Mecp2 trans-splicing molecules

Abstract

Provided are exon editor constructs comprising a binding domain that binds a target intron of a MeCP2 pre-mRNA, a half-intron, and a coding domain comprising one or more MeCP2 exons. Also provided are methods of expressing functional MeCP2 in a target cell, comprising contacting the target cell with an exon editor construct provided herein.

Description

I. Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 598,693, filed November 14, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] II. Sequence List This application contains a sequence list, which has been submitted electronically in XML format and is hereby incorporated herein by reference in its entirety. The XML sequence list was created on November 13, 2024, named ASC-003WO_SL.xml, and has a size of 329,544 bytes. Background Technology

[0003] Rett syndrome is a devastating neurodevelopmental disorder that primarily affects girls, and there is currently no effective treatment. Individuals with Rett syndrome typically utter their first few words, but soon after, they lose the ability to speak, walk, or use their hands purposefully. There are no treatments available to improve the condition. Even with the best current standards of care, they often suffer daily from vomiting, seizures, and uncontrollable crying. Summary of the Invention

[0004] Gene therapy holds immense promise for treating Rett syndrome (RTT), caused by mutations in the methyl CpG-binding protein 2 (MECP2) gene. However, conventional gene therapy methods, such as gene replacement, can lead to overproduction of MeCP2, triggering a condition known as MeCP2 duplication syndrome. Furthermore, with thousands of different mutations potentially causing Rett syndrome, single base editor drugs cannot address the needs of a large number of patients. Gene editing methods, such as CRISPR / Cas9-based editing, would also require the introduction of Cas9 into the brain, increasing the risk of immune rejection. Therefore, there is a need in the art for alternative strategies to correct mutations in the MeCP2 gene.

[0005] To address these unmet needs, this paper describes an RNA exon editing solution comprising the delivery of an exon editor construct encoding a therapeutic RNA exon editor designed for trans-splicing into an endogenous MeCP2 precursor mRNA containing at least one mutation. Trans-splicing replaces one or more exons of the MeCP2 precursor mRNA with exons from the therapeutic RNA exon editor encoding functional amino acid sequences present in a bioactive MeCP2 protein, thereby correcting one or more mutations in the endogenous MeCP2 precursor mRNA. Rett syndrome is a promising candidate disease for exon editing approaches. Exon editing using a single therapeutic RNA exon editor can correct mutations in ~95% of Rett patients. An exemplary MeCP2 RNA exon editor is described in this paper.

[0006] In some embodiments, an exon editor construct described herein comprises: (a) a binding domain that binds to a target intron of MeCP2 precursor mRNA; (b) a hemiintron; and (c) a coding domain that comprises one or more MeCP2 exons. In some embodiments, the binding domain comprises any binding domain described in Section VI.C.3 below. In some embodiments, the hemiintron comprises any hemiintron disclosed in Section VI.C.4. In some embodiments, the coding domain sequence comprises any CDS disclosed in Section VI.C.2. In some embodiments, the exon editor construct further comprises a 3' UTR sequence operatively linked to a 3' CDS. In some embodiments, the 3' UTR sequence comprises any 3' UTR sequence disclosed in Section VI.C.5. In some embodiments, the exon editor construct further comprises a 3' polyA tail operatively linked to a 3' CDS as disclosed herein. In some embodiments, the exon editor construct further comprises a 3' transcription terminator sequence operatively linked to a 3' CDS. In some implementations, the transcription terminator sequence includes any transcription terminator sequence disclosed in Section VI.C.6.

[0007] The embodiments disclosed herein include an exon editor construct encoding an RNA exon editor, wherein the exon editor construct comprises encoding the following sequences: (a) a binding domain that binds to a target intron of MeCP2 precursor mRNA; (b) a hemiintron; and (c) a coding domain comprising one or more MeCP2 exons. In some embodiments, the target intron of the MeCP2 precursor mRNA is intron 1 or intron 2. In some embodiments, the binding domain binds to a binding site comprising: nucleotides -100 to 1500 or -50 to 1449 of intron 1 (SEQ ID NO: 1); nucleotides 3950 to 4250 or 4020-4169 of intron 1; or nucleotides 1-300 of intron 2 (SEQ ID NO: 2 and 3). In some embodiments, the binding domain binds to binding sites comprising: nucleotides of intron 1 (SEQ ID NO: 1) of -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000; or nucleotides of intron 2 (SEQ ID NO: 2 and 3) of 50-300, 50-250, 75-300, 75-250, 80-300, 80-250, 90-300, 90-250, 95-300, 95-250, or 99-248. In some implementations, the size of the binding domain ranges from about 50-300 nucleotides; about 50-250 nucleotides; about 50-150 nucleotides; about 50-100 nucleotides; about 75-300 nucleotides; about 75-250 nucleotides; about 75-200 nucleotides; about 75-150 nucleotides; about 100-300 nucleotides; about 100-250 nucleotides; about 100-200 nucleotides; about 100-150 nucleotides; about 125-300 nucleotides; about 125-250 nucleotides; about 125-200 nucleotides; about 125-150 nucleotides; or about 150 nucleotides. In some implementations, the size of the binding domain ranges from 50 to 300 nucleotides; 50 to 250 nucleotides; 50 to 150 nucleotides; 50 to 100 nucleotides; 75 to 300 nucleotides; 75 to 250 nucleotides; 75 to 200 nucleotides; 75 to 150 nucleotides; 100 to 300 nucleotides; 100 to 250 nucleotides; 100 to 200 nucleotides; 100 to 150 nucleotides; 125 to 300 nucleotides; 125 to 250 nucleotides; 125 to 200 nucleotides; 125 to 150 nucleotides; or 150 nucleotides.In some embodiments, such as the exon editor construct described in any of the foregoing embodiments, the size of the binding domain ranges from about 100-200 nucleotides; about 100-150 nucleotides; about 125-200 nucleotides; about 125-150 nucleotides; or about 150 nucleotides; or the size ranges from 100-200 nucleotides; 100-150 nucleotides; 125-200 nucleotides; 125-150 nucleotides; or 150 nucleotides. In some embodiments, the binding domain is at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the binding site it binds to. In some embodiments, the binding domain comprises a continuous nucleotide chain segment that is 100% complementary to a portion of the binding site it binds to, wherein the length of the continuous nucleotide chain segment is at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, at least 70 nucleotides, at least 75 nucleotides, at least 80 nucleotides, at least 85 nucleotides, at least 90 nucleotides, at least 95 nucleotides, at least 100 nucleotides, at least 105 nucleotides, at least 110 nucleotides, at least 115 nucleotides, at least 120 nucleotides, at least 130 nucleotides, at least 135 nucleotides, at least 140 nucleotides, at least 145 nucleotides, or at least 150 nucleotides. In some embodiments, the sequence encoding the binding domain comprises, substantially comprises, or comprises any one of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 47. In some embodiments, the sequence encoding the binding domain comprises, substantially comprises, or comprises any one of SEQ ID NO: 55. In some embodiments, the sequence encoding the binding domain comprises, substantially comprises, or comprises any one of SEQ ID NO: 24, 28, 29, or 30.In some implementations, the coding structure domain comprises, is substantially composed of, or consists of: SEQ ID NO: 7 and 8 or sequences having 90% identity with SEQ ID NO: 7 and 8; SEQ ID NO: 76 or sequences having 90% identity with SEQ ID NO: 76; SEQ ID NO: 12; SEQ ID NO: 14; or SEQ ID NO: 9.

[0008] In some implementations, the encoded structure field contains, is substantially composed of, or is composed of any of SEQ ID NO: 76 or 14.

[0009] In some implementations, the combined structural domain, half-intron, and coding structural domain are operably connected in the 5' to 3' direction.

[0010] In some embodiments, the half-intron comprises a 3' splice site and optionally a branch point sequence and a polypyrimidine tract, wherein the binding domain, the half-intron containing the 3' splice site, and the coding domain are operatively connected in the 5' to 3' direction. In some embodiments, the half-intron comprises, substantially comprises, or consists of the following: a polypyrimidine tract, a branch point, and a 3' splice site. In some embodiments, the half-intron comprises, substantially comprises, or consists of the following: SEQ ID NO: 17 or a sequence having at least 90% identity with SEQ ID NO: 17.

[0011] In some embodiments, the exon editor construct further includes a 3' untranslated region (3' UTR), wherein the binding domain, half-introns, coding domain, and 3' UTR are operatively connected in the 5' to 3' direction. In some embodiments, the 3' UTR includes RDH1pA 3' UTR (SEQ ID NO: 19), mWPRE 3' UTR (SEQ ID NO: 20), or truncated forms thereof, such as WPRE3 3' UTR (SEQ ID NO: 87). In some embodiments, RDH1pA 3' UTR includes, substantially consists of, or consists of: SEQ ID NO: 19 or a sequence having at least 90% identity with SEQ ID NO: 19; or mWPRE 3' UTR includes, substantially consists of, or consists of: SEQ ID NO: 20 or a sequence having at least 90% identity with SEQ ID NO: 20. In some embodiments, the 3' UTR includes a truncated WPRE sequence. In some implementations, the truncated WPRE sequence comprises, is substantially composed of, or consists of: SEQ ID NO: 87 or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 87.

[0012] In some implementations, the exon editor construct comprises: a binding domain comprising, substantially comprising, or comprising SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 47, or 55; a half-intron comprising, substantially comprising, or comprising SEQ ID NO: 17; a coding domain comprising, substantially comprising, or comprising SEQ ID NO: 76; and a 3' UTR comprising, substantially comprising, or comprising SEQ ID NO: 20, wherein the binding domain, the half-intron, the coding domain, and the 3' UTR are operatively connected in the 5' to 3' direction.

[0013] In some embodiments, the exon editor builder comprises, is substantially composed of, or consists of: SEQ ID NO: 78, 80, 81, 82, 83, or 84. In some embodiments, the exon editor builder comprises, is substantially composed of, or consists of: SEQ ID NO: 78 or 80.

[0014] In some embodiments, the exon editor construct further includes a triple-helix terminator, wherein the binding domain, half-intron, coding domain, 3' UTR (when present), and triple-helix terminator are operatively connected in the 5' to 3' direction. In some embodiments, the triple-helix terminator comprises, substantially comprises, or comprises SEQ ID NO: 77 or a sequence having at least 90% identity with SEQ ID NO: 77. In some embodiments, the triple-helix terminator comprises, substantially comprises, or comprises SEQ ID NO: 22.

[0015] In some implementations, the exon editor also includes a sequence encoding a poly A sequence, for example, SEQ ID NO: 21.

[0016] In some embodiments, the exon editor construct further includes a sequence of encoding epitope tags, wherein the binding domain, half-intron, encoding domain, epitope tag, 3' UTR (if present), and triple-helix terminator (if present) are operatively connected in the 5' to 3' direction. In some embodiments, the exon editor construct comprises, substantially comprises, or comprises: SEQ ID NO: 78.

[0017] This paper also discloses an RNA exon editor, which is transcribed from an exon editor construct of any of the above embodiments.

[0018] In some embodiments, the MeCP2 precursor mRNA contains at least one mutation associated with Rett syndrome. In some embodiments, the at least one Rett syndrome-associated mutation includes at least one mutation in exon 3 of the MeCP2 gene allele or at least one mutation in exon 4 of the MeCP2 gene allele, or any combination thereof. In some embodiments, the at least one Rett syndrome-associated mutation is X-linked. In some embodiments, the MeCP2 protein containing at least one Rett syndrome-associated mutation is expressed in at least one of neural stem cells, neurons, astrocytes, or oligodendrocytes, or any combination thereof.

[0019] This document also discloses a vector comprising an exon editor construct of any of the above embodiments. In some embodiments, the vector comprises a 5' regulatory domain operatively linked to a binding domain at its 5'. In some embodiments, the 5' regulatory domain comprises a constitutive promoter or a tissue-specific promoter. In some embodiments, the constitutive promoter is a CMV promoter.

[0020] This paper also discloses a proviral plasmid containing an exon editor builder of any of the above implementations.

[0021] This document also discloses an adeno-associated virus (AAV) comprising an exon editor construct according to any of the above embodiments, wherein the AAV optionally includes a 5' regulatory domain operatively linked to the exon editor construct. In some embodiments, the AAV includes a 5' regulatory domain operatively linked to a binding domain. In some embodiments, the 5' regulatory domain includes a constitutive promoter. In some embodiments, the constitutive promoter is a CMV promoter. In some embodiments, the AAV exhibits neurotropic characteristics. In some embodiments, the AAV is AAV9, AAV8, AAV5, or AAV2.

[0022] A composition is also disclosed comprising an exon editor construct of any of the above embodiments, a vector of any of the above embodiments, a proviral plasmid of any of the above embodiments, or an AAV of any of the above embodiments. In some embodiments, the composition comprises a pharmaceutically acceptable excipient.

[0023] A method for expressing bioactive MeCP2 in target cells to restore the functional level of the MeCP2 protein in the target cells is also disclosed. The method includes transducing the target cells using an exon editor construct of any of the above embodiments, a vector of any of the above embodiments, a proviral plasmid of any of the above embodiments, an AAV of any of the above embodiments, or a composition of any of the above embodiments. In some embodiments, the target cells contain at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of at least one mutant MeCP2 precursor mRNA associated with Rett syndrome, which is modified to a transcript encoding a functional MeCP2, wherein at least one mutation is corrected. In some embodiments, the target cells contain at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of at least one mutant MeCP2 precursor mRNA associated with Rett syndrome, which is modified to a transcript encoding a functional MeCP2, wherein at least one mutation is corrected. In some embodiments, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the mutated MeCP2 precursor mRNA associated with Rett syndrome in the target cells is modified into a transcript encoding functional MeCP2, wherein at least one mutation is corrected. In some embodiments, the functional level of MeCP2 in the target cells is restored by expressing a biologically functional MeCP2 protein.

[0024] Also disclosed is a method for reducing the expression of MeCP2 containing at least one mutation associated with Rett syndrome in a subject, the method comprising transfecting or transducing target cells, more specifically neurons, in a subject with an exon editor construct, a vector, a proviral plasmid, an AAV, or a combination thereof, according to any of the above embodiments.

[0025] Also disclosed is a method for correcting at least one mutation in the MeCP2 exon sequence in the MeCP2 precursor mRNA of a subject, the method comprising administering to the subject an exon editor construct of any of the above embodiments, a vector of any of the above embodiments, a proviral plasmid of any of the above embodiments, an AAV of any of the above embodiments, or a combination of any of the above embodiments.

[0026] Also disclosed is a method for treating Rett syndrome in a subject in need, the method comprising administering to the subject an exon editor construct of any of the above embodiments, a vector of any of the above embodiments, a proviral plasmid of any of the above embodiments, an AAV of any of the above embodiments, or a combination of any of the above embodiments.

[0027] In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of the target cells containing at least one mutant MeCP2 precursor mRNA associated with Rett syndrome are modified to encode a transcript encoding functional MeCP2, wherein at least one mutation is corrected. In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of the target cells containing at least one mutant MeCP2 precursor mRNA associated with Rett syndrome are modified to encode a transcript encoding functional MeCP2, wherein at least one mutation is corrected. In some embodiments, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the target cells containing at least one mutant MeCP2 precursor mRNA associated with Rett syndrome are modified to encode a transcript encoding functional MeCP2, wherein at least one mutation is corrected. In some embodiments, the functional level of MeCP2 in target cells is restored by expressing a biologically functional MeCP2 protein. In some embodiments, the method includes administering to the brain of a subject an exon editor construct as described in any one of embodiments 1-29 or 31-34, a vector as described in any one of embodiments 35-38, a proviral plasmid as described in embodiment 39, an AAV as described in any one of embodiments 40-45, or a composition as described in any one of embodiments 46-47. In some embodiments, the subject is a mammal, preferably a rodent, a non-human primate, or a human. In some embodiments, the subject is genetically predisposed to Rett syndrome or has been diagnosed with Rett syndrome.

[0028] It also discloses exon editor constructs of any of the above embodiments, vectors of any of the above embodiments, proviral plasmids of any of the above embodiments, AAVs of any of the above embodiments, or compositions of any of the above embodiments, for the prevention or treatment of Rett syndrome in subjects in need.

[0029] It also discloses exon editor constructs of any of the above embodiments, vectors of any of the above embodiments, proviral plasmids of any of the above embodiments, AAVs of any of the above embodiments, or compositions of any of the above embodiments, for the preparation of medicaments for the treatment or prevention of Rett syndrome in subjects of need.

[0030] The exemplary RNA exon editors disclosed herein include the exon editors listed in embodiments 66 to 248. The exemplary constructs of the RNA exon editors disclosed herein include the exon editor constructs listed in embodiments 1 to 35 and 249 to 264. The exemplary vectors disclosed herein include the vectors listed in embodiments 36 to 39 and 265 to 268. The exemplary proviral plasmids disclosed herein include the proviral plasmids listed in embodiments 40 and 269. The exemplary AAVs disclosed herein include the AAVs listed in embodiments 41 to 46, 270, and 271. The exemplary compositions disclosed herein include the compositions listed in embodiments 47, 48, and 272. The exemplary methods for expressing biologically active MeCP2 include the methods listed in embodiments 49 to 53 and 273 to 275. The exemplary methods for reducing the expression of MeCP2 containing at least one mutation associated with Rett syndrome include the methods listed in embodiments 54 and 276. The exemplary methods for correcting at least one mutation in the MeCP2 exon sequence include the methods listed in embodiments 55 and 277. Exemplary methods for treating Rett syndrome in subjects in need include those listed in embodiments 56 to 63, 278 and 279. Attached Figure Description

[0031] Figure 1 A schematic representation depicting the frequency and location of missense mutations in MeCP2 associated with Rett syndrome (adapted from Ehrhart, F. et al., Scientific Data, 2021). The structural / functional domains of the MeCP2 protein include: the N-terminal domain (NTD); the methyl-binding domain (MBD); the intercalary domain (ID); the transcriptional repression domain (TRD); and the C-terminal domain (CTD). The exon editor described in this article corrects mutations in exons 3 and 4, thereby treating approximately 95% of patients with RTT.

[0032] Figure 2 An exemplary embodiment of exon editing for MeCP2 is depicted. The 3' exon editor (dark blue) is depicted as binding to the target precursor mRNA of MeCP2. In the illustrated embodiment, trans-splicing between the target precursor mRNA and the exon editor can correct mutations in exons 3 and 4.

[0033] Figure 3 The gene structure of human MeCP2 was depicted. The gene structure and sequence length of MeCP2 were depicted as having exons 1-4 and introns 1-3. The 3' UTR (included in exon 4) varies between MeCP2 subtypes, so only the length of the coding sequence of exon 4 is indicated.

[0034] Figure 4Examples of MeCP2 E1 and E2 mRNA isoforms are presented. The E1 isoform mRNA consists of exons 1, 3, and 4. The translation start codon for translating the E1 isoform protein is located in exon 1, as indicated. The E2 mRNA isoform consists of exons 1, 2, 3, and 4. The translation start codon for translating the E2 isoform protein is located in exon 2, as indicated.

[0035] Figure 5 The domains of an exemplary 3' MeCP2 exon editor are depicted. Each domain of the 3' exon editor is indicated. Promoter: A sequence necessary to drive the expression of the exon editor. Binding domain: A sequence antisense to the target precursor mRNA that recruits the exon editor to the target via base pairing. Hemiintron: A sequence containing a branch point, polypyrimidine bundle, and splice acceptor site. MeCP2 exon 3 and 4 coding domain sequences (CDS): Sequences encoding MeCP2 exons 3 and 4. Myc tag: An epitope tag used to detect proteins produced by exon editing. 3' Untranslated region (UTR): A regulatory sequence. Terminator: A sequence used to terminate transcription of the exon editor. In some embodiments, the expression of the RNA exon editor is driven by a CMV / CMV enhancer / promoter. In some embodiments, the RNA exon editor comprises a binding domain targeting MeCP2 intron 1, a half-intron, a splice site sequence, a replacement wild-type MeCP2 exon, a myc tag for protein detection, and a 3' UTR. In some embodiments, the RNA exon editor comprises a binding domain targeting MeCP2 intron 1, a half-intron, a splice site sequence, a replacement wild-type MeCP2 exon, and a 3' UTR. In some embodiments, the RNA exon editor comprises a binding domain targeting MeCP2 intron 1, a half-intron, a splice site sequence, a modified replacement wild-type MeCP2 exon, and a 3' UTR. In some embodiments, the 3' UTR is a native MeCP2 3' UTR or a truncated form thereof. In some embodiments, the 3' UTR is a heterologous 3' UTR (e.g., an artificial 3' UTR assembled through genetic engineering).

[0036] Figure 6 A scan of the binding domain of intron 1 in MeCP2 is depicted. The relative positions of the binding domain (depicted as the shape above the bar indicating intron 1) along the length of intron 1 in MeCP2 are shown.

[0037] Figure 7A diagram illustrating the identification of MeCP2 intron 1 hotspots is presented. HEK293 cells were transfected with a MeCP2 intron 1-targeting RNA exon editor targeting various regions of intron 1. Cells were harvested 48 hours post-transfection, and trans-splicing efficiency was determined by RT-qPCR. The nomenclature for binding domains is: (nucleotide base position in the intron at the 5' end of the binding domain) - (length of the binding domain (nt)). For example, 51_150 indicates that the binding domain is the reverse complementary sequence of bases 51-200 in intron 1, corresponding to position 51-200 in SEQ ID NO: 1. Position -50 represents the position 50 nucleotides upstream of the exon 1-intron junction. The sequence identifiers of the binding domains tested are shown in Table 4. The various binding domains tested included nucleotide substitutions that reduced cryptic splicing sites, as indicated in Table 4.

[0038] Figure 8 The verification of the intron-1 binding domain is demonstrated. (Source: [Original Source Name]) Figure 7 The selected exon editors were retested by two separate operators. HEK293 cells were transfected with MeCP2 intron 1-targeting RNA exon editors targeting various regions of intron 1. Cells were harvested 48 hours post-transfection, and trans-splicing efficiency was determined by RT-qPCR. The nomenclature for binding domains was: (nucleotide base position in the intron at the 5' start position of the binding domain) - (length of the binding domain (nt)). For example, 51_150 indicates that the binding domain is the reverse complementary sequence of bases 51-200 in intron 1. The substitution % was calculated as edited MeCP2 mRNA copy number / total MeCP2 mRNA x 100. The sequences of the binding domains tested are shown in Table 4.

[0039] Figure 9 A summary of the scanning profile of the binding domain of MeCP2 intron 1. The percentages represent the trans-splicing efficiency of the RNA exon editor with the binding domain at the indicated location.

[0040] Figure 10 A schematic diagram depicting an exemplary scanning profile of the binding domains of intron 2 of MeCP2. The relative positions of the binding domains (depicted in green) are shown at the 5' and 3' ends of intron 2.

[0041] Figure 11A graph illustrating the activity of the tested exemplary MeCP2 intron 2 binding domains is presented. HEK293 cells were transfected with a MeCP2 intron 2-targeting RNA exon editor targeting various regions of intron 2. Cells were harvested 48 hours post-transfection, and trans-splicing efficiency was determined by RT-qPCR. The nomenclature for the binding domains is: (nucleotide base position in the intron at the 5' start position of the binding domain) - (length of the binding domain (nt)). For example, 99_150 indicates that the binding domain is the reverse complementary sequence of bases 99-248 in intron 2, corresponding to position 99-248 in SEQ ID NO: 2. The substitution % is calculated as edited MeCP2 mRNA (E2 isotype) copy number / total MeCP2 RNA (E2 isotype) x 100. The sequence identifiers of the tested binding domains are shown in Table 5.

[0042] Figure 12 This diagram presents an overview of the binding domain scan of MeCP2 intron 2. The percentages represent the trans-splicing efficiency of the RNA exon editor with the binding domain at the indicated location.

[0043] Figure 13 An exemplary scanning profile of the binding domains of MeCP2 intron 3 is depicted. The relative positions of the binding domains (depicted in green) are shown along the length of intron 3.

[0044] Figure 14 A graph illustrating the activity of the tested exemplary MeCP2 intron 3 binding domains is presented. HEK293 cells were transfected with a MeCP2 intron 3-targeting RNA exon editor targeting various regions of intron 3. Cells were harvested 48 hours post-transfection, and trans-splicing efficiency was determined by RT-qPCR. The nomenclature for the binding domains is: (nucleotide base position in the intron at the 5' start position of the binding domain) - (length of the binding domain (nt)). For example, 26_150 indicates that the binding domain is the reverse complementary sequence of bases 26-175 in intron 3, corresponding to positions 26-175 in SEQ ID NO: 4. The substitution % was calculated as edited MeCP2 mRNA copy number / total MeCP2 RNA x 100. The sequence identifiers of the tested binding domains are shown in Table 7.

[0045] Figure 15 A summary of the performance of the intron 3 binding domain is presented. The percentages represent the trans-splicing efficiency of the RNA exon editor with the binding domain at the indicated location.

[0046] Figure 16The performance of the exon editor is shown by certain 3' end modifications. Proteins derived from the edited mRNA (left) and exon editor expression (right) are plotted for editors with the indicated elements at the 3' end. The figure represents the performance of the editor relative to editor 1. HEK293 cells were transfected with the MeCP2 exon editor. Cells were harvested 48 hours after transfection, and (1) proteins derived from the edited mRNA were determined by Western blotting, and (2) exon editor expression was determined by RT-qPCR.

[0047] Figure 17 The results showed that the reproducibility of MeCP2 exon editing was observed in different cell types. HEK293 and U-251 MG cells were transfected with the MeCP2 exon editor as instructed. Cells were harvested 48 hours post-transfection, and trans-splicing efficiency was determined by RT-qPCR. Replacement % was calculated as edited MeCP2 mRNA copy number / total MeCP2 RNA x 100.

[0048] Figure 18 Immunoblot analysis was performed to visualize proteins generated from MeCP2-edited mRNA. U-251 cells were transfected with plasmids encoding full-length chimeric (edited) mRNA mimics or exon editors. Cells were harvested 48 hours post-transfection and analyzed by Western blotting. Only exon editors with active splicing sites produced proteins of the expected size.

[0049] Figure 19 A graph illustrating the activity of exemplary MeCP2 intron 2 binding domains tested by two different operators is presented. HEK293 cells were transfected with a MeCP2 intron 2-targeting RNA exon editor targeting various regions of intron 2. Cells were harvested 48 hours post-transfection, and trans-splicing efficiency was determined by RT-qPCR. The nomenclature for the binding domains is: (nucleotide base position in the intron at the 5' start position of the binding domain) - (length of the binding domain (nt)). For example, 99_150 indicates that the binding domain is the reverse complementary sequence of bases 99-248 in intron 2, corresponding to position 99-248 in SEQ ID NO: 2. The various binding domains tested included nucleotide substitutions that reduced cryptic splicing sites, as indicated in Table 6. Figure 19The diagram shows a comparison between binding domains with nucleotide substitutions that reduce cryptic splicing sites (MeCP2_intron2_99_150SM (SEQ ID NO: 88)) and binding domains lacking these substitutions (MeCP2_intron2_99_150 (SEQ ID NO: 55)). The substitution percentage was calculated as edited MeCP2 mRNA (E2 isotype) copy number / total MeCP2 RNA (E2 isotype) x 100. Detailed Implementation

[0050] A. Introduction The following examples are provided to illustrate some embodiments of this disclosure, but are not intended to limit the scope of this disclosure; it should be understood that other procedures, methods or techniques known to those skilled in the art may be used alternatively by way of their exemplary nature.

[0051] Rett syndrome (RTT) is a rare neurological disorder, occurring in approximately 1 in 10,000 female infants worldwide. It is even rarer in boys. Diagnosis typically occurs between 6 and 18 months of age, when the child begins to miss developmental milestones or lose previously mastered skills. Rett syndrome is characterized by severe impairments in speech, walking, eating, and even breathing. A hallmark of Rett syndrome is almost constant, repetitive hand movements. Symptoms may include: seizures or Rett seizures, aphasia, loss of purposeful use of the hands, involuntary hand movements, loss of mobility, gait disturbances, loss of muscle tone, scoliosis, breathing problems, sleep disturbances, uncontrollable crying, and / or slowed growth of the head, feet, and / or hands.

[0052] Rett syndrome is an X-linked neurodevelopmental disorder, with some patients experiencing severe motor abnormalities and shortened lifespan. Rett syndrome is caused by loss-of-function mutations in MECP2 (a transcriptional regulator highly expressed in neurons). Inactivating mutations in MeCP2 alter the expression of many genes and ultimately lead to abnormalities in neuronal morphology and circuits. MeCP2 More than 900 different mutations were discovered in the gene, most of which are located in eight different "hotspots". See also Figure 1 Rett syndrome presents with widespread disability, ranging from mild to severe. The course and severity of Rett syndrome are determined by the location, type, and severity of the mutation and X-inactivation.

[0053] In addition to Rett syndrome, MeCP2 Mutations in genes are also associated with, for example MECP2 Duplication syndrome MECP2It is associated with severe neonatal encephalopathy and other conditions such as pyramidal tract signs, Parkinson's disease, and macroorchidism (PPM-X) syndrome. There is currently no treatment for [the condition / condition]. MeCP2 Approved treatments for any disease related to mutations in genes.

[0054] Gene therapy in the treatment of Rett syndrome and other related conditions MeCP2 It holds great promise for understanding diseases related to mutations in genes. However, the main obstacle is... MECP2 A two-fold increase in gene copy number and corresponding gene expression levels leads to a disease phenotype, thus creating a narrow therapeutic window for conventional gene replacement. This window may be even narrower due to random X chromosome inactivation, as seen in patients with Rett syndrome, where half of the cells express fully functional MeCP2 protein. Therefore, conventional gene replacement may not be able to deliver sufficient MeCP2 to cells that do require it while avoiding overexpression in cells that do not need it.

[0055] Exon editing molecules utilize RNA trans-splicing to remove and replace diseased exons. As described in this article, single exon editors can correct the 3' portion of mRNA by replacing the usual intramolecular cis-splicing with intermolecular trans-splicing. See also Figure 2 Exon editing for the treatment of Rett syndrome has at least two advantages. First, a single exon editor targeting MeCP2 intron 1 or intron 2 can correct ~95% of MECP2 patient mutations, while a single exon editor targeting MeCP2 intron 3 can correct ~75% of MECP2 patient mutations. Second, regardless of promoter or vector dosage, exon editing therapy inherently limits the maximum expression of corrected mRNA in individual and per-cell states to normal endogenous levels. This inherent control stems from the exon editor acting on the expressed precursor mRNA, thus correcting it only when the target precursor mRNA transcript is expressed. This level of control may be crucial for half of Rett patient cells that already express the correct amount of fully functional MECP2. In these cells, exon editing replaces one functional mRNA with another, whereas gene replacement therapy would lead to unintended, disease-causing overexpression of MECP2 mRNA.

[0056] Table 1: List of the most abundant pathogenic mutations associated with Rett syndrome.

[0057] The compositions and methods described herein relate to the treatment of [a condition caused by] [a specific condition]. MeCP2Trans-splicing molecules (e.g., precursor mRNA trans-splicing molecules) cause diseases or conditions due to mutations in genes. These mutations include point mutations and deletions, the vast majority of which have been identified. MeCP2 Identified in exon 3 or exon 4 of the gene. See also Figure 1 The compositions and methods described herein employ nucleic acid trans-splicing molecules encoding RNA trans-splicing molecules targeting precursor mRNA for gene therapy (e.g., in vivo gene therapy, such as delivery via adeno-associated virus) to treat diseases caused by at least one mutation in MeCP2, such as Rett syndrome. The compositions and methods described herein also employ nucleic acid trans-splicing molecules encoding RNA trans-splicing molecules targeting precursor mRNA for gene therapy (e.g., in vivo gene therapy) in combination with other therapeutic agents described herein to treat Rett syndrome.

[0058] As described in this article, Rett syndrome is a neurodevelopmental disorder associated with mutations in the methyl CpG-binding protein 2 (MECP2) gene. Rett syndrome is a progressive neurodevelopmental disorder and one of the most common causes of cognitive impairment in women.

[0059] MECP2 is a chromatin-associated protein that can both activate and repress transcription. It is essential for neuronal maturation and is developmentally regulated. The MeCP2 gene is a member of a family of nucleoproteins containing a methyl-CpG binding domain (MBD). MeCP2 can specifically bind to methylated CpG and can also interact with other co-regulatory complexes. MeCP2 is X-linked and affected by X inactivation.

[0060] Exon replacement via trans-splicing of MeCP2 precursor mRNA Therapeutic RNA exon editors have the potential to correct ~95% of Rett patient mutations with a single MeCP2 RNA exon editor, which replaces all exons in which ~95% of the mutations occur. The inventors have developed exemplary MeCP2 RNA exon editors capable of replacing a significant portion (e.g., at least about 15%) of the mutant MeCP2 protein in cultured cells with a biologically active, functional MeCP2 protein. The MeCP2 RNA exon editor described herein comprises a coding domain sequence including sequences encoding multiple MeCP2 exons of a functional (e.g., wild-type) MeCP2 amino acid sequence; a splicing domain sequence spliced ​​to native MeCP2 precursor mRNA; and a binding domain sequence annealed to introns of the native MeCP2 precursor mRNA. Such exemplary MeCP2 RNA exon editors will be administered to animal subjects, and it is anticipated that 15% or more of the mutant MeCP2 protein will be replaced by the functional MeCP2 protein, and the RNA exon editor will provide sufficient replacement of the mutant MeCP2 to achieve a therapeutic effect in vivo. While reluctant to be bound by theory, the exemplary MeCP2 RNA exon editor will confer therapeutic effects by achieving at least one of the following: restoring the functional level of transcriptional regulation, restoring the functional level of binding to methylated DNA, or restoring the functional level of interaction with co-regulatory factor complexes, or any combination thereof.

[0061] B. Definition As used herein, "trans-splicing" refers to the linking of a first RNA molecule containing one or more exons (e.g., exogenous exons or exons that are part of a coding domain of an RNA exon editor) to a second RNA molecule (e.g., an endogenous precursor mRNA molecule) via a spliceosome-mediated mechanism, replacing a portion of the second RNA molecule with a portion of the first RNA molecule. For example, Figure 2 The general mechanism of RNA trans-splicing is illustrated.

[0062] As used herein, the terms “RNA exon editor,” “precursor mRNA trans-splicing molecule,” “RNA trans-splicing molecule (RTM),” “nucleic acid trans-splicing molecule,” or “trans-splicing molecule” are used interchangeably. In some embodiments, an RNA exon editor comprises three main elements: (a) a binding domain that imparts specificity by tethering the trans-splicing molecule to its target gene (e.g., precursor mRNA); (b) a hemiintron containing a splicing site; and (c) a coding domain configured to trans-splice to a target nucleic acid, said coding domain being capable of replacing one or more exons in the target nucleic acid (e.g., containing at least one mutated exon). An RNA exon editor is an effector molecule that physically binds to a target precursor mRNA. The terms “DNA encoding an RNA exon editor,” “exon editor construct encoding an RNA exon editor,” or “DNA sequence encoding an RNA exon editor” can be used to refer to a DNA sequence, construct, or vector encoding an RNA exon editor. In some embodiments, the vector may be an AAV vector containing a DNA sequence encoding an RNA exon editor. In some embodiments, the DNA sequence encoding the RNA exon editor contains cDNA, for example, as part of a functional exon (e.g., a functional MeCP2 exon), to replace at least one mutated MeCP2 exon in a target gene (e.g., a precursor mRNA). In some cases, the sequence of the RNA exon editor component is disclosed herein in the form of a DNA sequence. For any sequence disclosed herein in the form of a DNA sequence, it is also contemplated to replace each T in the RNA sequence with U. Thus, if a given SEQ ID NO is identified as having a sequence that can be included in the RNA exon editor, the SEQ ID NO form of replacing each T with U is also contemplated.

[0063] As used herein, “trans-splicing efficiency” refers to the ratio of the detected expression level of the desired trans-spliced ​​RNA product (i.e., a chimeric RNA molecule produced by RNA trans-splicing, comprising a functional exon of an RNA exon editor operatively linked to an endogenous target precursor mRNA) to the amount of DNA encoding the RNA exon editor or the introduced RNA exon editor (or reference molecule). In some cases, the expression level of the trans-spliced ​​RNA product is detected based on RNA isolated from cells or tissues using RNA-seq.

[0064] As used in this article, “RNA replacement %” refers to the portion of the total target mRNA population that has been successfully trans-spliced ​​(TS), and is calculated using the following equation: Target (ONT) TS% = 100 * (ONT copy number / (ONT copy number + native copy number)).

[0065] As used herein, “relative trans-splicing efficiency” refers to the ratio of the tested trans-splicing efficiency to a reference trans-splicing efficiency, where the tested trans-splicing efficiency is the trans-splicing efficiency of the RNA exon editor described herein (the first RNA exon editor; the tested one), and the reference trans-splicing efficiency is the trans-splicing efficiency of a reference RNA exon editor (e.g., an RNA exon editor containing the same elements as the first RNA exon editor, but whose binding domains are replaced with out-of-order binding domains or non-target binding domains). The relative trans-splicing efficiency of the RNA exon editor can be given as the ratio (also known as a fold increase) of the efficiency of the first trans-spliced ​​RNA (test) tested under similar conditions to the reference trans-splicing efficiency.

[0066] As used herein, the term "operably linked" refers to an arrangement of elements in which the described components are configured to perform their usual function. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked" to the other nucleic acid sequence. Elements do not need to be adjacent to be operably linked. Thus, for example, an intermediary sequence may exist between operably linked sequences (e.g., a binding domain and a coding sequence may be separated by an intermediary sequence, and the binding domain is still considered "operably linked" to the coding sequence).

[0067] As used herein, the term "coding domain" refers to a nucleic acid sequence (e.g., an RNA sequence, a DNA sequence, or a combination of RNA and DNA) that encodes a portion of a protein (e.g., a target protein in which a mutation is being corrected). Therefore, a coding domain may include one or more functional exons (e.g., sequences of functional exons). In some cases, one or more functional exons of a coding domain are not separated by introns (e.g., as in endogenous precursor mRNA) but are adjacent to each other (e.g., as in cDNA). In some cases, a coding domain may include one or more introns (e.g., native introns), or untranslated regions (UTRs, such as native UTRs) located between exons or, in other cases, adjacent to exons (e.g., upstream or downstream).

[0068] As used herein, "native 3' MeCP2 untranslated region" or "native 3' MeCP2 UTR" refers to a sequence longer than 20 nucleotides that shares at least 90% sequence identity with the region at the 3' of the translation stop codon in the native MeCP2 gene (e.g., the human MeCP2 gene). In some embodiments, the RNA exon editor contains a native 3' MeCP2 untranslated region. The native 3' MeCP2 untranslated region contains an alternative polyA signal spanning from 0.1 kb to 8.5 kb for the MeCP2 3' UTR, corresponding to native MeCP2 3' UTRs of varying lengths that are differentially expressed in different tissues. The 8 kb 3' UTR is enriched in brain tissue compared to shorter MeCP2 3' UTRs. In some embodiments, the native 3' MeCP2 UTR is replaced by a shorter 3' UTR. In some embodiments, the 3' UTR is the RDH1pA 3' UTR, a synthetic 3' UTR containing a 110 bp highly conserved MeCP2 distal polyadenylation signal and an upstream miRNA binding panel containing sites for three additional miRNAs endogenous to the MECP2 3' UTR: miR-19, miR-22, and miR-132. The DNA sequence encoding the RDH1pA 3' UTR contains SEQ ID NO: 19.

[0069] As used herein, the “functional sequence of the 3' MeCP2 exon” refers to a nucleic acid sequence containing one or more of MeCP2 exons 2, 3, and 4 (e.g., exons 3 and 4) that encodes the functional (biologically active) portion of the MeCP2 protein. In some embodiments, the “functional sequence of the 3' MeCP2 exon” refers to a nucleic acid sequence containing exons 3-4 of MeCP2 that encodes the functional (biologically active) portion of the MeCP2 protein. In some embodiments, the “functional sequence of the 3' MeCP2 exon” refers to a nucleic acid sequence containing exons 2-4 of MeCP2 that encodes the functional (biologically active) portion of the MeCP2 protein. In some embodiments, the “functional sequence of the 3' MeCP2 exon” refers to a nucleic acid sequence containing exon 4 of MeCP2 that encodes the functional (biologically active) portion of the MeCP2 protein. The functional sequence of the 3' MeCP2 exon provides expression of a functional MeCP2 protein (e.g., a non-mutated MeCP2 protein) when 5' trans-splicing is performed at the binding site to an endogenous MeCP2 exon. In some cases, the functional sequence of the 3' MeCP2 exon includes the exon sequence adjacent to the exon that the RNA exon editor is trans-splicing (e.g., an RNA exon editor that binds to MeCP2 intron 1 and trans-splices with endogenous MeCP2 exon 1 may include the functional sequence of the 3' MeCP2 exon containing exons 3 and 4).

[0070] As used herein, the term "functional" when used in a protein context refers to a biologically active protein. The term "functional" can also be used to refer to the amount of protein activity necessary to support normal cellular function. For MeCP2, the term "functional" can be used to refer to the amount of MeCP2 protein activity necessary to restore MeCP2 activity levels to support normal cellular function, for example, in the neuronal context of the brain. Such levels are sufficient to reduce or prevent disruption of gene expression, neuronal development, synaptic maturation, plasticity, and / or other disease manifestations associated with reduced MeCP2 activity levels. More specifically, since defective (non-functional) MeCP2 proteins containing at least one mutation lead to synaptic and circuit-level defects in brain function, restoring the functional level of MeCP2 at least in part means increasing biologically active MeCP2 protein to reduce the amount or extent of gene expression defects, synaptic defects, circuit-level defects, and / or other brain functional defects associated with reduced MeCP2 activity levels. While reluctant to be bound by theory, the term “functional” can be used to refer to the amount of MeCP2 protein activity necessary to achieve at least one of the following: the level of functional restoration of transcriptional regulation, the level of functional restoration of binding to methylated DNA, or the level of functional restoration of interaction with co-regulatory factor complexes, or any combination thereof.

[0071] In the context of treating symptoms associated with pathogenic MeCP2 activity (e.g., Rett syndrome) or using therapeutic agents containing an RNA exon editor or a DNA sequence encoding an RNA exon editor as described herein, "functional" refers to restoring an amount of functional (biologically active) MeCP2 protein sufficient to alleviate or eliminate one or more symptoms associated with symptoms (e.g., Rett syndrome) of a condition containing at least one mutated defective (non-functional) MeCP2 protein and / or reduced MeCP2 activity levels. In some embodiments, such methods or uses result in an increase in MeCP2 protein activity. In some implementations, such an increase in MeCP2 protein activity, relative to control (normal / wild-type) cells expressing functional (bioactive) MeCP2 at levels supporting normal cellular function, would increase the level of functional (bioactive) MeCP2 activity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%). In some implementations, such an increase in MeCP2 protein activity, relative to cells where MeCP2 exists at normal wild-type levels (such as those present in cells expressing non-mutant MeCP2), restores the MeCP2 activity level to at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) of the MeCP2 activity.

[0072] As used herein, “cryptic splice site corrected,” “cryptic splice site mitigated,” or “crypticsplice site resistant” refers to an RNA exon editor or a nucleic acid molecule encoding an RNA exon editor, or a portion thereof (e.g., a coding domain sequence therein), that has been modified to alter individual nucleotides therein to reduce the splicing frequency occurring at cryptic splice sites identified in the context of an RNA exon editor. In some embodiments, the modification does not result in any change to the amino acid sequence thereby encoded. In some embodiments, the cryptic splice site resistant nucleic acid sequence within the RNA exon editor is a coding domain sequence (CDS). In some implementations, the cryptic splice site resistant MeCP2 CDS comprises, is substantially composed of, or consists of exons 3 and 4 or exon 4 of the MeCP2 gene, wherein cryptic splice sites have been identified in the context of a MeCP2 RNA exon editor, and wherein at least one of the cryptic splice sites has been modified to reduce the splicing frequency at at least one site without altering the amino acid encoded therefrom.

[0073] As used herein, a “half-intron” refers to a nucleic acid sequence containing a motif recognized by the spliceosome and mediating trans-splicing. In some embodiments, a half-intron contains a polypyrimidine bundle (pPy), a branch point (BP), and / or a 3' splice site, or any combination thereof. See, for example, SEQ ID NO: 17 as an exemplary half-intron. The 3' splice site may be a common 3' splice site. In some embodiments, a half-intron contains other regulatory elements that have one or more of the following effects: increasing the recruitment of splicing factors to the RNA exon editor, increasing the transcription / expression of functional MeCP2, increasing the efficiency of on-target (ONT) trans-splicing, reducing off-target (OFT) trans-splicing, or reducing cis-splicing on target precursor mRNA, or any combination thereof.

[0074] As used herein, the “binding domain” of an RNA exon editor or a nucleic acid molecule encoding the RNA exon editor is a multinucleotide sequence that binds to a target gene (e.g., endogenous precursor mRNA) at a binding site by hybridization (i.e., complete or partial complementarity with the binding site).

[0075] As used herein, the term "binding site" refers to an endogenous polynucleotide sequence in the target precursor mRNA (e.g., precursor mRNA of an endogenous gene, such as MeCP2 precursor mRNA) that binds to a binding domain of the RNA exon editor. The binding site extends from the 5'th nucleotide to which the binding domain binds to the 3'th nucleotide. In some embodiments, the length of the binding site is the same as that of the binding domain. In some embodiments, the binding site is 1-10 nucleotides longer or shorter than the binding domain (i.e., some nucleotides of the binding site or the binding domain do not hybridize). In embodiments involving a binding domain having at least two non-overlapping sequences that are at least 80% complementary to the binding site, the binding site may be substantially shorter than the binding domain.

[0076] As used herein, “complementarity” and its grammatical variations refer to the percentage of nucleotide bases in a given sequence that pair with a reference sequence via hydrogen bonds. In the absence of a given percentage of complementarity, the terms “complement” and “complementary” refer to 100% complementarity.

[0077] As used herein, if each nucleotide base of a given sequence (e.g., a binding domain sequence) pairs with a reference sequence (e.g., an endogenous precursor mRNA binding site) via hydrogen bonding to form a double-stranded sequence (e.g., via Watson-Crick base-pairing, such as each A pairing with T or U, and each C pairing with G), then the given sequence is “100% complementary” to the reference sequence, or has “100% complementarity”. For example, a binding domain antisense-oriented to the binding site is complementary to the binding site. RNA pairing includes G-U pairing; therefore, an RNA binding domain that pairs with its binding site via GU is 100% complementary to the binding site. Therefore, a binding domain that is completely antisense-complementary to its binding site (i.e., the A of the binding domain pairs with the U of the binding site) can be modified to replace any one or more A's with G without significantly affecting binding.

[0078] As used herein, if X% of the nucleotide bases of a given sequence (e.g., a binding domain sequence) pair with a reference sequence (e.g., an endogenous precursor mRNA binding site) via hydrogen bonding, for example, by hybridization to form a double-stranded sequence (e.g., via Watson-Crick base pairing, such as A pairing with T or U, and C pairing with G), then the given sequence is “at least X% complementary” to the reference sequence, or has “X% complementarity”. For example, if at least 135 of the 150 residues of a binding domain sequence of length 150 bases pair with a binding site of length 150 bases via hydrogen bonding through Watson-Crick base pairing, and the remaining 15 or fewer mismatched nucleotides, then the binding domain sequence is at least 90% complementary to the binding site.

[0079] As used herein, “binding” between a binding domain and an intron refers to a hydrogen bond (e.g., double helix formation or Watson-Crick pairing) between the binding domain and, for example, a target intron, sufficient to mediate trans-splicing by associating the RNA exon editor with the target (e.g., precursor mRNA). In some embodiments, the hydrogen bond between the binding domain and the target intron is located between complementary and antisense oriented (e.g., hybridizing) nucleotide bases.

[0080] As used herein, the term "mutation" can be used to refer to any abnormal nucleic acid sequence encoding a defective protein product (e.g., a nonfunctional protein product, a non-biologically active protein, a protein product with reduced function, a protein product with pathogenic or abnormal function, and / or a protein product produced in amounts less than or greater than normal). Mutations include base pair mutations (e.g., single nucleotide polymorphisms), duplications, missense mutations, frameshift mutations, deletions, insertions, and splicing mutations. In some embodiments, a mutation refers to a nucleic acid sequence that differs from one or more portions of the corresponding wild-type nucleic acid sequence or a functional variant thereof. In some embodiments, a mutation refers to a nucleic acid sequence encoding a protein having an amino acid sequence that differs from the corresponding wild-type protein or a functional variant thereof. A "mutated exon" (e.g., a mutated MeCP2 exon) refers to an exon containing a mutation or an exon sequence reflecting a mutation in a different region, such as a cryptic exon resulting from a mutation in an intron.

[0081] Unless otherwise stated, the term "MeCP2" (methylCpG-binding protein 2) refers to any native MeCP2 from any vertebrate source, including mammals such as primates (e.g., humans, African green monkeys, and cynomolgus monkeys) and rodents (e.g., mice and rats), as well as functionally equivalent or modified variants (e.g., natural or synthetic variants), mutants, mutant proteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments. Functionally equivalent and modified variants can be identified based on known MeCP2 signaling. MeCP2 encompasses full-length, unprocessed MeCP2, as well as any form of MeCP2 naturally produced in cells. Exemplary human MeCP2 sequences are provided as reference sequences from the National Center for Biotechnology Information (NCBI): NM_001110792.2 (specifically for the E1 isotype lacking exon 2) or NM_004992.4 (specifically for the E2 isotype containing exons 1-4). In some cases, the MeCP2 fragment is encoded by a therapeutic agent comprising a sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NO: 10, 11, or 12 or any combination thereof (e.g., a box containing exon 3 and exon 4 sequences, or a box containing exon 2, 3, and 4 sequences) or SEQ ID NO: 9 or a fragment thereof (e.g., a box containing exon 3 and exon 4 sequences) or a functional portion thereof, and / or a codon-modified variant thereof. Exemplary codon-modified variants of this type include codon-modified variants of exon 3 (e.g., SEQ ID NO: 13 and 85) and codon-modified variants of exon 4 (e.g., SEQ ID NO: 14 and 86), as well as combinations thereof (e.g., a box of codon-modified variants of exon 3 and exon 4 sequences; SEQ ID NO: 76). In some embodiments, the functional MeCP2 exon includes a codon-modified variant of exon 4 (e.g., SEQ ID NO: 14 or 86).

[0082] As used herein, a “variant” means a polynucleotide that differs from a reference polynucleotide sequence (such as a naturally occurring polynucleotide sequence) at at least one nucleic acid residue, or a polypeptide (e.g., an AAV capsid sequence) that differs from a reference polypeptide sequence (such as a naturally occurring polypeptide sequence or, for example, any rAAV sequence described herein) at at least one amino acid residue. In this context, a difference of at least one residue can include, for example, the substitution, deletion, or insertion of one nucleic acid residue by another nucleic acid, or the substitution of one amino acid residue by another amino acid. A variant can be a homolog, isotype, or transcriptomic variant of a polynucleotide as defined herein, wherein the homolog, isotype, or transcriptomic variant is characterized by the degree of identity or homology as defined herein. In some cases, variants of polynucleotides or polypeptides include at least one nucleic acid substitution (e.g., 1-100 nucleic acid or amino acid substitutions, 1-50 nucleic acid or amino acid substitutions, 1-20 nucleic acid or amino acid substitutions, 1-10 nucleic acid or amino acid substitutions, such as 1 nucleic acid or amino acid substitution, 2 nucleic acid or amino acid substitutions, 3 nucleic acid or amino acid substitutions, 4 nucleic acid or amino acid substitutions, 5 nucleic acid or amino acid substitutions, 6 nucleic acid or amino acid substitutions, 7 nucleic acid or amino acid substitutions, 8 nucleic acid or amino acid substitutions, 9 nucleic acid or amino acid substitutions, or 10 nucleic acid or amino acid substitutions). Nucleic acid substitutions that give the expressed polypeptide an amino acid exchanged from the same class are referred to herein as conserved substitutions. Specifically, these are amino acids having aliphatic side chains, positively or negatively charged side chains, or aromatic groups in the side chains, wherein the side chains can form hydrogen bridges, such as side chains with hydroxyl functional groups. Through conservative substitution, for example, an amino acid with a polar side chain can be replaced by another amino acid with a corresponding polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain can be replaced by another amino acid with a corresponding hydrophobic side chain (e.g., serine (threonine) is replaced by threonine (serine), or leucine (isoleucine) is replaced by isoleucine (leucine).

[0083] In some cases, the terminology also encompasses insertions, deletions, and / or non-conserved substitutions, such as at locations that do not result in substantial modifications to the protein's three-dimensional structure. Those skilled in the art can readily determine modifications to the three-dimensional structure through insertions or deletions, for example, using CD spectroscopy (circular dichroism spectroscopy).

[0084] The term "homology" refers to the degree of similarity between two nucleic acid sequences. Sequence homology is determined by comparing two sequences aligned under standard conditions in terms of length. The sequences to be compared in this paper may have additions or deletions (e.g., gaps) in the best alignment of the two sequences. In some implementations, sequence homology is calculated by creating alignments using, for example, the ClustalW algorithm (Nucleic Acid Res., 1994, 22(22): 4673 4680). Commonly available sequence analysis software, such as Vector NTI, GENTYX, BLAST, or analysis tools provided by public databases, can also be used.

[0085] As used herein, the term "heterogeneous" when used to describe a first element with reference to a second element means that the first and second elements do not exist in nature in the manner described. For example, a heterologous nucleic acid sequence (e.g., a heterologous 3'UTR) may refer to a nucleic acid sequence that is contained in a construct of this disclosure and is operatively linked to a nucleic acid sequence that is not found in nature to be operatively linked to.

[0086] As used herein, the term “AAV” or “AAV serotype” refers to dozens of naturally occurring and available adeno-associated viruses, as well as artificially created AAVs. Among the AAVs isolated from or engineered from humans or non-human primates (NHPs) and well characterized, human AAV2 was the first AAV developed as a gene transfer vector; it has been widely used for efficient gene transfer experiments in various target tissues and animal models.

[0087] As used herein, with respect to AAV, the term variant means any AAV sequence derived from a known AAV sequence, including those that share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or greater sequence identity on the amino acid or nucleic acid sequence. In another embodiment, the AAV capsid includes variants that may include up to about 10% variation with any described or known AAV capsid sequence. That is, the AAV capsid shares about 90% to about 99.9% identity, about 95% to about 99% identity, or about 97% to about 98% identity with AAV capsids provided herein and / or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with other AAV capsids. When determining the percentage of identity of the AAV capsid, comparisons can be made with any variable protein (e.g., vp1, vp2, or vp3).

[0088] ITRs or other AAV components can be readily isolated from or engineered from AAVs using techniques available to those skilled in the art. Such AAVs can be isolated, engineered, or obtained from academic, commercial, or public sources (e.g., Manassas, Va.). Alternatively, AAV sequences can be engineered using synthetic or other suitable methods, referencing published sequences (such as those in the literature or databases such as GenBank, PubMed, etc.). AAV viruses can be engineered using conventional molecular biology techniques, thereby optimizing these particles to achieve cell-specific delivery of nucleic acid sequences, minimize immunogenicity, adjust stability and particle lifetime, efficiently degrade, and accurately deliver to the cell nucleus, among other things.

[0089] As used herein, the terms “subject,” “individual,” or “patient” include any mammal, including primates such as humans, that requires these treatments or preventative measures. Other mammals requiring such treatments or preventative measures include non-human primates (NHPs; e.g., cynomolgus monkeys and African green monkeys), dogs, cats or other domesticated animals, horses, livestock, laboratory animals, etc. An individual may be male or female. In one embodiment, the individual suffers from a disease or condition caused by a mutation in the MeCP2 gene (e.g., Rett syndrome). In another embodiment, the individual is at risk of developing a disease or condition caused by a mutation in the MeCP2 gene. In yet another embodiment, the individual has shown clinical signs of a disease or condition caused by a mutation in the MeCP2 gene (such as Rett syndrome). The individual may be of any age at which the treatment or preventative therapy might be beneficial. For example, in some implementation schemes, individuals are 0-5 years old, 5-10 years old, 10-20 years old, 20-30 years old, 30-40 years old, 30-50 years old, 40-50 years old, 50-60 years old, 60-70 years old, or over 70 years old.

[0090] As used herein, the terms "mutation-related condition" or "mutation-related condition" refer to the correlation between a condition and a mutation. In some embodiments, a mutation-related condition is known or suspected to be wholly or partially, or directly or indirectly, caused by said mutation. For example, an individual with the mutation may be at risk of developing said condition, and said risk may also depend on other factors, such as other (e.g., independent) mutations (e.g., in the same or different genes) or environmental factors.

[0091] As used herein, the term "treatment" or its grammatical derivatives are defined as reducing disease progression, alleviating the severity of disease symptoms, slowing the progression of disease symptoms, removing disease symptoms, or delaying the onset of disease. In some implementations, the term "treatment" is used to refer to the sustained or durable effect of a therapeutic agent (such as the RNA exon editor described herein).

[0092] As used herein, the term "prevention" of a disease, or its grammatical derivatives, is defined as reducing the risk of disease onset, for example, preventative therapy for individuals at risk of developing a mutation-related disease. An individual can be characterized as "at risk of developing a disease" by identifying a mutation associated with the disease, according to any suitable method known in the art or described herein. In some embodiments, an individual at risk of developing a disease has one or more disease-related MeCP2 mutations. Alternatively or additionally, an individual may be characterized as "at risk of developing a disease" if they have a family history of the disease.

[0093] MeCP2 syndrome is inherited in an X-linked manner. Over 99% of MeCP2 cases are sporadic (i.e., only one case occurs in a family), inherited by newborns. de novo The MeCP2 variant can be caused by or possibly inherited from parents with germline mosaicism. In rare cases, the MeCP2 variant can be inherited from a heterozygous mother, where X chromosome inactivation results in very few or no clinical manifestations. For a known heterozygous mother, the risk of her child inheriting the MeCP2 variant is 50%.

[0094] Treatment or prevention of an individual's condition can be performed by directly administering DNA encoding an RNA exon editor (e.g., via a vector containing a DNA sequence encoding an RNA exon editor) or the RNA exon editor itself to the individual. In some embodiments, the vector containing the DNA sequence encoding an RNA exon editor includes an AAV vector. In some embodiments, the AAV vector is administered via AAV viral particles. Alternatively, host cells containing an RNA exon editor can be administered to the individual.

[0095] As used in the methods described herein, the term "application" or its grammatical derivatives refer to the delivery of an RNA exon editor or a DNA sequence encoding said RNA exon editor (e.g., within a vector (e.g., an AAV vector or AAV particles)) or a combination thereof, or ex vivo treated cells, to an individual in need, such as an individual with a MeCP2 mutation or defect. In some embodiments, the brain is targeted to deliver an RNA exon editor or a DNA sequence encoding said RNA exon editor (e.g., within a vector (e.g., an AAV vector or AAV particles)) or a combination thereof. In some embodiments, the targeted brain cells include, for example, neurons, neural stem cells, astrocytes, or oligodendrocytes, or any combination thereof.

[0096] In some embodiments, the method involves delivering an RNA exon editor or a DNA sequence encoding the RNA exon editor (e.g., within a vector (e.g., an AAV vector or AAV particles)) or a combination thereof to an individual via intravenous (IV), intracerebral (IC) delivery (e.g., slow delivery injection or convection-enhanced diffusion injection), intraventricular (ICV) delivery, intrathecal delivery, or intracerebellomedullary cistern delivery. In some embodiments, the target is a neuron, neural stem cell, astrocyte, or oligodendrocyte, or any combination thereof. In some embodiments, IC injection involves stereotactic implantation of a microinjection-guided cannula to improve delivery to a specific site in the brain. In some embodiments, the composition is administered systemically (e.g., intravenously). Other methods of administration may also be chosen by those skilled in the art in light of this disclosure.

[0097] As used herein, “regulating MeCP2 expression” refers to increasing the expression of functional MeCP2 protein produced after trans-splicing. Increased expression of functional MeCP2 protein produced after trans-splicing is associated with decreased expression of endogenous mutant (non-functional) MeCP2. Regulating MeCP2 expression can be used to refer, for example, to increasing the expression of MeCP2 protein produced after trans-splicing (e.g., MeCP2 transcripts or protein products with corrected mutation sites mediated by RNA exon editors, relative to their endogenous mutant transcripts or protein products). Regulating MeCP2 expression can also be used to refer, for example, to decreasing the expression of endogenous (e.g., mutant) MeCP2. Functional MeCP2 protein is expressed after trans-splicing of an endogenous MeCP2 exon containing at least one mutation.

[0098] As used herein, “codon optimization” refers to modifying a nucleic acid sequence to alter individual nucleic acids without altering the encoded amino acids. Sequences modified in this manner are referred to herein as “codon-optimized.” This process can be performed on any sequence described in this specification to enhance expression or stability. Codon optimization can be performed as described in, for example, U.S. Patent Nos. 7,561,972, 7,561,973, and 7,888,112, each of which is incorporated herein by reference in its entirety. Sequences around translation start sites can be converted into shared Kozak sequences according to known methods. See, for example, Kozak et al., 1987. Nucleic Acids Res. 15 (20): 8125-8148, which is incorporated herein by reference in its entirety.

[0099] The term "pharmaceuticalally acceptable" means that it is safe for use in mammals, such as humans. In some implementations, a pharmaceutically acceptable composition is approved by a federal or state regulatory agency, or is listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals and more specifically for use in humans.

[0100] The term "carrier" refers to a diluent, adjuvant, excipient, or mediator that is administered together with a therapeutic molecule (e.g., a trans-splicing molecule of this disclosure or a carrier or cell comprising a trans-splicing molecule). Examples of suitable drug carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18th edition.

[0101] The term “a (a / an)” means “one (species) or more (species)”. For example, “one gene” should be understood to represent one or more such genes. Therefore, the terms “a (a / an)”, “one or more of a (oran)”, and “at least one of a (or an)” are used interchangeably in this document.

[0102] Unless otherwise stated, as used herein, the term “about” means a value that varies within ±10% relative to a reference value.

[0103] C. RNA exon editor This document provides an RNA exon editor and a DNA sequence encoding the RNA exon editor (e.g., a vector containing a DNA sequence encoding an RNA exon editor or an AAV viral particle containing a DNA sequence encoding an RNA exon editor), which can be used to correct mutations in MeCP2 by replacing at least one MeCP2 exon containing at least one mutation with a functional MeCP2 exon (e.g., a MeCP2 exon with a 3' binding site, such as exons 3-4, 2-4, or 4 of MeCP2). In some embodiments, the RNA exon editor is referred to as a precursor RNA trans-splicing molecule (RTM). The RNA exon editor is designed to allow the replacement of defective or mutated portions of precursor mRNA exons with nucleic acid sequences, such as exons with functional (e.g., normal) sequences without mutations. The functional sequence can be a wild-type, naturally occurring sequence, or a corrected sequence with some other modifications (e.g., codon optimization).

[0104] In some embodiments, the RNA exon editor includes a binding domain, a half-intron, and a coding domain. In some embodiments, the RNA exon editor includes a binding domain, a splicing site, and a coding domain. In some embodiments, the RNA exon editor has a 3' regulatory domain containing a native 3' MeCP2 untranslated region (UTR) or a heterologous 3' UTR (e.g., a sequence having at least 80% sequence identity with SEQ ID NO: 19 or SEQ ID NO: 20). In some embodiments, the RNA exon editor has a 3' splicing site YAG / [where Y is a pyrimidine (cytosine or thymidine (which may be uracil in RNA)); " / " indicates the location of the 3' splicing site; for example, TAG]. In some embodiments, the RNA exon editor comprises a hemiintron that comprises, substantially comprises, or comprises the following: SEQ ID NO: 17 or a sequence having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 17. In some embodiments, the RNA exon editor further comprises at least one of an SV40 terminator and a poly A sequence (e.g., SEQ ID NO: 21) or a MALAT1 triple-helix terminator sequence (e.g., SEQ ID NO: 22), or a combination thereof. See, for example, Figure 5 and Figure 16 .

[0105] In some embodiments, the RNA exon editor described herein includes, operatively connected in the 5' to 3' direction, a binding domain, a half-intron, a sequence encoding a domain (e.g., a CDS, such as a functional sequence encoding a MeCP2 exon (e.g., the functional sequence of the MeCP2 exon at the binding site 3'), and a 3' untranslated region. In some embodiments, the RNA exon editor described herein includes, operatively connected in the 5' to 3' direction, a binding domain, a half-intron, a sequence encoding a domain (e.g., a CDS, such as a functional sequence encoding a MeCP2 exon (e.g., the functional sequence of the MeCP2 exon at the binding site 3'), a 3' untranslated region, a termination and poly A sequence, and a triple-helix terminator.

[0106] In some embodiments, the RNA exon editor described herein is configured to correct at least one mutation (e.g., two different mutations, each present in a single exon or two different exons of the MeCP2 gene in the subject) located in the 3' region of the MeCP2 gene (e.g., the 3' region of intron 1) by trans-splicing a coding domain having a functional sequence of the 3' MeCP2 exon to the endogenous MeCP2 exon of the target intron 5' of the MeCP2 gene, by binding to target intron 1 and mediating trans-splicing of the coding domain having a functional sequence of the 3' MeCP2 exon to the endogenous MeCP2 exon of the target intron. Such trans-splicing thereby repairs the defective MeCP2 gene in the individual's target cells by replacing the defective exon and removing the defective portion of the target precursor mRNA, resulting in functional MeCP2 mRNA capable of transcribing the functional MeCP2 protein in the cell.

[0107] 1. MeCP2 The MeCP2 gene targeted by the trans-splicing molecules described herein may contain one or more mutations associated with Rett syndrome. Exemplary human MeCP2 sequences are provided as reference sequences from the National Center for Biotechnology Information (NCBI): NM_001110792.2 (specific to the E1 subtype) or NM_004992.4 (specific to the E2 subtype). In addition to published sequences, all subsequently obtained corrections or naturally occurring conserved and non-pathogenic variant sequences present in humans or other mammalian populations are included. Those with additional conserved nucleotide substitutions or those leading to codon optimization are also included. Sequences provided via database accession numbers can also be used to search for homologous sequences in the same or another mammalian organism.

[0108] The MeCP2 protein contains the following well-defined structural / functional domains: N-terminal domain (NTD); methyl-binding domain (MBD); intercalary domain (ID); transcriptional repression domain (TRD); NCoR-interacting domain (NID); and C-terminal domain (CTD) (see [link to relevant documentation]). Figure 1 The TRD contains nuclear localization sequences (NLS). Mutations in the coding region of the X-chromosome-linked MeCP2 gene alter the ability of its encoded protein, MeCP2, to bind DNA against a chromatin background. Mutations affecting the MBD of MeCP2 influence its DNA-binding stability and affinity.

[0109] It is anticipated that the MeCP2 nucleic acid sequence and the resulting expressed protein will tolerate certain minor modifications at the nucleic acid level, including, for example, modifications to silent nucleotide bases (e.g., preferred codons). In other embodiments, nucleic acid base modifications that alter amino acids are envisioned, for example, to improve the expression of the resulting peptide / protein. In some embodiments, modifications based on allelic variations arising from the natural degeneracy of the genetic code are envisioned.

[0110] Modifications to the MeCP2 gene also include analogs or modified forms of the encoded amino acid sequence. Typically, these analogs differ from the specific identified protein by only one to four codon changes. Conserved substitutions occur within amino acid families that are related to side chains and chemical properties.

[0111] The nucleic acid sequence of the functional MeCP2 gene can be derived from any mammal that naturally expresses functional MeCP2 or its homologs. In other embodiments, the MeCP2 gene sequence is modified to enhance expression in target cells. Such modifications include codon optimization.

[0112] As described above, Rett syndrome is caused by mutations in the MeCP2 gene. Compositions containing the RNA exon editor described herein can correct approximately 95% of mutations associated with Rett syndrome because the RNA exon editor replaces all exons 3 and 4 of the MeCP2 gene, where approximately 95% of identified mutations are located.

[0113] The attached sequence listing provides various sequences of human MeCP2. SEQ ID NO: 1 is a partial nucleotide sequence of wild-type MeCP2 intron 1, starting at position 1 of intron 1. SEQ ID NO: 113 is the complete nucleotide sequence of wild-type MeCP2 intron 1. SEQ ID NO: 2 is a partial nucleotide sequence of wild-type MeCP2 intron 2, starting at position 1 of intron 2. SEQ ID NO: 112 is the complete nucleotide sequence of wild-type MeCP2 intron 2. SEQ ID NO: 3 is a partial nucleotide sequence of wild-type MeCP2 intron 2, ending at the last nucleotide of intron 2. SEQ ID NO: 4 is the complete nucleotide sequence of wild-type MeCP2 intron 3. SEQ ID NO: 5 is the nucleotide sequence of the open reading frame of wild-type MeCP2 subtype E1. SEQ ID NO: 6 is the sequence of the open reading frame of wild-type MeCP2 E1 exon 1. SEQ ID NO: 7 is the sequence of wild-type MeCP2 E1 exon 3. SEQ ID NO: 7 is the sequence of exon 4 of wild-type MeCP2 E1. SEQ ID NO: 9 is the nucleotide sequence of the open reading frame of wild-type MeCP2 isoform E2. SEQ ID NO: 10 is the sequence of the open reading frame of exon 2 of wild-type MeCP2 E2. SEQ ID NO: 11 is the sequence of exon 3 of wild-type MeCP2 E2. SEQ ID NO: 7 is the sequence of exon 4 of wild-type MeCP2 E2.

[0114] 2. Encoding Structure Domain In some embodiments, the coding domain of the 3' trans-splicing molecule includes all MeCP2 exons (e.g., functional MeCP2 exons) located at the 3' of the target MeCP2 intron (e.g., MeCP2 intron 1). In some embodiments where the 3' trans-splicing molecule targets MeCP2 intron 1, the coding domain may include functional MeCP2 exons 3-4. In some embodiments, functional MeCP2 exons 3-4 are encoded by sequences comprising SEQ ID NO: 11 and 12 or sequences having at least 95% sequence identity with sequences comprising SEQ ID NO: 11 and 12. In some embodiments where the 3' trans-splicing molecule targets MeCP2 intron 1, the coding domain may include functional MeCP2 exons 2-4. In some embodiments, functional MeCP2 exons 2-4 are encoded by sequences comprising SEQ ID NO: 9 or sequences having at least 95% sequence identity with SEQ ID NO: 9 or fragments thereof. In any sequence disclosed herein as a coding domain sequence of an RNA trans-splicing molecule, U may replace T in the sequence. Thus, for example, in some embodiments, functional MeCP2 exons 2-4 comprise an RNA form of SEQ ID NO: 9 in which each T is replaced by U, or a sequence having at least 95% sequence identity with SEQ ID NO: 9 in which each T is replaced by U. In some embodiments, the binding domain binds to intron 2, and the coding domain comprises functional MeCP2 exons 3-4. In some embodiments, functional MeCP2 exons 3-4 are encoded by sequences comprising SEQ ID NO: 11 and 12, or sequences having at least 95% sequence identity with sequences comprising SEQ ID NO: 11 and 12. In some embodiments, the binding domain binds to intron 3, and the coding domain comprises functional MeCP2 exon 4. In some embodiments, the functional MeCP2 exon 4 is encoded by a sequence comprising SEQ ID NO: 12 or a sequence having at least 95% sequence identity with SEQ ID NO: 12. In some embodiments, the functional MeCP2 exon includes codon-modified variants of exon 3 (e.g., SEQ ID NO: 13 or 85) and codon-modified variants of exon 4 (e.g., SEQ ID NO: 14 or 86), as well as combinations thereof (e.g., a box containing codon-modified variants of exon 3 and exon 4 sequences; SEQ ID NO: 76). In some embodiments, the functional MeCP2 exon includes codon-modified variants of exon 4 (e.g., SEQ ID NO: 14).

[0115] In some embodiments, the coding domain (e.g., for a transgene encoding an RNA exon editor) includes a cDNA sequence of a MeCP2 exon (e.g., MeCP2 exons 3-4) to replace a mutated MeCP2 exon. For example, one or more functional MeCP2 exons within the coding domain may be a cDNA sequence. In some embodiments, the entire coding domain is a cDNA sequence. Alternatively or concurrently, all or part of the coding domain, or one or more functional MeCP2 exons thereof, may be naturally occurring sequences (e.g., sequences with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with endogenous MeCP2 exons).

[0116] In some embodiments, all or a portion of the coding domain or the coding sequence of the coding domain, or one or more functional MeCP2 exons, are codon-optimized sequences, wherein the nucleic acid sequence has been modified, for example, to enhance expression or stability, without causing changes to the encoded amino acids. Codon optimization can be performed in a manner as described in U.S. Patent Nos. 7,561,972, 7,561,973, and 7,888,112, each of which is incorporated herein by reference in its entirety. A codon-optimized form of MeCP2 exon 3 is shown in SEQ ID NO: 13. A codon-optimized form of MeCP2 exon 4 is shown in SEQ ID NO: 14. For delivery via recombinant AAV as described herein, in one embodiment, the coding domain can be a nucleic acid sequence of up to 4,000 nucleotides in length (e.g., 3,000 to 4,000 nucleotides in length, 3,100 to 3,800 nucleotides in length, 3,200 to 3,700 nucleotides in length, or 3,300 to 3,500 nucleotides in length, e.g., 3,000 to 3,100 nucleotides in length, 3,100 to 3,200 nucleotides in length, 3,200 to 3,300 nucleotides in length, 3,300 to 3,400 nucleotides in length, 3,400 to 3,500 nucleotides in length, 3,500 to 3,300 nucleotides in length, 3,400 to 3,500 nucleotides in length, 3,500 to 3,300 nucleotides in length). 600 nucleotide bases, 3,600 to 3,700 nucleotide bases in length, 3,700 to 3,800 nucleotide bases in length, 3,800 to 3,900 nucleotide bases in length, or 3,900 to 4,000 nucleotide bases in length, for example, about 3,108 nucleotide bases in length, about 3,285 nucleotide bases in length, about 3,375 nucleotide bases in length, about 3,503 nucleotide bases in length, about 3,630 nucleotide bases in length, about 3,540 nucleotide bases in length, about 3,363 nucleotide bases in length, about 3,273 nucleotide bases in length, about 3,145 nucleotide bases in length, or about 3,018 nucleotide bases in length).

[0117] In some embodiments, this document describes an RNA exon editor comprising, in the 5' to 3' direction: (a) a binding domain sequence configured to bind introns of an endogenous RNA molecule; (b) a splice acceptor sequence; and (c) a cDNA coding domain sequence; wherein the coding domain sequence contains at least one nucleotide change relative to the endogenous RNA molecule sequence, wherein the at least one nucleotide change disrupts a cryptic splicing site within the coding domain sequence. In some embodiments, the nucleotide change is a synonymous nucleotide change. In some embodiments, the cryptic splicing site is identified experimentally. In some embodiments, the cryptic splicing site is predicted based on computer analysis.

[0118] This document also covers a method for modifying RNA molecules in cells, the method comprising providing cells with an exogenous RNA molecule (via, for example, a DNA vector encoding the exogenous RNA molecule or a viral particle containing such a DNA vector), said exogenous RNA molecule comprising, in the 5' to 3' direction: (a) a binding domain sequence configured to bind to an intron of an endogenous target RNA molecule; (b) a splice acceptor sequence configured to splice to a splice donor sequence of the endogenous target RNA molecule; and (c) a cDNA coding domain sequence containing one or more nucleotide changes different from those of the endogenous target RNA molecule in the cell; wherein the one or more nucleotide changes disrupt one or more cryptic splicing sites within the coding domain sequence of the exogenous RNA molecule. In some embodiments, the nucleotide changes are synonymous nucleotide changes. In some embodiments, the cryptic splicing sites are identified experimentally. In some embodiments, the cryptic splicing sites are predicted based on computer analysis.

[0119] This article also covers a method for increasing the trans-splicing efficiency of an RNA exon editor or improving the therapeutic performance of an RNA exon editor, comprising introducing nucleotide changes into the coding domain sequence of the RNA exon editor, wherein the nucleotide changes disrupt cryptic splicing sites in the coding domain sequence of the RNA exon editor. In some embodiments, the nucleotide changes are synonymous nucleotide changes. In some embodiments, the cryptic splicing sites are identified experimentally. In some embodiments, the cryptic splicing sites are predicted based on computer analysis.

[0120] Nucleotide changes that reduce cryptic splicing sites can include eliminating or reducing the ability of cryptic splicing sites (e.g., cryptic splicing sites within coding or binding domain sequences of an RNA exon editor) to be used in splicing reactions. For example, cryptic splicing sites identified in the context of an RNA exon editor typically include splice sites, polypyrimidine bundles, and branching points. In some embodiments, one or more nucleotide changes may be introduced into at least one or any combination of splice sites, polypyrimidine bundles, or branching points of cryptic splicing sites identified in the context of an RNA exon editor. In some embodiments, nucleotide changes are designed to minimize potential effects on the protein thereby encoded. Those skilled in the art will understand that conserved amino acid changes will be preferred over non-conserved amino acid changes if the nucleotide changes made to reduce the frequency of cryptic splicing site use also alter the amino acids encoded by the trans-spliced ​​RNA. Furthermore, those skilled in the art can readily analyze protein sequences and structures, focusing on functional domains and important sequences therein, to assess whether such changes can be reasonably expected to alter the function of the protein encoded by the trans-spliced ​​protein. Skilled personnel can also use assays known in the art to test proteins containing such amino acid changes to determine whether biological activity has changed. In some embodiments, more than one nucleotide changes within the identified cryptic splicing site. In some cases, the number of nucleotides that should change is determined empirically based on computer predictions and / or experimental results. In some embodiments, one or more (also referred to herein as at least one) synonymous nucleotide changes may be introduced into at least one or any combination of splicing sites, polypyrimidine bundles, or branching points of cryptic splicing sites identified in the context of an RNA exon editor. Synonymous nucleotide changes do not alter the amino acid sequence of proteins encoded by trans-spliced ​​RNA. In some embodiments, more than one synonymous nucleotide change may be introduced into at least one or any combination of splicing sites, polypyrimidine bundles, or branching points of cryptic splicing sites identified in the context of an RNA exon editor.

[0121] In addition to the above, experimental results and sequence information are typically analyzed as follows: Variations are made to remove experimentally identified cryptic splicing sites by searching for and replacing certain elements of the splice donor site. For introducing nucleotide variations, the AG site at the end of the splice donor site (and the stronger CAG site) is preferred. If the AG site is not found or cannot be changed without introducing non-synonymous nucleotide variations, the sequence upstream of the splice site (42-4 base pairs) is scanned for branching points (e.g., sequences matching YNAH, where Y is pyrimidine, N represents any nucleotide, H is adenine, cytosine, or thymine (or uracil in RNA), and A is the branching point adenine). Any such branching point sequences identified are then analyzed, and the introduction of one or more nucleotide variations to reduce the use of cryptic splicing sites at experimentally identified sites is considered. Furthermore, the sequence is scanned to determine the presence of polypyrimidine bundles (multiple Y (pyrimidine) immediately upstream of the terminal AG). Typically, such polypyrimidine bundles contain at least five pyrimidines within 10 base pairs upstream of the splice site. Once identified, these polypyrimidine bundles are then analyzed, and the introduction of one or more nucleotide variations is considered to reduce the use of hidden splice sites at experimentally identified hidden splice sites.

[0122] In some embodiments, the cryptic splicing sites that are modified to reduce off-target splicing, or the off-target splicing sites, are sites that have been empirically identified as off-target splicing sites. For example, the techniques described in Example 7 of PCT / US23 / 66969 (published as WO2023 / 220742) can be used to identify such sites, which are hereby incorporated herein by reference in their entirety. In some embodiments, the nucleotide changes that reduce cryptic splicing sites alter all cryptic splicing sites whose frequency of use is above a predetermined threshold.

[0123] In some implementations, the cryptic splice sites that are modified to reduce off-target splicing, or the off-target splice sites that are already predicted to be off-target splice sites, are modified. Such predictions can be made based on sequence analysis to identify canonical splice sites, polypyrimidine bundles, and / or branching points among the putative cryptic splice sites.

[0124] The inventors employ a proprietary combination of proprietary software and expertise to identify cryptic splicing sites that are highly likely to influence the activity of the RNA exon editor. This proprietary combination also relies on the operational sequence of the following cycles, which the inventors have determined affects the therapeutic performance of the resulting RNA exon editor. In short, the codon domain sequences are subjected to three rounds of iterative analysis and modification. In the first round, proprietary software is used to identify and modify high-ranking cryptic splicing sites to reduce / eliminate splicing site usage. In the second round, the sequences generated after the first round are further analyzed using a combination of proprietary software and expertise to identify and rank additional cryptic splicing sites and modify these sites to reduce / eliminate cryptic splicing site usage. And in the third round, the sequences generated after the second round are further analyzed using a combination of proprietary software and expertise to identify sequences requiring further modification, thereby improving and / or enhancing the therapeutic performance of the resulting RNA exon editor.

[0125] This is a modified version of the above process for analyzing and modifying binding domain sequences. In short, a combination of proprietary software and expertise is used to perform two rounds of iterative analysis and modification of the binding domain sequences. In the first round, hidden splicing sites are identified and ordered, and these sites are modified to reduce / eliminate their use. A second round is then performed, in which the sequences generated after the first round are further analyzed using the same combination of proprietary software and expertise to identify sequences requiring further modification, thereby improving or enhancing the therapeutic performance of the resulting RNA exon editor.

[0126] Through the above analysis, the inventors identified hidden splicing sites in the codon-optimized sequences of MeCP2 exons 3 (SEQ ID NO: 13) and 4 (SEQ ID NO: 14), and provided probability scores for the relative likelihood of these hidden splicing sites participating in off-target splicing reactions. The nucleotides present in codon-optimized exon 3 (SEQ ID NO: 13) that lead to hidden splicing sites and their relative probability scores are shown in Table 2. A relative probability score of 1 indicates that such sites have the highest relative probability of hidden site use in the scoring system presented herein.

[0127] Table 2: Exon 3 cryptic splicing sites

[0128] The nucleotides present in codon-optimized exon 4 (SEQ ID NO: 14) that lead to the hidden splicing site and their relative probability scores are shown in Table 3.

[0129] Table 3: Exon 4 cryptic splicing sites

[0130] In some embodiments, the coding domain sequence comprises the MeCP2 exon 3 nucleotide sequence, and one or more of the positions identified in Table 2 contain nucleotide variations relative to SEQ ID NO: 13 that reduce cryptic splicing sites. In some embodiments, the coding domain sequence has nucleotide variations relative to SEQ ID NO: 13 at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the positions shown in Table 2. In some embodiments, the coding domain sequence has nucleotide variations relative to SEQ ID NO: 13 at one or more of the positions shown in Table 2 with a relative probability of 1. In some embodiments, the coding domain sequence has nucleotide variations relative to SEQ ID NO: 13 at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the positions shown in Table 2 with a relative probability of 1.

[0131] In some embodiments, the coding domain sequence comprises the MeCP2 exon 4 nucleotide sequence, which contains nucleotide variations at one or more of the positions identified in Table 3 relative to SEQ ID NO: 14 that reduce cryptic splicing sites. In some embodiments, the coding domain sequence has nucleotide variations at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the positions shown in Table 3 relative to SEQ ID NO: 14. In some embodiments, the coding domain sequence has nucleotide variations at one or more of the positions shown in Table 3 relative to SEQ ID NO: 14 with a relative probability of 1. In some implementations, the coding domain sequence at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of the positions shown in Table 2 has a nucleotide change relative to SEQ ID NO: 14 with a relative probability of 1.

[0132] In some embodiments, the nucleotide change of reducing the cryptic splice site causes a nucleotide sequence that matches the canonical splice site common sequence to no longer match the canonical sequence. In some embodiments, the nucleotide change of reducing the cryptic splice site eliminates potential splice site nucleotides. In some embodiments, the nucleotide change of reducing the cryptic splice site eliminates potential polypyrimidine bundle nucleotides. In some embodiments, the nucleotide change of reducing the cryptic splice site eliminates potential branching point nucleotides. In some embodiments, the nucleotide change of reducing the cryptic splice site is a synonymous nucleotide change. In some embodiments, the nucleotide change of reducing the cryptic splice site causes a change in the amino acid encoded by the exon editor. In some embodiments, the amino acid change is a conserved amino acid substitution. The sequence of MeCP2 exon 3 with the nucleotide change of reducing the cryptic splice site is shown in SEQ ID NO: 85. The sequence of MeCP2 exon 4 with the nucleotide change of reducing the cryptic splice site is shown in SEQ ID NO: 86. The sequences of MeCP2 exons 3 and 4 with reduced nucleotide changes at hidden splicing sites are shown in SEQ ID NO: 76. The sequences in SEQ ID NO: 76, 85, and 86 also underwent nucleotide changes for codon optimization.

[0133] In some embodiments, the coding domain sequence of the MeCP2 trans-splicing molecule has nucleotide changes at one or more of the following nucleotides in MeCP2 exon 3, numbered according to SEQ ID NO: 7: 4A, 7A, 11T, 12C, 13A, 16A, 22G, 28C, 37C, 43T, 49G, 55A, 67C, 85G, 91G, 94A, 97A, 103C, 106A, 109G, 115A, 119T, 120C, 127T, 130T, 133G, 176T, 177C, 182T, 183C, 184G, 206T, 207C, 223A, 239C, 241T, 280A, 290C, 317C, 320T, 321C, or 331A. In some embodiments, the CDS has nucleotide changes at 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 of these positions. In some embodiments, U is used instead of T at the disclosed positions. In some embodiments, the coding domain sequence of the MeCP2 trans-splicing molecule has nucleotide changes at one or more of the following positions in MeCP2 exon 4, according to SEQ ID NO: Numbering: 7G, 10A, 13A, 19T, 20C, 22C, 23T, 24C, 25T, 28A, 35T, 40T, 49T, 55A, 61T, 67G, 94T, 106A, 107C, 130A, 286T, 304A, 595T, 683C, 740T, 741C, 806T, 807C, 809T, 810C, 824T, 825C, 851T, 852C 854T, 855C, 892G, 980C, 982C, 984C, 1034C, 1055C, 1072A, 1073C, 1075T, 1117T, 1118C, 1123T, 1124C, 1125G, 1147T, 1148C, 1164A, 1180C, 1182T, 1221A, 1231C, 1258C, 1261T, 1262C, or 1272A.In some implementations, CDS numbers 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, 3 in these positions are used. Nucleotide changes are present at 1, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61 sites. In some embodiments, U is used instead of T in the disclosed positions.

[0134] In some embodiments, the coding domain sequence of the MeCP2 trans-splicing molecule comprises an exon 3 nucleotide sequence having one or more of the following nucleotides or any combination thereof at a designated position: 43T, 49G, 55A, 67C, 85G, 91G, 94A, 97A, 103C, 106A, 109G, 115A, 118T, 119T, 120C, 130T, 133G, 176T, 177C, 182T, 183C, 184G, 206T, 207C, 223A, 239C, 241T, 280A, 290C, 317C, 320T, 321C, or 331A (numbered according to SEQ ID NO: 7). In some embodiments, the coding domain sequence comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 85, wherein the coding domain sequence contains one or more of the following nucleotides or any combination thereof at a designated position: 43T, 49G, 55A, 67C, 85G, 91G, 94A, 97A, 103C, 106A, 109G, 115A, 118T, 119T, 120C, 130T, 133G, 176T, 177C, 182T, 183C, 184G, 206T, 207C, 223A, 239C, 241T, 280A, 290C, 317C, 320T, 321C, or 331A (numbered according to SEQ ID NO: 85).

[0135] In some embodiments, the coding domain sequence of the MeCP2 trans-splicing molecule comprises an exon 4 nucleotide sequence having one or more of the following nucleotides or any combination thereof at a designated position: 49T, 55A, 61T, 67G, 94T, 106A, 107C, 130A, 286T, 304A, 595T, 683C, 740T, 741C, 806T, 807C, 808T, 809C, 824T, 825C, 851T, 852C, 854T, 855C, 892G, 980C, 982C, 1034C, 1055C, 1072A, 1073C, or 1075T (numbered according to SEQ ID NO: 8). In some embodiments, the coding domain sequence comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 86, wherein the coding domain sequence comprises one or more of the following nucleotides or any combination thereof at a designated position: 49T, 55A, 61T, 67G, 94T, 106A, 107C, 130A, 286T, 304A, 595T, 683C, 740T, 741C, 806T, 807C, 808T, 809C, 824T, 825C, 851T, 852C, 854T, 855C, 892G, 980C, 982C, 1034C, 1055C, 1072A, 1073C, or 1075T (numbered according to SEQ ID NO: 86).

[0136] 3. Combining structural domains The MeCP2 trans-splicing molecule described herein provides a binding domain (BD) configured to bind / anneal to target MeCP2 introns and / or exons. In some embodiments, the BD is configured to bind / anneal to target MeCP2 introns (e.g., intron 1, intron 2, or intron 3). In some embodiments, the binding domain is a nucleic acid sequence that is at least 75% complementary to the sequence of the target MeCP2 intron precursor mRNA (e.g., target MeCP2 intron; intron 1, intron 2, or intron 3). In some implementations, the binding domain is a nucleic acid sequence that is at least 75% complementary to, at least 76% complementary to, at least 77% complementary to, at least 78% complementary to, the sequence of the target MeCP2 intron and / or exon precursor mRNA (e.g., target MeCP2 intron; intron 1, intron 2, or intron 3), at least 75% complementary to, at least 76% complementary to, at least 77% complementary to, at least 79% complementary to, at least 80% complementary to, at least 81% complementary to, at least 82% complementary to, at least 83% complementary to, at least 84% complementary to, at least 85% complementary to, at least 86% complementary to, at least 87% complementary to, at least 88% complementary to, at least 89% complementary to, at least 90% complementary to, at least 91% complementary to, at least 92% complementary to, at least 93% complementary to, at least 94% complementary to, at least 95% complementary to, at least 96% complementary to, at least 97% complementary to, at least 98% complementary to, at least 99% complementary to, or 100% complementary to.

[0137] In addition to the above, the inventors also tested a mouse MeCP2 exon editor comprising a BD that binds at a similar position to mouse MeCP2 intron 1 pre-mRNA relative to human MeCP2 precursor mRNA. Exemplary mouse MeCP2 exon editors of this type, comprising BDs having at least 80% (e.g., 82%) identity with similar human binding domain sequences, target human MeCP2 precursor mRNA and have been shown to achieve 10% ONT trans-splicing to the human MeCP2 precursor mRNA target. Therefore, such exemplary mouse MeCP2 exon editors achieve a significant level of cross-species in-target trans-splicing. In contrast, positionally similar human MeCP2 exon editors achieve 15% ONT to the human MeCP2 precursor mRNA target. In some embodiments, the exemplary mouse MeCP2 exon editor comprises a segment of up to 35-40 nucleotides that is completely complementary to the human MeCP2 precursor mRNA. In some implementations, most mismatches between the mouse MeCP2 exon editor and the human MeCP2 precursor mRNA target are located at the 5' end of the binding domain.

[0138] The MeCP2 trans-splicing molecule described herein provides a binding domain (BD) configured to bind / anneal to a target MeCP2 intron and / or exon. In some embodiments, the target MeCP2 intron is MeCP2 intron 1. In some embodiments, the binding domain is a nucleic acid sequence that is at least 80% complementary (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary) to the sequence of the target MeCP2 intron precursor mRNA (e.g., the target MeCP2 intron). MeCP2 trans-splicing molecules containing such binding domains can inhibit endogenous target cis-splicing while enhancing trans-splicing between the trans-splicing molecule and the target MeCP2 precursor mRNA (e.g., by creating chimeric molecules having a portion of endogenous MeCP2 mRNA and a coding domain having one or more functional MeCP2 exons encoding wild-type MeCP2 amino acid sequences). In some embodiments involving trans-splicing molecule coding sequences (e.g., vectors encoding trans-splicing molecules), the binding domain coding sequence encodes a nucleic acid sequence that is at least 80% complementary to the sequence of the target MeCP2 intron precursor mRNA (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary).

[0139] In some embodiments, this disclosure provides a trans-splicing molecule (or a vector encoding said trans-splicing molecule) that binds MeCP2 at intron 1, for example, wherein the nucleic acid trans-splicing molecule is configured to trans-splice a coding domain to endogenous MeCP2 exon 1. In some embodiments, the trans-splicing molecule binds MeCP2 precursor mRNA at a location across the exon 1-intron 1 junction. In some embodiments, the trans-splicing molecule described herein includes a binding domain that binds to an intron 1 or SEQ ID NO: 1 nucleotide -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 (e.g., six or more, eight or more, ten or more, or twelve or more, twenty-five or more, fifty or more, 75 or more, 100 or more, 150 or more, 200 or more, or 250 or more). Those binding sites of multiple, or in the range of 6 to 12, 12 to 25, 25 to 50, 25 to 75, 25 to 100, 25 to 150, 25 to 200, 50 to 75, 50 to 100, 50 to 150, 50 to 200, 50 to 250, 100 to 150, 100 to 200, or 100 to 250, wherein nucleotide position-1 is defined as the last nucleotide of MeCP2 exon 1 (position 114 of SEQ ID NO: 110 or SEQ ID NO: 111), and position 1 is defined as... MeCP2The first nucleotide of intron 1 (position 115 of SEQ ID NO: 111 or position 1 of SEQ ID NO: 1), with negative numbers increasing from position -1 along the 5' direction and positive numbers increasing from position 1 along the 3' direction. In some embodiments, the trans-splicing molecule described herein includes nucleotides 1-100, 1-200, 1-300, 1-399, 1-500, 1-600, 1-649, 1-700, 1-800, 1-900, and 1-1000 in which the binding domain binds to SEQ ID NO: 1. 1-1100, 1-1200, 1-1300, 1-1400, 1-1449, 51-201, 51-399, 51-524, 51-649, 51-809, 51-969, 51-1129, 51-1289, 51-1449, 125-201, 125-399, 125-524, 125-649, 125-8 09, 125-969, 125-1129, 125-1289, 125-1449, 250-399, 250-524, 250-649, 250-809, 250-969, 250-1129, 250-1289, 250-1449, 375-399, 375-524, 375-649, 375-809, 3 75-969, 375-1129, 375-1289, 375-1449, 500-524, 500-649, 500-809, 500-969, 500-1129, 500-1289, 500-1449, 660-809, 660-969, 660-1129, 660-1289, 660-1449, 82 Those binding sites within the range of 0-969, 820-1129, 820-1289, 820-1449, 980-1129, 980-1289, 980-1449, 1140-1289, 1140-1449, 1300-1449, 2100-2250, 3950-4169, 3950-4250, or 4020-4169.In some embodiments, the trans-splicing molecule described herein includes those wherein the binding domain binds nucleotides comprising SEQ ID NO:1, namely 1-100, 1-200, 1-300, 1-399, 1-500, 1-600, 1-649, 1-700, 1-800, 1-900, 1-1000, 1-1100, 1-1200, 1-1300, 1-1400, 1-1449, 51-201, 51-399, 51-524, 51-649, 51-809, 51-969, 51-1129, 51-1289, 51-1449, 125-201, 125-399, 125-524, 125-649, 125-809. 125-969, 125-1129, 125-1289, 125-1449, 250-399, 250-524, 250-649, 250-809, 250-969, 250-1129, 250-1289, 250-1449, 375-399, 375-524, 375-649, 375-809, 375-969 375-1129, 375-1289, 375-1449, 500-524, 500-649, 500-809, 500-969, 500-1129, 500-1289, 500-1449, 660-809, 660-969, 660-1129, 660-1289, 660-1449, 820-969, 820 Those with at least 100 consecutive nucleotide binding sites within the range of -1129, 820-1289, 820-1449, 980-1129, 980-1289, 980-1449, 1140-1290, 1140-1449, 1300-1449, 2100-2250, 3950-4169, 3950-4250, or 4020-4169. In some embodiments, the trans-splicing molecules described herein include those wherein the binding domain comprises about 50-300 nucleotides; about 50-250 nucleotides; about 50-200 nucleotides; about 50-150 nucleotides; about 50-100 nucleotides; about 75-300 nucleotides; about 75-250 nucleotides; about 75-200 nucleotides; about 75-150 nucleotides; about 100-300 nucleotides; about 100-250 nucleotides; about 100-200 nucleotides; about 100-150 nucleotides; about 125-300 nucleotides; about 125-250 nucleotides; about 125-200 nucleotides; about 125-150 nucleotides; about 50 nucleotides; about 100 nucleotides; about 125 nucleotides; about 150 nucleotides; about 200 nucleotides; or about 250 nucleotides in length.In some embodiments, the trans-splicing molecules described herein include those wherein the binding domain comprises 50-300 nucleotides; 50-250 nucleotides; 50-200 nucleotides; 50-150 nucleotides; 50-100 nucleotides; 75-300 nucleotides; 75-250 nucleotides; 75-200 nucleotides; 75-150 nucleotides; 100-300 nucleotides; 100-250 nucleotides; 100-200 nucleotides; 100-150 nucleotides; 125-300 nucleotides; 125-250 nucleotides; 125-200 nucleotides; 125-150 nucleotides; 50 nucleotides; 100 nucleotides; 125 nucleotides; 150 nucleotides; 200 nucleotides; or 250 nucleotides in length. In some embodiments, the binding domain comprises 50 or more consecutive nucleic acid residues, said nucleic acid residues being 100% complementary to 50 or more consecutive nucleotides at the binding site. In some embodiments, the binding domain comprises 50 consecutive nucleic acid residues, said nucleic acid residues being 100% complementary to 50 consecutive nucleotides at the binding site. In some embodiments, the binding domain comprises 50 consecutive nucleic acid residues, said nucleic acid residues being at least 90% complementary to 50 consecutive nucleotides at the binding site (e.g., 90-100% complementary). In some embodiments, the binding domain comprises 50 consecutive nucleic acid residues, said nucleic acid residues being at least 80% complementary to 50 consecutive nucleotides at the binding site (e.g., 80-100% complementary). In some embodiments, the binding domain comprises a sequence of 100 to 200 nucleotides in length and binds MeCP2 at a binding site within nucleotides 1-1449 of SEQ ID NO:1, wherein the binding domain comprises 50 consecutive nucleotides, said nucleotides being 100% complementary to 50 consecutive nucleotides at the binding site. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and binds MeCP2 at a binding site within nucleotides 1-1449 of SEQ ID NO:1, wherein the binding domain comprises 50 consecutive nucleotides, said nucleotides being 90% to 100% complementary to the 50 consecutive nucleotides at the binding site. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 100% complementary to a binding site comprising at least 100 consecutive nucleotides within nucleotides 1-1449 of SEQ ID NO:1. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 100% complementary to a binding site comprising at least 75 consecutive nucleotides within nucleotides 1-1449 of SEQ ID NO:1.In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 90% to 100% complementary to a binding site comprising at least 100 consecutive nucleotides from nucleotides 1 to 1449 of SEQ ID NO:1. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and binds MeCP2 at a binding site from nucleotides 3950 to 4250 of SEQ ID NO:1, wherein the binding domain comprises 50 consecutive nucleotides, said nucleotides being 100% complementary to the 50 consecutive nucleotides at the binding site. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and binds MeCP2 at a binding site from nucleotides 3950 to 4250 of SEQ ID NO:1, wherein the binding domain comprises 50 consecutive nucleotides, said nucleotides being 90% to 100% complementary to the 50 consecutive nucleotides at the binding site. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 100% complementary to the binding site of at least 75 consecutive nucleotides within nucleotides 3950-4250 of SEQ ID NO:1. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 100% complementary to the binding site of at least 100 consecutive nucleotides within nucleotides 3950-4250 of SEQ ID NO:1. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 90% to 100% complementary to the binding site of at least 100 consecutive nucleotides within nucleotides 3950-4250 of SEQ ID NO:1.

[0140] In some cases, the binding domain includes any six or more consecutive nucleotides within the range of -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of the nucleotides in MeCP2 intron 1 (e.g., any eight or more consecutive nucleic acids within the nucleotides in MeCP2 intron 1, or any ten or more consecutive nucleotides within the range of -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of the nucleotides in MeCP2 intron 1). Multiple consecutive nucleic acids, any 12 or more consecutive nucleic acids within the range of -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of nucleotides in MeCP2 intron 1, and any 20 or more consecutive nucleic acids within the range of -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of nucleotides in MeCP2 intron 1, and any nucleotides within the range of -100 to 1500, -50 to 1450, or -50 to 1450. Any 30 or more consecutive nucleic acids within the ranges of -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000; any 40 or more consecutive nucleic acids within the ranges of -100 to 1500, -50 to 1450, 3950 to 4250, 4020 to 4170, ... or -50 to 1000; any 40 or more consecutive nucleic acids within the ranges of -100 to 1500, -50 to 1450, 3950 to 4250, 4020 to 4170, -100 to 1300, or -100 to 1000; any nucleotides within the ranges of -100 to 1500, -50 to 1450, 3950 to 4250, 4020 to 4170, -100 to 1300, or -100 to 1000; any nucleotides within the ranges of -100 to 1500, -50 to 1450, 3950 to 4250, 4020 to 4170, -100 to 1300, or -100 to 1000; any nucleotides within the ranges of -100 to 1500, -50 to Any 50 or more consecutive nucleic acids within the range of -50 to 1300 or -50 to 1000; any 75 or more consecutive nucleic acids within the range of -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300 or -50 to 1000; any 100 or more consecutive nucleic acids within the range of -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300 or -50 to 1000.MeCP2 intron 1 contains any 150 or more consecutive nucleic acids within the ranges of -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000. Any 200 or more consecutive nucleic acids within the range of -50 to 1300 or -50 to 1000, or any 250 or more consecutive nucleic acids within the range of -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300 or -50 to 1000, wherein nucleotide position -1 is defined as the last nucleotide of MeCP2 exon 1 (position 114 of SEQ ID NO: 111) and position 1 is defined as... MeCP2 The first nucleotide of intron 1 (SEQ ID NO: 111, position 115) is negatively incremented from position -1 along the 5' direction and positively incremented from position 1 along the 3' direction.

[0141] In some cases, the binding site includes any six or more consecutive nucleotides within the range of -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of the nucleotides in MeCP2 intron 1 (e.g., any eight or more consecutive nucleic acids within the range of -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of the nucleotides in MeCP2 intron 1). Any ten or more consecutive nucleic acids within the range of -50 to 1300 or -50 to 1000; any 12 or more consecutive nucleic acids within the range of -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 in MeCP2 intron 1; any 20 or more consecutive nucleic acids within the range of -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 in MeCP2 intron 1; any 100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000 in MeCP2 intron 1. Any 30 or more consecutive nucleic acids within the range of -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000; any 40 or more consecutive nucleic acids within the range of -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000; any 5 ...300, -100 to 1000, -50 to 1300, or -50 to 1000; any 50 or more consecutive nucleic acids within the range of -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000; any 40 or more consecutive nucleic acids within the range of -100 to 1300, -100 to 1300, -100 to 1300, -100 to 1300, -50 to 1300, or -50 to 1000; any 50 or more consecutive nucleic acids within the range of -100 to 1300, -100 to 1450, -100 to 1300, -100 to 1300, -100 to 1300, -50 to 1300, or -50 to 1300; any 50 or more consecutive nucleic acids within the range of -100 to 130 Any 75 or more consecutive nucleic acids within the range of 500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000; any 100 or more consecutive nucleic acids within the range of -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000; any 150 or more consecutive nucleic acids within the range of -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000.The nucleotides of MeCP2 intron 1 can be any 200 or more consecutive nucleic acids within the range of -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000, or any 250 or more consecutive nucleic acids within the range of -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000, wherein nucleotide position -1 is defined as the last nucleotide of MeCP2 exon 1 (position 114 of SEQ ID NO: 111) and position 1 is defined as... MeCP2 The first nucleotide of intron 1 (position 115 of SEQ ID NO: 111) is negatively incremented from position -1 along the 5' direction and positively incremented from position 1 along the 3' direction.

[0142] In some implementations, the binding domain has at least two non-overlapping sequences that are at least 80% complementary to the binding site.

[0143] In some embodiments, the binding domain comprises a nucleic acid sequence having at least 80% identity with any of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 47 (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity). In any binding domain sequence disclosed herein, U may replace all Ts in the sequence. Therefore, for example, in some embodiments, the binding domain comprises a nucleic acid sequence having at least 80% identity with any of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 47 (each T in the U-substituted sequence) (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity; e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity).

[0144] In some embodiments, the binding domain is a DNA sequence having at least 80% identity with any of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 47 (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity). In some embodiments, the binding domain comprises or consists of the following: SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 47.

[0145] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 24, wherein the binding domain comprises nucleotides 23C and / or 72C, numbered according to SEQ ID NO: 24.

[0146] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 25, wherein the binding domain comprises nucleotides 97C and / or 146C, numbered according to SEQ ID NO: 25.

[0147] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 27, wherein the binding domain comprises nucleotides 8C, 56C, 72C, or 85C, or any combination thereof, numbered according to SEQ ID NO: 27.

[0148] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 28, wherein the binding domain contains nucleotide 133C, numbered according to SEQ ID NO: 28.

[0149] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 30, wherein the binding domain comprises nucleotides 4C, 71C, or 101C, or any combination thereof, numbered according to SEQ ID NO: 30.

[0150] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 32, wherein the binding domain contains nucleotide 119A, numbered according to SEQ ID NO: 32.

[0151] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 33, wherein the binding domain contains nucleotide 133C, numbered according to SEQ ID NO: 33.

[0152] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 47, wherein the binding domain contains nucleotide 149C, numbered according to SEQ ID NO: 47.

[0153] In some embodiments, the binding domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity with any of SEQ ID NO: 116 to 137, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, N is a nucleotide other than G. In some embodiments, the binding domain comprises or consists of any of SEQ ID NO: 116 to 137, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, any nucleotide at the position designated as N is not complementary to the corresponding position in the native MeCP2 precursor mRNA.

[0154] In some embodiments, the binding domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 116, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, the N at position 23 of SEQ ID NO: 116 is C. In some embodiments, the N at position 72 of SEQ ID NO: 116 is C.

[0155] In some embodiments, the binding domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 117, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, the N at position 97 of SEQ ID NO: 117 is C. In some embodiments, the N at position 146 of SEQ ID NO: 117 is C.

[0156] In some embodiments, the binding domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 118, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, the N at position 8 of SEQ ID NO: 118 is C. In some embodiments, the N at position 56 of SEQ ID NO: 118 is C. In some embodiments, the N at position 82 of SEQ ID NO: 118 is C. In some embodiments, the N at position 85 of SEQ ID NO: 118 is C.

[0157] In some embodiments, the binding domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 119, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, the N at position 133 of SEQ ID NO: 119 is C.

[0158] In some embodiments, the binding domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 120, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, the N at position 4 of SEQ ID NO: 120 is C. In some embodiments, the N at position 71 of SEQ ID NO: 120 is C. In some embodiments, the N at position 101 of SEQ ID NO: 120 is C.

[0159] In some embodiments, the binding domain comprises a nucleotide sequence having at least 50 consecutive nucleotides, said consecutive nucleotides having 100% sequence identity with a consecutive nucleotide chain of any one of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 47. In some embodiments, the binding domain comprises a nucleic acid sequence having at least 75 consecutive nucleotides, said consecutive nucleotides having 100% sequence identity with a consecutive nucleotide chain of any one of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 47. In some embodiments, the binding domain comprises a nucleic acid sequence having at least 100 consecutive nucleotides, said consecutive nucleotides having 100% sequence identity with a consecutive nucleotide chain of any one of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 47.

[0160] Some embodiments of the MeCP2 trans-splicing molecule described herein provide a binding domain (BD) configured to bind / anneal to a target MeCP2 intron and / or exon. In some embodiments, the target MeCP2 intron is MeCP2 intron 2. In some embodiments, the binding domain is a nucleic acid sequence that is at least 80% complementary (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary) to the sequence of the target MeCP2 intron precursor mRNA (e.g., the target MeCP2 intron). MeCP2 trans-splicing molecules containing such binding domains can inhibit endogenous target cis-splicing while enhancing trans-splicing between the trans-splicing molecule and the target MeCP2 precursor mRNA (e.g., by creating chimeric molecules having a portion of endogenous MeCP2 mRNA and a coding domain having one or more functional MeCP2 exons encoding wild-type MeCP2 amino acid sequences). In some embodiments involving trans-splicing molecule coding sequences (e.g., vectors encoding trans-splicing molecules), the binding domain coding sequence encodes a nucleic acid sequence that is at least 80% complementary to the sequence of the target MeCP2 intron precursor mRNA (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary).

[0161] In some embodiments, this disclosure provides a trans-splicing molecule (or a vector encoding the trans-splicing molecule) that binds MeCP2 at intron 2, for example, wherein the nucleic acid trans-splicing molecule is configured to trans-splice a coding domain to endogenous MeCP2 exon 1 or exon 2. In some embodiments, the trans-splicing molecules described herein include those in which the binding domain binds to any one or more of the following nucleotides of SEQ ID NO: 2: 1-300, 50-300, 50-250, 75-300, 75-250, 80-300, 80-250, 90-300, 90-250, 95-300, 95-250, or 99-248 (e.g., six or more, eight or more, ten or more, or twelve or more, twenty-five or more, fifty or more, 75 or more, 100 or more, 150 or more, 200 or more, or 250 or more). In some embodiments, the trans-splicing molecules described herein include those wherein the binding domain comprises about 50-300 nucleotides; about 50-250 nucleotides; about 50-200 nucleotides; about 50-150 nucleotides; about 50-100 nucleotides; about 75-300 nucleotides; about 75-250 nucleotides; about 75-200 nucleotides; about 75-150 nucleotides; about 100-300 nucleotides; about 100-250 nucleotides; about 100-200 nucleotides; about 100-150 nucleotides; about 125-300 nucleotides; about 125-250 nucleotides; about 125-200 nucleotides; about 125-150 nucleotides; about 50 nucleotides; about 100 nucleotides; about 125 nucleotides; about 150 nucleotides; about 200 nucleotides; or about 250 nucleotides in length. In some embodiments, the trans-splicing molecules described herein include those wherein the binding domain comprises 50-300 nucleotides; 50-250 nucleotides; 50-200 nucleotides; 50-150 nucleotides; 50-100 nucleotides; 75-300 nucleotides; 75-250 nucleotides; 75-200 nucleotides; 75-150 nucleotides; 100-300 nucleotides; 100-250 nucleotides; 100-200 nucleotides; 100-150 nucleotides; 125-300 nucleotides; 125-250 nucleotides; 125-200 nucleotides; 125-150 nucleotides; 50 nucleotides; 100 nucleotides; 125 nucleotides; 150 nucleotides; 200 nucleotides; or 250 nucleotides in length. In some implementations, the binding domain comprises 50 or more consecutive nucleic acid residues that are 100% complementary to 50 or more consecutive nucleotides at the binding site.In some embodiments, the binding domain comprises 50 consecutive nucleic acid residues that are 100% complementary to 50 consecutive nucleotides at the binding site. In some embodiments, the binding domain comprises 50 consecutive nucleic acid residues that are at least 90% complementary to 50 consecutive nucleotides at the binding site (e.g., 90-100% complementary). In some embodiments, the binding domain comprises 50 consecutive nucleic acid residues that are at least 80% complementary to 50 consecutive nucleotides at the binding site (e.g., 80-100% complementary). In some embodiments, the binding domain comprises a sequence of 100 to 200 nucleotides in length and binds MeCP2 at a binding site within nucleotides 1-450 of SEQ ID NO: 2, wherein the binding domain comprises 50 consecutive nucleotides that are 100% complementary to 50 consecutive nucleotides at the binding site. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and binds MeCP2 at a binding site within nucleotides 1-450 of SEQ ID NO: 2, wherein the binding domain comprises 50 consecutive nucleotides, said nucleotides being 90% to 100% complementary to the 50 consecutive nucleotides at the binding site. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and binds MeCP2 at a binding site within nucleotides 1-450 of SEQ ID NO: 2, wherein the binding domain comprises 75 consecutive nucleotides, said nucleotides being 90% to 100% complementary to the 75 consecutive nucleotides at the binding site. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 100% complementary to a binding site comprising at least 100 consecutive nucleotides within nucleotides 1-450 of SEQ ID NO: 2. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 90% to 100% complementary to a binding site comprising at least 100 consecutive nucleotides within nucleotides 1-450 of SEQ ID NO: 2. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and binds MeCP2 at a binding site within nucleotides 99-248 of SEQ ID NO: 2, wherein the binding domain comprises 50 consecutive nucleotides, said nucleotides being 100% complementary to the 50 consecutive nucleotides at the binding site. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and binds MeCP2 at a binding site within nucleotides 99-248 of SEQ ID NO: 2, wherein the binding domain comprises 50 consecutive nucleotides, said nucleotides being 90% to 100% complementary to the 50 consecutive nucleotides at the binding site.In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 100% complementary to a binding site comprising at least 100 consecutive nucleotides within nucleotides 99-248 of SEQ ID NO: 2. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 90% to 100% complementary to a binding site comprising at least 100 consecutive nucleotides within nucleotides 99-248 of SEQ ID NO: 2. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and binds MeCP2 at a binding site within nucleotides 149-300, 199-350, 249-400, or 299-450 of SEQ ID NO: 2, wherein the binding domain comprises 50 consecutive nucleotides, said nucleotides being 100% complementary to the 50 consecutive nucleotides at the binding site. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and binds MeCP2 at a binding site within nucleotides 149-300, 199-350, 249-400, or 299-450 of SEQ ID NO: 2, wherein the binding domain comprises 50 consecutive nucleotides, said nucleotides being 90% to 100% complementary to the 50 consecutive nucleotides at the binding site. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 100% complementary to a binding site comprising at least 100 consecutive nucleotides within nucleotides 149-300, 199-350, 249-400, or 299-450 of SEQ ID NO: 2. In some embodiments, the binding domain comprises a sequence in the range of 100 to 200 nucleotides and is 90% to 100% complementary to the binding site of at least 100 consecutive nucleotides within nucleotides 149-300, 199-350, 249-400, or 299-450 of SEQ ID NO: 2.

[0162] In some cases, the binding domain includes any six or more consecutive nuclei within the range of nucleotides 1-300, 50-300, 50-250, 75-300, 75-250, 80-300, 80-250, 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450 of the MeCP2 intron 2. nucleotides (e.g., any eight or more consecutive nucleic acids within the nucleotides of MeCP2 intron 2, nucleotides of MeCP2 intron 2 1-300, 50-300, 50-250, 75-300, 75-250, 80-300, 80-250, 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400 or Any ten or more consecutive nucleic acids within the range of 199-450, and any nucleotides of MeCP2 intron 2 within the range of 1-300, 50-300, 50-250, 75-300, 75-250, 80-300, 80-250, 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450. Two or more consecutive nucleic acids, any 20 or more consecutive nucleic acids within the range of nucleotides 1-300, 50-300, 50-250, 75-300, 75-250, 80-300, 80-250, 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450 of MeCP2 intron 2.MeCP2 intron 2 consists of any 30 or more consecutive nucleic acids within the range of 1-300, 50-300, 50-250, 75-300, 75-250, 80-300, 80-250, 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450. MeCP2 intron 2 consists of any 40 or more consecutive nucleic acids within the range of nucleotides 1-300, 50-300, 50-250, 75-300, 75-250, 80-300, 80-250, 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450. MeCP2 intron 2 consists of any 50 or more consecutive nucleic acids within the range of 1-300, 50-300, 50-250, 75-300, 75-250, 80-300, 80-250, 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450. MeCP2 intron 2 consists of any 75 or more consecutive nucleic acids within the range of nucleotides 1-300, 50-300, 50-250, 75-300, 75-250, 80-300, 80-250, 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450.MeCP2 intron 2 consists of any 100 or more consecutive nucleic acids within the range of 1-300, 50-300, 50-250, 75-300, 75-250, 80-300, 80-250, 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450. MeCP2 intron 2 consists of any 150 or more consecutive nucleic acids within the range of 1-300, 50-300, 50-250, 75-300, 75-250, 80-300, 80-250, 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450. The eCP2 intron 2 contains any 200 or more consecutive nucleic acids within the range of 1-300, 50-300, 50-250, 75-300, 75-250, 80-300, 80-250, 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450. MeCP2 intron 2 consists of any 250 or more consecutive nucleic acids within the range of 1-300, 50-300, 50-250, 75-300, 75-250, 80-300, 80-250, 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450.

[0163] In some cases, the binding site contains any six or more consecutive nucleotides within the range of 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450 of the MeCP2 intron 2 (e.g., 90-300, 90-250, 95-300, 95-250, or 99-248). Any eight or more consecutive nucleic acids within the range of 9-248; any ten or more consecutive nucleic acids within the range of 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450 within the range of 90-300, 90-250, 95-300, 95-250 within the range of 95-248 within the range of 99-248; any ten or more consecutive nucleic acids within the range of 90-300, 90-250, 95-300, 95-250 within the range of 95-248 within the range of 99-248 within the range of 99-300, 90-250, 95-300, 95-250 within the range of 99-248 within the range of 199-248; any ten or more consecutive nucleic acids within the range of 90-300, 90-250, 95-300, 95-250 within the range of 99-248 within the range of 99-248 within the range of 99-248 within the range of 99-248 within the range of 99-250 ... Any 12 or more consecutive nucleic acids within the range of 0, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450, and the nucleotides of MeCP2 intron 2 within the range of 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 19 Any 20 or more consecutive nucleic acids within the range of 9-450, 249-450, 99-350, 99-400, 199-400, or 199-450; any 30 or more consecutive nucleic acids within the range of 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450 of MeCP2 intron 2.Any 40 or more consecutive nucleic acids within the range of nucleotides 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450 in MeCP2 intron 2, or any nucleotides within the range of 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, or 299-450 in MeCP2 intron 2. Any 50 or more consecutive nucleic acids within the range of 0, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450; any 75 or more consecutive nucleic acids within the range of 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450; M Any 100 or more consecutive nucleic acids within the range of nucleotides 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450 in intron 2 of eCP2; or any nucleotides within the range of 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, or 299-450 in intron 2 of MeCP2. Any 150 or more consecutive nucleic acids within the range of 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450; any 200 or more consecutive nucleic acids within the range of 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450 of MeCP2 intron 2.Or any 250 or more consecutive nucleic acids within the range of 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450 of intron 2 of MeCP2.

[0164] In some implementations, the binding domain has at least two non-overlapping sequences that are at least 80% complementary to the binding site.

[0165] In some implementations, the binding domain comprises a nucleic acid sequence having at least 80% identity with SEQ ID NO: 55, 56, 57, 58, 59, 88, 89, 90, 91 or 92 (including the type in which each T is replaced by U in SEQ ID NO: 55, 56, 57, 58, 59, 88, 89, 90, 91 or 92) (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity; e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity). In some embodiments, the binding domain comprises a nucleic acid sequence having at least 50 consecutive nucleotides, said nucleotides being 100% complementary to 50 consecutive nucleotides of SEQ ID NO: 55, 56, 57, 58, 59, 88, 89, 90, 91, or 92. In some embodiments, the binding domain comprises a nucleic acid sequence having at least 100 consecutive nucleotides, said nucleotides being 100% complementary to 100 consecutive nucleotides of SEQ ID NO: 55, 56, 57, 58, 59, 88, 89, 90, 91, or 92.

[0166] In some embodiments, the binding domain comprises a nucleotide sequence having at least 50 consecutive nucleotides, said nucleotides having 100% sequence identity with a consecutive nucleotide chain of any one of SEQ ID NO: 55, 56, 57, 58, 59, 88, 89, 90, 91, or 92. In some embodiments, the binding domain comprises a nucleic acid sequence having at least 75 consecutive nucleotides, said nucleotides having 100% sequence identity with a consecutive nucleotide chain of any one of SEQ ID NO: 55, 56, 57, 58, 59, 88, 89, 90, 91, or 92. In some embodiments, the binding domain comprises a nucleic acid sequence having at least 100 consecutive nucleotides, said nucleotides having 100% sequence identity with a consecutive nucleotide chain of any one of SEQ ID NO: 55, 56, 57, 58, 59, 88, 89, 90, 91, or 92.

[0167] In some embodiments, the binding domain comprises one or more nucleotide substitutions relative to the endogenous MeCP2 mRNA sequence, wherein the one or more nucleotide substitutions disrupt cryptic splicing sites within the binding domain sequence. In some embodiments, the binding domain comprises a nucleic acid sequence having one or more variations of the following relative to SEQ ID NO: 55: G25C, G59C, G63C, G99C, or G120C. In some embodiments, the binding domain comprises a nucleic acid sequence having at least 80% identity with SEQ ID NO: 88 (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), wherein the binding domain comprises the following nucleotides, numbered according to SEQ ID NO: 88: 25C, 59C, 63C, 99C, or 120C.

[0168] In some embodiments, the binding domain is a DNA sequence having at least 80% identity with SEQ ID NO: 55 (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity).

[0169] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 90, wherein the binding domain comprises nucleotides 84A and / or 106C, numbered according to SEQ ID NO: 90.

[0170] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 91, wherein the binding domain comprises nucleotides 136C and / or 138C, numbered according to SEQ ID NO: 91.

[0171] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 92, wherein the binding domain comprises nucleotide 51C or any combination thereof, numbered according to SEQ ID NO: 92.

[0172] In some embodiments, the binding domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity with any of SEQ ID NO: 138 to 152, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, N is a nucleotide other than G. In some embodiments, the binding domain comprises or consists of any of SEQ ID NO: 139 to 152, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, any nucleotide at the position designated as N is not complementary to the corresponding position on the native MeCP2 precursor mRNA.

[0173] In some embodiments, the binding domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 138, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, the N at position 25 of SEQ ID NO: 138 is C. In some embodiments, the N at position 59 of SEQ ID NO: 138 is C. In some embodiments, the N at position 63 of SEQ ID NO: 138 is C. In some embodiments, the N at position 99 of SEQ ID NO: 138 is C. In some embodiments, the N at position 120 of SEQ ID NO: 138 is C.

[0174] In some embodiments, the binding domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 139, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, the N at position 84 of SEQ ID NO: 139 is A. In some embodiments, the N at position 106 of SEQ ID NO: 139 is C.

[0175] In some embodiments, the binding domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 140, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, the N at position 136 of SEQ ID NO: 140 is C. In some embodiments, the N at position 138 of SEQ ID NO: 140 is C. In some embodiments, the N at position 82 of SEQ ID NO: 118 is C. In some embodiments, the N at position 85 of SEQ ID NO: 118 is C.

[0176] In some embodiments, the binding domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 141, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, the N at position 51 of SEQ ID NO: 141 is C.

[0177] The MeCP2 trans-splicing molecule described herein provides a binding domain (BD) configured to bind / anneal to a target MeCP2 intron and / or exon. In some embodiments, the target MeCP2 intron is MeCP2 intron 3 (SEQ ID NO: 4). In some embodiments, the binding domain is a nucleic acid sequence that is at least 80% complementary (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary) to the sequence of the target MeCP2 intron precursor mRNA (e.g., the target MeCP2 intron). MeCP2 trans-splicing molecules containing such binding domains can inhibit endogenous target cis-splicing while enhancing trans-splicing between the trans-splicing molecule and the target MeCP2 precursor mRNA (e.g., by creating chimeric molecules having a portion of endogenous MeCP2 mRNA and a coding domain having one or more functional MeCP2 exons encoding wild-type MeCP2 amino acid sequences). In some embodiments involving trans-splicing molecule coding sequences (e.g., vectors encoding trans-splicing molecules), the binding domain coding sequence encodes a nucleic acid sequence that is at least 80% complementary to the sequence of the target MeCP2 intron precursor mRNA (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary).

[0178] In some embodiments, the binding domain is a DNA sequence having at least 80% identity with any of SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 74 or 75 (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity; e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity).

[0179] As detailed herein, the first stage of MeCP2 exon editor design begins with screening and selecting highly efficient BD sequences that are complementary to the targeted precursor mRNA introns. As shown herein, BDs targeting intron 1 of MeCP2 exhibit the highest level of trans-splicing and have therefore been identified as effective elements of an exemplary MeCP2-targeted exon editor for treating the Rett syndrome patient population. See also Figures 6 to 9 .

[0180] Any binding domain disclosed herein may contain one or more nucleotide changes (e.g., nucleotide substitutions) relative to the native precursor mRNA sequence, wherein one or more nucleotide changes disrupt cryptic splicing sites within the binding domain sequence. Reducing nucleotide changes at cryptic splicing sites can result in a lack of complementarity between the binding domain and the binding site on the precursor mRNA at the location of the nucleotide change. However, despite the lack of complementarity at the location of one or more nucleotide changes, the binding domain can still bind to the binding site and support efficient trans-splicing. This is in Figure 19 As shown, an exon editor containing a 150-nucleotide binding domain (BD sequence: SEQ ID NO:88) with five reduced cryptic splicing sites provides efficient trans-splicing to MeCP2 intron 2.

[0181] In some embodiments, the RNA exon editor of this disclosure includes a binding domain that specifically binds to a binding site within MeCP2 introns 1, 2, or 3. In some embodiments, the RNA exon editor of this disclosure includes a binding domain that specifically binds to any of the binding sites disclosed herein.

[0182] In some embodiments, the binding domain of the RNA exon editor of this disclosure binds to the same sequence of the MeCP2 precursor mRNA bound to any of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 47, 55, 56, 57, 58, 59, 88, 89, 90, 91, or 92. In some embodiments, such binding domains are 100% complementary to the binding site.

[0183] 4. Semi-introns In some embodiments, the hemiintron includes a 3' splice site. In some embodiments, the hemiintron includes a polypyrimidine bundle (pPy), a branch point (BP), and / or a 3' splice site to mediate trans-splicing. In some embodiments, the hemiintron has a single splice site, meaning that due to the lack of a corresponding splice site, the splice site is designed to preferentially perform trans-splicing rather than cis-splicing.

[0184] Those skilled in the art can select alternative hemiintrons based on known methods and principles. In some embodiments, the 3' splice site concordant sequence is the nucleic acid sequence YAG / [where Y is a pyrimidine (cytosine or thymidine); " / " indicates the location of the 3' splice site; for example, TAG]. In some embodiments, the RNA exon editor includes the 5' sequence TAG in the 5' to 3' direction, immediately adjacent to the first nucleotide of the coding domain sequence (e.g., the coding domain sequence of MeCP2 exon 1, exon 2, exon 3, or exon 4). In some embodiments, endogenous splice sites corresponding to exons and introns proximal to the splice site can be used to maintain any splice regulatory signals. Therefore, in some embodiments, the hemiintron may comprise a sequence of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides from the 3' end of MeCP2 intron 1, intron 2, or intron 3. In some embodiments, the hemiintron comprises BP and pPy. In some embodiments, the hemiintron comprises BP, pPy, and a 3' splice site (splicing acceptor). In some embodiments, the hemiintron comprises BP, pPy, and a 3' splice site operatively linked in the 5' to 3' direction. In some embodiments, BP has the sequence YUNAY, where Y is a pyrimidine (C or U) and N is any nucleotide; A is a branching site. In some embodiments, BP has the sequence YNYTRAC, where Y is a pyrimidine (C or U), N is any nucleotide, and R is a purine; A is a branching site. In some embodiments, BP has the sequence YNAH, where Y is pyrimidine, N represents any nucleotide, H is adenine, cytosine, or thymine, and A is a branching site. In some embodiments, BP is located at least 10 nucleotides upstream of the 3' splice site. In some embodiments, BP is located 10-12, 10-15, 10-17, or 10-20 nucleotides upstream of the 3' splice site. In some embodiments, BP is separated from the 3' splice site by 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides of pPy (i.e., BP and the 3' splice site are separated by a total of 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides). In some embodiments, at least 60%, 70%, 80%, or 90% of the nucleotides (i.e., pPy) between the BP sequence and the 3' splice site are pyrimidines. In some embodiments, all nucleotides (i.e., pPy) between the BP sequence and the 3' splice site are pyrimidines.

[0185] In some embodiments, a suitable 3' splice site includes a TAG. In some embodiments, the hemiintron comprises the nucleotide sequence shown in SEQ ID NO: 17 or a sequence having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any of SEQ ID NO: 17.

[0186] The half-intron can be operatively linked to the 5' of the coding structure domain sequence (CDS) (e.g., directly linked to the CDS or having an intermediary sequence connecting the 3' end of the half-intron and the 5' end of the CDS).

[0187] 5. 3' Non-translated area In some embodiments, the nucleic acid trans-splicing molecule includes a 3' untranslated region. In some embodiments, the 3' untranslated region comprises, is substantially composed of, or consists of a heterologous 3' untranslated region. In some embodiments, the 3' UTR is the RDH1pA 3' UTR, which is a synthetic 3' UTR containing a 110 bp highly conserved MeCP2 distal polyadenylation signal and an upstream miRNA binding site containing sites for three additional miRNAs endogenous to the MeCP2 3' UTR: miR-19, miR-22, and miR-132. The DNA sequence encoding the RDH1pA 3' UTR contains SEQ ID NO: 19. In some embodiments, the 3' UTR is the mWPRE 3' UTR. The DNA sequence encoding the mWPRE 3' UTR contains SEQ ID NO: 20. In some embodiments, the 3' UTR comprises a truncated WPRE sequence. The DNA sequence encoding the truncated WPRE 3' UTR comprises SEQ ID NO: 87. In some embodiments, the truncated WPRE sequence comprises, substantially comprises, or comprises SEQ ID NO: 87 or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 87. In some embodiments, the 3' untranslated region comprises, substantially comprises, or comprises SEQ ID NO: 19 or 20. In some embodiments, the 3' untranslated region contains a sequence having at least 80% sequence identity with SEQ ID NO: 19 or 20 (e.g., at least 85% sequence identity, at least 86% sequence identity, at least 87% sequence identity, at least 88% sequence identity, at least 89% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity).

[0188] In some implementations, the 3' untranslated region can be operatively connected to the 3' of the coding structure domain (e.g., directly connected to the coding structure domain or having an intermediary sequence connecting the 5' end of the 3' untranslated region and the 3' end of the coding structure domain).

[0189] In addition to the above, nucleic acid transsplicing molecules may include semi-intronic domains located at one or more sites within the molecule. In some embodiments, the semi-intronic domain is operatively linked to the 3' of a binding domain (e.g., directly linked to the binding domain). The semi-intronic domain can be of any suitable size. In some embodiments, the semi-intronic domain is longer than 20 nucleotides (e.g., between 20 and 100 nucleotides or between 20 and 85 nucleotides). In some cases, the semi-intronic domain comprises, is substantially composed of, or consists of a nucleic acid sequence having at least 80% identity with SEQ ID NO: 17 (e.g., at least 85% sequence identity, at least 86% sequence identity, at least 87% sequence identity, at least 88% sequence identity, at least 89% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity).

[0190] The inventors discovered that truncated variants of the WPRE 3' UTR sequence can be used to enhance mid-target splicing in RNA exon editors. Two otherwise identical RNA exon editors were compared: one with the WPRE 3' UTR sequence of SEQ ID NO: 20, and the other with a truncated variant of that sequence, in which 203 internal nucleotides were deleted (“WPRE3”; SEQ ID NO: 87). The trans-splicing efficiency of both RNA exon editors was tested. In two replicates of this experiment, the RNA exon editor with the truncated WPRE 3' UTR achieved 18–19% mid-target splicing, while the exon editor with the full-length WPRE 3' UTR achieved 13–14%. Surprisingly, the truncated WPRE 3' UTR sequence, which is 42.6% the length of the full-length WPRE sequence, performed as well as or better than the full-length sequence.

[0191] Some embodiments of an RNA exon editor include the following components operatively linked in the 5' to 3' direction: a binding domain of a binding site configured to bind to a target region of a precursor mRNA of a target gene, a half-intron, a coding domain sequence encoding one or more exons of the target gene, and a truncated WPRE 3' UTR sequence. In some embodiments, the truncated WPRE 3' UTR sequence lacks at least 50, 75, 100, 125, 150, 175, or 200 consecutive nucleotides of SEQ ID NO: 20. In some embodiments, the truncated WPRE 3' UTR sequence lacks 50-200, 50-150, 50-100, 50-203, 100-203, 150-203, or 175-203 consecutive nucleotides of SEQ ID NO: 20. In some embodiments, the truncated WPRE 3' UTR sequence comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 87. In some embodiments, the truncated WPRE 3' UTR sequence comprises or consists of SEQ ID NO: 87. In some embodiments, the target gene is MeCP2. In some embodiments, the target gene is not MeCP2. In some embodiments, the RNA exon editor further comprises one or more of a poly A sequence, a triple-helix terminator sequence, or a 3x UBS sequence, or any combination thereof, operatively linked to the 3' of the truncated WPRE 3' UTR sequence.

[0192] 6. 3' transcription terminator domain In some cases, the trans-splicing molecule includes a 3' transcription terminator domain. In some embodiments, such a 3' transcription terminator domain forms a triple helix structure that effectively caps the 3' end of the trans-splicing molecule. In some cases, the 3' transcription terminator domain is derived from human long non-coding RNA MALAT1 (e.g., wild-type MALAT1). In some embodiments, the 3' transcription terminator domain includes an tRNA-like domain. A 3' transcription terminator domain that can be used as part of the MeCP2 trans-splicing molecule of the present invention is described in International Patent Publication No. WO 2020 / 214973, which is incorporated herein by reference in its entirety. For example, in some embodiments, the region of the RNA exon editor operatively linked to the 3' end of the CDS includes a terminator domain comprising, substantially comprising, or comprising a MALAT1+mascRNA domain such as SEQ ID NO: 77, or a variant thereof such as SEQ ID NO: 22, the variant being an antimut1 variant as described in PCT / US2023 / 066969.

[0193] In some implementations, the exemplary RNA exon editors described herein include those containing a binding domain that binds to intron 1 of MeCP2, wherein such exemplary RNA exon editors binding intron 1 may contain a binding domain, a half-intron, a coding domain sequence (e.g., SEQ ID NO: 76 or SEQ ID NO: 9), and a 3' transcription terminator.

[0194] In some implementations, the exemplary RNA exon editors described herein include those containing a binding domain that binds to intron 2 of MeCP2, wherein such exemplary RNA exon editors binding intron 2 may include a binding domain, a half-intron, a coding sequence (e.g., SEQ ID NO: 76), and a 3' transcription terminator.

[0195] In some implementations, the exemplary RNA exon editors described herein include those containing a binding domain that binds to intron 3 of MeCP2, wherein such exemplary RNA exon editors binding intron 3 may include a binding domain, a half-intron, a coding sequence (e.g., SEQ ID NO: 14), and a 3' transcription terminator.

[0196] In some embodiments, the binding of trans-splicing molecules to target precursor mRNA is mediated by a complementarity percentage (i.e., based on the base-pairing characteristics of nucleic acids), triple helix formation, or protein-nucleic acid interactions (as described in the literature cited herein), or any combination thereof. In one embodiment, the nucleic acid trans-splicing molecule comprises DNA, RNA, or a DNA / RNA hybrid molecule, wherein the DNA or RNA is single-stranded or double-stranded. This document also includes RNA or DNA that can hybridize with one of the aforementioned RNAs or DNAs, preferably under stringent conditions, such as at 60°C in 2.5x SSC buffer, and several washes at 37°C in a lower buffer concentration (e.g., 0.5x SSC buffer). When trans-splicing molecules are synthesized in vitro, such trans-splicing molecules can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve the stability of the molecule, hybridization with target mRNA, transport to cells, stability against enzymatic cleavage in cells, etc. For example, modifying trans-splicing molecules to reduce total charge can enhance cellular uptake of the molecule. Furthermore, modifications can be made to reduce susceptibility to nucleases or chemical degradation. The nucleic acid molecule can be synthesized by conjugation to another molecule (e.g., a peptide, a hybridization-triggered cross-linking agent, a transporter, a hybridization-triggered cleavage agent, etc.).

[0197] Various other well-known modifications to nucleic acid molecules can be introduced as a means of increasing intracellular stability and half-life (see above for oligonucleotides). Possible modifications are known in the art. Modifications that can be made to the structure of synthetic trans-splicing molecules include backbone modifications.

[0198] 7. Connector In some embodiments, the trans-splicing molecule of this disclosure comprises a nucleotide linker located between one or more of its components. In some embodiments, the linker comprises one or more nucleotides. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides.

[0199] 8. Cell line assay In some cases, the trans-splicing molecules described herein are tested in cultured cell lines. To screen, select, and improve the functionality of RNA exon editors, cultured cell lines can be obtained or engineered to express the targeted MeCP2 precursor mRNA at adequate levels.

[0200] 9. Animal models The exemplary exon editors described herein target human intron 1 or intron 2. To test their in vivo efficacy, the inventors will use mice with the human MeCP2 gene knocked into an autosome: FVB / N-Tg(MECP2*R270X / GFP)AHzo / J. The resulting mouse mutant MeCP2 (tm1.1) reproduces many phenotypes observed in humans and is a standard model used in this field. Therefore, this mouse model will be used to evaluate the ability of the RNA exon editors described herein to rescue MeCP2 mutant defects in the in vivo context.

[0201] Further details regarding FVB / N-Tg(MECP2*R270X / GFP)AHzo / J are available on The Jackson Laboratory website. In short, as described above, MeCP2-R270X transgenic mice express human MeCP2 truncated at amino acid R270 and C-terminally labeled with EGFP. As determined by immunohistochemistry, the transgene expression reproduces the expression of the human MECP2 gene in the cortex, hippocampus, cerebellum, hypothalamus, and brainstem. The MeCP2 protein is entirely localized in the nucleus and concentrated in heterochromatin foci, similar to endogenous proteins. MeCP2-R270X transgenic mice did not exhibit a distinct phenotype prior to combination with the mouse Mecp2 knockout allele. When combined with the mouse Mecp2 knockout (stock code 003890), the median lifespan of the mice was 85 days, slightly longer than mice carrying the Mecp2 knockout alone (76 days).

[0202] In some implementations, AAV9 containing the exemplary RNA exon editor described herein is administered via intracerebroventricular injection into P0-P1 neonatal FVB / N-Tg(MECP2*R270X / GFP)AHzo / J mice.

[0203] 10. RNA Exon Editor Screening Platform As described herein, 3' RNA exon editors contain several functional sequence elements, such as binding domains (BDs) for precursor mRNA targeting and hemiintrons that provide splice acceptor sites. In some embodiments, the hemiintrons contain branching points and polypyrimidine bundles in addition to the 3' splice acceptor sites. When an RNA exon editor is engineered for a given gene target, the ability of multiple sequence options for each of these elements to promote high trans-splicing (TS) efficiency is tested. Such tests can be performed by cloning and transfecting individual RNA exon editor variants and analyzing their efficiency using RT-qPCR / ddPCR and Western blot analysis, as described below.

[0204] 11. Screening with RNA exon editors in individual formats: This approach can be applied to test small-scale variable elements within RNA exon editor sequences prior to initiating library-based multiplex screening, to validate the performance of RNA exon editors identified in multiplex screening, and / or to improve the performance of lead candidates. TS efficiency is assessed at both the RNA and protein levels.

[0205] At the RNA level, TS activity is assessed by isolating total RNA from cells, followed by reverse transcription and real-time quantitative PCR (RT-qPCR). The RT-qPCR measures the copy number of RNA targeting, for example, the following: RNF20 (a housekeeping gene for normalization); native (MeCP2) mRNA; exon editor RNA; target- and exon-edited RNA (ONT), which is a product of positive TS; and ONT + exon editor + OFT (off-target), a single assay capturing all three targets. OFT indicates incorrect RNA molecules that can be trans-spliced ​​by the RNA exon editor.

[0206] ONT TS efficiency, also known as substitution percentage, represents the portion of the total MeCP2 mRNA population that has successfully undergone TS, and is calculated using the following equation: ONT TS% = 100 * (ONT copy count / (ONT copy count + native copy count)).

[0207] RNA exon editor TS efficiency is the portion of the RNA exon editor transcript population that has been correctly trans-spliced ​​into MeCP2 RNA, and is calculated using the following equation: Exon editor TS% = 100 * (ONT copy number / (ONT copy number + exon editor copy number + OFT copy number)).

[0208] At the protein level, TS activity is measured by applying Western blot analysis to proteins extracted from cell or tissue samples. β-actin (a cytoskeletal protein) can be used as a loading control. ONT protein levels are measured using MeCP2-specific antibodies (Abs). For constructs that include tags (e.g., C-terminal Myc tags), Western blots can be used to assess ONT protein levels using Abs specific to that tag (e.g., Myc-specific Abs).

[0209] 12. Exemplary Combinations Some embodiments of the RNA exon editor include a combination of a binding domain, a half-intron, and a 3' untranslated region, as well as the 3' transcription terminator domain disclosed above. In some embodiments, the RNA exon editor comprises a binding domain selected from any of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 47, 55, 56, 57, 58, 59, 88, 89, 90, 91, or 92, or a functional variant thereof; a half-intron of SEQ ID NO: 17, or a functional variant thereof; a coding domain sequence selected from one of SEQ ID NO: 7-14, 76, 85, or 86, or a combination thereof, or a functional variant thereof; a 3' UTR selected from SEQ ID NO: 19, 20, or 87, or a functional variant thereof; and a 3' transcription terminator domain of SEQ ID NO: 22, or a functional variant thereof. Some embodiments do not include one or more of the elements disclosed above. For example, some embodiments do not include one or more of the half-intron of SEQ ID NO: 17, the 3' UTR of SEQ ID NO: 19, 20 or 87, or the MALAT1 terminator of SEQ ID NO: 22 or 77. In some embodiments, the RNA exon editor includes a binding domain selected from one of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 47, 55, 56, 57, 58, 59, 88, 89, 90, 91 or 92 or a functional variant thereof, and a splicing site. In some embodiments, the RNA exon editor comprises a binding domain selected from SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 47, 55, 56, 57, 58, 59, 88, 89, 90, 91, or 92, or a functional variant thereof, and a half-intron. In some embodiments, the RNA exon editor comprises a binding domain selected from SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 47, 55, 56, 57, 58, 59, 88, 89, 90, 91, or 92, or a functional variant thereof, and a binding domain selected from SEQ ID NO: 17, or a functional variant thereof. In some implementations, the RNA exon editor comprises a binding domain selected from SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 47, 55, 56, 57, 58, 59, 88, 89, 90, 91 or 92 or a functional variant thereof, and SEQ ID NO: 19 or a functional variant thereof.In some embodiments, the RNA exon editor comprises a binding domain selected from SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 47, 55, 56, 57, 58, 59, 88, 89, 90, 91, or 92, or a functional variant thereof, and SEQ ID NO: 19, or 20, or a functional variant thereof. In some embodiments, the RNA exon editor comprises a binding domain selected from SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 47, 55, 56, 57, 58, 59, 88, 89, 90, 91, or 92, or a functional variant thereof, and SEQ ID NO: 22, or a functional variant thereof. In some implementations, the RNA exon editor comprises a binding domain selected from one of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 47, 55, 56, 57, 58, 59, 88, 89, 90, 91 or 92 or a functional variant thereof, and a coding domain sequence comprising one or more of SEQ ID NO: 7-14, 76, 85 or 86.

[0210] An exemplary construction of the RNA exon editor disclosed herein includes the following (in the 5' to 3' orientation): Exon Editor A: [BD]-[Half-Intron]-[CDS]; Exon Editor B: [BD]-[Half-Intron]-[CDS]-[3' UTR]; Exon editor C: [BD]-[Half-introns]-[CDS]-[Terminators]; Exon editor D: [BD]-[half-intron]-[CDS]-[3' UTR]-[terminator]; Exon editor E: [BD]-[BP]-[pPy]-[3' splice site]-[CDS]; Exon editor F: [BD]-[BP]-[pPy]-[3' splice site]-[CDS]-[3' UTR]; Exon editor G: [BD]-[BP]-[pPy]-[3' splice site]-[CDS]-[terminator]; and Exon editor H: [BD]-[BP]-[pPy]-[3' splice site]-[CDS]-[3' UTR]-[terminator]; Wherein, “BD” represents any BD disclosed in Section VI.C.3; “half-intron” represents any half-intron disclosed in Section VI.C.4; “CDS” represents any CDS disclosed in Section VI.C.2; “3' UTR” represents any 3' UTR disclosed in Section VI.C.5; “terminator” represents any terminator disclosed in Section VI.C.6; “BP” represents any branch point sequence disclosed in Section VI.C.4; “pPy” represents any polypyrimidine bundle sequence disclosed in Section VI.C.4; “3' splice site” represents the nucleotide sequence YAG, where Y is pyrimidine; and “]-[” represents a phosphodiester bond or linker. In some embodiments, an exon editor construct encoding any of exon editors 1 to 8 includes a promoter sequence operatively linked upstream of the BD of any of exon editors 1 to 8.

[0211] In some embodiments, the RNA exon editor of this disclosure includes, in the 5' to 3' direction, the following operatively linked to the 3' end of the coding domain sequence: a WPRE sequence having at least 90% identity with SEQ ID NO: 87, a MALAT1 terminator anti-Mut1 variant having at least 90% identity with SEQ ID NO: 22, and a 3xUBS sequence having at least 90% identity with SEQ ID NO: 79. In some embodiments, the RNA exon editor of this disclosure includes, in the 5' to 3' direction, the following operatively linked to the 3' end of the coding domain sequence: the WPRE sequence of SEQ ID NO: 87, the MALAT1 terminator anti-Mut1 variant of SEQ ID NO: 22, and the 3xUBS sequence of SEQ ID NO: 79.

[0212] Further exemplary combinations of the RNA exon editor components are described below in embodiments 1 through 279.

[0213] D. Carrier Various techniques can be used, such as recombinant adeno-associated virus (AAV) vectors or other vector modalities (e.g., non-viral vectors), to deliver trans-splicing molecules to target cells of an individual. Therefore, this document provides vectors containing / encoding trans-splicing molecules (e.g., viral or non-viral vectors containing / encoding trans-splicing molecules, such as DNA vectors containing / encoding trans-splicing molecules). Any suitable nucleic acid vector can be used in conjunction with the compositions and methods of this invention to design and assemble components of the trans-splicing molecules and recombinant AAV. In one embodiment, the vector is a promoter-driven recombinant AAV carrying a trans-splicing molecule, the promoter expressing the trans-splicing molecule in selected cells of an individual. Methods for assembling recombinant vectors are known in the art. See, for example, Ausubel et al. Current Protocols in Molecular Biology , John Wiley&Sons, New York, 1989; Kay, MA et al., Nat. Medic ,2001, 7(l):33-40; and Walther W. and Stein U., Drugs 2000, 60(2):249-71.

[0214] In some embodiments described herein, the trans-splicing molecule is delivered to selected cells requiring treatment, such as neurons, using an AAV vector. A variety of naturally occurring AAV serotypes are available. AAV capsids exist in a variety of natural variants, allowing the identification and use of AAVs with properties particularly suitable for neurons. Artificial AAV vectors can be engineered using conventional molecular biology techniques, making it possible to optimize these particles to specifically deliver the trans-splicing molecule nucleic acid sequence to cells, minimize immunogenicity, adjust stability and particle lifetime, efficiently degrade, and accurately deliver to the cell nucleus, etc. Such artificial capsids can be generated, for example, using any suitable technique, using a combination of a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) and a heterologous sequence obtainable from different selected AAVs, discontinuous portions of the same AAV, or non-AAV viral or non-viral sources. Artificial AAVs can be, but are not limited to, pseudotyped AAVs, chimeric AAV capsids, recombinant AAV capsids, or “humanized” AAV capsids. A pseudotyped vector in which the capsid of one AAV is replaced by a heterologous capsid protein can be used to deliver the trans-splicing molecule described herein.

[0215] The expression of the trans-splicing molecules described herein can be achieved in selected cells by delivery using recombinantly engineered AAVs or artificial AAVs containing sequences that encode the desired trans-splicing molecules. The use of AAVs is a common method for exogenous DNA delivery because it is relatively non-toxic, provides efficient gene transfer, and can be optimized for specific purposes. Among the well-characterized AAV serotypes isolated from humans or non-human primates, human serotype 2 has been widely used for efficient gene transfer experiments in various target tissues and animal models.

[0216] In some implementations, the AAV is AAV1 or a variant thereof (e.g., SEQ ID NO: 6 or 64 of US20030138772 or SEQ ID NO: 11 or 27 of US20150159173), AAV2 or a variant thereof (e.g., SEQ ID NO: 7 or 70 of US20030138772, SEQ ID NO: 7 or 23 of US20150159173 or SEQ ID NO: 7 of US20150159173), AAV2G9 or a variant thereof, AAV3 or a variant thereof (e.g., SEQ ID NO: 8 or 71 of US20030138772), AAV3a or a variant thereof, AAV3b or a variant thereof (e.g., SEQ ID NO: 1 and 10 of US Patent No. 6,156,303), AAV3-3 or a variant thereof (e.g., SEQ ID NO: WO2005033321). SEQ ID NO: 200 and 217), AAV4 or variants thereof (e.g., SEQ ID NO: 63 of US20030138772), AAV4-4 or variants thereof (e.g., SEQ ID NO: 201 or 218 of WO2005033321), AAV5 or variants thereof (e.g., SEQ ID NO: 114 of US20030138772), AAV6 or variants thereof (e.g., SEQ ID NO: 65 of US20030138772), AAV6.1 or variants thereof (e.g., SEQ ID NO: 29 of US20150159173), AAV6.2 or variants thereof, AAV6.1.2 or variants thereof, AAV7 or variants thereof (e.g., SEQ ID NO: 1-3 of US20030138772), AAV7.2 or variants thereof, AAV8 or variants thereof (e.g., SEQ ID NO: 200 and 217 of US20030138772). 4 and 95 or AAV8(b) (as described in U.S. Patent No. 9,567,376, which is incorporated herein by reference in its entirety), AAV9 or variants thereof (e.g., SEQ ID NO: 5 and 100 of US20030138772), AAV9.9 or variants thereof, AAV9.11 or variants thereof, AAV9.13 or variants thereof, AAV9.16 or variants thereof, AAV9.24 or variants thereof, AAV9.45 or variants thereof, AAV9.47 or variants thereof, AAV9.61 or variants thereof, AAV9.68 or variants thereof, AAV9.84 or variants thereof (see, for example, N. Pulicherla et al.) Molecular Therapy19(6):1070-1078 (2011), which is incorporated herein by reference in its entirety, AAV10 or variants thereof (e.g., SEQ ID NO: 117 of US20030138772), AAV11 or variants thereof (e.g., SEQ ID NO: 118 of US20030138772), AAV12 or variants thereof (e.g., SEQ ID NO: 118 of US20030138772). 119), AAV16.3 or a variant thereof, AAV24.1 or a variant thereof, AAV27.3 or a variant thereof, AAV42.12 or a variant thereof, AAV42-1b or a variant thereof, AAV42-2 or a variant thereof, AAV42-3a or a variant thereof, AAV42-3b or a variant thereof, AAV42-4 or a variant thereof, AAV42-5a or a variant thereof, AAV42-5b or a variant thereof, AAV42-6b or a variant thereof, AAV42-8 or a variant thereof, AAV42-10 or a variant thereof, AAV42-11 or a variant thereof, AAV42-12 or a variant thereof, AAV42-13 or a variant thereof, AAV42-15 or a variant thereof, AAV42-aa or a variant thereof, AAV43-1 or a variant thereof, AAV43-12 or a variant thereof, AAV43-20 or a variant thereof, AAV43-21 or a variant thereof, AAV43-23 or a variant thereof, AAV43-25 or a variant thereof, AAV43-5 or a variant thereof, A AV44.1 or its variants, AAV44.2 or its variants, AAV44.5 or its variants, AAV223.1 or its variants, AAV223.2 or its variants, AAV223.4 or its variants, AAV223.5 or its variants, AAV223.6 or its variants, AAV223.7 or its variants, AAV1-7 / rh.48 or its variants, AAV1-8 / rh.49 or its variants, AAV2-15 / rh.62 or its variants, AAV AAV2-3 / rh.61 or a variant thereof, AAV2-4 / rh.50 or a variant thereof, AAV2-5 / rh.51 or a variant thereof, AAV3.1 / hu.6 or a variant thereof, AAV3.1 / hu.9 or a variant thereof, AAV3-9 / rh.52 or a variant thereof, AAV3-11 / rh.53 or a variant thereof, AAV4-8 / rh.64 or a variant thereof, AAV4-9 / rh.54 or a variant thereof (e.g., SEQ of WO2005033321) SEQ ID NO: 116), AAV4-19 / rh.55 or its variants (e.g., SEQ ID NO: 117 of WO2005033321), AAV5-3 / rh.57 or its variants, AAV5-22 / rh.58 or its variants, AAV7.3 / hu.7 or its variants, AAV16.8 / hu.10 or its variants, AAV16.12 / hu.11 or its variants, AAV29.3 / bb.1 or its variants, AAV29.5 / bb.2 or its variants, AAV106.1 / hu.37 or its variants, AAV114.3 / hu.40 or its variants, AAV127.2 / hu.41 or its variants, AAV127.5 / hu.42 or its variants, AAV128.3 / hu.44 or its variants, AAV130.4 / hu.48 or its variants, AAV145.1 / hu.53 or its variants, AAV145.5 / hu.54 or its variants, AAV145.6 / hu.55 or its variants, AAV161.10 / hu.60 or its variants, AAV161.6 / h u.61 or a variant thereof, AAV33.12 / hu.17 or a variant thereof, AAV33.4 / hu.15 or a variant thereof, AAV33.8 / hu.16 or a variant thereof, AAV52 / hu.19 or a variant thereof, AAV52.1 / hu.20 or a variant thereof, AAV58.2 / hu.25 or a variant thereof, AAVA3.3 or a variant thereof, AAVA3.4 or a variant thereof, AAVA3.5 or a variant thereof, AAVA3.7 or a variant thereof, AAVC1 or a variant thereof, AAVC2 or a variant thereof, AAVC5 or a variant thereof, AAV-DJ or a variant thereof (e.g., SEQ of US20140359799) SEQ ID NO: 2 or 3), AAV-DJ8 or a variant thereof, AAVF3 or a variant thereof, AAVF5 or a variant thereof, AAVH2 or a variant thereof, AAVH6 or a variant thereof, AAVLK03 or a variant thereof, AAVH-1 / hu.1 or a variant thereof, AAVH-5 / hu.3 or a variant thereof, AAVLG-10 / rh.40 or a variant thereof, AAVLG-4 / rh.38 or a variant thereof, AAVLG-9 / hu.39 or a variant thereof, AAVN721-8 / rh.43 or a variant thereof, AAVCh.5 or a variant thereof (e.g., SEQ ID NO 46 of US20150159173), AAVCh.5R1 or a variant thereof, AAVcy.2 or a variant thereof, AAVcy.3 or a variant thereof, AAVcy.4 or a variant thereof, AAVcy.5 or a variant thereof (e.g., SEQ ID NO: 46 of US20150159173). 8 and 24), AAVcy.5R1 or variants thereof, AAVcy.5R2 or variants thereof, AAVcy.5R3 or variants thereof, AAVcy.5R4 or variants thereof, AAVcy.6 or variants thereof, AAVhu.1 or variants thereof (e.g., SEQ ID NO: 144 of WO2005033321), AAVhu.2 or variants thereof (e.g., SEQ ID NO: 143 of WO2005033321), AAVhu.3 or variants thereof (e.g., SEQ ID NO: 145 of WO2005033321), AAVhu.4 or variants thereof (e.g., SEQ ID NO: 141 of WO2005033321), AAVhu.5 or variants thereof, AAVhu.6 or variants thereof (e.g., SEQ ID NO: 84 of WO2005033321), AAVhu.7 or variants thereof (e.g., SEQ ID NO: 150 of WO2005033321), AAVhu.9 or variants thereof (e.g., SEQ ID NO: 155 of WO2005033321), AAVhu.10 or variants thereof (e.g., SEQ ID NO: 156 of WO2005033321), AAVhu.11 or variants thereof (e.g., SEQ ID NO: 153 of WO2005033321), AAVhu.13 or variants thereof (SEQ ID NO: 153 of US20150159173). 16 and 32), AAVhu.15 or variants thereof (e.g., SEQ ID NO: 147 of WO2005033321), AAVhu.16 or variants thereof (e.g., SEQ ID NO: 148 of WO2005033321), AAVhu.17 or variants thereof (e.g., SEQ ID NO: 83 of WO2005033321), AAVhu.18 or variants thereof (e.g., SEQ ID NO: 149 of WO2005033321), AAVhu.19 or variants thereof (e.g., SEQ ID NO: 133 of WO2005033321), AAVhu.20 or variants thereof (e.g., SEQ ID NO: 134 of WO2005033321), AAVhu.21 or variants thereof (e.g., SEQ ID NO: 147 of WO2005033321). 135), AAVhu.22 or variants thereof (e.g., SEQ ID NO: 138 of WO2005033321), AAVhu.23.2 or variants thereof (e.g., SEQ ID NO: 137 of WO2005033321), AAVhu.24 or variants thereof (e.g., SEQ ID NO: 136 of WO2005033321), AAVhu.25 or variants thereof (e.g., SEQ ID NO: 146 of WO2005033321), AAVhu.26 or variants thereof (e.g., SEQ ID NO: 17 and 33 of US20150159173), AAVhu.27 or variants thereof (e.g., SEQ ID NO: 140 of WO2005033321), AAVhu.28 or variants thereof (e.g., SEQ ID NO: 138 of US20150159173). 42) AAVhu.29 or its variants (e.g., SEQ ID NO: 132 of WO2005033321), AAVhu.29R or variants thereof, AAVhu.31 or variants thereof (e.g., SEQ ID NO: 121 of WO2005033321), AAVhu.32 or variants thereof (SEQ ID NO: 122 of WO2005033321), AAVhu.34 or variants thereof (e.g., SEQ ID NO: 125 of WO2005033321), AAVhu.35 or variants thereof (e.g., SEQ ID NO: 164 of WO2005033321), AAVhu.37 or variants thereof (e.g., SEQ ID NO: 18 and 34 of US20150159173), AAVhu.39 or variants thereof (e.g., SEQ ID NO: 102 of WO2005033321), AAVhu.40 or variants thereof (e.g., SEQ ID NO: 102 of WO2005033321). 87), AAVhu.41 or variants thereof (e.g., SEQ ID NO: 91 of WO2005033321), AAVhu.42 or variants thereof (e.g., SEQ ID NO: 85 of WO2005033321), AAVhu.43 or variants thereof (e.g., SEQ ID NO: 160 of WO2005033321), AAVhu.44 or variants thereof (e.g., SEQ ID NO: 45 of US20150159173), AAVhu.44R1 or variants thereof, AAVhu.44R2 or variants thereof, AAVhu.44R3 or variants thereof, AAVhu.45 or variants thereof (e.g., SEQ ID NO: 127 of WO2005033321), AAVhu.46 or variants thereof (e.g., SEQ ID NO: 91 of WO2005033321). AAVhu.47 or variants thereof (e.g., SEQ ID NO: 128 of WO2005033321), AAVhu.48 or variants thereof (e.g., SEQ ID NO: 38 of US20150159173), AAVhu.48R1 or variants thereof, AAVhu.48R2 or variants thereof, AAVhu.48R3 or variants thereof, AAVhu.49 or variants thereof (e.g., SEQ ID NO: 189 of WO2005033321), AAVhu.51 or variants thereof (e.g., SEQ ID NO: 190 of WO2005033321), AAVhu.52 or variants thereof (e.g., SEQ ID NO: 191 of WO2005033321), AAVhu.53 or variants thereof (e.g., SEQ ID NO: 159 of US20150159173). NO:19 and 35), AAVhu.54 or its variants (e.g., SEQ ID NO: 188 of WO2005033321), AAVhu.55 or variants thereof (e.g., SEQ ID NO: 187 of WO2005033321), AAVhu.56 or variants thereof (e.g., SEQ ID NO: 192 of WO2005033321), AAVhu.57 or variants thereof (e.g., SEQ ID NO: 193 of WO2005033321), AAVhu.58 or variants thereof (e.g., SEQ ID NO: 194 of WO2005033321), AAVhu.60 or variants thereof (e.g., SEQ ID NO: 184 of WO2005033321), AAVhu.61 or variants thereof (e.g., SEQ ID NO: 185 of WO2005033321), AAVhu.63 or variants thereof (e.g., SEQ ID NO: 195 of WO2005033321), AAVhu.64 or variants thereof (e.g., SEQ ID NO: 195 of WO2005033321). SEQ ID NO: 196), AAVhu.66 or variants thereof (e.g., SEQ ID NO: 197 of WO2005033321), AAVhu.67 or variants thereof (e.g., SEQ ID NO: 198 of WO2005033321), AAVhu.14 / 9 or variants thereof, AAVhu.t 19 or variants thereof, AAVrh.2 or variants thereof (e.g., SEQ ID NO: 39 of US20150159173), AAVrh.2R or variants thereof, AAVrh.8 or variants thereof (e.g., SEQ ID NO: 41 of US20150159173), AAVrh.8R or variants thereof, AAVrh.10 or variants thereof (e.g., SEQ ID NO: 197 of US20150159173). 9 and 25), AAVrh.12 or variants thereof, AAVrh.13 or variants thereof (e.g., SEQ ID NO: 10 and 26 of US20150159173), AAVrh.13R or variants thereof, AAVrh.14 or variants thereof, AAVrh.17 or variants thereof, AAVrh.18 or variants thereof, AAVrh.19 or variants thereof, AAVrh.20 or variants thereof (e.g., SEQ ID NO: 10 and 26 of US20150159173). 1) AAVrh.21 or variants thereof, AAVrh.22 or variants thereof, AAVrh.23 or variants thereof, AAVrh.24 or variants thereof, AAVrh.25 or variants thereof, AAVrh.31 or variants thereof, AAVrh.32 or variants thereof, AAVrh.33 or variants thereof, AAVrh.34 or variants thereof, AAVrh.35 or variants thereof, AAVrh.36 or variants thereof, AAVrh.37 or variants thereof (e.g., SEQ ID NO: 40 of US20150159173), AAVrh.37R2 or variants thereof, AAVrh.38 or variants thereof (e.g., SEQ ID NO: 86 of WO2005033321), AAVrh.39 or variants thereof (e.g., SEQ ID NO: 3, 20, or 36 of US20150159173), AAVrh.40 or variants thereof (e.g., SEQ ID NO: 92 of WO2005033321), AAVrh.43 or variants thereof (e.g., SEQ ID NO: 21 and 37 of US20150159173), AAVrh.46 or variants thereof (e.g., SEQ ID NO: 4 and 22 of US20150159173), AAVrh.48 or variants thereof (e.g., SEQ ID NO: 44 of US20150159173), AAVrh.48.1 or variants thereof (e.g., SEQ ID NO: 86 of US20150159173). 44) AAVrh.48.1.2 or variants thereof, AAVrh.48.2 or variants thereof, AAVrh.49 or variants thereof (e.g., SEQ ID NO: 103 of WO2005033321), AAVrh.50 or variants thereof (e.g., SEQ ID NO: 108 of WO2005033321), AAVrh.51 or variants thereof (e.g., SEQ ID NO: 104 of WO2005033321), AAVrh.52 or variants thereof (e.g., SEQ ID NO: 96 of WO2005033321), AAVrh.53 or variants thereof (e.g., SEQ ID NO: 97 of WO2005033321), AAVrh.54 or variants thereof (e.g., SEQ ID NO: 97 of US20150159173). 49) AAVrh.56 or variants thereof (e.g., SEQ ID NO: 152 of WO2005033321), AAVrh.57 or variants thereof (e.g., SEQ ID NO: 105 of WO2005033321), AAVrh.58 or variants thereof (e.g., SEQ ID NO: 48 of US20150159173), AAVrh.61 or variants thereof (e.g., SEQ ID NO: 107 of WO2005033321), AAVrh.62 or variants thereof (e.g., SEQ ID NO: 114 of WO2005033321), AAVrh.64 or variants thereof (e.g., SEQ ID NO: 152 of US20150159173). 43) AAVrh.64R1 or variants thereof, AAVrh.64R2 or variants thereof, AAVrh.67 or variants thereof (e.g., SEQ ID NO: 47 of US20150159173), AAVrh.73 or a variant thereof (e.g., SEQ ID NO: 5 of US20150159173) or AAVrh.74 or a variant thereof (e.g., SEQ ID NO: 6 of US2015015917). Non-limiting examples of variations include SEQ ID Nos: 9, 27-45, 47-62, 66-69, 73-81, 84-94, 96, 97, 99 and 101-113 of US20030138772 (the contents of which are incorporated herein by reference in their entirety), and SEQ ID Nos: 1, 2, 4-82, 89, 90, 93-95, 98, 100, 101, 109-113, 118-120, 124, 126, 131, 139, 142, 151, 154, 158, 161, 162, 165-183, 202, 204-212, 215, 219 and 224-236 of WO2005033321 (the contents of which are incorporated herein by reference in their entirety). In one embodiment, the AAV serotype is any of those described in US 2021 / 0189430 (the contents of which are incorporated herein by reference in their entirety). The amino acid sequence of the AAV may include one or more amino acid substitutions at one or more positions in the AAV capsid protein that interact with heparan sulfate proteoglycans or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588 (numbered based on the VP1 number of AAV2).

[0217] Unless otherwise stated, the AAV ITR and other selected AAV components described herein can be readily selected from any AAV serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or other known and unknown AAV serotypes. In one embodiment, the ITR is derived from AAV2. These ITRs or other AAV components can be readily isolated from AAV serotypes using techniques available to those skilled in the art. Such AAVs can be isolated or obtained from academic, commercial, or public sources (e.g., Manassas, VA). Alternatively, AAV sequences can be obtained by synthesis or other suitable means referencing published sequences (e.g., available in the literature or databases such as GenBank, PubMed, etc.).

[0218] The AAV fragments required for assembly into a vector include cap proteins (including vp1, vp2, vp3, and hypervariable regions), rep proteins (including rep 78, rep 68, rep 52, and rep 40), and sequences encoding these proteins. These fragments are readily available for use in a variety of vector systems and host cells. Such fragments can be used alone, in combination with other AAV serotype sequences or fragments, or in combination with elements derived from other AAV or non-AAV viral sequences. As used herein, artificial AAV serotypes include, but are not limited to, AAVs having non-naturally occurring capsid proteins. Such artificial capsids can be generated using any suitable technique, using a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) combined with a heterologous sequence that can be obtained from different selected AAV serotypes, discontinuous portions of the same AAV serotype, or non-AAV viral or non-viral sources. Artificial AAV serotypes can be, but are not limited to, pseudotyped AAVs, chimeric AAV capsids, recombinant AAV capsids, or “humanized” AAV capsids. A pseudotype vector, as described herein, is useful for use with an AAV capsid accompanied by an ITR of an AAV having a different capsid protein. In one embodiment, the AAV is AAV2 / 5 (i.e., an AAV with an AAV2 ITR and an AAV5 capsid). In another embodiment, the AAV is AAV2 / 8 (i.e., an AAV with an AAV2 ITR and an AAV8 capsid). In one embodiment, the AAV comprises an AAV8 capsid. Such an AAV8 capsid comprises an amino acid sequence found at NCBI reference sequence: YP_077180.1. In another embodiment, the AAV8 capsid comprises a capsid encoded by nt 2121 to 4337 of GenBank accession: AF513852.1.

[0219] In one embodiment, the vector useful in the compositions and methods described herein contains at least a sequence encoding a selected AAV serotype capsid (e.g., AAV2 capsid) or a fragment thereof. In another embodiment, the useful vector contains at least a sequence encoding a selected AAV serotype rep protein (e.g., AAV2 rep protein) or a fragment thereof. Optionally, such vectors may contain both AAV cap and rep protein. In vectors providing both AAV rep and cap, the AAV rep and AAV cap sequences may both be derived from a single serotype source, such as AAV2.

[0220] Alternatively, a vector in which the rep sequence originates from an AAV serotype different from the AAV serotype providing the cap sequence can be used. In one embodiment, the rep and cap sequences are expressed from different sources (e.g., different vectors, or host cells and vectors). In another embodiment, these rep sequences are fused in-frame with cap sequences of different AAV serotypes to form chimeric AAV vectors, such as those described in U.S. Patent No. 7,282,199 (incorporated herein by reference).

[0221] Suitable recombinant AAV (rAAV) is generated by culturing host cells containing a nucleic acid sequence encoding an AAV serotype capsid protein or a fragment thereof as defined herein; a functional rep gene; a small gene consisting of, for example, an AAV ITR and a trans-splicing molecule nucleic acid sequence; and sufficient helper functions to allow the small gene to be packaged into the AAV capsid protein. The components required for culturing in host cells to package the AAV small gene into the AAV capsid may be provided trans-present to the host cells. Alternatively, any one or more of the required components (e.g., the small gene, rep sequence, cap sequence, and / or helper functions) may be provided by stable host cells that have been engineered to contain one or more of the required components using methods known to those skilled in the art.

[0222] In one embodiment, the AAV includes a promoter (or a functional fragment of a promoter). The promoter to be used in the rAAV can be selected from a large number of constitutive or inducible promoters capable of expressing the selected transgene in desired target cells. See, for example, the list of promoters identified in International Patent Publication No. WO 2014 / 012482 (incorporated herein by reference). In some embodiments, the promoter is specific for expression in the brain (e.g., the brainstem). In some embodiments, the promoter is cell-specific. The term "cell-specific" means that the specific promoter selected for the recombinant vector can guide the expression of the selected transgene in a specific cell type. In some embodiments, the promoter is specific for the expression of the transgene in neuronal cells.

[0223] In another embodiment, the promoter is a native promoter of the target gene to be expressed. Useful promoters include, but are not limited to, the CAGGS promoter and neuron-specific promoters, including, but not limited to, the human synapsein 1 gene promoter, neuron-specific enolase (NSE) promoter, human synapsein 1 promoter, CaMK kinase promoter, or MeCP2 promoter. Other suitable promoters include inducible promoters, which initiate transcription only when the host cell is exposed to a stimulus that acts as a trigger for activating the promoter.

[0224] Other commonly used regulatory sequences contained in small genes or rAAVs are also disclosed in literature such as WO 2014 / 124282 and other documents cited herein and incorporated herein by reference. Those skilled in the art can make selections among these and other expression control sequences without departing from the scope described herein.

[0225] The selected genetic element can be delivered by any suitable method, including those described herein. Methods for constructing any of the embodiments described herein are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for generating rAAV viral particles are well known, and the choice of appropriate method does not limit the methods and constructs described herein. See, for example, K. Fisher et al. J. Virol ., 1993 70: 520-532 and U.S. Patent 5,478,745, each of which is incorporated herein by reference.

[0226] In some embodiments, the trans-splicing molecule is included in a proviral plasmid, such as those disclosed in International Patent Publication No. WO 2012 / 158757 (incorporated herein by reference). Such proviral plasmids contain a modular recombinant AAV genome comprising: a wild-type 5' AAV2 ITR sequence, with side-joinings allowing easy removal or replacement of unique restriction sites of the ITR; and a promoter comprising a cytomegalovirus (CMV) sequence or a neuron-specific promoter / enhancer upstream of a 49-nucleotide sequence of chicken β-actin, with side-joinings allowing easy removal or replacement of unique restriction sites of the entire promoter sequence, and upstream sequence side-joinings allowing easy removal or replacement of unique restriction sites of the upstream CMV or enhancer sequence from the promoter sequence. The trans-splicing molecules described herein are insertable into polyclonal multi-connector sites, wherein the trans-splicing molecules are operatively linked to and regulated by the promoter. The plasmid is flanked by unique restriction sites that allow for easy removal or substitution of the bovine growth hormone polyadenylated sequence of the poly A sequence; and also by unique restriction sites flanking the 3' ITR wild-type 3' AAV2 ITR sequence that allows for easy removal or substitution. The plasmid backbone contains elements essential for replication in bacterial cells, such as the kanamycin resistance gene, and is itself flanked by transcription terminator / insulator sequences.

[0227] In some embodiments, the proviral plasmid comprises: (a) a modular recombinant AAV genome containing operatively associated: (i) a wild-type 5' AAV2 ITR sequence, with side-attached unique restriction sites that allow for easy removal or replacement of the ITR; and (ii) a promoter comprising (A) a CMV sequence of 49 nucleotides upstream of a CMV-chicken β-actin sequence, or (B) a neuron-specific promoter / enhancer. The promoter side-attached sequence allows for easy removal or replacement of unique restriction sites throughout the promoter sequence, and the upstream sequence side-attached sequence allows for easy removal or replacement of unique restriction sites from the promoter sequence only from or in relation to the upstream CMV or enhancer sequence. Part of this proviral plasmid is also a polyclonal multi-adaptor sequence that allows the insertion of trans-splicing molecule sequences, including any of those described herein, wherein the trans-splicing molecules are operatively linked to and regulated by a promoter; a bovine growth hormone polyadenylated sequence, flanked to allow for easy removal or replacement of the unique restriction site of the poly A sequence; and a wild-type 3' AAV2ITR sequence, flanked to allow for easy removal or replacement of the unique restriction site of the 3' ITR. The proviral plasmid also contains a plasmid backbone containing elements essential for replication in bacterial cells and a kanamycin resistance gene, flanked by transcription terminator / insulator sequences. The proviral plasmids described herein may also contain a 5.1 kb non-coding λ phage padding sequence in the plasmid backbone to increase backbone length and prevent reverse packaging of the non-functional AAV genome.

[0228] On the other hand, the promoter of the proviral plasmid is modified to reduce its size, thereby allowing for the insertion of larger trans-splicing molecular sequences into rAAV. In one embodiment, the CMV / CBA hybrid promoter (which typically comprises approximately 1,000 base pairs of non-coding exons and introns) is replaced by a 130-base-pair chimeric intron, as described in International Patent Publication No. WO 2017 / 087900 (which is incorporated herein by reference in its entirety).

[0229] Next, these proviral plasmids are used with currently conventional packaging methods to produce recombinant viruses that express trans-splicing molecules carried by the proviral plasmids. Those skilled in the art can readily select suitable production cell lines. For example, suitable host cells can be selected from any organism, including prokaryotic (e.g., bacterial) cells and eukaryotic cells, including insect cells, yeast cells, and mammalian cells. Simply put, the proviral plasmid is transfected into the selected packaging cells, where it may exist transiently. Alternatively, the small gene or gene expression cassette with its flanking ITR is stably integrated into the host cell's genome in chromosomal form or as an episome. Suitable transfection techniques are known and readily available for delivering the recombinant AAV genome to host cells. Typically, the proviral plasmid is cultured in host cells expressing cap and / or rep proteins. In the host cells, the small gene, consisting of the trans-splicing molecule and the flanking AAV ITR, is rescued and packaged into capsid or envelope proteins, forming infectious viral particles. Therefore, recombinant AAV infectious particles can be generated by culturing packaging cells carrying proviral plasmids in the presence of sufficient viral sequences to allow the packaging of the gene expression cassette viral genome into the infectious AAV envelope or capsid.

[0230] Alternatively, non-AAV vectors (e.g., nonviral vectors) can be used to deliver trans-splicing molecules. Any suitable nonviral vector technology known in the art or described herein can be used. Suitable nonviral vectors for delivering trans-splicing molecules include liposomes (e.g., cationic liposomes, monolayer liposomes, or multilayer liposomes), nanoparticles (e.g., polymer nanoparticles, lipid nanoparticles (LNPs), PEGylated nanoparticles (e.g., PEGylated LNPs), peptide nanoparticles, metal nanoparticles, etc.), dendritic polymers (e.g., cationic dendritic polymers, such as polypropyleneimine dendritic polymers), and exosomes (e.g., immune-inert and / or targeted exosomes, such as those used in Alvarez-Erviti et al., 2011). Nat. Biotechnol. (Prepared using the techniques described in 29:341) and microvesicles. In some cases, cell-penetrating peptides (CPPs) can be used to deliver the trans-splicing molecules described herein, which can cause transmembrane translocation of target cells and facilitate the delivery of trans-splicing molecules into the target cell interior.

[0231] E. Pharmaceutical compositions and packaging This document provides pharmaceutical compositions comprising nucleic acid trans-splicing molecules, proviral plasmids comprising any MeCP2 nucleic acid trans-splicing molecule described herein, or rAAV. In some embodiments, the pharmaceutical composition comprises any 3' trans-splicing molecule described herein.

[0232] Such pharmaceutical compositions can be prepared to be pure / uncontaminated and suitable for in vivo administration. Contamination of the pharmaceutical compositions described herein can be assessed using conventional methods, and then formulated into pharmaceutical compositions intended for a suitable route of administration. Other compositions containing trans-splicing molecules (e.g., naked DNA) can be similarly formulated using suitable carriers. Such formulations involve the use of pharmaceutically and / or physiologically acceptable mediators or carriers, particularly for administration to target cells (e.g., neurons). In one embodiment, suitable carriers for administration to target cells include buffered saline, isotonic sodium chloride solution, or other buffers (e.g., HEPES) to maintain pH at appropriate physiological levels, and optionally other pharmaceutical agents, agents, stabilizers, buffers, carriers, adjuvants, diluents, etc.

[0233] In some embodiments, the carrier is an injectable liquid. Exemplary physiologically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free phosphate-buffered saline. A variety of such known carriers are provided in U.S. Patent No. 7,629,322 (incorporated herein by reference). In one embodiment, the carrier is an isotonic sodium chloride solution. In another embodiment, the carrier is a balanced salt solution. In one embodiment, the carrier includes Tween. If long-term storage of the virus is intended, it can be frozen in the presence of glycerol or TWEEN® 20.

[0234] In other embodiments, compositions containing the trans-splicing molecule described herein include a surfactant. Useful surfactants, such as Pluronic F68 (Poloxamer 188, also known as LUTROL® F68), may be included because they prevent AAV adhesion to inert surfaces and thus ensure the delivery of the desired dose. For example, an exemplary composition designed for the treatment of Rett syndrome as described herein comprises a recombinant gland-associated vector carrying a nucleic acid sequence encoding a 3' trans-splicing molecule as described herein under the control of a regulatory sequence, and a pharmaceutically acceptable carrier that expresses the trans-splicing molecule in the neuronal cells of a mammalian subject. The carrier is an isotonic sodium chloride solution and includes the surfactant Pluronic F68. In one embodiment, the trans-splicing molecule is any of those described herein.

[0235] In another exemplary embodiment, the composition comprises an rAAV virus containing any MeCP2 trans-splicing molecule described herein for MeCP2 gene correction, a nucleic acid sequence under the control of a promoter that directs the expression of the trans-splicing molecule in neurons, neural stem cells, astrocytes, or oligodendrocytes, or any combination thereof (e.g., neurons in the brainstem), wherein the composition is formulated together with a carrier and additional components suitable for intracerebral delivery (e.g., by slow delivery or convection-enhanced infusion) or intraventricular delivery. In another embodiment, the composition or components for producing or assembling such a composition (including a carrier, rAAV particles, surfactants, and / or components for producing rAAV) and laboratory hardware suitable for preparing the composition may be incorporated into a kit. Such a kit may also include instructions for administering the composition to an individual, for example, as a treatment for Rett syndrome.

[0236] This document also provides a kit containing a pharmaceutical composition comprising a 3' trans-splicing molecule (e.g., wherein the trans-splicing molecule is packaged in any AAV carrier described herein). In some embodiments, the kit includes instructions on mixing the pharmaceutical composition prior to administration.

[0237] F. Methods and Applications The aforementioned nucleic acid transsplicing molecules (e.g., nucleic acid transsplicing molecules and vectors encoding nucleic acid transsplicing molecules) and compositions can be used to express functional MeCP2 and / or regulate MeCP2 expression in target cells of an individual (e.g., neurons, neural stem cells, astrocytes, or oligodendrocytes, or any combination thereof), for example, in methods for treating diseases or conditions associated with mutations in the MeCP2 gene (such as Rett syndrome), including delaying or improving symptoms associated with Rett syndrome.

[0238] In some implementations, symptoms of Rett syndrome include, but are not limited to, developmental delay, cognitive problems, slow brain and head growth, abnormal hand movements, hyperventilation, language disorders, motor and coordination disorders (e.g., difficulty walking, tiptoeing, or wide-base gait), repetitive movements, uncontrollable crying or screaming, seizures, and impaired social interaction.

[0239] The aforementioned nucleic acid trans-splicing molecules (e.g., nucleic acid trans-splicing molecules and vectors encoding nucleic acid trans-splicing molecules) and compositions may also be used to express functional MeCP2 and / or regulate MeCP2 expression in target cells of an individual (e.g., neurons, neural stem cells, astrocytes, or oligodendrocytes, or any combination thereof), for example, as applied to their use in treating diseases or conditions associated with mutations in the MeCP2 gene (such as Rett syndrome), including delaying or improving symptoms associated with Rett syndrome as described herein, or their use in the preparation of medicaments for treating diseases or conditions associated with mutations in the MeCP2 gene (such as Rett syndrome), including delaying or improving symptoms associated with Rett syndrome as described herein. Such methods and uses involve contacting a target MeCP2 molecule (e.g., a MeCP2 precursor mRNA) with a trans-splicing molecule as described herein [e.g., a 3' trans-splicing molecule as described herein, a composition containing said molecule (e.g., a pharmaceutical composition), or a drug containing said molecule] under conditions where the coding domain of a trans-splicing molecule is spliced ​​to the target MeCP2 precursor mRNA, to replace a portion of the target precursor mRNA carrying one or more defects or mutations with the biologically functional (i.e., healthy) or normal or wild-type or corrected mRNA of the target gene, thereby correcting MeCP2 expression in target cells. Therefore, the methods and compositions are intended for treating Rett syndrome lesions and symptoms associated with specific mutations.

[0240] In some embodiments, this document provides methods for expressing functional MeCP2 in target cells by contacting (e.g., transducing) the target cells with any of the nucleic acid transsplicing molecules, vectors (e.g., AAV vectors) or compositions described herein. In one embodiment, contact involves directly administering a composition (e.g., a pharmaceutical composition) to a diseased individual. In another embodiment, contact can be performed ex vivo, wherein cultured cells (e.g., neuronal cells or precursors thereof) and treated neuronal cells are re-implanted into the individual. In another embodiment, the method involves administering rAAV containing any of the 3' MeCP2 transsplicing molecules described herein. These methods include administering an effective concentration of any of the compositions described herein to the individual in need.

[0241] In some embodiments, the method includes selecting one or more trans-splicing molecules for use in treating an individual with a condition associated with a mutation in MeCP2. In some embodiments, this document covers the use of one or more trans-splicing molecules for treating an individual with a condition associated with a mutation in MeCP2, or their use in the preparation of a medicament for treating an individual with a condition associated with a mutation in MeCP2. Such methods and uses include selecting one or more trans-splicing molecules for treating an individual with a condition associated with a mutation in MeCP2, or the use of one or more trans-splicing molecules selected in this way in treating an individual with a condition associated with a mutation in MeCP2, or the use of such selected one or more trans-splicing molecules in the preparation of a medicament for treating an individual with a condition associated with a mutation in MeCP2. Such selection may be based on the individual's genotype. In some embodiments, the condition associated with MeCP2 may be an X-linked condition. Methods for screening and identifying specific mutations in MeCP2 are known in the art.

[0242] The method disclosed herein includes selecting a single trans-splicing molecule based on the location of a single mutation in MeCP2 (e.g., a mutation in one allele of an individual). As described herein, pathogenic mutations associated with Rett syndrome include those listed in Table 1, and most mutations identified to date are located in exon 3 or 4 of MeCP2. Therefore, in some embodiments, the method disclosed herein includes applying a single trans-splicing molecule to correct at least one pathological mutation in exon 3 or exon 4 of the MeCP2 gene, e.g., regardless of the location of any other mutations that may be present in other alleles.

[0243] The nucleic acid transsplicing molecules described herein, and vectors, proviral plasmids, and AAVs containing said molecules, as well as compositions containing such nucleic acid transsplicing molecules and vectors, proviral plasmids, and AAVs, can be used for medical treatment, particularly for the treatment of Rett syndrome. In some embodiments, when using, for example, an AAV vector (or other gene therapy vector), the AAV vector can be administered systemically to deliver to the whole brain. In some embodiments, when using, for example, an AAV vector (or other gene therapy vector), the AAV vector can be administered systemically to deliver to the whole brain and enteric nervous system. In some embodiments, when using, for example, an AAV vector (or other gene therapy vector), the AAV vector can be administered by direct infusion into the brain. In some embodiments, direct infusion includes intrathecal infusion of the AAV vector into the cerebrospinal fluid. Intrathecal infusion provides an efficient mode of delivery to the CNS, where neurons can be targeted, for example. In some embodiments, the injectable can be delivered to the brainstem, midbrain, or cortex, or any combination thereof, and / or vicinity via convection-enhanced diffusion (CED), thereby targeting at least one of the brainstem, midbrain, or cortex, or any combination thereof. In some embodiments, the injection may target the brainstem, midbrain, or cortex, or any combination thereof, to provide greater coverage of brain structures associated with Rett syndrome. Such injections can be performed using magnetic resonance imaging-guided injection. This type of treatment is particularly useful for human subjects with Rett syndrome. Such treatment involves human subjects with Rett syndrome, including those with a genetic predisposition to develop Rett syndrome but who do not exhibit symptoms of Rett syndrome. Therefore, in some embodiments, treatment of human subjects with Rett syndrome may include treatment of any subject carrying a pathological mutation associated with Rett syndrome.

[0244] In some implementations, the effective concentration of recombinant adeno-associated virus carrying the trans-splicing molecule as described herein is between approximately 10 ppm. 8 One and 10 13 The range of individual vector genomes (vg / mL). rAAV infection units, such as those of McLaughlin et al., J. Virol The measurement is performed as described in 1988, 62: 1963. In another embodiment, the concentration is between 10... 9 With 10 13 The effective concentration is within the range of vg / mL. In another embodiment, the effective concentration is approximately 1.5 x 10⁻⁶. 11 vg / mL. In another embodiment, the effective concentration is approximately 5 x 10⁻⁶ vg / mL. 11 vg / mL. In one implementation, the effective concentration is approximately 1.5 x 10⁻⁶ vg / mL. 10 vg / mL. In another embodiment, the effective concentration is approximately 2.8 x 10⁻⁶. 11vg / mL. In another embodiment, the effective concentration is approximately 1.5 x 10⁻⁶ vg / mL. 12 vg / mL. In another embodiment, the effective concentration is approximately 1.5 x 10⁻⁶ vg / mL. 13 vg / mL.

[0245] The aim is to use the lowest effective dose (total genome copies delivered) of virus to reduce the risk of adverse effects, such as toxicity and other problems associated with administration to the brain. The effective dose of recombinant adeno-associated virus carrying the trans-splicing molecules described herein is approximately 10 [units of measurement missing] per dose (i.e., per injection). 8 One and 10 13 Within the range of vector genomes (vg). In one embodiment, the dose is between 10 9 With 10 13 The effective dose is within the range of vg. In another embodiment, the effective dose is approximately 1.5 x 10⁻⁶. 11 vg. In another embodiment, the effective dose is approximately 5 x 10 11 vg. In one implementation, the effective dose is approximately 1.5 x 10 10 vg. In another embodiment, the effective dose is approximately 2.8 x 10 11 vg. In another embodiment, the effective dose is approximately 1.5 × 10⁻⁶. 12 vg. In another embodiment, the effective concentration is approximately 1.5 x 10⁻⁶. 13 vg. The attending physician may also choose other doses within these ranges or other units, taking into account the individual's physical condition, including the individual's age; the composition applied and the specific condition; the targeted cells and the degree of development of the condition (if progressive).

[0246] In some embodiments, the composition can be delivered in volumes of about 50 μL to about 1 mL, including all figures within that range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 70 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 250 μL. In another embodiment, the volume is about 300 μL. In another embodiment, the volume is about 350 μL. In another embodiment, the volume is about 400 μL. In another embodiment, the volume is about 450 μL. In another embodiment, the volume is about 500 μL. In another embodiment, the volume is about 600 μL. In another embodiment, the volume is approximately 750 μL. In another embodiment, the volume is approximately 850 μL. In yet another embodiment, the volume is approximately 1,000 μL.

[0247] In some embodiments, the treatments and uses described herein replace 10% or more of the target MeCP2 mRNA in the target cells (e.g., 11% or more of the target MeCP2 mRNA in the target cells, 12% or more of the target MeCP2 mRNA in the target cells, 13% or more of the target MeCP2 mRNA in the target cells, 14% or more of the target MeCP2 mRNA in the target cells, 15% or more of the target MeCP2 mRNA in the target cells, 16% or more of the target MeCP2 mRNA in the target cells, 17% or more of the target MeCP2 mRNA in the target cells, 18% or more of the target MeCP2 mRNA in the target cells, 19% or more of the target MeCP2 mRNA in the target cells). In some embodiments, the treatments and uses described herein replace 20% or more of the target MeCP2 mRNA in the target cells (e.g.,21% or more of the target MECP2 mRNA in target cells; 22% or more of the target MECP2 mRNA in target cells; 23% or more of the target MECP2 mRNA in target cells; 24% or more of the target MECP2 mRNA in target cells; 25% or more of the target MECP2 mRNA in target cells; 26% or more of the target MECP2 mRNA in target cells; 27% or more of the target MECP2 mRNA in target cells; 28% or more of the target MECP2 mRNA in target cells; 29% or more of the target MECP2 mRNA in target cells; 30% or more of the target MECP2 mRNA in target cells; 31% or more of the target MECP2 mRNA in target cells; 32% or more of the target MECP2 mRNA in target cells; 33% or more of the target MECP2 mRNA in target cells; 34% or more of the target MECP2 mRNA in target cells; 35% or more of the target MECP2 mRNA in target cells. The target MECP2 mRNA concentrations are: 36% or more of the target MECP2 mRNA in the target cells; 37% or more of the target MECP2 mRNA in the target cells; 38% or more of the target MECP2 mRNA in the target cells; 39% or more of the target MECP2 mRNA in the target cells; 40% or more of the target MECP2 mRNA in the target cells; 41% or more of the target MECP2 mRNA in the target cells; 42% or more of the target MECP2 mRNA in the target cells; 43% or more of the target MECP2 mRNA in the target cells; 44% or more of the target MECP2 mRNA in the target cells; 45% or more of the target MECP2 mRNA in the target cells; 46% or more of the target MECP2 mRNA in the target cells; 47% or more of the target MECP2 mRNA in the target cells; 48% or more of the target MECP2 mRNA in the target cells; 49% or more of the target MECP2 mRNA in the target cells; or 50% or more of the target MECP2 mRNA in the target cells. In some implementations, the treatments and uses described herein replace 50% or more of the target MECP2 mRNA in the target cells (e.g.,51% or more of the target MECP2 mRNA in target cells; 52% or more of the target MECP2 mRNA in target cells; 53% or more of the target MECP2 mRNA in target cells; 54% or more of the target MECP2 mRNA in target cells; 55% or more of the target MECP2 mRNA in target cells; 56% or more of the target MECP2 mRNA in target cells; 57% or more of the target MECP2 mRNA in target cells; 58% or more of the target MECP2 mRNA in target cells; 59% or more of the target MECP2 mRNA in target cells; 60% or more of the target MECP2 mRNA in target cells; 61% or more of the target MECP2 mRNA in target cells; 62% or more of the target MECP2 mRNA in target cells; 63% or more of the target MECP2 mRNA in target cells; 64% or more of the target MECP2 mRNA in target cells; 65% or more of the target MECP2 mRNA in target cells. mRNA, 66% or more of the target MECP2 mRNA in target cells, 67% or more of the target MECP2 mRNA in target cells, 68% or more of the target MECP2 mRNA in target cells, 69% or more of the target MECP2 mRNA in target cells, 70% or more of the target MECP2 mRNA in target cells, 71% or more of the target MECP2 mRNA in target cells, 72% or more of the target MECP2 mRNA in target cells, 73% or more of the target MECP2 mRNA in target cells, 74% or more of the target MECP2 mRNA in target cells, 75% or more of the target MECP2 mRNA in target cells, 76% or more of the target MECP2 mRNA in target cells, 77% or more of the target MECP2 mRNA in target cells, 78% or more of the target MECP2 mRNA in target cells, 79% or more of the target MECP2 mRNA in target cells, or 80% or more of the target MECP2 mRNA in target cells (e.g.,81% or more of the target MECP2 mRNA in target cells; 82% or more of the target MECP2 mRNA in target cells; 83% or more of the target MECP2 mRNA in target cells; 84% or more of the target MECP2 mRNA in target cells; 85% or more of the target MECP2 mRNA in target cells; 86% or more of the target MECP2 mRNA in target cells; 87% or more of the target MECP2 mRNA in target cells; 88% or more of the target MECP2 mRNA in target cells; 89% or more of the target MECP2 mRNA in target cells; 90% or more of the target MECP2 mRNA in target cells; 91% or more of the target MECP2 mRNA in target cells; 92% or more of the target MECP2 mRNA in target cells; 93% or more of the target MECP2 mRNA in target cells; 94% or more of the target MECP2 mRNA in target cells. The target MECP2 mRNA concentrations are: 95% or more of the target MECP2 mRNA in the target cells; 96% or more of the target MECP2 mRNA in the target cells; 97% or more of the target MECP2 mRNA in the target cells; 98% or more of the target MECP2 mRNA in the target cells; 99% or more of the target MECP2 mRNA in the target cells; or 100% or more of the target MECP2 mRNA in the target cells.

[0248] For each described method and use, the treatment or use may be used to prevent further damage or to save tissue with mild, moderate, or advanced disease. As used herein, the term “save” means to stop disease progression, prevent damage from spreading to undamaged cells, and / or improve damage to damaged cells.

[0249] Therefore, in one embodiment, the composition is administered before the onset of disease. In another embodiment, the composition is administered before the appearance of symptoms. In another embodiment, the composition is administered after the appearance of symptoms. In another embodiment, the composition is administered when, for example, less than 90% of the target cells are functional or remainder compared to a reference tissue. In another embodiment, the composition is administered when, for example, more than 10% of the target cells are functional or remainder compared to a reference tissue. In another embodiment, the composition is administered when more than 20% of the target cells are functional or remainder. In another embodiment, the composition is administered when more than 30% of the target cells are functional or remainder. In another embodiment, the composition is administered when more than 40% of the target cells are functional or remainder. In another embodiment, the composition is administered when more than 50% of the target cells are functional or remainder. In another embodiment, the composition is administered when more than 60% of the target cells are functional or remainder. In another embodiment, the composition is administered when more than 70% of the target cells are functional or remainder. In another embodiment, the composition is applied when more than 80% of the target cells are functional or remain. In another embodiment, the composition is applied when more than 90% of the target cells are functional or remain. In another embodiment, the composition is applied when more than 95% of the target cells are functional or remain.

[0250] In yet another embodiment, any of the above methods or uses are performed in combination with another therapy or secondary therapy. The therapy can be any now-known or not-yet-known therapy that helps prevent, halt, or improve these mutations or defects or any associated effects. The secondary therapy can be administered before, simultaneously with, or after the administration of the above-described pharmaceutical composition. In one embodiment, the secondary therapy involves a non-specific method for maintaining neuronal cell health, such as the administration of neurotrophic factors, antioxidants, and / or anti-apoptotic agents. This non-specific method is achieved by injecting proteins, recombinant DNA, recombinant viral vectors, stem cells, fetal tissue, or genetically modified cells. The latter may include encapsulated genetically modified cells.

[0251] For use in these methods, the volume and viral titer of each injection are determined individually and may be the same as or different from other injections performed, for example, in the brain. The dosage, administration, and protocol may be determined by the attending physician in accordance with the teachings of this disclosure.

[0252] In some implementations, the methods disclosed herein do not involve altering the reproductive genetic characteristics of any person or other animal.

[0253] The following embodiments do not limit the scope of the implementations described herein. Those skilled in the art will recognize that modifications can be made to the following embodiments without departing from the spirit or scope of the invention.

[0254] G. Specific Implementation Plan This disclosure is illustrated by the following specific examples of implementation schemes: 1. An exon editor construct encoding an RNA exon editor, wherein the exon editor construct comprises encoding the following sequence: (a) Binding domain, which binds to the target intron of MeCP2 precursor mRNA; (b) Hemiintrons; and (c) Encoding structure field, which contains one or more MeCP2 exons.

[0255] 2. The exon editor construct as described in embodiment 1, wherein the target intron of the MeCP2 precursor mRNA is intron 1 or intron 2.

[0256] 3. The exon editor construct as described in embodiment 1 or embodiment 2, wherein the binding domain binds to a binding site comprising: Nucleotides of intron 1 (SEQ ID NO: 1) -100 to 1500 or -50 to 1449; Nucleotides of intron 1 are 3950 to 4250 or 4020-4169; or Nucleotides 1-300 of intron 2 (SEQ ID NO: 2 and 3).

[0257] 4. The exon editor construct as described in any of the foregoing embodiments, wherein the binding domain binds to a binding site comprising: Nucleotides of intron 1 (SEQ ID NO: 1) -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000; or Nucleotides of intron 2 (SEQ ID NO: 2) are 50-300, 50-250, 75-300, 75-250, 80-300, 80-250, 90-300, 90-250, 95-300, 95-250 or 99-248.

[0258] 5. The exon editor construct as described in any of the preceding embodiments, wherein the size of the binding domain ranges from about 50-300 nucleotides; about 50-250 nucleotides; about 50-150 nucleotides; about 50-100 nucleotides; about 75-300 nucleotides; about 75-250 nucleotides; about 75-200 nucleotides; about 75-150 nucleotides; about 100-300 nucleotides; about 100-250 nucleotides; about 100-200 nucleotides; about 100-150 nucleotides; about 125-300 nucleotides; about 125-250 nucleotides; about 125-200 nucleotides; about 125-150 nucleotides; or about 150 nucleotides.

[0259] 6. The exon editor construct as described in any of the preceding embodiments, wherein the size of the binding domain ranges from 50-300 nucleotides; 50-250 nucleotides; 50-150 nucleotides; 50-100 nucleotides; 75-300 nucleotides; 75-250 nucleotides; 75-200 nucleotides; 75-150 nucleotides; 100-300 nucleotides; 100-250 nucleotides; 100-200 nucleotides; 100-150 nucleotides; 125-300 nucleotides; 125-250 nucleotides; 125-200 nucleotides; 125-150 nucleotides; or 150 nucleotides.

[0260] 7. The exon editor construct as described in any of the preceding embodiments, wherein the size of the binding domain is in the range of about 100-200 nucleotides; about 100-150 nucleotides; about 125-200 nucleotides; about 125-150 nucleotides; or about 150 nucleotides; or in the range of 100-200 nucleotides; 100-150 nucleotides; 125-200 nucleotides; 125-150 nucleotides; or 150 nucleotides.

[0261] 8. The exon editor construct as described in any of the preceding embodiments, wherein the binding domain is at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the binding site to which it is bound.

[0262] 9. An exon editor construct as described in any of the preceding embodiments, wherein the binding domain comprises a continuous nucleotide chain 100% complementary to a portion of the binding site to which it binds, wherein the length of the continuous nucleotide chain is at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, at least 70 nucleotides, at least 75 nucleotides, at least 80 nucleotides, at least 85 nucleotides, at least 90 nucleotides, at least 95 nucleotides, at least 100 nucleotides, at least 105 nucleotides, at least 110 nucleotides, at least 115 nucleotides, at least 120 nucleotides, at least 130 nucleotides, at least 135 nucleotides, at least 140 nucleotides, at least 145 nucleotides, or at least 150 nucleotides.

[0263] 10. The exon editor construct as described in any of the preceding embodiments, wherein the sequence encoding the binding domain comprises, substantially comprises, or comprises any one of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 47.

[0264] 11. An exon editor construct as described in any of the preceding embodiments, wherein the sequence encoding the binding domain comprises, substantially comprises, or comprises SEQ ID NO: 55.

[0265] 12. An exon editor construct as described in any of the foregoing embodiments, wherein the sequence encoding the binding domain comprises, substantially comprises, or comprises any one of SEQ ID NO: 24, 28, 29, or 30.

[0266] 13. The exon editor construct as described in any of the foregoing embodiments, wherein the coding structure domain comprises, substantially comprises, or comprises the following: SEQ ID NO: 7 and 8 or sequences having 90% identity with SEQ ID NO: 7 and 8; SEQ ID NO: 76 or a sequence having 90% identity with SEQ ID NO: 76; SEQ ID NO: 12; SEQ ID NO: 14; or SEQ ID NO: 9.

[0267] 14. An exon editor construct as described in any of the foregoing embodiments, wherein the coding structure domain comprises, is substantially composed of, or is composed of any one of SEQ ID NO: 76 or 14.

[0268] 15. An exon editor construct as described in any of the preceding embodiments, wherein the binding domain, the half-intron, and the coding domain are operatively connected in the 5' to 3' direction.

[0269] 16. An exon editor construct as described in any of the preceding embodiments, wherein the half-intron comprises a 3' splice site and optional branch point sequences and a polypyrimidine bundle, wherein the binding domain, the half-intron comprising the 3' splice site, and the coding domain are operatively connected in the 5' to 3' direction.

[0270] 17. The exon editor construct as described in embodiment 16, wherein the half-intron comprises, substantially comprises, or comprises a polypyrimidine bundle, a branch point, and a 3' splice site.

[0271] 18. An exon editor construct as described in any one of embodiments 16 or 17, wherein the semi-introns comprise, substantially consist of, or consist of: SEQ ID NO: 17 or a sequence having at least 90% identity with SEQ ID NO: 17.

[0272] 19. The exon editor construct as described in any of the preceding embodiments, further comprising a 3' untranslated region (3' UTR), wherein the binding domain, the half-intron, the coding domain and the 3' UTR are operatively connected in the 5' to 3' direction.

[0273] 20. The exon editor construct as described in embodiment 19, wherein the 3' UTR comprises RDH1pA 3'UTR (SEQ ID NO: 19) or mWPRE 3' UTR (SEQ ID NO: 20).

[0274] 21. The exon editor builder as described in implementation scheme 20, wherein The RDH1pA 3' UTR comprises, substantially comprises, or comprises: SEQ ID NO: 19 or a sequence having at least 90% identity with SEQ ID NO: 19; or The mWPRE 3' UTR comprises, is substantially composed of, or is composed of: SEQ ID NO: 20 or a sequence having at least 90% identity with SEQ ID NO: 20.

[0275] 22. The exon editor builder as described in any of the foregoing embodiments, wherein the exon editor builder comprises: Combined with the structural domain, it comprises, is substantially composed of, or is composed of: SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 47 or 55; A hemiintron, comprising, substantially consisting of, or consisting of: SEQ ID NO: 17; The encoding structure field contains, is substantially composed of, or is composed of: SEQ ID NO: 76; And 3' UTR, which comprises, substantially comprises, or comprises SEQ ID NO: 20, wherein the binding domain, the half-intron, the coding domain and the 3' UTR are operatively connected in the 5' to 3' direction.

[0276] 23. The exon editor construct as described in embodiment 22, wherein the exon editor construct comprises, is substantially composed of, or is composed of: SEQ ID NO: 78, 80, 81, 82, 83 or 84.

[0277] 24. The exon editor builder as described in embodiment 22, wherein the exon editor builder comprises, is substantially composed of, or is composed of: SEQ ID NO: 78 or 80.

[0278] 25. An exon editor construct as described in any one of embodiments 1-19, further comprising a triple helix terminator, wherein the binding domain, the half-intron, the coding domain, the 3' UTR (if present), and the triple helix terminator are operatively connected in the 5' to 3' direction.

[0279] 26. The exon editor construct as described in embodiment 25, wherein the triple helix terminator comprises, substantially comprises, or comprises SEQ ID NO: 77 or a sequence having at least 90% identity with SEQ ID NO: 77.

[0280] 27. The exon editor construct as described in embodiment 25, wherein the triple helix terminator comprises, substantially comprises, or comprises SEQ ID NO: 22.

[0281] 28. The exon editor construct as described in any of the preceding embodiments further comprises a sequence encoding a poly A sequence, for example, SEQ ID NO: 21.

[0282] 29. An exon editor construct as described in any of the preceding embodiments, further comprising a sequence of encoded epitope tags, wherein the binding domain, the half-intron, the encoded domain, the epitope tags, the 3'UTR (if present), and the triple-helix terminator (if present) are operatively connected in the 5' to 3' direction.

[0283] 30. The exon editor builder as described in embodiment 28, wherein the exon editor builder comprises, is substantially composed of, or is composed of: SEQ ID NO: 78.

[0284] 31. An RNA exon editor, which is transcribed from an exon editor construct according to any one of embodiments 1-30.

[0285] 32. An exon editor construct as described in any one of embodiments 1-30, wherein the MeCP2 precursor mRNA contains at least one mutation associated with Rett syndrome.

[0286] 33. An exon editor construct as described in any one of embodiments 1-30, wherein the at least one mutation associated with Rett syndrome comprises at least one mutation in exon 3 of the MeCP2 gene allele or at least one mutation in exon 4 of the MeCP2 gene allele, or any combination thereof.

[0287] 34. An exon editor construct as described in any one of embodiments 32-33, wherein at least one mutation associated with Rett syndrome is X-linked.

[0288] 35. An exon editor construct as described in any one of embodiments 32-34, comprising the expression of at least one of the mutated MeCP2 protein associated with Rett syndrome in at least one or any combination of neural stem cells, neurons, astrocytes or oligodendrocytes.

[0289] 36. A carrier comprising an exon editor construct according to any one of embodiments 1-30 or 32-35.

[0290] 37. The carrier as described in embodiment 36, wherein the carrier includes a 5' control domain operatively connected to the 5' of the binding domain.

[0291] 38. The vector of any one of embodiments 36-37, wherein the 5' regulatory domain comprises a constitutive promoter or a tissue-specific promoter.

[0292] 39. The carrier as described in embodiment 38, wherein the constitutive promoter is a CMV promoter.

[0293] 40. A proviral plasmid comprising an exon editor construct according to any one of embodiments 1-30 or 32-35.

[0294] 41. An adeno-associated virus (AAV) comprising an exon editor construct according to any one of embodiments 1-30 or 32-35, wherein the AAV optionally comprises a 5' regulatory domain operatively linked to the 5' of the exon editor construct.

[0295] 42. The AAV as described in embodiment 41, wherein the AAV includes a 5' control domain operatively connected to the 5' of the binding domain.

[0296] 43. The AAV as described in any one of embodiments 41-42, wherein the 5' regulatory domain comprises a constitutive promoter.

[0297] 44. The AAV as described in embodiment 43, wherein the constitutive promoter is a CMV promoter.

[0298] 45. The AAV as described in any one of embodiments 41-44, wherein the AAV exhibits neuronal tropism.

[0299] 46. ​​The AAV as described in any one of embodiments 41-45, wherein the AAV is AAV9, AAV8, AAV5 or AAV2, or a variant of AAV9, AAV8, AAV5 or AAV2.

[0300] 47. A composition comprising an exon editor construct according to any one of embodiments 1-30 or 32-35, a vector according to any one of embodiments 36-39, a proviral plasmid according to embodiment 40, or an AAV according to any one of embodiments 41-46.

[0301] 48. The composition as described in embodiment 47, comprising a pharmaceutically acceptable excipient.

[0302] 49. A method for expressing bioactive MeCP2 in target cells to restore the functional level of the MeCP2 protein in the target cells, the method comprising transducing the target cells with an exon editor construct of any one of embodiments 1-30 or 32-35, a vector of any one of embodiments 36-39, a proviral plasmid of embodiment 40, an AAV of any one of embodiments 41-46, or a composition of any one of embodiments 47-48.

[0303] 50. The method of embodiment 49, wherein at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of the MeCP2 precursor mRNA containing at least one mutation associated with Rett syndrome is modified to encode a transcript of functional MeCP2, wherein the at least one mutation is corrected.

[0304] 51. The method of embodiment 50, wherein at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of the MeCP2 precursor mRNA containing at least one mutation associated with Rett syndrome is modified to a transcript encoding a functional MeCP2, wherein the at least one mutation is corrected.

[0305] 52. The method of embodiment 51, wherein at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the MeCP2 precursor mRNA containing at least one mutation associated with Rett syndrome is modified to a transcript encoding a functional MeCP2, wherein the at least one mutation is corrected.

[0306] 53. The method of any one of embodiments 49-52, wherein the functional level of MeCP2 in the target cells is restored by expressing a biologically functional MeCP2 protein.

[0307] 54. A method for reducing the expression of MeCP2 containing at least one mutation associated with Rett syndrome in a subject, the method comprising transfecting or transducing target cells, more specifically neurons, in the subject with an exon editor construct of any one of embodiments 1-30 or 32-35, a vector of any one of embodiments 36-39, a proviral plasmid of embodiment 40, an AAV of any one of embodiments 41-46, or a composition of any one of embodiments 47-48.

[0308] 55. A method for correcting at least one mutation in the MeCP2 exon sequence in the MeCP2 precursor mRNA of a subject, the method comprising administering to the subject an exon editor construct of any one of embodiments 1-30 or 32-35, a vector of any one of embodiments 36-39, a proviral plasmid of embodiment 40, an AAV of any one of embodiments 41-46, or a composition of any one of embodiments 47-48.

[0309] 56. A method for treating Rett syndrome in a subject in need, the method comprising administering to the subject a therapeutically effective amount of an exon editor construct of any one of embodiments 1-30 or 32-35, a vector of any one of embodiments 36-39, a proviral plasmid of embodiment 40, an AAV of any one of embodiments 41-46, or a composition of any one of embodiments 47-48.

[0310] 57. The method of any one of embodiments 54-56, wherein at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of the MeCP2 precursor mRNA containing at least one mutation associated with Rett syndrome is modified to encode a transcript of functional MeCP2, wherein the at least one mutation is corrected.

[0311] 58. The method of embodiment 57, wherein at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of the MeCP2 precursor mRNA containing at least one mutation associated with Rett syndrome is modified to a transcript encoding a functional MeCP2, wherein the at least one mutation is corrected.

[0312] 59. The method of embodiment 58, wherein at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the MeCP2 precursor mRNA containing at least one mutation associated with Rett syndrome is modified to a transcript encoding a functional MeCP2, wherein the at least one mutation is corrected.

[0313] 60. The method of any one of embodiments 54-59, wherein the functional level of MeCP2 in the target cells is restored by expressing a biologically functional MeCP2 protein.

[0314] 61. The method of any one of embodiments 54-60, the method comprising administering to the brain of the subject an exon editor construct as described in any one of embodiments 1-30 or 32-35, a vector as described in any one of embodiments 36-39, a proviral plasmid as described in embodiment 40, an AAV as described in any one of embodiments 41-46, or a composition as described in any one of embodiments 47-48.

[0315] 62. The method of any one of embodiments 54-61, wherein the subject is a mammal, preferably a rodent, a non-human primate, or a human.

[0316] 63. The method of any one of embodiments 54-62, wherein the subject is genetically predisposed to Rett syndrome or has been diagnosed with Rett syndrome.

[0317] 64. An exon editor construct as described in any one of embodiments 1-30 or 32-35, a vector as described in any one of embodiments 36-39, a proviral plasmid as described in embodiment 40, an AAV as described in any one of embodiments 41-46, or a composition as described in any one of embodiments 47-48, for the prevention or treatment of Rett syndrome in a subject of need.

[0318] 65. An exon editor construct as described in any one of embodiments 1-30 or 32-35, a vector as described in any one of embodiments 36-39, a proviral plasmid as described in embodiment 40, an AAV as described in any one of embodiments 41-46, or a composition as described in any one of embodiments 47-48, for the preparation of a medicament for the treatment or prevention of Rett syndrome in a subject of need.

[0319] 66. An RNA exon editor comprising: operably linked in the 5' to 3' direction: (a) A binding domain configured to bind to a binding site within the target region of the MeCP2 precursor mRNA; (b) Hemiintrons; and (c) Encoding structure domain, which contains a sequence encoding one or more MeCP2 exons.

[0320] 67. An RNA exon editor as described in embodiment 66, wherein the target region comprises or consists of nucleotides of MeCP2 intron 1 - 100 to 1500 (positions 15 to 1615 of SEQ ID NO: 111).

[0321] 68. An RNA exon editor as described in embodiment 66, wherein the target region comprises or consists of nucleotides of MeCP2 intron 1 - 100 to 100 (positions 15 to 215 of SEQ ID NO: 111).

[0322] 69. An RNA exon editor as described in embodiment 66, wherein the target region comprises or consists of nucleotides 50 to 1449 of MeCP2 intron 1 (positions 65 to 1564 of SEQ ID NO: 111).

[0323] 70. An RNA exon editor as described in embodiment 66, wherein the target region comprises or consists of nucleotides 50 to 99 of MeCP2 intron 1 (positions 65 to 214 of SEQ ID NO: 111).

[0324] 71. An RNA exon editor as described in embodiment 66, wherein the target region comprises or consists of nucleotides 51 to 649 of MeCP2 intron 1 (positions 51 to 649 of SEQ ID NO: 1).

[0325] 72. An RNA exon editor as described in embodiment 66, wherein the target region comprises or consists of nucleotides 51 to 524 of MeCP2 intron 1 (positions 51 to 524 of SEQ ID NO: 1).

[0326] 73. An RNA exon editor as described in embodiment 66, wherein the target region comprises or consists of nucleotides 51 to 1449 of MeCP2 intron 1 (positions 51 to 1449 of SEQ ID NO: 1).

[0327] 74. An RNA exon editor as described in embodiment 66, wherein the target region comprises or consists of nucleotides 125 to 524 of MeCP2 intron 1 (positions 125 to 524 of SEQ ID NO: 1).

[0328] 75. An RNA exon editor as described in embodiment 66, wherein the target region comprises or consists of nucleotides 125 to 649 of MeCP2 intron 1 (positions 125 to 649 of SEQ ID NO: 1).

[0329] 76. The RNA exon editor as described in embodiment 66, wherein the target region comprises or consists of nucleotides 820 to 1449 of MeCP2 intron 1 (positions 820 to 1449 of SEQ ID NO: 1).

[0330] 77. An RNA exon editor as described in embodiment 66, wherein the target region comprises or consists of nucleotides 3950 to 4250 of MeCP2 intron 1 (positions 3950 to 4250 of SEQ ID NO: 1).

[0331] 78. An RNA exon editor as described in embodiment 66, wherein the target region comprises or consists of nucleotides 4020 to 4169 of MeCP2 intron 1 (positions 4020 to 4169 of SEQ ID NO: 1).

[0332] 79. An RNA exon editor as described in embodiment 66, wherein the target region comprises or consists of nucleotides 1 to 300 of MeCP2 intron 2 (positions 1 to 300 of SEQ ID NO: 2).

[0333] 80. An RNA exon editor as described in embodiment 66, wherein the target region comprises or consists of nucleotides 99 to 248 of MeCP2 intron 2 (positions 99 to 248 of SEQ ID NO: 2).

[0334] 81. An RNA exon editor as described in any one of embodiments 66 to 80, wherein the binding site comprises or consists of at least 25 consecutive nucleotides of the target region.

[0335] 82. An RNA exon editor as described in any one of embodiments 66 to 80, wherein the binding site comprises or consists of at least 50 consecutive nucleotides of the target region.

[0336] 83. An RNA exon editor as described in any one of embodiments 66 to 80, wherein the binding site comprises or consists of 25 to 100 consecutive nucleotides of the target region.

[0337] 84. An RNA exon editor as described in any one of embodiments 66 to 80, wherein the binding site comprises or consists of 50 to 100 consecutive nucleotides of the target region.

[0338] 85. An RNA exon editor as described in any one of embodiments 66 to 80, wherein the binding site comprises or consists of 50 to 150 consecutive nucleotides of the target region.

[0339] 86. An RNA exon editor as described in any one of embodiments 66 to 80, wherein the binding site comprises or consists of 100 to 200 consecutive nucleotides of the target region.

[0340] 87. An RNA exon editor as described in any one of embodiments 66 to 80, wherein the binding site comprises or consists of 100 to 150 consecutive nucleotides of the target region.

[0341] 88. An RNA exon editor as described in any one of embodiments 66 to 80, wherein the binding site comprises or consists of 150 consecutive nucleotides of the target region.

[0342] 89. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 80% complementary to the entire binding site.

[0343] 90. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 90% complementary to the entire binding site.

[0344] 91. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is 100% complementary to the entire binding site.

[0345] 92. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 80% complementary to 25 consecutive nucleotides within the binding site.

[0346] 93. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 90% complementary to 25 consecutive nucleotides within the binding site.

[0347] 94. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is 100% complementary to 25 consecutive nucleotides within the binding site.

[0348] 95. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 80% complementary to 50 consecutive nucleotides within the binding site.

[0349] 96. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 90% complementary to 50 consecutive nucleotides within the binding site.

[0350] 97. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is 100% complementary to 50 consecutive nucleotides within the binding site.

[0351] 98. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 80% complementary to 75 consecutive nucleotides within the binding site.

[0352] 99. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 90% complementary to 75 consecutive nucleotides within the binding site.

[0353] 100. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is 100% complementary to 75 consecutive nucleotides within the binding site.

[0354] 101. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 80% complementary to 100 consecutive nucleotides within the binding site.

[0355] 102. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 90% complementary to 100 consecutive nucleotides within the binding site.

[0356] 103. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is 100% complementary to 100 consecutive nucleotides within the binding site.

[0357] 104. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 80% complementary to 150 consecutive nucleotides within the binding site.

[0358] 105. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 90% complementary to 150 consecutive nucleotides within the binding site.

[0359] 106. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is 100% complementary to 150 consecutive nucleotides within the binding site.

[0360] 107. An RNA exon editor as described in any one of embodiments 66 to 106, wherein the binding domain is at least 25 nucleotides in length.

[0361] 108. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain is at least 50 nucleotides in length.

[0362] 109. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain is at least 100 nucleotides in length.

[0363] 110. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain is at least 150 nucleotides in length.

[0364] 111. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain is 25 to 150 nucleotides in length.

[0365] 112. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain is 50 to 100 nucleotides in length.

[0366] 113. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain is 50 to 150 nucleotides in length.

[0367] 114. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain is 100 to 150 nucleotides in length.

[0368] 115. An RNA exon editor as described in any one of embodiments 66 to 88, wherein the binding domain is 100 to 200 nucleotides in length.

[0369] 116. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 23 (where each T is replaced by U).

[0370] 117. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 23 (where each T is replaced by U).

[0371] 118. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 23 (where each T is replaced by U).

[0372] 119. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 24 (where each T is replaced by U).

[0373] 120. An RNA exon editor as described in embodiment 119, wherein the binding domain comprises nucleotides 23C and / or 72C, numbered according to SEQ ID NO: 24 (where each T is replaced by U).

[0374] 121. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 24 (where each T is replaced by U).

[0375] 122. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 24 (where each T is replaced by U).

[0376] 123. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 25 (where each T is replaced by U).

[0377] 124. An RNA exon editor as described in embodiment 123, wherein the binding domain comprises nucleotides 97C and / or 146C, numbered according to SEQ ID NO: 25 (where each T is replaced by U).

[0378] 125. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 25 (where each T is replaced by U).

[0379] 126. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 25 (where each T is replaced by U).

[0380] 127. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 26 (where each T is replaced by U).

[0381] 128. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 26 (where each T is replaced by U).

[0382] 129. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 26 (where each T is replaced by U).

[0383] 130. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 27 (where each T is replaced by U).

[0384] 131. An RNA exon editor as described in embodiment 130, wherein the binding domain comprises nucleotides 8C, 56C, 72C, or 85C, or any combination thereof, numbered according to SEQ ID NO: 27 (where each T is replaced by U).

[0385] 132. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 27 (where each T is replaced by U).

[0386] 133. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 27 (where each T is replaced by U).

[0387] 134. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 28 (where each T is replaced by U).

[0388] 135. An RNA exon editor as described in embodiment 134, wherein the binding domain comprises nucleotide 133C, numbered according to SEQ ID NO: 28 (where each T is replaced by U).

[0389] 136. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 28 (where each T is replaced by U).

[0390] 137. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 28 (where each T is replaced by U).

[0391] 138. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 29 (where each T is replaced by U).

[0392] 139. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 29 (where each T is replaced by U).

[0393] 140. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 29 (where each T is replaced by U).

[0394] 141. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 30 (where each T is replaced by U).

[0395] 142. An RNA exon editor as described in embodiment 141, wherein the binding domain comprises nucleotides 4C, 71C, or 101C, or any combination thereof, numbered according to SEQ ID NO: 30 (where each T is replaced by U).

[0396] 143. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 30 (where each T is replaced by U).

[0397] 144. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 30 (where each T is replaced by U).

[0398] 145. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 31 (where each T is replaced by U).

[0399] 146. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 31 (where each T is replaced by U).

[0400] 147. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 31 (where each T is replaced by U).

[0401] 148. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 32 (where each T is replaced by U).

[0402] 149. An RNA exon editor as described in embodiment 148, wherein the binding domain comprises nucleotide 119A, numbered according to SEQ ID NO: 32 (where each T is replaced by U).

[0403] 150. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 32 (where each T is replaced by U).

[0404] 151. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 32 (where each T is replaced by U).

[0405] 152. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 33 (where each T is replaced by U).

[0406] 153. An RNA exon editor as described in embodiment 152, wherein the binding domain comprises nucleotide 133C, numbered according to SEQ ID NO: 33 (where each T is replaced by U).

[0407] 154. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 33 (where each T is replaced by U).

[0408] 155. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 33 (where each T is replaced by U).

[0409] 156. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 47 (where each T is replaced by U).

[0410] 157. An RNA exon editor as described in embodiment 156, wherein the binding domain comprises nucleotide 149C, numbered according to SEQ ID NO: 47 (where each T is replaced by U).

[0411] 158. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 47 (where each T is replaced by U).

[0412] 159. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 47 (where each T is replaced by U).

[0413] 160. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 55 (where each T is replaced by U).

[0414] 161. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 55 (where each T is replaced by U).

[0415] 162. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 55 (where each T is replaced by U).

[0416] 163. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 88, wherein the binding domain has a C at one or more of positions 25, 59, 63, 99 or 120, and is numbered according to SEQ ID NO: 88 (where each T is replaced by U).

[0417] 164. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 88, wherein the binding domain has C at positions 25, 59, 63, 99 and 120, and is numbered according to SEQ ID NO: 88 (where each T is replaced by U).

[0418] 165. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 88, wherein the binding domain has a C at one or more of positions 25, 59, 63, 99 or 120, and is numbered according to SEQ ID NO: 88 (where each T is replaced by U).

[0419] 166. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 88, wherein the binding domain has C at positions 25, 59, 63, 99 and 120, and is numbered according to SEQ ID NO: 88 (where each T is replaced by U).

[0420] 167. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 88 (where each T is replaced by U).

[0421] 168. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 89 (where each T is replaced by U).

[0422] 169. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 89 (where each T is replaced by U).

[0423] 170. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 89 (where each T is replaced by U).

[0424] 171. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 90, wherein the binding domain has an A at position 84 or a C at position 106, and is numbered according to SEQ ID NO: 90 (where each T is replaced by U).

[0425] 172. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 90, wherein the binding domain has an A at position 84 and a C at position 106, and is numbered according to SEQ ID NO: 90 (where each T is replaced by U).

[0426] 173. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 90, wherein the binding domain has an A at position 84 or a C at position 106, and is numbered according to SEQ ID NO: 90 (where each T is replaced by U).

[0427] 174. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 90, wherein the binding domain has an A at position 84 or a C at position 106, and is numbered according to SEQ ID NO: 90 (where each T is replaced by U).

[0428] 175. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 90 (where each T is replaced by U).

[0429] 176. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 91, wherein the binding domain has a C at position 136 or 138, numbered according to SEQ ID NO: 91 (where each T is replaced by U).

[0430] 177. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 91, wherein the binding domain has C at positions 136 and 138, numbered according to SEQ ID NO: 91 (where each T is replaced by U).

[0431] 178. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 91, wherein the binding domain has a C at position 136 or 138, numbered according to SEQ ID NO: 91 (where each T is replaced by U).

[0432] 179. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 91, wherein the binding domain has C at positions 136 and 138, numbered according to SEQ ID NO: 91 (where each T is replaced by U).

[0433] 180. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 91 (where each T is replaced by U).

[0434] 181. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 92, wherein the binding domain has a C at position 51, numbered according to SEQ ID NO: 92 (where each T is replaced by U).

[0435] 182. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 92, wherein the binding domain has a C at position 51, numbered according to SEQ ID NO: 92 (where each T is replaced by U).

[0436] 183. An RNA exon editor as described in embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 92 (where each T is replaced by U).

[0437] 184. An RNA exon editor as described in any one of embodiments 66 to 183, wherein the binding domain comprises one or more nucleotide substitutions relative to the endogenous MeCP2 mRNA sequence, wherein the one or more nucleotide substitutions disrupt cryptic splicing sites within the binding domain sequence.

[0438] 185. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence encoding a functional MeCP2 exon 4 amino acid sequence.

[0439] 186. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence encoding the 4-amino acid sequence of wild-type MeCP2 exon.

[0440] 187. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence encoding a functional MeCP2 exon 3 amino acid sequence and a functional MeCP2 exon 4 amino acid sequence.

[0441] 188. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence encoding the amino acid sequence of wild-type MeCP2 exon 3 and the amino acid sequence of wild-type MeCP2 exon 4.

[0442] 189. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence encoding a functional MeCP2 exon 2 amino acid sequence, a functional MeCP2 exon 3 amino acid sequence, and a functional MeCP2 exon 4 amino acid sequence.

[0443] 190. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence encoding the amino acid sequence of wild-type MeCP2 exon 2, the amino acid sequence of wild-type MeCP2 exon 3, and the amino acid sequence of wild-type MeCP2 exon 4.

[0444] 191. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises, in the 5' to 3' direction: a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 7 (where each T is replaced by U) and a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 8 (where each T is replaced by U).

[0445] 192. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises, in the 5' to 3' direction, the nucleotide sequence of SEQ ID NO: 7 (where each T is replaced by U) and the nucleotide sequence of SEQ ID NO: 8 (where each T is replaced by U).

[0446] 193. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises, in the 5' to 3' direction: a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 11 (where each T is replaced by U) and a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 12 (where each T is replaced by U).

[0447] 194. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises, in the 5' to 3' direction, the nucleotide sequence of SEQ ID NO: 11 (where each T is replaced by U) and the nucleotide sequence of SEQ ID NO: 12 (where each T is replaced by U).

[0448] 195. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises, in the 5' to 3' direction: a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 10 (where each T is replaced by U), a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 11 (where each T is replaced by U), and a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 12 (where each T is replaced by U).

[0449] 196. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 12 (where each T is replaced by U).

[0450] 197. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises the nucleotide sequence of SEQ ID NO: 12 (where each T is replaced by U).

[0451] 198. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises, in the 5' to 3' direction, the nucleotide sequence of SEQ ID NO: 10 (where each T is replaced by U), the nucleotide sequence of SEQ ID NO: 11 (where each T is replaced by U), and the nucleotide sequence of SEQ ID NO: 12 (where each T is replaced by U).

[0452] 199. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 9 (where each T is replaced by U).

[0453] 200. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence of SEQ ID NO: 9 (where each T is replaced by U).

[0454] 201. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises, in the 5' to 3' direction: a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 13 (where each T is replaced by U) and a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 14 (where each T is replaced by U).

[0455] 202. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises, in the 5' to 3' direction, the nucleotide sequence of SEQ ID NO: 13 (where each T is replaced by U) and the nucleotide sequence of SEQ ID NO: 14 (where each T is replaced by U).

[0456] 203. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 14 (where each T is replaced by U).

[0457] 204. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises the nucleotide sequence of SEQ ID NO: 14 (where each T is replaced by U).

[0458] 205. An RNA exon editor as described in any one of embodiments 66 to 204, wherein the coding domain comprises at least one nucleotide substitution relative to the endogenous RNA molecule sequence, wherein the at least one nucleotide substitution disrupts a hidden splicing site within the coding domain sequence.

[0459] 206. The RNA exon editor of claim 205, wherein the at least one nucleotide substitution is a synonymous nucleotide substitution.

[0460] 207. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises, in the 5' to 3' direction: a MeCP2 exon 3 nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 85 (where each T is replaced by U) and a sequence with SEQ ID NO: 86 (where each T is replaced by U) a MeCP2 exon 4 nucleotide sequence having at least 90% sequence identity, wherein the MeCP2 exon 3 nucleotide sequence comprises one or more of the following nucleotides: 43T, 49G, 55A, 67C, 85G, 91G, 94A, 97A, 103C, 106A, 109G, 115A, 118T, 119T, 120C, 130T, 133G, 176T, 177C, 182T, 183C, 184G, 206T, 207C, 223A, 239C, 241T, 280A, 290C, 317C, 320T, 321C, or 331A, according to SEQ ID NO: 7 is numbered, and the MeCP2 exon 4 nucleotide sequence contains one or more of the following nucleotides: 49T, 55A, 61T, 67G, 94T, 106A, 107C, 130A, 286T, 304A, 595T, 683C, 740T, 741C, 806T, 807C, 808T, 809C, 824T, 825C, 851T, 852C, 854T, 855C, 892G, 980C, 982C, 1034C, 1055C, 1072A, 1073C, or 1075T, as numbered according to SEQ ID NO: 8.

[0461] 208. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises, in the 5' to 3' direction, the nucleotide sequence of SEQ ID NO: 85 (where each T is replaced by U) and the nucleotide sequence of SEQ ID NO: 86 (where each T is replaced by U).

[0462] 209. An RNA exon editor as described in any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence containing a MeCP2 exon 3 sequence and a MeCP2 exon 4 sequence, wherein the nucleotide sequence is consistent with SEQ ID NO: 76 (where each T is replaced by U) has at least 90% sequence identity, and wherein the MeCP2 exon 3 nucleotide sequence comprises one or more of the following nucleotides: 43T, 49G, 55A, 67C, 85G, 91G, 94A, 97A, 103C, 106A, 109G, 115A, 118T, 119T, 120C, 130T, 133G, 176T, 177C, 182T, 183C, 184G, 206T, 207C, 223A, 239C, 241T, 280A, 290C, 317C, 320T, 321C, or 331A, according to SEQ ID NO: 7 is numbered, and the MeCP2 exon 4 nucleotide sequence therein contains one or more of the following nucleotides: 49T, 55A, 61T, 67G, 94T, 106A, 107C, 130A, 286T, 304A, 595T, 683C, 740T, 741C, 806T, 807C, 808T, 809C, 824T, 825C, 851T, 852C, 854T, 855C, 892G, 980C, 982C, 1034C, 1055C, 1072A, 1073C, or 1075T, as numbered according to SEQ ID NO: 8.

[0463] 210. An RNA exon editor as described in any one of embodiments 66 to 209, wherein the hemiintron comprises a branch point, a polypyrimidine bundle, and a 3' splice site operably connected in a 5' to 3' direction.

[0464] 211. An RNA exon editor as described in embodiment 210, wherein the branch point is located at least 15 to 25 nucleotides upstream of the 3' splice site.

[0465] 212. An RNA exon editor as described in embodiment 210 or 211, wherein the 3' splicing site comprises the nucleotide sequence YAG, where Y is a pyrimidine.

[0466] 213. An RNA exon editor as described in any one of embodiments 210 to 212, wherein the polypyrimidine bundle comprises at least 10 consecutive pyrimidine nucleotides.

[0467] 214. An RNA exon editor as described in any one of embodiments 210 to 213, wherein the hemiintrons comprise a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 17 (where each T is replaced by U).

[0468] 215. An RNA exon editor as described in any one of embodiments 210 to 214, wherein the half-introns comprise a nucleotide sequence of SEQ ID NO: 17 (where each T is replaced by U).

[0469] 216. An RNA exon editor as described in any one of embodiments 66 to 215, further comprising a 3' untranslated region (3' UTR) operatively linked to the 3' of the coding domain.

[0470] 217. An RNA exon editor as described in any one of embodiments 66 to 215, wherein the 3' UTR comprises a nucleotide sequence (RHD1pA3' UTR) having at least 90% sequence identity with SEQ ID NO: 19 (where each T is replaced by U).

[0471] 218. An RNA exon editor as described in embodiment 216 or 217, wherein the 3' UTR comprises the nucleotide sequence of SEQ ID NO: 19 (where each T is replaced by U).

[0472] 219. An RNA exon editor as described in embodiment 216, wherein the 3' UTR comprises a nucleotide sequence (mWPRE 3' UTR) having at least 90% sequence identity with SEQ ID NO: 20 (where each T is replaced by U).

[0473] 220. An RNA exon editor as described in embodiment 219, wherein the 3' UTR comprises the nucleotide sequence of SEQ ID NO:20 (where each T is replaced by U).

[0474] 221. An RNA exon editor as described in embodiment 216, wherein the 3' UTR comprises a nucleotide sequence (truncated mWPRE 3'UTR) having at least 90% sequence identity with SEQ ID NO: 87 (where each T is replaced by U).

[0475] 222. An RNA exon editor as described in embodiment 221, wherein the 3' UTR comprises the nucleotide sequence of SEQ ID NO:87 (where each T is replaced by U).

[0476] 223. An RNA exon editor as described in any one of embodiments 66 to 222, further comprising a polyadenylated sequence (Sv40pA element) having at least 90% sequence identity with SEQ ID NO: 21 (where each T is replaced by U).

[0477] 224. An RNA exon editor as described in embodiment 223, wherein the polyadenylated sequence comprises the nucleotide sequence of SEQ ID NO: 21 (where each T is replaced by U).

[0478] 225. An RNA exon editor as described in any one of embodiments 66 to 222, further comprising a 3' transcription terminator domain operatively linked to a 3' UTR.

[0479] 226. An RNA exon editor as described in embodiment 225, wherein the transcription terminator domain comprises a triple-helix terminator.

[0480] 227. An RNA exon editor as described in embodiment 225 or 226, wherein the transcription terminator domain comprises the MALAT1 terminator.

[0481] 228. An RNA exon editor as described in any one of embodiments 225 to 227, wherein the transcription terminator domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 77 (where each T is replaced by U).

[0482] 229. An RNA exon editor as described in any one of embodiments 225 to 228, wherein the transcription terminator domain comprises the nucleotide sequence of SEQ ID NO: 77 (where each T is replaced by U).

[0483] 230. An RNA exon editor as described in any one of embodiments 225 to 227, wherein the transcription terminator domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 22 (where each T is replaced by U).

[0484] 231. An RNA exon editor as described in any one of embodiments 225 to 227 or 230, wherein the transcription terminator domain comprises the nucleotide sequence of SEQ ID NO: 22 (where each T is replaced by U).

[0485] 232. An RNA exon editor as described in any one of embodiments 225 to 231, further comprising a 3xUBS sequence operatively linked to the 3' of the transcription terminator domain.

[0486] 233. An RNA exon editor as described in embodiment 232, wherein the 3xUBS sequence comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 79 (where each T is replaced by U).

[0487] 234. An RNA exon editor as described in embodiment 232 or 233, wherein the 3xUBS sequence comprises the nucleotide sequence of SEQ ID NO: 79 (where each T is replaced by U).

[0488] 235. An RNA exon editor comprising a nucleotide sequence having at least 90% sequence identity with nucleotides 601-2522 of SEQ ID NO: 78 (where each T is replaced by U).

[0489] 236. An RNA exon editor comprising the nucleotide sequence of nucleotides 601-2522 of SEQ ID NO: 78 (where each T is replaced by U).

[0490] 237. An RNA exon editor comprising a nucleotide sequence having at least 90% sequence identity with nucleotides 601-2483 of SEQ ID NO: 80 (where each T is replaced by U).

[0491] 238. An RNA exon editor comprising the nucleotide sequence of nucleotides 601-2483 of SEQ ID NO: 80 (where each T is replaced by U).

[0492] 239. An RNA exon editor comprising a nucleotide sequence having at least 90% sequence identity with nucleotides 601-2522 of SEQ ID NO: 81 (where each T is replaced by U).

[0493] 240. An RNA exon editor comprising the nucleotide sequence of nucleotides 601-2522 of SEQ ID NO: 81 (where each T is replaced by U).

[0494] 241. An RNA exon editor comprising a nucleotide sequence having at least 90% sequence identity with nucleotides 601-3011 of SEQ ID NO: 82 (where each T is replaced by U).

[0495] 242. An RNA exon editor comprising the nucleotide sequence of nucleotides 601-3011 of SEQ ID NO: 82 (where each T is replaced by U).

[0496] 243. An RNA exon editor comprising a nucleotide sequence having at least 90% sequence identity with nucleotides 601-3250 of SEQ ID NO: 83 (where each T is replaced by U).

[0497] 244. An RNA exon editor comprising the nucleotide sequence of nucleotides 601-3250 of SEQ ID NO: 83 (where each T is replaced by U).

[0498] 245. An RNA exon editor comprising a nucleotide sequence having at least 90% sequence identity with nucleotides 601-3128 of SEQ ID NO: 84 (where each T is replaced by U).

[0499] 246. An RNA exon editor comprising the nucleotide sequence of nucleotides 601-3128 of SEQ ID NO: 84 (where each T is replaced by U).

[0500] 247. An RNA exon editor comprising: operably linked in the 5' to 3' direction: (a) A binding domain configured to bind to a binding site within the target region of the MeCP2 precursor mRNA; (b) Hemiintron; (c) A coding structure field containing a sequence encoding one or more MeCP2 exons; and (d) A truncated WPRE 3' UTR containing a sequence that has at least 90% sequence identity with SEQ ID NO: 87 (where each T is replaced by U).

[0501] 248. The RNA exon editor as described in embodiment 247, wherein the truncated WPRE 3' UTR comprises or consists of the following: SEQ ID NO: 87.

[0502] 249. An exon editor construct encoding an RNA exon editor according to any one of embodiments 66 to 248.

[0503] 250. The exon editor construct as described in embodiment 249, further comprising a promoter operatively connected to the 5' of the binding structure domain.

[0504] 251. The exon editor construct as described in embodiment 249, further comprising a sequence operatively connected to the 5' of the binding domain and having at least 90% sequence identity with SEQ ID NO: 15.

[0505] 252. The exon editor construct as described in embodiment 249, further comprising a sequence of SEQ ID NO: 15 operatively linked to 5' of the binding domain.

[0506] 253. An exon editor construct encoding the RNA exon editor of embodiment 235, wherein the exon editor construct comprises a sequence having at least 90% identity with SEQ ID NO: 78.

[0507] 254. An exon editor construct encoding the RNA exon editor of embodiment 236, wherein the exon editor construct comprises the sequence of SEQ ID NO: 78.

[0508] 255. An exon editor construct encoding the RNA exon editor of embodiment 237, wherein the exon editor construct comprises a sequence having at least 90% identity with SEQ ID NO: 80.

[0509] 256. An exon editor construct encoding the RNA exon editor of embodiment 238, wherein the exon editor construct comprises the sequence of SEQ ID NO: 80.

[0510] 257. An exon editor construct encoding the RNA exon editor of embodiment 239, wherein the exon editor construct comprises a sequence having at least 90% identity with SEQ ID NO: 81.

[0511] 258. An exon editor construct encoding the RNA exon editor of embodiment 240, wherein the exon editor construct comprises the sequence of SEQ ID NO: 81.

[0512] 259. An exon editor construct encoding the RNA exon editor of embodiment 241, wherein the exon editor construct comprises a sequence having at least 90% identity with SEQ ID NO: 82.

[0513] 260. An exon editor construct encoding the RNA exon editor of embodiment 242, wherein the exon editor construct comprises the sequence of SEQ ID NO: 82.

[0514] 261. An exon editor construct encoding the RNA exon editor of embodiment 243, wherein the exon editor construct comprises a sequence having at least 90% identity with SEQ ID NO: 83.

[0515] 262. An exon editor construct encoding the RNA exon editor of embodiment 244, wherein the exon editor construct comprises the sequence of SEQ ID NO: 83.

[0516] 263. An exon editor construct encoding the RNA exon editor of embodiment 245, wherein the exon editor construct comprises a sequence having at least 90% identity with SEQ ID NO: 84.

[0517] 264. An exon editor construct encoding the RNA exon editor of embodiment 246, wherein the exon editor construct comprises the sequence of SEQ ID NO: 84.

[0518] 265. A carrier comprising an exon editor builder according to any one of embodiments 249 to 264.

[0519] 266. The carrier as described in embodiment 265, wherein the carrier includes a 5' control domain operatively connected to the 5' of the binding domain.

[0520] 267. The carrier as described in embodiment 266, wherein the 5' regulatory domain comprises a constitutive promoter.

[0521] 268. The vector as described in embodiment 266, wherein the 5' regulatory domain comprises a tissue-specific promoter.

[0522] 269. A proviral plasmid comprising an exon editor construct according to any one of embodiments 249 to 264.

[0523] 270. An adeno-associated virus (AAV) comprising an exon editor construct according to any one of embodiments 249 to 264.

[0524] 271. The AAV as described in embodiment 270, wherein the AAV exhibits neuronal orientation.

[0525] 272. A composition comprising an exon editor construct according to any one of embodiments 249 to 264, a vector according to any one of embodiments 265 to 268, a proviral plasmid according to embodiment 269, or an AAV according to embodiments 270 to 271, and a pharmaceutically acceptable excipient.

[0526] 273. A method for expressing functional MeCP2 in target cells, the method comprising transducing the target cells using an exon editor construct according to any one of embodiments 249 to 264, a vector according to any one of embodiments 265 to 268, a proviral plasmid according to embodiment 269, or an AAV according to embodiments 270 to...

Claims

1. An exon editor construct encoding an RNA exon editor, wherein the exon editor construct comprises encoding the following sequence: (a) A binding domain that binds to a target intron of a MeCP2 precursor mRNA, wherein the target intron of the MeCP2 precursor mRNA is intron 1 or intron 2. (b) Hemiintrons; and (c) Encoding structure field, which contains one or more MeCP2 exons.

2. The exon editor construct of claim 1, wherein the binding domain binds to a site comprising: Intron 1 contains nucleotides ranging from -100 to 1500 or -50 to 1449. Intron 1 contains nucleotides 3950 to 4250 or 4020-4169.

3. The exon editor construct of claim 1, wherein the binding domain binds to the binding sites of nucleotides 1-300 containing intron 2.

4. The exon editor construct of claim 2, wherein the binding domain binds to a binding site comprising nucleotides -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 containing intron 1.

5. The exon editor construct of claim 3, wherein the binding domain binds to a binding site of nucleotides 50-300, 50-250, 75-300, 75-250, 80-300, 80-250, 90-300, 90-250, 95-300, 95-250, or 99-248 containing intron 2.

6. The exon editor construct according to any one of claims 1 to 5, wherein the size of the binding domain ranges from 50 to 300 nucleotides; 50 to 250 nucleotides; 50 to 150 nucleotides; 50 to 100 nucleotides; 75 to 300 nucleotides; 75 to 250 nucleotides; 75 to 200 nucleotides; 75 to 150 nucleotides; 100 to 300 nucleotides; 100 to 250 nucleotides; 100 to 200 nucleotides; 100 to 150 nucleotides; 125 to 300 nucleotides; 125 to 250 nucleotides; 125 to 200 nucleotides; 125 to 150 nucleotides; or 150 nucleotides.

7. The exon editor construct according to any one of claims 1 to 5, wherein the size of the binding domain ranges from about 100-200 nucleotides; about 100-150 nucleotides; about 125-200 nucleotides; about 125-150 nucleotides; or about 150 nucleotides; or the size ranges from 100-200 nucleotides; 100-150 nucleotides; 125-200 nucleotides; 125-150 nucleotides; or 150 nucleotides.

8. The exon editor construct according to any one of claims 1 to 7, wherein the binding domain is at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the binding site to which it is bound.

9. The exon editor construct of any one of claims 1 to 8, wherein the binding domain comprises a continuous nucleotide chain 100% complementary to a portion of the binding site to which it binds, wherein the length of the continuous nucleotide chain is at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, at least 70 nucleotides, at least 75 nucleotides, at least 80 nucleotides, at least 85 nucleotides, at least 90 nucleotides, at least 95 nucleotides, at least 100 nucleotides, at least 105 nucleotides, at least 110 nucleotides, at least 115 nucleotides, at least 120 nucleotides, at least 130 nucleotides, at least 135 nucleotides, at least 140 nucleotides, at least 145 nucleotides, or at least 150 nucleotides.

10. The exon editor construct of any one of claims 1 to 9, wherein the sequence encoding the binding domain comprises, substantially comprises, or comprises: SEQ ID NO: any one of 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 47, 116, 117, 118, 119, 120 or 121.

11. The exon editor construct of any one of claims 1 to 9, wherein the sequence encoding the binding domain comprises, substantially comprises, or comprises: SEQ ID NO: 55, 56, 57, 58, 59, 88 or 138.

12. The exon editor construct of any one of claims 1 to 9, wherein the sequence encoding the binding domain comprises, substantially comprises, or comprises: SEQ ID NO: any one of 24, 28, 29, 30, 116, 119 or 120.

13. The exon editor construct as described in any one of claims 1 to 12, wherein the coding structure domain comprises, substantially comprises, or comprises the following: SEQ ID NO: 7 and 8 or sequences having 90% identity with SEQ ID NO: 7 and 8; SEQ ID NO: 76 or a sequence having 90% identity with SEQ ID NO: 76; SEQ ID NO: 13 and 14 or sequences having 90% identity with SEQ ID NO: 13 and 14; SEQ ID NO: 12; SEQ ID NO: 14; or SEQ ID NO:

9.

14. The exon editor construct of any one of claims 1 to 12, wherein the coding structure domain comprises, substantially comprises, or comprises: Either SEQ ID NO: 76 or 14.

15. The exon editor construct of any one of claims 1 to 14, wherein the binding domain, the half-intron, and the coding domain are operatively connected in the 5' to 3' direction.

16. The exon editor construct of any one of claims 1 to 15, wherein the half-intron comprises a 3' splice site and optional branch point sequences and a polypyrimidine bundle, wherein the binding domain, the half-intron comprising the 3' splice site, and the coding domain are operatively connected in the 5' to 3' direction.

17. The exon editor construct of claim 16, wherein the semi-introns comprise, substantially consist of, or consist of: Polypyrimidine bundles, branch points, and 3' splice sites.

18. The exon editor construct of any one of claims 16 or 17, wherein the semi-introns comprise, substantially consist of, or consist of: SEQ ID NO: 17 or a sequence having at least 90% identity with SEQ ID NO:

17.

19. The exon editor construct of any one of claims 1 to 18, further comprising a 3' untranslated region (3'UTR), wherein the binding domain, the half-intron, the coding domain, and the 3' UTR are operatively connected in the 5' to 3' directions.

20. The exon editor construct of claim 19, wherein the 3' UTR comprises RDH1pA 3' UTR (SEQ ID NO: 19) or mWPRE 3' UTR (SEQ ID NO: 20).

21. The exon editor builder of claim 20, wherein... The RDH1pA 3' UTR comprises, is substantially composed of, or is composed of the following: SEQ ID NO: 19 or a sequence having at least 90% identity with SEQ ID NO: 19; or The mWPRE 3' UTR comprises, is substantially composed of, or is composed of: SEQ ID NO: 20 or a sequence having at least 90% identity with SEQ ID NO:

20.

22. The exon editor builder as described in any one of claims 1 to 21, wherein the exon editor builder comprises: Combined with a structural domain, it contains, is essentially composed of, or is composed of the following: SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 47, 55, 88, 116, 117, 118, 119, 120, 121 or 138; A hemiintron, comprising, substantially consisting of, or consisting of: SEQ ID NO: 17; The encoding structure field contains, is substantially composed of, or is composed of: SEQ ID NO: 76; And 3' UTR, which comprises, substantially comprises, or comprises SEQ ID NO: 20, wherein the binding domain, the half-intron, the coding domain and the 3' UTR are operatively connected in the 5' to 3' direction.

23. The exon editor builder of claim 22, wherein the exon editor builder comprises, substantially comprises, or comprises: SEQ ID NO: 78, 80, 81, 82, 83 or 84.

24. The exon editor builder of claim 22, wherein the exon editor builder comprises, substantially comprises, or comprises the following: SEQ ID NO: 78, 80 or 83.

25. The exon editor construct of any one of claims 1 to 24, further comprising a triple helix terminator, wherein the binding domain, the half-intron, the coding domain, the 3' UTR (if present), and the triple helix terminator are operatively connected in the 5' to 3' directions.

26. The exon editor construct of claim 25, wherein the triple-helix terminator comprises, substantially comprises, or comprises: SEQ ID NO: 77 or a sequence having at least 90% identity with SEQ ID NO:

77.

27. The exon editor construct of claim 25, wherein the triple-helix terminator comprises, substantially comprises, or comprises: SEQ ID NO:

22.

28. The exon editor construct of any one of claims 1 to 27, further comprising a sequence encoding a poly A sequence, for example, SEQ ID NO:

21.

29. The exon editor construct of any one of claims 1 to 28, further comprising a sequence of encoded epitope tags, wherein the binding domain, the half-intron, the encoded domain, the epitope tags, the 3' UTR (if present), and the triple-helix terminator (if present) are operatively connected in the 5' to 3' directions.

30. The exon editor builder of claim 28, wherein the exon editor builder comprises, substantially comprises, or comprises: SEQ ID NO:

78.

31. An RNA exon editor, which is transcribed from the exon editor construct of any one of claims 1-30.

32. The exon editor construct of any one of claims 1 to 30, wherein the MeCP2 precursor mRNA contains at least one mutation associated with Rett syndrome.

33. The exon editor construct of any one of claims 1 to 30, wherein the at least one mutation associated with Rett syndrome comprises at least one mutation in exon 3 of the MeCP2 gene allele or at least one mutation in exon 4 of the MeCP2 gene allele, or any combination thereof.

34. The exon editor construct of claim 32 or 33, wherein the at least one mutation associated with Rett syndrome is X-linked.

35. The exon editor construct of any one of claims 32 to 34, wherein at least one of the Rett syndrome-associated mutated MeCP2 proteins is expressed in at least one or any combination of neural stem cells, neurons, astrocytes, or oligodendrocytes.

36. A carrier comprising an exon editor construct according to any one of claims 1 to 30 or 32 to 35.

37. The carrier of claim 36, wherein the carrier includes a 5' control domain operatively connected to the 5' of the binding domain.

38. The vector of claim 36 or 37, wherein the 5' regulatory domain comprises a constitutive promoter or a tissue-specific promoter.

39. The carrier of claim 38, wherein the constitutive promoter is a CMV promoter.

40. A proviral plasmid comprising an exon editor construct according to any one of claims 1 to 30 or 32 to 35.

41. An adeno-associated virus (AAV) comprising an exon editor construct according to any one of claims 1 to 30 or 32 to 35, wherein the AAV optionally comprises a 5' regulatory domain operatively linked to the 5' of the exon editor construct.

42. The AAV of claim 41, wherein the AAV includes a 5' control domain operatively connected to the 5' of the binding domain.

43. The AAV of claim 41 or 42, wherein the 5' regulatory domain comprises a constitutive promoter.

44. The AAV of claim 43, wherein the constitutive promoter is a CMV promoter.

45. The AAV of any one of claims 41 to 44, wherein the AAV exhibits neurotropic characteristics.

46. ​​The AAV of any one of claims 41 to 45, wherein the AAV is AAV9, AAV8, AAV5 or AAV2, or a variant of AAV9, AAV8, AAV5 or AAV2.

47. A composition comprising an exon editor construct according to any one of claims 1 to 30 or 32 to 35, a vector according to any one of claims 36 to 39, a proviral plasmid according to claim 40, or an AAV according to any one of claims 41 to 46.

48. The composition of claim 47, wherein it comprises a pharmaceutically acceptable excipient.

49. A method for expressing bioactive MeCP2 in target cells to restore the functional level of the MeCP2 protein in the target cells, the method comprising transfecting or transducing the target cells with an exon editor construct of any one of claims 1 to 30 or 32 to 35, a vector of any one of claims 36 to 39, a proviral plasmid of claim 40, an AAV of any one of claims 41 to 46, or a composition of claims 47 to 48.

50. The method of claim 49, wherein at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of the MeCP2 precursor mRNA containing at least one mutation associated with Rett syndrome is modified to become a transcript encoding a functional MeCP2, wherein the at least one mutation is corrected.

51. The method of claim 50, wherein at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of the MeCP2 precursor mRNA containing at least one mutation associated with Rett syndrome is modified to encode a transcript of functional MeCP2, wherein the at least one mutation is corrected.

52. The method of claim 51, wherein at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the MeCP2 precursor mRNA containing at least one mutation associated with Rett syndrome is modified to encode a transcript of functional MeCP2, wherein the at least one mutation is corrected.

53. The method of any one of claims 49 to 52, wherein the functional level of MeCP2 in the target cells is restored by expressing a biologically functional MeCP2 protein.

54. A method for reducing the expression of MeCP2 containing at least one mutation associated with Rett syndrome in a subject, the method comprising transfecting or transducing target cells, more particularly neurons, in the subject with an exon editor construct of any one of claims 1 to 30 or 32 to 35, a vector of any one of claims 36 to 39, a proviral plasmid of claim 40, an AAV of any one of claims 41 to 46, or a composition of claim 47 or 48.

55. A method for correcting at least one mutation in the MeCP2 exon sequence in the MeCP2 precursor mRNA of a subject, the method comprising administering to the subject an exon editor construct of any one of claims 1 to 30 or 32 to 35, a vector of any one of claims 36 to 39, a proviral plasmid of claim 40, an AAV of any one of claims 41 to 46, or a composition of claims 47 to 48.

56. A method for treating Rett syndrome in a subject in need, the method comprising administering to the subject a therapeutically effective amount of any one of claims 1 to 30 or 32 to 35 of an exon editor construct, any one of claims 36 to 39 of a vector, any one of claims 40 of a proviral plasmid, any one of claims 41 to 46 of an AAV, or any one of claims 47 or 48 of a composition.

57. The method of any one of claims 54 to 56, wherein at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of the MeCP2 precursor mRNA containing at least one mutation associated with Rett syndrome is modified to encode a transcript of functional MeCP2, wherein the at least one mutation is corrected.

58. The method of claim 57, wherein at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of the MeCP2 precursor mRNA containing at least one mutation associated with Rett syndrome is modified to form a transcript encoding a functional MeCP2, wherein the at least one mutation is corrected.

59. The method of claim 58, wherein at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the MeCP2 precursor mRNA containing at least one mutation associated with Rett syndrome is modified to encode a transcript of functional MeCP2, wherein the at least one mutation is corrected.

60. The method of any one of claims 54 to 59, wherein the functional level of MeCP2 in the target cells is restored by expressing a biologically functional MeCP2 protein.

61. The method of any one of claims 54 to 60, the method comprising administering to the brain of the subject an exon editor construct as claimed in any one of claims 1 to 30 or 32 to 35, a vector as claimed in any one of claims 36 to 39, a proviral plasmid as claimed in claim 40, an AAV as claimed in any one of claims 41 to 46, or a composition as claimed in any one of claims 47 to 48.

62. The method of any one of claims 54 to 61, wherein the subject is a mammal, preferably a rodent, a non-human primate, or a human.

63. The method of any one of claims 54 to 62, wherein the subject is genetically predisposed to Rett syndrome or has been diagnosed with Rett syndrome.

64. An exon editor construct as claimed in any one of claims 1 to 30 or 32 to 35, a vector as claimed in any one of claims 36 to 39, a proviral plasmid as claimed in claim 40, an AAV as claimed in any one of claims 41 to 46, or a composition as claimed in any one of claims 47 to 48, for the prevention or treatment of Rett syndrome in a subject of need.

65. The exon editor construct of any one of claims 1 to 30 or 32 to 35, the vector of any one of claims 36 to 39, the proviral plasmid of claim 40, the AAV of any one of claims 41 to 46, or the composition of any one of claims 47 to 48, for the preparation of a medicament for the treatment or prevention of Rett syndrome in a subject of need.

66. An RNA exon editor comprising: operably linked in the 5' to 3' direction: (a) A binding domain configured to bind to a binding site within the target region of the MeCP2 precursor mRNA; (b) Hemiintrons; and (c) A coding structure field containing a sequence that encodes one or more MeCP2 exons; The coding domain contains at least one nucleotide variation at a hidden splicing site relative to the wild-type MeCP2 sequence.

67. An RNA exon editor comprising: operably linked in the 5' to 3' direction: (a) A binding domain configured to bind to a binding site within the target region of the MeCP2 precursor mRNA; (b) Hemiintrons; and (c) A coding structure field containing a sequence that encodes one or more MeCP2 exons; The binding domain contains at least one nucleotide variation at a hidden splicing site relative to the corresponding wild-type MeCP2 precursor mRNA sequence.

68. An RNA exon editor comprising: operably linked in the 5' to 3' direction: (a) A binding domain configured to bind to a binding site within the target region of the precursor mRNA of the target gene; (b) Hemiintron; (c) A coding domain comprising a sequence encoding one or more exons of the target gene; and (d) A 3' truncated WPRE 3' UTR sequence, which contains or consists of the following: A sequence that has at least 90% sequence identity with SEQ ID NO:

87.

69. The RNA exon editor of claim 68, wherein the 3' truncated WPRE 3' UTR sequence comprises or consists of the following: SEQ ID NO: 87.

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