a splicing modifier that promotes SMN2 exon 7 inclusion, wherein the splicing modifier that promotes SMN2 exon 7 inclusion is risdiplum.

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