MECP2 trans-splicing molecule

Abstract

An exon editor construct is provided comprising a binding domain that binds to a target intron of MeCP2 pre-mRNA, a hemi-intron, and a coding domain comprising one or more MeCP2 exons. A method for expressing functional MeCP2 in target cells is also provided, comprising contacting target cells with the exon editor construct provided herein.

Description

Technology Field

[0001] I. Cross-reference regarding related applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 598,693 filed November 14, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] II. List of sequences

[0004] The present application is submitted electronically in XML format and includes a list of sequences, the entirety of which is incorporated herein by reference. The XML list of sequences, created on November 13, 2024, is named ASC-003WO_SL.xml and has a size of 329,544 bytes. Background Technology

[0005] III. Background

[0006] Rett syndrome is a destructive neurodevelopmental disorder that primarily afflicts girls, and there is no effective treatment for it. Patients with Rett syndrome typically speak a few words at first but soon lose the ability to speak, walk, or use their hands intentionally. There is no "disease-modifying" treatment available for these patients. Even with the best current treatment standards, they often suffer daily from vomiting, seizures, and uncontrollable crying.

[0007] IV. Overview

[0008] Gene therapy holds great potential for treating Rett syndrome (RTT), which is caused by mutations in the methyl CpG-binding protein 2 (MECP2) gene. However, conventional gene therapy approaches, such as gene replacement, can lead to the overproduction of MeCP2, causing a condition known as MeCP2 duplication syndrome. Furthermore, with thousands of different mutations potentially causing Rett syndrome, individual base editor drugs will not be able to address the needs of a significant number of patients. Gene editing approaches, such as CRISPR / Cas9-based editing, also require the introduction of Cas9 into the brain, which increases the risk of immune rejection. Therefore, alternative strategies to correct mutations in the MeCP2 gene are needed in the field.

[0009] To address these unmet needs, an RNA exon editing solution is described herein, comprising delivering an exon editor construct encoding a therapeutic RNA exon editor designed to transsplice into endogenous MeCP2 pre-mRNA containing at least one mutation. Transsplicing replaces one or more exons of the MeCP2 pre-mRNA with exons from the therapeutic RNA exon editor encoding a functional amino acid sequence present in the biologically active MeCP2 protein, thereby correcting one or more mutations in the endogenous MeCP2 pre-mRNA. Rett syndrome is a promising candidate for the exon editing approach. Exon editing can correct approximately 95% of mutations in Rett patients with a single therapeutic RNA exon editor. An exemplary MeCP2 RNA exon editor is described herein.

[0010] In some embodiments, an exon editor construct is described herein comprising: (a) a binding domain that binds to a target intron of MeCP2 pre-mRNA; (b) a hemi-intron; and (c) a coding domain comprising one or more MeCP2 exons. In some embodiments, the binding domain comprises any of the binding domains described in Section VI.C.3 below. In some embodiments, the hemi-intron comprises any of the hemi-introns disclosed in Section VI.C.4. In some embodiments, the coding domain sequence comprises any of the CDSs disclosed in Section VI.C.2. In some embodiments, the exon editor construct further comprises a 3' UTR sequence operably linked to the CDS at 3'. In some embodiments, the 3' UTR sequence comprises any of the 3' UTR sequences disclosed in Section VI.C.5. In some embodiments, the exon editor construct further comprises the poly-A tail disclosed herein operably linked to the CDS at 3'. In some embodiments, the exon editor construct further comprises a transcription terminator sequence operably linked to the CDS at 3'. In some embodiments, the transcription terminator sequence comprises any of the transcription terminator sequences disclosed in Section VI.C.6.

[0011] Embodiments disclosed herein comprise an exon editor construct encoding an RNA exon editor, wherein the exon editor construct comprises (a) a binding domain that binds to a target intron of MeCP2 pre-mRNA; (b) a hemi-intron; and (c) a sequence encoding a coding domain comprising one or more MeCP2 exons. In some embodiments, the target intron of MeCP2 pre-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 (SEQN: 1); nucleotides 3950 to 4250 or 4020 to 4169 of intron 1; or nucleotides 1 to 300 of intron 2 (SEQN: 2 and 3). In some embodiments, the binding domain binds to a binding site comprising nucleotides -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of intron 1 (SEQN: 1); or nucleotides 50-300, 50-250, 75-300, 75-250, 80-300, 80-250, 90-300, 90-250, 95-300, 95-250, or 99-248 of intron 2 (SEQN: 2 and 3). In some embodiments, the binding domain has a size of about 50-300 nucleotides; about 50-250 nucleotides; about 50-150 nucleotides; about 50-100 nucleotides; Approximately 75-300 nucleotides; approximately 75-250 nucleotides; approximately 75-200 nucleotides; approximately 75-150 nucleotides; approximately 100-300 nucleotides; approximately 100-250 nucleotides; approximately 100-200 nucleotides; approximately 100-150 nucleotides; approximately 125-300 nucleotides; approximately 125-250 nucleotides; approximately 125-200 nucleotides; approximately 125-150 nucleotides; or a range of approximately 150 nucleotides.In some embodiments, the binding domain is in the size range of 50-300 nucleotides; 50-250 nucleotides; 50-150 nucleotides; 50-100 nucleotides; 75-300 nucleotides; 75-250 nucleotides; 75 to 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. In some embodiments, an exon editor construct of any one of the prior embodiments, wherein 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 about 150 nucleotides; in the range of 100-200 nucleotides; 100-150 nucleotides; 125-200 nucleotides; 125-150 nucleotides; or in the range of 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 to which it binds.In some embodiments, the binding domain comprises a stretch of adjacent nucleotides that is 100% complementary to the portion of the binding site to which it binds, wherein the stretch of adjacent nucleotides is at least 5 nucleotide lengths, at least 10 nucleotide lengths, at least 15 nucleotide lengths, at least 20 nucleotide lengths, at least 25 nucleotide lengths, at least 30 nucleotide lengths, at least 35 nucleotide lengths, at least 40 nucleotide lengths, at least 45 nucleotide lengths, at least 50 nucleotide lengths, at least 55 nucleotide lengths, at least 60 nucleotide lengths, at least 65 nucleotide lengths, at least 70 nucleotide lengths, at least 75 nucleotide lengths, at least 80 nucleotide lengths, at least 85 nucleotide lengths, at least 90 nucleotide lengths, at least 95 nucleotide lengths, at least 100 nucleotide lengths, and at least 105 The nucleotide length is at least 110 nucleotide lengths, at least 115 nucleotide lengths, at least 120 nucleotide lengths, at least 130 nucleotide lengths, at least 135 nucleotide lengths, at least 140 nucleotide lengths, at least 145 nucleotide lengths, or at least 150 nucleotide lengths. In some embodiments, the sequence encoding the binding domain comprises, is essentially made of, or is made of 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, is essentially made of, or is made of SEQ ID NO: 55. In some embodiments, the sequence encoding the binding domain comprises, is essentially made of, or is made of any one of SEQ ID NO: 24, 28, 29, or 30.In some embodiments, the coding domain comprises, is essentially made up of, or is made up of SEQ ID NO: 7 and 8 or a sequence that is 90% identical to SEQ ID NO: 7 and 8; SEQ ID NO: 76 or a sequence that is 90% identical to SEQ ID NO: 76; SEQ ID NO: 12; SEQ ID NO: 14; or SEQ ID NO: 9.

[0012] In some embodiments, the coding domain comprises either SEQ ID NO: 76 or 14, is essentially made up of, or is made up of.

[0013] In some embodiments, the binding domain, hemi-intron, and coding domain are operably connected in the 5'-to-3' direction.

[0014] In some embodiments, the hemi-intron comprises a 3' splice site and optionally, a fork sequence and a polypyrimidine track, wherein the binding domain, the hemi-intron comprising the 3' splice site, and the coding domain are operably connected in the 5'-to-3' direction. In some embodiments, the hemi-intron comprises, is essentially composed of, or consists of a polypyrimidine track, a fork, and a 3' splice site. In some embodiments, the hemi-intron comprises, is essentially composed of, or consists of SEQ ID NO: 17 or a sequence having at least 90% identity with SEQ ID NO: 17.

[0015] In some embodiments, the exon editor construct further comprises a 3' untranslated region (3' UTR), wherein a binding domain; a hemi-intron; a coding domain; and the 3' UTR are operably connected in the 5'-to-3' direction. In some embodiments, the 3' UTR comprises an RDH1pA 3' UTR (SEQN: 19), an mWPRE 3' UTR (SEQN: 20), or a truncated version thereof, e.g., a WPRE3 3' UTR (SEQN: 87). In some embodiments, the RDH1pA 3' UTR comprises, is essentially composed of, or is composed of SEQN: 19 or a sequence having at least 90% identity with SEQN: 19; and the mWPRE 3' UTR comprises, is essentially composed of, or is composed of SEQN: 20 or a sequence having at least 90% identity with SEQN: 20. In some embodiments, the 3' UTR comprises a truncated WPRE sequence. In some embodiments, the truncated WPRE sequence comprises, is essentially composed of, or is composed of a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 87 or SEQ ID NO: 87.

[0016] In some embodiments, the exon editor construct comprises a binding domain comprising, essentially consisting of, or consisting of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 47, or 55; a hemi-intron comprising, essentially consisting of, or consisting of SEQ ID NO: 17; a coding domain comprising, essentially consisting of, or consisting of, SEQ ID NO: 76; and a 3' UTR comprising, essentially consisting of, or consisting of, SEQ ID NO: 20, wherein the binding domain, the hemi-intron, the coding domain, and the 3' UTR are operably connected in the 5'-to-3' direction.

[0017] In some embodiments, the exon editor composition comprises, is essentially composed of, or is composed of SEQ ID NO: 78, 80, 81, 82, 83, or 84. In some embodiments, the exon editor composition comprises, is essentially composed of, or is composed of SEQ ID NO: 78 or 80.

[0018] In some embodiments, the exon editor construct further comprises a triple helix terminator, wherein a binding domain; a hemi-intron; a coding domain; a 3' UTR, if present; and the triple helix terminator is operably connected in the 5'-to-3' direction. In some embodiments, the triple helix terminator comprises, is essentially composed of, or is composed of 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, is essentially composed of, or is composed of SEQ ID NO: 22.

[0019] In some embodiments, the exon editor further includes a sequence encoding a poly A sequence, e.g., sequence number: 21.

[0020] In some embodiments, the exon editor construct further comprises a sequence encoding an epitope tag, wherein a binding domain; a hemi-intron; a coding domain; an epitope tag; if present, a 3' UTR; and if present, a triple helix terminator are operably connected in the 5'-to-3' direction. In some embodiments, the exon editor construct comprises, is essentially composed of, or is composed of SEQ ID NO: 78.

[0021] An RNA exon editor transcribed from an exon editor composition of any of the embodiments described above is also disclosed herein.

[0022] In some embodiments, MeCP2 pre-mRNA contains at least one mutation associated with Rett syndrome. In some embodiments, 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. In some embodiments, the at least one mutation associated with Rett syndrome is X-linked. In some embodiments, the MeCP2 protein containing at least one mutation associated with Rett syndrome is expressed in at least one of neural stem cells, neurons, astrocytes, or oligodendrocytes, or any combination thereof.

[0023] A vector comprising an exon editor construct of any of the embodiments described above is also disclosed herein. In some embodiments, the vector comprises a 5' regulatory domain operably connected to a binding domain. 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.

[0024] A provirus plasmid comprising an exon editor composition of any of the embodiments described above is also disclosed herein.

[0025] An adeno-associated virus (AAV) comprising an exon editor construct of any of the embodiments described above is also disclosed herein, wherein the AAV optionally comprises a 5' regulatory domain operably linked to the exon editor construct at 5'. In some embodiments, the AAV comprises a 5' regulatory domain operably linked to a binding domain at 5'. In some embodiments, the 5' regulatory domain comprises a constitutive promoter. In some embodiments, the constitutive promoter is a CMV promoter. In some embodiments, the AAV exhibits neuronal orientation. In some embodiments, the AAV is AAV9, AAV8, AAV5, or AAV2.

[0026] A composition comprising an exon editor construct of any one of the embodiments described above, a vector of any of the embodiments described above, a provirus plasmid of any of the embodiments described above, or an AAV of any of the embodiments described above is also disclosed. In some embodiments, the composition comprises a pharmaceutically acceptable excipient.

[0027] A method for restoring the functional level of MeCP2 protein in a target cell by expressing biologically active MeCP2 in the target cell is also disclosed, comprising transfecting the target cell with an exon editor construct of any of the embodiments described above, a vector of any of the embodiments described above, a proviral plasmid of any of the embodiments described above, an AAV of any of the embodiments described above, or a composition of any of the embodiments described above. 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 MeCP2 pre-mRNA containing at least one mutation associated with Rett syndrome in the target cell is modified to become a transcript encoding functional MeCP2 in which 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 MeCP2 pre-mRNA containing at least one mutation associated with Rett syndrome in target cells is modified to become a transcript encoding functional MeCP2 in which 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 MeCP2 pre-mRNA containing at least one mutation associated with Rett syndrome in target cells is modified to become a transcript encoding functional MeCP2 in which at least one mutation is corrected. In some embodiments, the functional level of MeCP2 is restored in target cells by expressing a biologically functional MeCP2 protein.

[0028] A method for reducing the expression of MeCP2 containing at least one mutation associated with Rett syndrome in a subject is also disclosed, comprising transfecting or transducing a target cell, more particularly a neuron, in a subject with an exon editor construct of any of the embodiments described above, a vector of any of the embodiments described above, a proviral plasmid of any of the embodiments described above, an AAV of any of the embodiments described above, or a composition of any of the embodiments described above.

[0029] A method for correcting at least one mutation in a MeCP2 exon sequence within MeCP2 pre-mRNA in a target cell of a subject is also disclosed, comprising administering to a subject an exon editor construct of any of the embodiments described above, a vector of any of the embodiments described above, a provirus plasmid of any of the embodiments described above, an AAV of any of the embodiments described above, or a composition of any of the embodiments described above.

[0030] A method for treating Rett syndrome in a subject requiring treatment is also disclosed, comprising administering to the subject an exon editor construct of any of the embodiments described above, a vector of any of the embodiments described above, a provirus plasmid of any of the embodiments described above, an AAV of any of the embodiments described above, or a composition of any of the embodiments described above.

[0031] 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 MeCP2 pre-mRNA containing at least one mutation associated with Rett syndrome in target cells is modified to become a transcript encoding functional MeCP2 in which 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 MeCP2 pre-mRNA containing at least one mutation associated with Rett syndrome in target cells is modified to become a transcript encoding functional MeCP2 in which 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 MeCP2 pre-mRNA containing at least one mutation associated with Rett syndrome in target cells is modified to become a transcript encoding functional MeCP2 in which at least one mutation is corrected. In some embodiments, the functional level of MeCP2 is restored in target cells by expressing a biologically functional MeCP2 protein. In some embodiments, the method comprises administering to the brain of a subject an exon editor construct of any one of embodiments 1-29 or 31-34, a vector of any one of embodiments 35-38, a proviral plasmid of embodiment 39, an AAV of any one of embodiments 40-45, or a composition of 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 has a genetic predisposition to Rett syndrome or has been diagnosed with Rett syndrome.

[0032] An exon editor construct of any of the embodiments described above, a vector of any of the embodiments described above, a provirus plasmid of any of the embodiments described above, an AAV of any of the embodiments described above, or a composition of any of the embodiments described above is also disclosed for use in the prevention or treatment of Rett syndrome in subjects requiring treatment.

[0033] An exon editor construct of any of the embodiments described above, a vector of any of the embodiments described above, a provirus plasmid of any of the embodiments described above, an AAV of any of the embodiments described above, or a composition of any of the embodiments described above are also disclosed for use in the manufacture of a drug for the treatment or prevention of Rett syndrome in subjects requiring treatment.

[0034] The exemplary RNA exon editors disclosed herein include the exon editors of listed embodiments 66 through 248. Exemplary constructs encoding the RNA exon editors disclosed herein include the exon editor constructs of listed embodiments 1 through 35 and 249 through 264. The exemplary vectors disclosed herein include the vectors of listed embodiments 36 through 39 and 265 through 268. The exemplary provirus plasmids disclosed herein include the provirus plasmids of listed embodiments 40 and 269. The exemplary AAVs disclosed herein include the AAVs of listed embodiments 41 through 46, 270, and 271. The exemplary compositions disclosed herein include the compositions of listed embodiments 47, 48, and 272. An exemplary method for expressing biologically active MeCP2 includes the methods of listed embodiments 49 to 53 and 273 to 275. An exemplary method for reducing the expression of MeCP2 containing one or more mutations associated with Rett syndrome includes the methods of listed embodiments 54 and 276. An exemplary method for correcting at least one mutation within the MeCP2 exon sequence includes the methods of listed embodiments 55 and 277. An exemplary method for treating Rett syndrome in subjects requiring treatment includes the methods of listed embodiments 56 to 63, 278, and 279. Brief explanation of the drawing

[0035] V. Brief Description of the Drawing Figure 1 is a schematic diagram depicting the frequency and location of missense mutations in MeCP2 associated with Rett syndrome (modified from Ehrhart, F., et. al., Scientific Data, 2021). The structural / functional domains of the MeCP2 protein include an N-terminal domain (NTD); a methyl-binding domain (MBD); an intervention domain (ID); a transcriptional repressive domain (TRD); and a C-terminal domain (CTD). The exon editor described herein corrects mutations in exons 3 and 4, which will treat approximately 95% of RTT patients. FIG. 2 depicts an exemplary embodiment of exon editing of MeCP2. A 3' exon editor (dark blue) is depicted bound to the target pre-mRNA of MeCP2. In the illustrated embodiment, trans-splicing between the target pre-mRNA and the exon editor can correct mutations in exons 3 and 4. Figure 3 depicts the genetic structure of human MeCP2. The genetic structure and sequence length of MeCP2 are depicted in exons 1-4 and introns 1-3. The 3' UTR (included within exon 4) differs among MeCP2 isoforms, so only the length of the coding sequence of exon 4 is shown. Figure 4 shows examples of MeCP2 E1 and E2 mRNA isoforms. The E1 isoform mRNA consists of exons 1, 3, and 4. The translation start codon for the translation of 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 the translation of the E2 isoform protein is located in exon 2, as indicated. Figure 5 depicts the domains of an exemplary 3' MeCP2 exon editor. Each domain of the 3' exon editor is indicated. Promoter: A sequence required to induce expression of the exon editor. Binding domain: A sequence antisense to the target pre-mRNA that recruits the exon editor to the target via base pairing. Hemi-intron: A sequence containing a fork, a poly-pyrimidine track, and a splice acceptor site. MeCP2 exon 3 and 4 coding domain sequence (CDS): The coding sequence of MeCP2 exons 3 and 4. Myc tag: An epitope tag used to detect the protein produced by exon editing. 3' untranslated region (UTR): A regulatory sequence. Terminator: A sequence for terminating the transcription of the exon editor. In some embodiments, expression of the RNA exon editor is induced by a CMV / CMV enhancer / promoter. In some embodiments, the RNA exon editor comprises a binding domain targeting MeCP2 intron 1, a hemi-intron, a splice site sequence, an alternate 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 hemi-intron, a splice site sequence, an alternate wild-type MeCP2 exon, and a 3' UTR. In some embodiments, the RNA exon editor comprises a binding domain targeting MeCP2 intron 1, a hemi-intron, a splice site sequence, a modified alternate wild-type MeCP2 exon, and a 3' UTR. In some embodiments, the 3' UTR is the original MeCP2 3' UTR or a truncated version thereof. In some embodiments, the 3' UTR is a heterogeneous 3' UTR (e.g., an artificial 3' UTR assembled through genetic engineering). Figure 6 depicts a scan of the MeCP2 intron 1 binding domain. The relative positions of the binding domain (depicted as a shape on a bar indicating the intron 1) along the length of the MeCP2 intron 1. Figure 7 shows a plot illustrating the identification of MeCP2 intron 1 hot spots. HEK293 cells were transfected with a MeCP2 intron 1-targeting RNA exon editor targeting various regions of intron 1. Cells were harvested 48 hours after transfection and evaluated for trans-splicing efficiency by RT-qPCR. The nomenclature for the binding domain is (nucleotide position in the intron at the 5' end start of the binding domain)_(length of the binding domain (nt)). For example, 51_150 indicates that the binding domain is the inverse complement sequence of bases 51-200 within intron 1, corresponding to position 51-200 of SEQ ID NO: 1. Position -50 indicates a 50-nucleotide position upstream of the exon 1-intron junction. The sequence identifiers of the tested binding domains are presented in Table 4. The various binding domains tested included cryptographic splice site-reduction nucleotide substitutions, as shown in Table 4. Figure 8 demonstrates the verification of the intron 1 binding domain. The exon editor selected from Figure 7 was re-tested by two individual operators. HEK293 cells were transfected with a MeCP2 intron 1-targeting RNA exon editor targeting various regions of intron 1. Cells were harvested 48 hours after transfection and evaluated for trans-splicing efficiency by RT-qPCR. The nomenclature for the binding domain is (nucleotide base position in the intron at the 5' end start position of the binding domain)_(length of the binding domain (nt)). For example, 51_150 indicates that the binding domain is the reverse complement sequence of bases 51-200 within intron 1. % substitution is calculated as the number of copies of edited MeCP2 mRNA / total MeCP2 mRNA x 100. The sequences of the tested binding domains are presented in Table 4. Figure 9 Summary of the binding domain scanning profile across MeCP2 intron 1. The percentages represent the trans-splicing efficiency of the RNA exon editor with binding domains at the indicated locations. Figure 10 illustrates a schematic diagram of an exemplary binding domain scanning profile across MeCP2 intron 2. The relative positions of the binding domains (depicted in green) are shown at the 5' and 3' ends of intron 2. Figure 11 shows a plot illustrating the activity of an exemplary MeCP2 intron 2 binding domain tested. HEK293 cells were transfected with a MeCP2 intron 2-targeting RNA exon editor targeting various regions of intron 2. Cells were harvested 48 hours after transfection and evaluated for trans-splicing efficiency by RT-qPCR. The nomenclature for the binding domain is (nucleotide base position in the intron at the 5' end start position of the binding domain)_(length of the binding domain (nt)). For example, 99_150 indicates that the binding domain is the inverse complement sequence of bases 99-248 within intron 2, corresponding to positions 99-248 of SEQ ID NO: 2. % substitution is calculated as the number of copies of edited MeCP2 mRNA (E2 isoform) / total MeCP2 RNA (E2 isoform) x 100. The sequence identifiers of the tested binding domains are presented in Table 5. Figure 12 shows a schematic diagram of the binding domain scanning profile across MeCP2 intron 2. The percentages represent the trans-splicing efficiency of the RNA exon editor having binding domains at the indicated locations. Figure 13 depicts an exemplary binding domain scanning profile across MeCP2 intron 3. The relative positions of the binding domains (depicted in green) are shown along the length of intron 3. Figure 14 shows a plot illustrating the activity of an exemplary MeCP2 intron 3 binding domain tested. HEK293 cells were transfected with a MeCP2 intron 3-targeting RNA exon editor targeting various regions of intron 3. Cells were harvested 48 hours after transfection and evaluated for trans-splicing efficiency by RT-qPCR. The nomenclature for the binding domain is (nucleotide base position in the intron at the 5' end start position of the binding domain)_(length of the binding domain (nt)). For example, 26_150 indicates that the binding domain is the inverse complement sequence of bases 26-175 within intron 3, corresponding to positions 26-175 of SEQ ID NO: 4. % substitution is calculated as the number of copies of edited MeCP2 mRNA / total MeCP2 RNA x 100. The sequence identifiers of the tested binding domains are presented in Table 7. Figure 15 shows a summary of the intron 3 binding domain performance. The percentages represent the trans-splicing efficiency of the RNA exon editor having the binding domain at the indicated positions. Figure 16 illustrates that specific 3' end modifications improve exon editor performance. Protein from edited mRNA (left) and exon editor expression (right) are plotted against an editor with the element indicated at the 3' end. The plot shows the performance of the editor against editor 1. HEK293 cells were transfected with the MeCP2 exon editor. Cells were harvested 48 hours after transfection and were evaluated for (1) protein produced from edited mRNA by Western blot, and (2) exon editor expression by RT-qPCR. Figure 17 illustrates relative MeCP2 exon editing in different cell types. HEK293 and U-251 MG cells were transfected with the MeCP2 exon editor as indicated. Cells were harvested 48 hours after transfection and trans-splicing efficiency was evaluated by RT-qPCR. % substitution is calculated as the number of copies of edited MeCP2 mRNA / total MeCP2 RNA x 100. Figure 18 shows the immunoblot of proteins produced from MeCP2-edited mRNA. U-251 cells were transfected with a full-length chimeric (edited) mRNA mimic or a plasmid encoding an exon editor. Cells were harvested 48 hours after transfection and analyzed by Western blot. Only exon editors with an active splice site produce proteins of the expected size. Figure 19 shows a plot illustrating the activity of exemplary MeCP2 intron 2 binding domains tested by two different operators. HEK293 cells were transfected with a MeCP2 intron 2-targeting RNA exon editor targeting various regions of intron 2. Cells were harvested 48 hours after transfection and evaluated for trans-splicing efficiency by RT-qPCR. The nomenclature for the binding domain is (nucleotide base position in the intron at the 5' end start position of the binding domain)_(length of the binding domain (nt)). For example, 99_150 indicates that the binding domain is the inverse complement sequence of bases 99-248 within intron 2, corresponding to positions 99-248 of SEQ ID NO: 2. The various binding domains tested included coding splice site-reducing nucleotide substitutions, as shown in Table 6. Figure 19 illustrates a comparison between a binding domain with a cryptic splice site-reduction nucleotide substitution (MeCP2_intron2_99_150SM (SEQN: 88)) and a binding domain without substitution (MeCP2_intron2_99_150 (SEQN: 55)). % substitution is calculated as the number of copies of edited MeCP2 mRNA (E2 isoform) / total MeCP2 RNA (E2 isoform) x 100. Specific details for implementing the invention

[0036] VI. Detailed Description

[0037] A. Introduction

[0038] The following examples are provided to illustrate some embodiments of the present disclosure, but are not intended to limit the scope of the present disclosure; it will be understood from their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may be used alternatively.

[0039] Rett syndrome (RTT) is a rare neurological disorder occurring in 1 in 10,000 female births worldwide. It is even rarer in boys. Diagnosis is typically made between 6 and 18 months of age when a child misses developmental milestones or begins to lose abilities they had already mastered. Rett syndrome is characterized by severe impairments related to speaking, walking, eating, and even breathing. A defining feature of Rett syndrome is persistent, repetitive hand movements. Symptoms may include seizures or Rett episodes, loss of speech, loss of intentional use of the hands, involuntary hand movements, loss of mobility, gait disturbances, loss of muscle tone, scoliosis, breathing problems, sleep disturbances, unsettled crying, and / or slow growth rates of the head, feet, and / or hands.

[0040] Rett syndrome is an X-linked neurodevelopmental disorder associated with severe motor abnormalities and reduced lifespan in a proportion of patients. Rett syndrome is caused by loss-of-function mutations in MECP2, a transcription regulator highly expressed in neurons. Inactivation mutations in MeCP2 alter the expression of many genes and ultimately lead to abnormalities in neuronal morphology and circuitry. MeCP2 There are over 900 mutations found in the gene, most of which are found in 8 different "hot spots." See Fig. 1. Rett syndrome presents with a wide range of impairments, from mild to severe. The course and severity of Rett syndrome are determined by the location, type, and severity of mutations and X-inactivations.

[0041] In addition to Rett syndrome, MeCP2 Mutations in genes can lead to other diseases, for example. MECP2 Overlapping syndrome, MECP2 - It is also associated with severe neonatal encephalopathy, pyramidal sign, parkinsonism, and macrochrysophagia (PPM-X) syndrome. MeCP2 There is no approved therapy to treat any of the diseases associated with gene mutations.

[0042] Gene therapy is for Rett syndrome and MeCP2 It holds great potential for treating other diseases related to gene mutations. However, the major barrier is MECP2 The doubling of gene copy numbers and corresponding gene expression levels induces a disease phenotype, creating a narrow therapeutic window for traditional gene replacement. This window can be further narrowed, for example, because half of the cells in patients with Rett syndrome express fully functional MeCP2 protein due to random X-chromosome inactivation. Therefore, it may not be possible with traditional gene replacement to deliver the appropriate MeCP2 to cells that need it while avoiding the overexpression phenotype in cells that do not need it.

[0043] Exon-editing molecules utilize RNA trans-splicing to excise and replace diseased exons. As described herein, a single exon-editor can correct the 3' portion of mRNA by substituting an intramolecular "cis" splicing reaction with an intermolecular "trans" splicing reaction (see Fig. 2). Exon-editing for the treatment of Rett syndrome offers at least two advantages. First, a single exon-editor targeting MeCP2 intron 1 or intron 2 can correct approximately 95% of MECP2 patient mutations, whereas a single exon-editor targeting MeCP2 intron 3 can correct approximately 75% of MECP2 patient mutations. Second, exon-editing therapy implicitly limits the maximum expression of the corrected mRNA to normal endogenous levels in each and all cells, regardless of promoter or vector dose. This intrinsic control occurs because the exon editor acts on the expressed pre-mRNA, correcting the target pre-mRNA transcript only when it is expressed. This degree of control may be essential for half of the rat patient cells that already express a precise amount of fully functional MECP2. In these cells, exon editing replaces one functional mRNA with another, whereas gene replacement therapy would result in undesirable, disease-induced overexpression of MECP2 mRNA.

[0044]

[0045] The compositions and methods described herein MeCP2 It includes trans-splicing molecules (e.g., pre-mRNA trans-splicing molecules) for treating diseases or disorders caused by mutations in genes. These mutations include point mutations and deletions, and the majority of these MeCP2It has been identified in exon 3 or exon 4 of the gene. See Fig. 1. The compositions and methods described herein are for gene therapy (e.g., to treat a disease caused by at least one mutation in MeCP2, e.g., Rett syndrome) (e.g., In vivo For gene therapy (e.g., when delivered by an adeno-associated virus), a nucleic acid trans-splicing molecule encoding an RNA trans-splicing molecule that targets pre-mRNA is used. The compositions and methods described herein are used in combination with other therapeutic agents described herein to treat Rett syndrome for gene therapy (e.g., In vivo For gene therapy, nucleic acid trans-splicing molecules encoding RNA trans-splicing molecules that target pre-mRNA are also used.

[0046] As described herein, Rett syndrome refers to a neurodevelopmental disorder associated with a mutation in the methyl CpG-binding protein 2 (MECP2) gene. Rett syndrome is a progressive neurodevelopmental disorder and is one of the most common causes of cognitive impairment in women.

[0047] MECP2 is a chromatin-associated protein capable of activating and repressing transcription. It is necessary for neuronal maturation and is developmentally regulated. The MeCP2 gene is a member of a family of nuclear proteins containing a methyl-CpG binding domain (MBD). MeCP2 can specifically bind to methylated CpGs and can also interact with other co-regulator complexes. MeCP2 is X-associated and processed by X-inactivation.

[0048] Exon replacement by MeCP2 pre-mRNA trans-splicing

[0049] Therapeutic RNA exon editors can potentially correct approximately 95% of rat patient mutations by replacing all exons in which approximately 95% of mutations occur, using a single MeCP2 RNA exon editor. The inventors have developed an exemplary MeCP2 RNA exon editor capable of replacing a significant portion (e.g., at least about 15%) of mutated MeCP2 proteins in cultured cells with biologically active, functional MeCP2 proteins. The MeCP2 RNA exon editor described herein comprises a coding domain sequence containing a sequence of multiple MeCP2 exons encoding a functional (e.g., wild-type) MeCP2 amino acid sequence, a splice domain sequence for splicing to natural MeCP2 pre-mRNA, and a binding domain sequence for annealing to the introns of natural MeCP2 pre-mRNA. This exemplary MeCP2 RNA exon editor In vivo It will be administered to animal subjects, and more than 15% of the mutated MeCP2 protein will be replaced with functional MeCP2 protein, and the RNA exon editor provides sufficient replacement of the mutated MeCP2 In vivo It is expected to achieve therapeutic effects. While not bound by theory, an exemplary MeCP2 RNA exon editor will confer therapeutic effects by achieving at least one of the following: the restoration of the functional level of transcriptional regulation, the restoration of the functional level of binding to methylated DNA, or the restoration of the functional level of interaction with the co-regulator complex, or any combination thereof.

[0050] B. Definition

[0051] As used herein, “trans-splicing” refers to binding a first RNA molecule containing one or more exons (e.g., exogenous exons or exons that are part of the coding domain of an RNA exon editor) to a second RNA molecule (e.g., endogenous pre-mRNA molecule) and replacing a portion of the second RNA molecule with a portion of the first RNA molecule through a splicosome-mediated mechanism. A general mechanism for an RNA trans-splicing reaction is illustrated, for example, in FIG. 2.

[0052] As used herein, the terms “RNA exon editor,” “pre-mRNA trans-splicing molecule,” “RNA trans-splicing molecule (RTM),” “nucleic acid trans-splicing molecule,” or “trans-splicing molecule” may be used interchangeably. In some embodiments, the RNA exon editor comprises three main elements: (a) a binding domain that confers specificity by tethering the trans-splicing molecule to its target gene (e.g., pre-mRNA); (b) a hemi-intron containing a splice site; and (c) a coding domain configured to be trans-spliced ​​to a target nucleic acid, which can replace one or more exons (e.g., an exon containing at least one mutation) in the target nucleic acid. The RNA exon editor is an effector molecule that physically binds to the target pre-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” may 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 comprising a DNA sequence encoding an RNA exon editor. In some embodiments, the DNA sequence encoding an RNA exon editor comprises cDNA as part of, for example, a functional exon (e.g., a functional MeCP2 exon) for the replacement of at least one MeCP2 exon containing a mutation in a target gene (e.g., pre-mRNA). In some cases, the sequence of the RNA exon editor component is disclosed herein as a DNA sequence. For any sequence disclosed herein as a DNA sequence, an RNA sequence in which each T in the sequence is substituted with U is also considered. Therefore, if a given sequence number is identified as having a sequence that can be included in an RNA exon editor, a version of the sequence number in which each T is substituted with U is also considered.

[0053] As used herein, “trans-splicing efficiency” refers to the ratio of the amount of DNA encoding the RNA exon editor or the introduced RNA exon editor (or reference molecule) to the detected expression level of the desired trans-spliced ​​RNA product (i.e., a chimeric RNA molecule containing functional exon(s) of the RNA exon editor operably linked to the endogenous target pre-mRNA generated by the RNA trans-splicing reaction). 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.

[0054] As used herein, "% RNA replacement" refers to the portion of the total target mRNA population that has undergone successful transsplicing (TS) and is calculated using the following equation: % Intra-target (ONT) TS = 100 * (ONT copy number / (ONT copy number + Natural copy number)).

[0055] As used herein, “relative trans-splicing efficiency” refers to the ratio of test trans-splicing efficiency to reference trans-splicing efficiency, wherein test trans-splicing efficiency is the trans-splicing efficiency of the RNA exon editor described herein (first RNA exon editor; test) and reference trans-splicing efficiency is the trans-splicing efficiency of the reference RNA exon editor (e.g., an RNA exon editor containing the same elements as the first RNA exon editor except that the binding domain is replaced with a scrambled binding domain or a non-targeted binding domain). The relative trans-splicing efficiency of the RNA exon editor may be given as the ratio (also known as a multiplier) of the efficiency of the first trans-splicing RNA (test) to the reference trans-splicing efficiency tested under similar conditions.

[0056] As used herein, the term "operably linked" or "operably linked" refers to an arrangement of elements, and the components described herein are configured to perform their general functions. A nucleic acid is "operably linked" to another nucleic acid sequence when it is placed in a functional relationship with another nucleic acid sequence. Elements do not need to be adjacent to be operable linked. Thus, for example, an intervening sequence may exist between operable linked sequences (for example, a binding domain and a coding sequence may be separated by an intervening sequence, and the binding domain is still considered "operably linked" to the coding sequence).

[0057] As used herein, the term “coding domain” refers to a nucleic acid sequence (e.g., RNA sequence, DNA sequence, or combination of RNA and DNA) that encodes a portion of a protein (e.g., a target protein for which a mutation is being corrected). Thus, 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 pre-mRNA) but are adjacent to each other (e.g., as cDNA). In some cases, a coding domain may include one or more introns (e.g., natural introns) or untranslated regions (UTR, e.g., natural UTR) located between exons or otherwise adjacent to exons (e.g., upstream or downstream of exons).

[0058] As used herein, "natural 3' MeCP2 untranslated region" or "natural 3' MeCP2 UTR" refers to a sequence of length exceeding 20 nucleotides that has at least 90% sequence identity with the region of the natural MeCP2 gene (e.g., human MeCP2 gene) at the 3' end of the translation termination codon. In some embodiments, the RNA exon editor includes the natural 3' MeCP2 untranslated region. The natural 3' MeCP2 untranslated region includes alternative poly-A signals ranging from 0.1 kb to 8.5 kb of the MeCP2 3' UTR, corresponding to various lengths of the natural MeCP2 3' UTR that are differentially expressed in different tissues. The 8 kb 3' UTR is abundant in brain tissue compared to the shorter MeCP2 3' UTR. In some embodiments, the natural 3' MeCP2 UTR is replaced by a shorter length 3' UTR. In some embodiments, the 3' UTR is the RDH1pA 3' UTR, which is a synthetic 3' UTR comprising a highly conserved MeCP2 distal polyadenylation signal of 110 bp and an upstream miRNA-binding panel comprising 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 comprises SEQ ID NO: 19.

[0059] As used herein, "functional sequence of 3' MeCP2 exons" refers to a nucleic acid sequence comprising one or more of MeCP2 exons 2, 3, and 4 (e.g., exon 3 and exon 4) that encode the functional (biologically active) portion of the MeCP2 protein. In some embodiments, "functional sequence of 3' MeCP2 exons" refers to a nucleic acid sequence comprising exons 3-4 of MeCP2 that encode the functional (biologically active) portion of the MeCP2 protein. In some embodiments, "functional sequence of 3' MeCP2 exons" refers to a nucleic acid sequence comprising exons 2-4 of MeCP2 that encode the functional (biologically active) portion of the MeCP2 protein. In some embodiments, "functional sequence of 3' MeCP2 exons" refers to a nucleic acid sequence comprising exon 4 of MeCP2 that encodes the functional (biologically active) portion of the MeCP2 protein. When trans-spliced ​​to the binding site with endogenous MeCP2 exon 5', the functional sequence of the 3' MeCP2 exon provides for the expression of a functional MeCP2 protein (e.g., a non-mutated MeCP2 protein). In some cases, the functional sequence of the 3' MeCP2 exon includes the sequence of an exon adjacent to the exon being trans-spliced ​​by the RNA exon editor (for example, an RNA exon editor that binds MeCP2 intron 1 and trans-splices with endogenous MeCP2 exon 1 may include the functional sequence of a 3' MeCP2 exon containing exons 3 and 4).

[0060] As used herein, the term "functional," when used in the context of proteins, refers to a biologically active protein. The term "functional" may also be used to refer to the amount of protein activity required to support normal cellular function. With respect to MeCP2, the term "functional" may be used to refer to the amount of MeCP2 protein activity required to restore MeCP2 activity levels to support normal cellular function, for example, in the context of neurons within the brain. Such levels are sufficient to reduce or prevent the disruption of gene expression, neuronal development, synaptic maturation, plasticity, and / or the manifestation of other diseases associated with reduced levels of MeCP2 activity. More specifically, because defective (non-functional) MeCP2 proteins containing one or more mutations cause synaptic and circuit-level defects in brain function, restoring the functional level of MeCP2 refers to an increase in biologically active MeCP2 protein to reduce, at least in part, the amount or degree of defects in brain function associated with defects in gene expression, synaptic defects, circuit-level defects, and / or reduced levels of MeCP2 activity. Without being bound by theory, the term “functional” may be used to refer to the amount of MeCP2 protein activity required to achieve at least one of the restoration of the functional level of transcriptional regulation, the restoration of the functional level of binding to methylated DNA, or the restoration of the functional level of interaction with co-regulator complexes, or any combination thereof.

[0061] In the context of the treatment of a pathological condition associated with pathogenic MeCP2 activity (e.g., Rett syndrome) or the use of a therapeutic agent comprising the RNA exon editor or a DNA sequence encoding the RNA exon editor described herein, “functional” refers to restoring an amount of functional (biologically active) MeCP2 protein sufficient to reduce or eliminate one or more symptoms of a pathological condition associated with a defective (non-functional) MeCP2 protein (e.g., Rett syndrome) comprising at least one mutation and / or reduced level of MeCP2 activity. In some embodiments, such methods or use lead to an increase in MeCP2 protein activity. In some embodiments, this increase in MeCP2 protein activity increases the level of functional (biologically active) 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%) compared to that of control (normal / wild-type) cells in which functional (biologically active) MeCP2 is expressed at a level supporting normal cellular function. In some embodiments, this increase in MeCP2 protein activity 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%) compared to that of cells in which MeCP2 is present at normal, wild-type levels, such as those in cells expressing non-mutated MeCP2.

[0062] As used herein, “code splice site corrected,” “code splice site reduced,” or “code splice site resistant” refers to an RNA exon editor or a nucleic acid molecule encoding an RNA exon editor, or a part of either one thereof (e.g., a coding domain sequence therein), modified to change individual nucleotides within it to reduce the frequency of splicing occurring at a code splice site identified in the context of an RNA exon editor. In some embodiments, the modification does not result in any change in the amino acid sequence encoded by it. In some embodiments, the code splice site resistant nucleic acid sequence within the RNA exon editor is a coding domain sequence (CDS). In some embodiments, the coding splice site-resistant MeCP2 CDS comprises exon 3 and exon 4 or exon 4 of the MeCP2 gene, is essentially composed of, or is composed of, wherein the coding splice sites are identified in the context of a MeCP2 RNA exon editor and at least one of the coding splice sites is modified to reduce the frequency of splicing at at least one site, but the amino acid encoded by is not changed.

[0063] As used herein, “hemi-intron” refers to a nucleic acid sequence containing a motif recognized by a splicosome and mediating trans-splicing. In some embodiments, the hemi-intron comprises a polypyrimidine track (pPy), a fork (BP), and / or a 3’ splice site, or any combination thereof. For example, see SEQ ID NO: 17 as an exemplary hemi-intron. The 3’ splice site may be a common 3’ splice site. In some embodiments, the hemi-intron comprises other regulatory elements having one or more of the following effects: increased recruitment of splicing factors to an RNA exon editor, increased transcription / expression of functional MeCP2, increased in-target (ONT) trans-splicing efficiency, decreased out-target (OFT) trans-splicing, or decreased cis-splicing on target pre-mRNA, or any combination thereof.

[0064] As used herein, the “binding domain” of an RNA exon editor or a nucleic acid molecule encoding it is a polynucleotide sequence that binds a target gene (e.g., endogenous pre-mRNA) to a binding site through hybridization (i.e., complete or partial complementarity to the binding site).

[0065] As used herein, the term “binding site” refers to the endogenous polynucleotide sequence of a target pre-mRNA (e.g., pre-mRNA of an endogenous gene, e.g., MeCP2 pre-mRNA) that is bound by the binding domain of an RNA exon editor. The binding site extends from the 5’-shortest nucleotide bound by the binding domain to the 3’-shortest nucleotide bound by the binding domain. In some embodiments, the binding site is the same length as the binding domain. In some embodiments, the binding site is 1 to 10 nucleotides longer or shorter than the binding domain (i.e., some nucleotides of the binding site or the binding domain are not hybridized). In an embodiment comprising 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.

[0066] As used herein, “complementary,” and its grammatical variations refer to the percentage of nucleotide bases of a given sequence that are paired with a reference sequence through hydrogen bonding. Where no percentage of complementary is given, the terms “complement” and “complementary” refer to 100% complementary.

[0067] As used herein, a given sequence (e.g., a binding domain sequence) is "100% complementary" or "100% complementary" to a reference sequence (e.g., an endogenous pre-mRNA binding site) when each nucleotide base of the given sequence pairs with the reference sequence through hydrogen bonds and hybridizes accordingly to form a double-stranded sequence (e.g., through Watson-Crick base pairs, e.g., each A pairs with T or U and each C pairs with G). For example, a binding domain that is in the antisense direction with respect to the binding site is complementary to the binding site. RNA pairs contain G pairs paired with U; therefore, an RNA binding domain having GU pairs as its binding site can be 100% complementary to the binding site. Thus, a binding domain that is exactly the anti-complement of 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 with G without substantially affecting binding.

[0068] As used herein, a given sequence (e.g., a binding domain sequence) is at least X% complementary to or has "X% complementary" to a reference sequence (e.g., an endogenous pre-mRNA binding site) when X% of the nucleotide bases of the given sequence are paired with the reference sequence via hydrogen bonds, e.g., when hybridized to form a double-stranded sequence (e.g., via Watson-Crick base pairs, e.g., A is paired with T or U and C is paired with G). For example, a binding domain sequence having a length of 150 bases is at least 90% complementary to a binding site having a length of 150 bases when at least 135 of its 150 residues are paired with the binding site via hydrogen bonds via Watson-Crick base pairs, leaving 15 or fewer mismatched nucleotides.

[0069] The “bonding” between the binding domain and the intron, as used herein, refers to hydrogen bonding (e.g., double helix formation, or Watson-Crick weaving) between the binding domain and, for example, the target intron to an extent sufficient to mediate trans-splicing by associating the RNA exon editor with a target (e.g., pre-mRNA). In some embodiments, the hydrogen bonding between the binding domain and the target intron is between nucleotide bases that are complementary to each other and oriented in the antisense direction (e.g., hybridizing to each other).

[0070] As used herein, the term “mutation” may be used to refer to any abnormal nucleic acid sequence encoding a defective protein product (e.g., non-functional protein product, abiotically active protein, protein product with reduced function, protein product with pathogenic or abnormal function, and / or protein product produced in smaller or larger quantities than normal). Mutations include base pair mutations (e.g., single nucleotide polymorphisms), duplication, missense mutations, frameshift mutations, deletions, insertions, and splice mutations. In some embodiments, a mutation refers to a nucleic acid sequence that differs from the corresponding wild-type nucleic acid sequence or its functional variant in one or more parts of the sequence. In some embodiments, a mutation refers to a nucleic acid sequence encoding a protein having an amino acid sequence different from the corresponding wild-type protein or its functional variant. "A mutated exon (e.g., a mutated MeCP2 exon) refers to an exon containing an exon sequence or mutation that reflects a mutation in a different region, such as a coding exon resulting from a mutation in an intron.

[0071] The term "MeCP2 (methyl CpG-binding protein 2)" refers to any natural 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), unless otherwise indicated, as well as functionally equivalent or improved variants thereof (e.g., natural or synthetic variants), mutants, mutains, analogs, subunits, receptor complexes, isoforms, splice variants, and fragments. Functionally equivalent and improved variants may be determined based on known MeCP2 signaling. MeCP2 includes full-length, untreated MeCP2, as well as any form of MeCP2 derived from natural processing in cells. Exemplary human MeCP2 sequences are provided as National Center for Biotechnology Information (NCBI) reference sequences: NM_001110792.2 (specific to the E1 isoform lacking exon 2) or NM_004992.4 (specific to the E2 isoform containing exons 1-4). In some cases, the MeCP2 fragment is encoded by a therapeutic agent comprising a sequence or fragment thereof having at least 95% sequence identity with any one of SEQ ID NO: 10, 11, or 12, or any combination thereof (e.g., an exon 3 and exon 4 sequence or a cassette of exon 2, 3, and 4 sequence) or SEQ ID NO: 9 (e.g., SEQ ID NO: 10, 11, or 12, or any combination thereof (e.g., a cassette of exon 3 and exon 4 sequence)) or SEQ ID NO: 9, having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 9, or a functional portion thereof, and / or a codon-modified variant thereof.Exemplary such codon-modified variants include codon-modified variants of exon 3 (e.g., SEQ ID NOs. 13 and 85) and codon-modified variants of exon 4 (e.g., SEQ ID NOs. 14 and 86) and combinations thereof (e.g., a cassette 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).

[0072] As used herein, “variant” refers to a polynucleotide that differs from a reference polynucleotide sequence in that at least one nucleic acid residue is different from a naturally occurring polynucleotide sequence, such as a naturally occurring polypeptide sequence or, for example, any of the rAAV sequences described herein, a polypeptide that differs from a reference polypeptide sequence in that at least one amino acid residue is different (e.g., an AAV capsid sequence). In this context, the difference of at least one residue may include, for example, substitution, deletion, or insertion of a nucleic acid residue with another nucleic acid, or substitution of an amino acid residue with another amino acid. The variant may be a homologue, isoform, or transcript variant of a polynucleotide as defined herein, wherein the homologue, isoform, or transcript variant is characterized by a degree of identity or homology as defined herein. In some cases, variants of polynucleotides or polypeptides comprise 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, e.g., 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 resulting in an expressed polypeptide having amino acids exchanged from the same class are referred herein as conservative substitutions. In particular, these are amino acids having an aliphatic side chain, a positively or negatively charged side chain, or an aromatic group in the side chain, and their side chains may form a hydrogen bridge, for example, their side chains have a hydroxyl function.By conservative substitution, for example, an amino acid having a polar side chain may be replaced by another amino acid having a corresponding polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain may be replaced by another amino acid having a corresponding hydrophobic side chain (e.g., serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine)).

[0073] In some cases, insertions, deletions, and / or non-conservative substitutions are also included in the term variant, for example, modifications at positions that do not cause substantial changes to the three-dimensional structure of the protein. Changes to the three-dimensional structure caused by insertions(s) or deletions can be easily determined by a person skilled in the art, for example, using a CD spectrum (circular dichroism spectrum).

[0074] The term “homology” refers to the degree of identity between the sequences of two nucleic acid sequences. Homology of sequences is determined by comparing two sequences aligned under standard conditions over the length of the sequences to be compared. The sequences to be compared herein may have additions or deletions (e.g., gaps and others) in the optimal alignment of the two sequences. In some embodiments, 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, GENETYX, BLAST, or analysis tools provided by public databases may also be used.

[0075] As used herein, the term “heterogeneous” indicates that when used to describe the first element in relation to the second element, the first element and the second element are not present in the properties arranged as described. For example, a heterogeneous nucleic acid sequence (e.g., a heterogeneous 3’ UTR) may refer to a nucleic acid sequence included in the composition of the present disclosure that is operably linked to a nucleic acid sequence that is not operably linked in nature.

[0076] As used herein, the terms "AAV" or "AAV serotype" refer to dozens of naturally occurring and available adeno-associated viruses, as well as artificial AAVs. Among the AAVs isolated from or engineered from human or non-human primates (NHPs) and widely characterized, human AAV2 was the first AAV developed as a gene transfer vector; it has been widely used for efficient gene transfer experiments in different target tissues and animal models.

[0077] As used herein with respect to AAV, the term variant means any AAV sequence derived from a known AAV sequence, including sharing 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 more of sequence identity over an amino acid or nucleic acid sequence. In another embodiment, the AAV capsid comprises a variant that may include up to about 10% variation from 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 the AAV capsid provided herein and / or known in the art. In some embodiments, the AAV capsid shares at least 95% identity with the AAV capsid. When determining the percentage identity of the AAV capsid, comparisons may be performed across any variable protein (e.g., vp1, vp2, or vp3).

[0078] ITR or other AAV components can be easily isolated or manipulated from AAV using techniques available to those skilled in the art. Such AAV may be isolated, manipulated, or obtained from academic, commercial, or public sources (e.g., American Type Culture Collection, Manassas, Virginia, USA). Alternatively, AAV sequences may be manipulated by synthesis or other suitable means by reference to published sequences, which are available, for example, in the literature or in databases such as GenBank, PubMed, or others. AAV viruses can be manipulated by conventional molecular biology techniques, making it possible to optimize these particles for cell-specific delivery of nucleic acid sequences, to minimize immunogenicity, to tune stability and particle lifetime, for efficient degradation, for precise delivery to the nucleus, etc.

[0079] As used herein, the terms “subject,” “individual,” or “patient” include any mammal requiring these treatment or prevention methods, including primates such as humans. Other mammals requiring such treatment or prevention include non-human primates (NHPs; e.g., cynomolgus monkeys and African green monkeys), dogs, cats, or other domesticated animals, horses, livestock, laboratory animals, etc. The individual may be male or female. In one embodiment, the individual has a disease or disorder 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 disorder caused by a mutation in the MeCP2 gene. In another embodiment, the individual has exhibited clinical signs of a disease or disorder caused by a mutation in the MeCP2 gene, e.g., Rett syndrome. The individual may be of any age for which treatment or preventive therapy may be beneficial. For example, in some embodiments, the individual is 0-5 years, 5-10 years, 10-20 years, 20-30 years, 30-40 years, 30-50 years, 40-50 years, 50-60 years, or 60-70 years, or over 70 years.

[0080] As used herein, the terms “disorder associated with mutation” or “disorder associated with mutation” refer to a correlation between a disorder and a mutation. In some embodiments, a disorder associated with mutation is known or suspected to be caused by a mutation wholly or partially, or directly or indirectly. For example, an individual with a mutation may be at risk of developing a disorder, and the risk may additionally depend on other factors, such as other (e.g., independent) mutations (e.g., in the same or different genes), or environmental factors.

[0081] As used herein, the term “therapeutic,” or its grammatical derivatives, is defined as reducing the progression of a disease, reducing the severity of disease symptoms, delaying the progression of disease symptoms, eliminating disease symptoms, or delaying the onset of a disease. In some embodiments, the term “therapeutic” is used to refer to a sustained or enduring effect of a therapeutic agent, such as the RNA exon editor described herein.

[0082] As used herein, the term “prevention” of a disorder, or its grammatical derivatives, is defined as reducing the risk of disease onset, for example, as a prophylactic treatment for an individual at risk of developing a disorder associated with a mutation. An individual may be characterized as “at risk” of developing a disorder by identifying a mutation associated with the disorder according to any suitable method known in the art or described herein. In some embodiments, an individual at risk of developing a disorder has one or more MeCP2 mutations associated with the disorder. Additionally, or alternatively, if an individual has a family history of the disorder, the individual may be characterized as “at risk” of developing the disorder.

[0083] MeCP2 disorders are inherited in an X-linked manner. More than 99% of MeCP2 disorders are De Novo It is a simple case (i.e., a single occurrence in the family) likely resulting from the pathogenic variant or the inheritance of a pathogenic variant from a parent with germline mosaicism. In rare cases, the MeCP2 variant can be inherited from a heterozygous mother in whom X-chromosome inactivation results in minimal or no clinical manifestations. For known heterozygous mothers, the risk for children inheriting the MeCP2 variant is 50%.

[0084] Treating or preventing a disease in an individual may be performed by administering DNA encoding an RNA exon editor (e.g., via a vector containing a DNA sequence encoding an RNA exon editor) or by administering the RNA exon editor directly to the individual. In some embodiments, a vector containing a DNA sequence encoding an RNA exon editor comprises an AAV vector. In some embodiments, the AAV vector is administered via an AAV virus particle. Alternatively, a host cell containing an RNA exon editor may be administered to the individual.

[0085] The term “administering” or its grammatical derivatives refer to delivering an RNA exon editor or a DNA sequence encoding it (e.g., within a vector, e.g., an AAV vector or an AAV particle) or a composition thereof, or extracellularly treated cells, to an individual requiring treatment, e.g., an individual having a mutation or defect in MeCP2, as used in the methods described herein. In some embodiments, the brain is targeted for delivery of the RNA exon editor or a DNA sequence encoding it (e.g., within a vector, e.g., an AAV vector or an AAV particle) or a composition thereof. In some embodiments, the brain cells targeted for delivery include, e.g., neurons, neural stem cells, astrocytes, or oligodendrocytes, or any combination thereof.

[0086] In some embodiments thereof, the method comprises delivering an RNA exon editor or a DNA sequence encoding it (e.g., within a vector, e.g., an AAV vector or an AAV particle) or a composition thereof to an individual by intravenous (IV) delivery, intracerebral (IC) delivery (e.g., slow delivery injection or convection-enhanced diffusion injection), intraventricular (ICV) delivery, intraspinal delivery, or intracerebral delivery. In some embodiments thereof, neurons, neural stem cells, astrocytes, or oligodendrocytes, or any combination thereof, are targeted. In some embodiments, IC injection involves the stereocoordinate implantation of a microinjection guide sleeve to improve delivery to a specific locus in the brain. In some embodiments, the composition is administered systemically (e.g., intravenously). Considering the present disclosure, a person skilled in the art may still select other methods of administration.

[0087] As used herein, "regulating the expression of MeCP2" refers to increasing the expression of the functional MeCP2 protein generated after trans-splicing. The increase in the expression of the functional MeCP2 protein generated after trans-splicing is associated with a decrease in the expression of the endogenous mutated (non-functional) MeCP2. Regulating the expression of MeCP2 may be used to refer to increasing the expression of the MeCP2 protein generated after trans-splicing (e.g., a MeCP2 transcript or protein product having an RNA exon editor-mediated corrected mutant site relative to its endogenous mutated transcript or protein product). Regulating the expression of MeCP2 may also be used to refer to decreasing the expression of the endogenous (e.g. mutated) MeCP2. Upon replacement of endogenous MeCP2 exons containing at least one mutation via trans-splicing, the functional MeCP2 protein is expressed.

[0088] As used herein, “codon optimization” refers to modifying a nucleic acid sequence to alter individual nucleic acids without any resulting changes in the encoded amino acids. A sequence modified in this manner is referred to herein as “codon-optimized.” This process may be performed on any sequence described herein to enhance expression or stability. Codon optimization may be performed in the manner described, for example, in the manner described in U.S. Patents No. 7,561,972, 7,561,973, and 7,888,112, each of which is incorporated herein by reference in its entirety. Sequences surrounding the translation start site may be converted into common Kozak sequences according to known methods. For example, see Kozak et al, 1987. Nucleic Acids Res. 15 (20): 8125-8148, which is incorporated herein by reference in its entirety.

[0089] The term "pharmaceuticalally acceptable" means safe for administration to mammals, e.g., humans. In some embodiments, pharmaceutically acceptable compositions are approved by federal or state regulatory agencies for use in animals, more particularly in humans, or are listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias.

[0090] The term “carrier” refers to a diluent, ajuvant, excipient, or vehicle to which a therapeutic molecule (e.g., a trans-splicing molecule or a trans-splicing molecule comprising a vector or cell of the present disclosure) is administered. Examples of suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA., 18th edition.

[0091] The term "one" means "one or more." For example, "gene" is understood to refer to one or more such genes. Accordingly, the terms "one," "one or more," and "at least one" are used interchangeably herein.

[0092] As used herein, the term "approximately" refers to a value within ±10% variability from a reference value unless otherwise specified.

[0093] C. RNA Exon Editor

[0094] An RNA exon editor useful for correcting mutations in MeCP2 by replacing at least one MeCP2 exon containing at least one mutation with a functional MeCP2 exon (e.g., MeCP2 exon 3' for the binding site of MeCP2, e.g., exon 3-4, exon 2-4, or exon 4) and a DNA sequence encoding the same (e.g., a vector containing a DNA sequence encoding the RNA exon editor or an AAV virus particle containing a DNA sequence encoding the RNA exon editor) are provided herein. In some embodiments, the RNA exon editor is referred to as a pre-RNA trans-splicing molecule (RTM). The design of the RNA exon editor allows for replacing a defective or mutated portion of a pre-mRNA exon(s) with a nucleic acid sequence, e.g., an exon(s) having a functional (e.g., normal) sequence without mutations. The functional sequence may be some other variation, for example, a wild-type, naturally occurring sequence with codon optimization, or a corrected sequence.

[0095] In some embodiments, the RNA exon editor comprises a binding domain, a hemi-intron, and a coding domain. In some embodiments, the RNA exon editor comprises a binding domain, a splice site, and a coding domain. In some embodiments, the RNA exon editor has a 3' regulatory domain comprising a natural 3' MeCP2 untranslated region (UTR) or a heterogeneous 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' splice site of YAG / [wherein Y is pyrimidine (cytosine or thymidine (which may be uracil in RNA)); and " / " indicates the location of the 3' splice site; e.g., TAG]. In some embodiments, the RNA exon editor comprises a sequence having at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with SEQ ID NO: 17, or comprises a hemi-intron that is essentially composed of or composed of such a sequence. In some embodiments, the RNA exon editor further comprises at least one of an SV40 termination and poly-A sequence (e.g., SEQ ID NO: 21) or a MALAT1 triple helix termination sequence (e.g., SEQ ID NO: 22), or a combination thereof. See, for example, FIG. 5 and 16.

[0096] In some embodiments, the RNA exon editor described herein comprises a binding domain, a hemi-intron, a coding domain sequence (e.g., a CDS, for example, a sequence encoding the functional sequence of a MeCP2 exon, for example, the functional sequence of MeCP2 exon 3' for the binding site), and a 3' untranslated region, operably connected in the 5' to 3' direction. In some embodiments, the RNA exon editor described herein comprises a binding domain, a hemi-intron, a coding domain sequence (e.g., a CDS, for example, a sequence encoding the functional sequence of a MeCP2 exon, for example, the functional sequence of MeCP2 exon 3' for the binding site), a 3' untranslated region, a termination and a poly-A sequence, and a triple helix terminator, operably connected in the 5' to 3' direction.

[0097] In some embodiments, the RNA exon editor described herein is configured to correct at least one mutation (e.g., two different mutations, where each mutation is present in a single exon or in two different exons of the MeCP2 gene within the subject) located in the 3' region of the MeCP2 gene (e.g., the region at 3' of intron 1) by binding to target intron 1 and mediating the trans-splicing of a coding domain having the functional sequence of the 3' MeCP2 exon to an endogenous MeCP2 exon at 5' of the target intron. This trans-splicing thus repairs the defective MeCP2 gene in the subject's target cells by replacing the defective exons / s and removing the defective portion of the target pre-mRNA, thereby yielding functional MeCP2 mRNA capable of transcribing a functional MeCP2 protein in the cells.

[0098] 1. MeCP2

[0099] 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 by the National Center for Biotechnology Information (NCBI): NM_001110792.2 (specific to the E1 isoform) or NM_004992.4 (specific to the E2 isoform). In addition to the disclosed sequences, all subsequently obtained corrections or naturally occurring conservative and non-disease-inducing variant sequences occurring in humans or other mammalian populations are also included. Those causing additional conservative nucleotide substitutions or codon optimizations are also included. Sequences provided by database accession numbers may also be used to search for homologous sequences in the same or another mammalian organism.

[0100] The MeCP2 protein contains several well-defined structural / functional domains as follows: the N-terminal domain (NTD); the methyl binding domain (MBD); the intervention domain (ID); the transcription repressive domain (TRD); the NCoR interaction domain (NID); and the C-terminal domain (CTD) (see Fig. 1). The TRD contains the nuclear localization sequence (NLS). Genetic mutations in the coding region of the X-chromosome-associated MeCP2 gene alter the ability of its encoded protein, MeCP2, to bind DNA within the context of chromatin. Mutations affecting the MBD of MeCP2 affect the stability and affinity of its DNA binding.

[0101] It is expected that the MeCP2 nucleic acid sequence and the expressed resulting protein may allow for specific minor modifications at the nucleic acid level, such as modifications to silenced nucleotide bases, for example, to preferred codons. In other embodiments, nucleic acid base modifications that alter amino acids to improve the expression of the resulting peptide / protein are envisioned, for example. In some embodiments, modifications of allelic fluctuations caused by the natural degeneracy of the genetic code are envisioned.

[0102] Analogues or modified versions of the encoded amino acid sequence are also included as modifications of the MeCP2 gene. Typically, these analogues differ from the specifically identified protein by only one to four codon changes. Conservative substitutions occur within the amino acid family related in their side chains and chemical properties.

[0103] The nucleic acid sequence of the functional MeCP2 gene may be derived from any mammal that naturally expresses functional MeCP2 or its homologues. In other embodiments, specific modifications are made to the MeCP2 gene sequence to enhance expression in target cells. These modifications include codon optimization.

[0104] As described in the above, Rett syndrome is caused by mutations in the MeCP2 gene. Since the RNA exon editor replaces the entirety of exons 3 and 4 of the MeCP2 gene, the composition comprising the RNA exon editor described herein can correct about 95% of the mutations associated with Rett syndrome, and about 95% of the mutations identified here are found.

[0105] The attached sequence list provides various sequences of human MeCP2. Sequence No. 1 is a partial nucleotide sequence of wild-type MeCP2 intron 1 starting at position 1 of intron 1. Sequence No. 113 is a complete nucleotide sequence of wild-type MeCP2 intron 1. Sequence No. 2 is a partial nucleotide sequence of wild-type MeCP2 intron 2 starting at position 1 of intron 2. Sequence No. 112 is a complete nucleotide sequence of wild-type MeCP2 intron 2. Sequence No. 3 is a partial nucleotide sequence of wild-type MeCP2 intron 2 ending at the final nucleotide of intron 2. Sequence No. 4 is a complete nucleotide sequence of wild-type MeCP2 intron 3. Sequence No. 5 is a nucleotide sequence of an open read frame of wild-type MeCP2 isoform E1. Sequence No. 6 is the sequence of an open read frame of wild-type MeCP2 E1 exon 1. Sequence No. 7 is the sequence of wild-type MeCP2 E1 exon 3. Sequence No. 7 is the sequence of wild-type MeCP2 E1 exon 4. Sequence No. 9 is the nucleotide sequence of an open read frame of wild-type MeCP2 isoform E2. Sequence No. 10 is the sequence of an open read frame of wild-type MeCP2 E2 exon 2. Sequence No. 11 is the sequence of wild-type MeCP2 E2 exon 3. Sequence No. 7 is the sequence of wild-type MeCP2 E2 exon 4.

[0106] 2. Coding Domain

[0107] In some embodiments, the coding domain of the 3' trans-splicing molecule comprises 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 comprise functional MeCP2 exons 3-4. In some embodiments, functional MeCP2 exons 3-4 are encoded by a sequence comprising SEQ ID NOs: 11 and 12 or a sequence having at least 95% sequence identity with the sequence comprising SEQ ID NOs: 11 and 12. In some embodiments where the 3' trans-splicing molecule targets MeCP2 intron 1, the coding domain may comprise functional MeCP2 exons 2-4. In some embodiments, functional MeCP2 exons 2-4 are encoded by a sequence comprising SEQ ID NO: 9 or a sequence having at least 95% sequence identity with SEQ ID NO: 9 or a fragment thereof. In any sequence disclosed herein as a coding domain sequence for an RNA trans-splicing molecule, U may substitute T in the sequence. Thus, for example, in some embodiments, functional MeCP2 exons 2-4 comprise an RNA version of SEQ ID NO: 9 having U substituted for each T of SEQ ID NO: 9 or a sequence having at least 95% sequence identity with SEQ ID NO: 9 having U substituted for each T of SEQ ID NO: 9. 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 a sequence comprising SEQ ID NOs. 11 and 12 or a sequence having at least 95% sequence identity with a sequence comprising SEQ ID NOs. 11 and 12. In some embodiments, a binding domain binds to intron 3, and a 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 comprises a codon-modified variant of exon 3 (e.g., SEQ ID NO: 13 or 85) and a codon-modified variant of exon 4 (e.g., SEQ ID NO: 14 or 86) and a combination thereof (e.g., a cassette of codon-modified variants of exon 3 and exon 4 sequences; SEQ ID NO: 76). In some embodiments, the functional MeCP2 exon comprises a codon-modified variant of exon 4 (e.g., SEQ ID NO: 14).

[0108] In some embodiments, the coding domain-encoding sequence (e.g., of a transplant gene encoding an RNA exon editor) comprises cDNA of MeCP2 exons (e.g., MeCP2 exons 3 to 4) for replacement of a mutated MeCP2 exon. For example, one or more functional MeCP2 exons within the coding domain may be cDNA sequences. In some embodiments, the entire coding domain is a cDNA sequence. Additionally, or alternatively, all or part of the coding domain, or one or more of its functional MeCP2 exons, may be naturally occurring sequences (e.g., sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with endogenous MeCP2 exons).

[0109] In some embodiments, all or part of the coding domain or the coding domain-encoding sequence, or one or more functional MeCP2 exons thereof, is a codon-optimized sequence in which the nucleic acid sequence is modified to improve, for example, expression or stability, without causing a change in the encoded amino acid. Codon optimization may be performed in the manner described, for example, in U.S. Patents No. 7,561,972, No. 7,561,973, and No. 7,888,112, each of which is incorporated herein by reference in its entirety. A codon-optimized version of MeCP2 exon 3 is presented in SEQ ID NO: 13. A codon-optimized version of MeCP2 exon 4 is presented in SEQ ID NO: 14.For delivery via recombinant AAV, as described herein, in one embodiment, the coding domain is up to 4,000 nucleotide base lengths (e.g., 3,000 to 4,000 nucleotide base lengths, 3,100 to 3,800 nucleotide base lengths, 3,200 to 3,700 nucleotide base lengths, or 3,300 to 3,500 nucleotide base lengths, e.g., 3,000 to 3,100 nucleotide base lengths, 3,100 to 3,200 nucleotide base lengths, 3,200 to 3,300 nucleotide base lengths, 3,300 to 3,400 nucleotide base lengths, 3,400 to 3,500 nucleotide base lengths, 3,500 to 3,600 nucleotide base lengths, It may be a nucleic acid sequence of 3,600 to 3,700 nucleotide base lengths, 3,700 to 3,800 nucleotide base lengths, 3,800 to 3,900 nucleotide base lengths, or 3,900 to 4,000 nucleotide base lengths, for example, about 3,108 nucleotide base lengths, about 3,285 nucleotide base lengths, about 3,375 nucleotide base lengths, about 3,503 nucleotide base lengths, about 3,630 nucleotide base lengths, about 3,540 nucleotide base lengths, about 3,363 nucleotide base lengths, about 3,273 nucleotide base lengths, about 3,145 nucleotide base lengths, or about 3,018 nucleotide base lengths.

[0110] In some embodiments, an RNA exon editor is described herein comprising (a) a binding domain sequence configured to bind to an intron of an endogenous RNA molecule in the 5' to 3' direction; (b) a splice acceptor sequence; and (c) a cDNA coding domain sequence; wherein the coding domain sequence comprises at least one nucleotide mutation relative to the endogenous RNA molecule sequence, and wherein at least one nucleotide change disrupts a coding splice site within the coding domain sequence. In some embodiments, the nucleotide change is a synonymous nucleotide change. In some embodiments, the coding splice site is identified experimentally. In some embodiments, the coding splice site is predicted based on in silico analysis.

[0111] A method for modifying an RNA molecule in a cell, 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 recipient sequence configured to splice to a splice donor sequence of an endogenous target RNA molecule; and (c) providing an exogenous RNA molecule to a cell (e.g., via a DNA vector encoding the exogenous RNA molecule or a viral particle containing such a DNA vector), wherein the one or more nucleotide changes disrupt one or more coding splice 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 coding splice sites are identified experimentally. In some embodiments, the coding splice sites are predicted based on in silico analysis.

[0112] 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 a nucleotide change into the coding domain sequence of the RNA exon editor, is also included herein, wherein the nucleotide change disrupts a coding splice site in the coding domain sequence of the RNA exon editor. In some embodiments, the nucleotide change is a synonymous nucleotide change. In some embodiments, the coding splice site is identified experimentally. In some embodiments, the coding splice site is predicted based on in silico analysis.

[0113] Cognitive splice site-reducing nucleotide changes may include changes that eliminate or reduce the ability of a coding splice site (e.g., the coding domain or binding domain sequence of an RNA exon editor) to be used in a splicing reaction. For example, a coding splice site identified in the context of an RNA exon editor typically includes a splice site, a polypyrimidine track, and a fork. In some embodiments, one or more nucleotide changes may be introduced into at least one of the splice site, polypyrimidine track, or fork of a coding splice site identified in the context of an RNA exon editor, or any combination thereof. In some embodiments, the nucleotide changes are designed to minimize potential effects on the protein encoded by them. Those skilled in the art will understand that if a nucleotide change made to reduce the frequency of use of a coding splice site also alters the amino acid encoded by the trans-spliced ​​RNA, a conservative amino acid change will be preferred over a non-conservative amino acid change. Furthermore, those skilled in the art can easily analyze the protein sequence and structure by focusing on the functional domain and the important sequences within it to evaluate whether these changes can be reasonably expected to alter the function of the protein encoded by the trans-spliced ​​protein. Those skilled in the art can also test proteins containing these amino acid changes and determine whether biological activity is altered using assays known in the art. In some embodiments, more than one nucleotide is changed within the identified coding splice site. Under some circumstances, the determination of how many nucleotides should be changed is made empirically based on in silico prediction 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 of a splice site, polypyrimidine track, or fork point, or any combination thereof, of a coding splice site identified in the context of an RNA exon editor. Synnonymous nucleotide changes do not alter the amino acid sequence of the protein encoded by the trans-spliced ​​RNA. In some embodiments, more than one synonymous nucleotide change may be introduced into at least one of a splice site, polypyrimidine track, or fork point, or any combination thereof, of a coding splice site identified in the context of an RNA exon editor.

[0114] In addition to the above, experimental results and sequence information were generally analyzed as follows. Changes to remove experimentally identified coding splice sites are carried out by searching for and replacing specific elements of the splice donor site. AG sites (and more strongly CAG sites) at the ends of the splice donor site are prioritized for the introduction of nucleotide changes. If an AG site is not found or cannot be changed without introducing a non-synonymous nucleotide change, sequence 42-4 base pairs upstream of the splice site are scanned for a fork (e.g., a sequence matching YNAH, where Y is pyrimidine, N represents any nucleotide, H is adenine, cytosine, or thymine (or uracil in RNA), and A is the fork adenine). Any such identified fork sequence is then analyzed and considered for the introduction of one or more nucleotide changes to reduce coding splice site utilization in the experimentally identified coding splice site. In addition, the sequence is also scanned for the presence of polypyrimidine tracks (multiple Ys (pyrimidines) immediately upstream of the terminal AG). Typically, these polypyrimidine tracks contain at least five pyrimidines within 10 base pairs upstream of the splice site. Once identified, these polypyrimidine tracks are then analyzed, and the introduction of one or more nucleotide changes is considered to reduce coding splice site utilization at the experimentally identified coding splice site.

[0115] In some embodiments, the cryptographic splice site that is modified to alleviate off-target splicing, or the off-target splice site, is a site empirically identified as a site of off-target splicing. Such sites may be identified, for example, using the technique described in Example 7 of PCT / US23 / 66969 (published as WO 2023 / 220742), the entirety of which is incorporated herein by reference. In some embodiments, all cryptographic splice sites having a utilization frequency above a predetermined threshold are modified by a cryptographic splice site-alleviating nucleotide change.

[0116] In some embodiments, the cryptographic splice site modified to mitigate off-target splicing, or the off-target splice site, is a site predicted to be the site of off-target splicing. This prediction is performed based on sequence analysis to identify the regular splice site, the polypyrimidine track, and / or the branching point of the presumed cryptographic splice site within it.

[0117] The inventors used a proprietary combination of proprietary software and know-how to identify cryptographic splice sites likely to affect RNA exon editor activity. The proprietary combination also relies on the sequence of operations regarding the cycles listed below, which were determined to affect the therapeutic performance of the RNA exon editors generated by the inventors. Briefly, three iterations of analysis and modification were performed on codon domain sequences, thereby in the first cycle, high-ranking cryptographic splice sites were identified using proprietary software and splice site utilization was reduced / eliminated; thereby in the second cycle, sequences generated after the first cycle were further analyzed using a combination of proprietary software and know-how to identify and rank additional cryptographic splice sites and modify these sites to reduce / eliminate cryptographic splice site utilization; Accordingly, in the third cycle, the sequences generated after the second cycle were further analyzed using a combination of proprietary software and know-how to identify sequences requiring additional modifications to improve and / or enhance the therapeutic performance of the RNA exon editor generated by this.

[0118] A modified version of the above process was carried out in relation to the analysis and modification of binding domain sequences. Briefly, two iterations of the analysis and modification of binding domain sequences were performed using a combination of proprietary software and know-how to identify and rank cryptographic splice sites in the first cycle and to modify these sites to reduce / eliminate cryptographic splice site utilization in these sites, and subsequently, in the second cycle, the combination of proprietary software and know-how was further analyzed to identify sequences that require additional modification to improve and / or enhance the therapeutic performance of the RNA exon editor generated therefrom.

[0119] By performing the above analysis, the inventors identified cryptographic splice sites in the codon-optimized versions of the sequences of MeCP2 exon 3 (SEQ No. 13) and exon 4 (SEQ No. 14), along with probability scores providing the relative likelihood of cryptographic splice sites involved in non-target splicing reactions. The nucleotides contributing to the cryptographic splice sites present in codon-optimized exon 3 (SEQ No. 13) and their relative probability scores are presented in Table 2. The relative probability score of 1 indicates that these sites have the highest relative probability of cryptographic site usage in the scoring system presented herein.

[0120]

[0121] The nucleotides and relative probability scores contributing to the cryptographic splice site in codon-optimized exon 4 (sequence number: 14) are presented in Table 3.

[0122]

[0123] In some embodiments, the coding domain sequence comprises a MeCP2 exon 3 nucleotide sequence containing a cryptographic splice site that reduces nucleotide changes compared to SEQ NO: 13 at one or more of the positions identified in Table 2. In some embodiments, the coding domain sequence has nucleotide changes compared to SEQ NO: 13 at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the positions presented in Table 2. In some embodiments, the coding domain sequence has nucleotide changes having a relative probability of 1 compared to SEQ NO: 13 at one or more of the positions presented in Table 2. In some embodiments, the coding domain sequence has a nucleotide change having a relative probability of 1 compared to SEQ ID NO: 13 at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 among the positions presented in Table 2.

[0124] In some embodiments, the coding domain sequence comprises a MeCP2 exon 4 nucleotide sequence containing a cryptographic splice site that reduces nucleotide changes compared to SEQ NO: 14 at one or more of the positions identified in Table 3. In some embodiments, the coding domain sequence has nucleotide changes compared to SEQ NO: 14 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 presented in Table 3. In some embodiments, the coding domain sequence has nucleotide changes having a relative probability of 1 compared to SEQ NO: 14 at one or more of the positions presented in Table 3. In some embodiments, the coding domain sequence has a nucleotide change having a relative probability of 1 compared to SEQ ID NO: 14 at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 among the positions presented in Table 2.

[0125] In some embodiments, a coded splice site-reduction nucleotide change causes a nucleotide sequence matching a regular splice site common sequence to no longer match the regular sequence. In some embodiments, a coded splice site-reduction nucleotide change removes potential splice site nucleotides. In some embodiments, a coded splice site-reduction nucleotide change removes potential polypyrimidine track nucleotides. In some embodiments, a coded splice site-reduction nucleotide change removes potential fork nucleotides. In some embodiments, a coded splice site-reduction nucleotide change is a synonymous nucleotide change. In some embodiments, a coded splice site-reduction nucleotide change causes a change in an amino acid encoded by an exon editor. In some embodiments, the amino acid change is a conservative amino acid substitution. The sequence of MeCP2 exon 3 having a coded splice site-reduction nucleotide change is presented in SEQ ID NO: 85. The sequence of MeCP2 exon 4 having a cryptic splice site-reduction nucleotide change is presented in SEQ NO: 86. The sequences of MeCP2 exons 3 and 4 having a cryptic splice site-reduction nucleotide change are presented in SEQ NO: 76. The sequences of SEQ NO: 76, 85, and 86 also have nucleotide changes performed for the purpose of codon optimization.

[0126] In some embodiments, the coding domain sequence of a MeCP2 trans-splicing molecule has a nucleotide change in 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, CDS has a nucleotide change 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 substituted for T at the disclosed positions. In some embodiments, the coding domain sequence of a MeCP2 trans-splicing molecule has nucleotide changes at one or more of the following positions in MeCP2 exon 4 numbered according to SEQ ID NO: 8: 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 embodiments, the CDS is 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, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61 of these positions It has a nucleotide change. In some embodiments, U is substituted instead of T at the initiation position.

[0127] In some embodiments, the coding domain sequence of a MeCP2 trans-splicing molecule comprises an exon 3 nucleotide sequence having one or more of the following nucleotides, or any combination thereof, at indicated positions: 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) Numbering). 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 comprises one or more of the following nucleotides or any combination thereof at indicated positions: 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 (numbering according to sequence number 85).

[0128] In some embodiments, the coding domain sequence of a MeCP2 trans-splicing molecule comprises an exon 4 nucleotide sequence having one or more of the following nucleotides, or any combination thereof, at indicated positions: 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 (Sequence No.: Numbering according to 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 indicated positions: 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 (numbering according to sequence number: 86).

[0129] 3. Combined domain

[0130] The MeCP2 trans-splicing molecule described herein features a binding domain (BD) configured to bind to / anneal a target MeCP2 intron and / or exon. In some embodiments, the BD is configured to bind to / anneal a target MeCP2 intron (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 pre-mRNA (e.g., target MeCP2 intron; intron 1, intron 2, or intron 3). In some embodiments, the binding domain is at least 75% complementary, at least 76% complementary, at least 77% complementary, at least 78% complementary, at least 79% complementary, at least 80% complementary, at least 81% complementary, at least 82% complementary, at least 83% complementary, at least 84% complementary, at least 85% complementary, at least 86% complementary, at least 87% complementary, at least 88% complementary, at least 89% complementary, at least 90% complementary, at least 91% complementary, at least 92% complementary, at least 93% complementary, at least 94% complementary, and at least 95% complementary to the sequence of a target MeCP2 intron and / or exon pre-mRNA (e.g., target MeCP2 intron; intron 1, intron 2, or intron 3). It is a nucleic acid sequence that is complementary, at least 96% complementary, at least 97% complementary, at least 98% complementary, at least 99% complementary, or 100% complementary.

[0131] In addition to the above, the inventors tested a mouse MeCP2 exon editor comprising a BD that binds to a similar position in mouse MeCP2 intron 1 pre-mRNA compared to human MeCP2 pre-mRNA. An exemplary mouse MeCP2 exon editor comprising a BD that is at least 80% identical (e.g., 82% identical) to a similar human binding domain sequence was shown to target human MeCP2 pre-mRNA and achieve 10% ONT trans-splicing to the human MeCP2 pre-mRNA target. This exemplary mouse MeCP2 exon editor, therefore, achieves a significant level of trans-splicing within the cross-species target. For comparison, a positionally similar human MeCP2 exon editor achieves 15% ONT to the human MeCP2 pre-mRNA target. In some embodiments, the exemplary mouse MeCP2 exon editor comprises a stretch of up to 35–40 nucleotides that is completely complementary to the human MeCP2 pre-mRNA. In some embodiments, most of the mismatch between the mouse MeCP2 exon editor and the human MeCP2 pre-mRNA target is located toward the 5' end of the binding domain.

[0132] The MeCP2 trans-splicing molecule described herein features a binding domain (BD) configured to bind / anneal 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% complementary, at least 90% complementary, at least 91% complementary, at least 92% complementary, at least 93% complementary, at least 94% complementary, at least 95% complementary, at least 96% complementary, at least 97% complementary, at least 98% complementary, at least 99% complementary, or 100% complementary) to the sequence of the target MeCP2 intron pre-mRNA (e.g., target MeCP2 intron). MeCP2 trans-splicing molecules containing such binding domains can promote trans-splicing between the trans-splicing molecule and the target MeCP2 pre-mRNA while inhibiting endogenous target cis-splicing (e.g., by creating a chimeric molecule having a coding domain with one or more functional MeCP2 exons encoding a wild-type MeCP2 amino acid sequence and a portion of the endogenous MeCP2 mRNA). In some embodiments relating to a trans-splicing molecule-encoding sequence (e.g., a vector encoding a trans-splicing molecule), the binding domain-encoding sequence encodes a nucleic acid sequence that is at least 80% complementary (e.g., at least 85% complementary, at least 86% complementary, at least 87% complementary, at least 88% complementary, at least 89% complementary, at least 90% complementary, at least 91% complementary, at least 92% complementary, at least 93% complementary, at least 94% complementary, at least 95% complementary, at least 96% complementary, at least 97% complementary, at least 98% complementary, at least 99% complementary, or 100% complementary) to the sequence of the target MeCP2 intron pre-mRNA.

[0133] In some embodiments, the present disclosure provides a trans-splicing molecule (or a vector encoding the same) that binds MeCP2 at intron 1, for example, wherein the nucleic acid trans-splicing molecule is configured to trans-splicate a coding domain to endogenous MeCP2 exon 1. In some embodiments, the trans-splicing molecule binds MeCP2 pre-mRNA at a site affecting the exon 1-intron 1 junction. In some embodiments, the trans-splicing molecule described herein has a binding domain comprising any one or more (e.g., 6 or more, 8 or more, 10 or more, or 12 or more, 25 or more, 50 or more, 75 or more, 100 or more, 150 or more, 200 or more, or 250 or more, or 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) nucleotides -100 to 1500, -50 to 1450, 3950 It comprises binding to an intron 1 of 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000, or to a binding site having SEQ ID NO: 1, 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 MeCP2It is defined as the first nucleotide of intron 1 (position 115 of sequence number 111 or position 1 of sequence number 1), and the negative number proceeding in the 5' direction from position -1 increases and the positive number proceeding in the 3' direction from position 1 increases. In some embodiments, the trans-splicing molecule described herein has a binding domain of nucleotides 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 of SEQ ID NO: 1 125-649, 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, It includes combining the joining sites within 500-1129, 500-1289, 500-1449, 660-809, 660-969, 660-1129, 660-1289, 660-1449, 820-969, 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 has a binding domain of nucleotides 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 of SEQ ID NO: 1 125-649, 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, It includes binding a binding site comprising at least 100 consecutive nucleotides within 500-1129, 500-1289, 500-1449, 660-809, 660-969, 660-1129, 660-1289, 660-1449, 820-969, 820-1129, 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 molecule described herein has a binding domain comprising 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; It comprises a length of about 50 nucleotides; about 100 nucleotides; about 125 nucleotides; about 150 nucleotides; about 200 nucleotides; or about 250 nucleotides. In some embodiments, the trans-splicing molecule described herein comprises a binding domain having a length of 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; It includes a length of 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 some embodiments, the binding domain comprises 50 or more consecutive nucleic acid residues that are 100% complementary to 50 or more consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises 50 consecutive nucleic acid residues that are 100% complementary to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises 50 consecutive nucleic acid residues that are at least 90% complementary (e.g., 90-100% complementary) to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises 50 consecutive nucleic acid residues that are at least 80% complementary (e.g., 80-100% complementary) to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises a sequence in the range of 100 to 200 nucleotide lengths and binds MeCP2 to a binding site within nucleotides 1 to 1449 of SEQ ID NO: 1, wherein the binding domain comprises 50 consecutive nucleotides that are 100% complementary to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises a sequence in the range of 100 to 200 nucleotide lengths and binds MeCP2 to a binding site within nucleotides 1 to 1449 of SEQ ID NO: 1, wherein the binding domain comprises 50 consecutive nucleotides that are 90% to 100% complementary to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises a sequence in the range of 100 to 200 nucleotide lengths and is 100% complementary to a binding site comprising at least 100 consecutive nucleotides within nucleotides 1 to 1449 of SEQ ID NO: 1. In some embodiments, the binding domain comprises a sequence ranging in length from 100 to 200 nucleotides and is 100% complementary to a binding site comprising at least 75 consecutive nucleotides within nucleotide 1-1449 of SEQ ID NO: 1.In some embodiments, the binding domain comprises a sequence in the range of 100 to 200 nucleotide lengths and is 90% to 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 in the range of 100 to 200 nucleotide lengths and binds MeCP2 to a binding site within nucleotides 3950-4250 of SEQ ID NO: 1, wherein the binding domain comprises 50 consecutive nucleotides that are 100% complementary to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises a sequence in the range of 100 to 200 nucleotide lengths and binds MeCP2 to a binding site within nucleotides 3950 to 4250 of SEQ ID NO: 1, wherein the binding domain comprises 50 consecutive nucleotides that are 90% to 100% complementary to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises a sequence in the range of 100 to 200 nucleotide lengths and is 100% complementary to a binding site comprising at least 75 consecutive nucleotides within nucleotides 3950 to 4250 of SEQ ID NO: 1. In some embodiments, the binding domain comprises a sequence in the range of 100 to 200 nucleotide lengths and is 100% complementary to a binding site comprising at least 100 consecutive nucleotides within nucleotides 3950 to 4250 of SEQ ID NO: 1. In some embodiments, the binding domain comprises a sequence of 100 to 200 nucleotide lengths and is 90% to 100% complementary to a binding site comprising at least 100 consecutive nucleotides within nucleotides 3950-4250 of SEQ ID NO:1.

[0134] In some cases, the binding domain is any six or more consecutive nucleotides within nucleotides -100 to 1500, -50 to 1450, 3950 to 4250, 4020 to 4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of MeCP2 intron 1 (e.g., any eight or more consecutive nucleotides within MeCP2 intron 1, any ten or more consecutive nucleotides within nucleotides -100 to 1500, -50 to 1450, 3950 to 4250, 4020 to 4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of MeCP2 intron 1). Nucleic acid, any 12 or more consecutive nucleic acids within nucleotides of MeCP2 intron 2 -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 20 or more consecutive nucleic acids within nucleotides of MeCP2 intron 1 -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000; nucleotides of MeCP2 intron 1 -100 to 1500, -50 to Any 30 or more consecutive nucleic acids within 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000, and any 40 or more consecutive nucleic acids within the nucleotides of MeCP2 intron 1 -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 50 or more consecutive nucleic acids within nucleotides of MeCP2 intron 1 -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 75 or more consecutive nucleic acids within nucleotides of MeCP2 intron 1 -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000; nucleotides of MeCP2 intron 1 -100 to 1500, -50 to Any 100 or more consecutive nucleic acids within 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000, nucleotides of MeCP2 intron 1 -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 150 or more consecutive nucleic acids within MeCP2 intron 1 nucleotides -100 to 1500, -50 to 1450, 3950 to 4250, It comprises any 200 or more consecutive nucleic acids within 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000, or any 250 or more consecutive nucleic acids within nucleotides of MeCP2 intron 1 -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, MeCP2 It is defined as the first nucleotide of intron 1 (position 115 of sequence number 111), and the negative number proceeding in the 5' direction from position -1 increases and the positive number proceeding in the 3' direction from position 1 increases.

[0135] In some cases, the binding domain is any six or more consecutive nucleotides within nucleotides -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of MeCP2 intron 1 (e.g., any eight or more consecutive nucleotides within nucleotides -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of MeCP2 intron 1, nucleotides -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1300 of MeCP2 intron 1, or Any 10 or more consecutive nucleic acids within -50 to 1000, nucleotides of MeCP2 intron 1 -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300, or any 12 or more consecutive nucleic acids within -50 to 1000, nucleotides of MeCP2 intron 1 -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300, or any 20 or more consecutive nucleic acids within -50 to 1000, nucleotides of MeCP2 intron 1 -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, Any 30 or more consecutive nucleic acids within -50 to 1300, or -50 to 1000, nucleotides of MeCP2 intron 1 -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300, or any 40 or more consecutive nucleic acids within -50 to 1000, nucleotides of MeCP2 intron 1 -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300,or any 50 or more consecutive nucleic acids within -50 to 1000, nucleotides of MeCP2 intron 1 -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300, or any 75 or more consecutive nucleic acids within -50 to 1000, nucleotides of MeCP2 intron 1 -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300, or any 100 or more consecutive nucleic acids within -50 to 1000, nucleotides of MeCP2 intron 1 -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, Comprising any 150 or more consecutive nucleic acids within -50 to 1300, or -50 to 1000, nucleotides of MeCP2 intron 1 -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300, or any 200 or more consecutive nucleic acids within -50 to 1000, or nucleotides of MeCP2 intron 1 -100 to 1500, -50 to 1450, -100 to 1300, -100 to 1000, -50 to 1300, or any 250 or more consecutive nucleic acids within -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 Location 1 is, MeCP2 It is defined as the first nucleotide of intron 1 (position 115 of sequence number 111), and the negative number proceeding in the 5' direction from position -1 increases and the positive number proceeding in the 3' direction from position 1 increases.

[0136] In some embodiments, the binding domain has at least two non-overlapping sequences that are at least 80% complementary to the binding site.

[0137] In some embodiments, the binding domain is at least 80% identical with any one of SEQ ID NOs: 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% identical; e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or It includes a nucleic acid sequence having 99% identity. In any binding domain sequence disclosed herein, U can substitute all T in the sequence. Thus, for example, in some embodiments, the binding domain has at least 80% identity with any one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 47 having a U substituting each T in the 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%, It includes a nucleic acid sequence having 96%, 97%, 98%, or 99% identity.

[0138] In some embodiments, the binding domain is at least 80% identical with any one of SEQ ID NOs: 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% identical; e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or It is a DNA sequence having 99% identity. In some embodiments, the binding domain comprises or consists of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 47.

[0139] 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.

[0140] 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.

[0141] 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 numbered according to SEQ ID NO: 27.

[0142] 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 comprises nucleotide 133C numbered according to SEQ ID NO: 28.

[0143] 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 numbered according to SEQ ID NO: 30, or any combination thereof.

[0144] 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 comprises nucleotide 119A numbered according to SEQ ID NO: 32.

[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: 33, wherein the binding domain comprises nucleotide 133C numbered according to SEQ ID NO: 33.

[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: 47, wherein the binding domain comprises nucleotide 149C numbered according to SEQ ID NO: 47.

[0147] 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 one of SEQ ID NOs: 116 to 137, wherein each N is independently A, T (or U for 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 one of SEQ ID NOs: 116 to 137, wherein each N is independently A, T (or U for RNA), C, or G. In some embodiments, the nucleotide at any of the positions designated as N is non-complementary to the corresponding position in natural MeCP2 pre-mRNA.

[0148] 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, N at position 23 of SEQ ID NO: 116 is C. In some embodiments, N at position 72 of SEQ ID NO: 116 is C.

[0149] 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, N at position 97 of SEQ ID NO: 117 is C. In some embodiments, N at position 146 of SEQ ID NO: 117 is C.

[0150] 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, N at position 8 of SEQ ID NO: 118 is C. In some embodiments, N at position 56 of SEQ ID NO: 118 is C. In some embodiments, N at position 82 of SEQ ID NO: 118 is C. In some embodiments, N at position 85 of SEQ ID NO: 118 is C.

[0151] 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, N at position 133 of SEQ ID NO: 119 is C.

[0152] 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, N at position 4 of SEQ ID NO: 120 is C. In some embodiments, N at position 71 of SEQ ID NO: 120 is C. In some embodiments, N at position 101 of SEQ ID NO: 120 is C.

[0153] In some embodiments, the binding domain comprises a nucleotide sequence having at least 50 consecutive nucleotides having 100% sequence identity with a continuous stretch of any one of SEQ ID NOs: 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 having 100% sequence identity with a continuous stretch of any one of SEQ ID NOs: 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 having 100% sequence identity with a continuous stretch of nucleotides of any one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 47.

[0154] Some embodiments of the MeCP2 trans-splicing molecule described herein feature 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% complementary, at least 90% complementary, at least 91% complementary, at least 92% complementary, at least 93% complementary, at least 94% complementary, at least 95% complementary, at least 96% complementary, at least 97% complementary, at least 98% complementary, at least 99% complementary, or 100% complementary) to the sequence of the target MeCP2 intron pre-mRNA (e.g., target MeCP2 intron). MeCP2 trans-splicing molecules containing such binding domains can promote trans-splicing between the trans-splicing molecule and the target MeCP2 pre-mRNA while inhibiting endogenous target cis-splicing (e.g., by creating a chimeric molecule having a coding domain with one or more functional MeCP2 exons encoding a wild-type MeCP2 amino acid sequence and a portion of the endogenous MeCP2 mRNA). In some embodiments relating to a trans-splicing molecule-encoding sequence (e.g., a vector encoding a trans-splicing molecule), the binding domain-encoding sequence encodes a nucleic acid sequence that is at least 80% complementary (e.g., at least 85% complementary, at least 86% complementary, at least 87% complementary, at least 88% complementary, at least 89% complementary, at least 90% complementary, at least 91% complementary, at least 92% complementary, at least 93% complementary, at least 94% complementary, at least 95% complementary, at least 96% complementary, at least 97% complementary, at least 98% complementary, at least 99% complementary, or 100% complementary) to the sequence of the target MeCP2 intron pre-mRNA.

[0155] In some embodiments, the present disclosure provides a trans-splicing molecule (or a vector encoding the same) that binds MeCP2 at intron 2, for example, wherein the nucleic acid trans-splicing molecule is configured to trans-splicate a coding domain to endogenous MeCP2 exon 1 or 2. In some embodiments, the trans-splicing molecule described herein comprises a binding domain that binds to a binding site having any one or more of the nucleotides 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 of SEQ ID NO: 2 (e.g., 6 or more, 8 or more, 10 or more, or 12 or more, 25 or more, 50 or more, 75 or more, 100 or more, 150 or more, 200 or more, or 250 or more). In some embodiments, the trans-splicing molecule described herein comprises a binding domain having about 50 to 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; It includes a length of about 150 nucleotides; about 200 nucleotides; or about 250 nucleotides.In some embodiments, the trans-splicing molecule described herein has a binding domain comprising 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; It includes a length of 125 nucleotides; 150 nucleotides; 200 nucleotides; or 250 nucleotides. In some embodiments, the binding domain comprises 50 or more consecutive nucleic acid residues that are 100% complementary to 50 or more consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises 50 consecutive nucleic acid residues that are 100% complementary to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises 50 consecutive nucleic acid residues that are at least 90% complementary (e.g., 90 to 100% complementary) to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises 50 consecutive nucleic acid residues that are at least 80% complementary (e.g., 80 to 100% complementary) to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises a sequence ranging in length from 100 to 200 nucleotides and binds MeCP2 to 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 of the binding site.In some embodiments, the binding domain comprises a sequence in the range of 100 to 200 nucleotide lengths and binds MeCP2 to a binding site within nucleotides 1-450 of SEQ ID NO: 2, wherein the binding domain comprises 50 consecutive nucleotides that are 90% to 100% complementary to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises a sequence in the range of 100 to 200 nucleotide lengths and binds MeCP2 to a binding site within nucleotides 1-450 of SEQ ID NO: 2, wherein the binding domain comprises 75 consecutive nucleotides that are 90% to 100% complementary to 75 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises a sequence in the range of 100 to 200 nucleotide lengths 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 in the range of 100 to 200 nucleotide lengths 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 in the range of 100 to 200 nucleotide lengths and binds MeCP2 to a binding site within nucleotides 99-248 of SEQ ID NO: 2, wherein the binding domain comprises 50 consecutive nucleotides that are 100% complementary to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises a sequence ranging in length from 100 to 200 nucleotides and binds MeCP2 to a binding site within nucleotides 99-248 of SEQ ID NO: 2, wherein the binding domain comprises 50 consecutive nucleotides that are 90% to 100% complementary to 50 consecutive nucleotides of the binding site.In some embodiments, the binding domain comprises a sequence in the range of 100 to 200 nucleotide lengths 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 in the range of 100 to 200 nucleotide lengths 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 in the range of 100 to 200 nucleotide lengths and binds MeCP2 to 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 that are 100% complementary to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises a sequence in the range of 100 to 200 nucleotide lengths and binds MeCP2 to 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 that are 90% to 100% complementary to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises a sequence in the range of 100 to 200 nucleotide lengths 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 nucleotide lengths and is 90% to 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.

[0156] In some cases, the binding domain is any six or more consecutive nucleotides within 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 (e.g., any eight or more nucleotides within MeCP2 intron 2). Any 10 or more consecutive nucleic acids within 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, nucleotides 1-300 of MeCP2 intron 2, Any 12 or more consecutive nucleic acids within 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, nucleotides 1-300, 50-300, 50-250, 75-300 of MeCP2 intron 2, 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,Any 20 or more consecutive nucleic acids within 199-400 or 199-450, or any 30 nucleotides of MeCP2 intron 2 within 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 Any 40 or more consecutive nucleic acids within 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, nucleotides 1-300 of MeCP2 intron 2, Any 50 or more consecutive nucleic acids within 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, nucleotides 1-300, 50-300, 50-250, 75-300 of MeCP2 intron 2, 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 75 or more consecutive nucleic acids within 199-450, 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 any 100 or more consecutive nucleic acids within 199-450, MeCP2 Any 150 or more consecutive nucleic acids within 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 intron 2, nucleotides 1-300 of MeCP2 intron 2, Any 200 or more consecutive nucleic acids within 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, or nucleotides 1-300, 50-300, 50-250 of MeCP2 intron 2, 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 contains any 250 or more consecutive nucleic acids within 199-450.

[0157] In some cases, the binding domain is any six or more consecutive nucleotides within 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 (e.g., any eight or more consecutive nucleotides within nucleotides 90-300, 90-250, 95-300, 95-250, or 99-248 of MeCP2 intron 2, of MeCP2 intron 2 Any 10 or more consecutive nucleic acids within 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, nucleotides 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, of MeCP2 intron 2 Any 12 or more consecutive nucleic acids within 99-450, 149-450, 199-450, 249-450, 99-350, 99-400, 199-400, or 199-450, any 20 or more consecutive nucleic acids within nucleotides of MeCP2 intron 2 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, Nucleotides of MeCP2 intron 2 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,Any 30 or more consecutive nucleic acids within 99-400, 199-400, or 199-450, 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, any 40 or more consecutive nucleic acids within 90-300, 90-250, 95-300, MeCP2 intron 2, Any 50 or more consecutive nucleic acids within 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, nucleotides of MeCP2 intron 2 90-300, 90-250, 95-300, 95-250, 99-248, 149-300, 199-350, 249-400, 299-450, 99-450, 149-450, 199-450, Any 75 or more consecutive nucleic acids within 249-450, 99-350, 99-400, 199-400, or 199-450, 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 any 100 or more consecutive nucleic acids within 199-450, nucleotides 90-300 of MeCP2 intron 2, 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 150 or more consecutive nucleic acids within 199-450, or 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 any 200 or more consecutive nucleic acids within 199-450, or nucleotides 90-300, 90-250, 95-300, 95-250, 99-248 of MeCP2 intron 2, Includes any 250 or more consecutive nucleic acids within 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.

[0158] In some embodiments, the binding domain has at least two non-overlapping sequences that are at least 80% complementary to the binding site.

[0159] In some embodiments, the binding domain is at least 80% identical (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 96%, at least 97%, at least 98%, at least 99%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 55, 56, 57, 58, 59, 88, 89, 90, 91%, or 92, including a version of SEQ ID NO: 55, 56, 57, 58, 59, 88, 89, 90, 91%, or 92 in which each T is substituted with U; e.g., It comprises a nucleic acid sequence having 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 that are 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 that are 100% complementary to 100 consecutive nucleotides of SEQ ID NO: 55, 56, 57, 58, 59, 88, 89, 90, 91, or 92.

[0160] In some embodiments, the binding domain comprises a nucleotide sequence having at least 50 consecutive nucleotides having 100% sequence identity with a continuous stretch of any one of SEQ ID NOs: 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 having 100% sequence identity with a continuous stretch of any one of SEQ ID NOs: 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 having 100% sequence identity with a continuous stretch of nucleotides of any one of SEQ ID NOs: 55, 56, 57, 58, 59, 88, 89, 90, 91, or 92.

[0161] In some embodiments, the binding domain comprises one or more nucleotide substitutions relative to the endogenous MeCP2 mRNA sequence, wherein one or more nucleotide substitutions disrupt coding splice sites within the binding domain sequence. In some embodiments, the binding domain comprises a nucleic acid sequence having one or more of the following variations 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, Includes 99C or 120C.

[0162] 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).

[0163] 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.

[0164] 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.

[0165] 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 numbered according to SEQ ID NO: 92 or any combination thereof.

[0166] 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 one of SEQ ID NOs: 138 to 152, wherein each N is independently A, T (or U for 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 one of SEQ ID NOs: 139 to 152, wherein each N is independently A, T (or U for RNA), C, or G. In some embodiments, the nucleotide at any of the positions designated as N is non-complementary to the corresponding position in natural MeCP2 pre-mRNA.

[0167] 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, N at position 25 of SEQ ID NO: 138 is C. In some embodiments, N at position 59 of SEQ ID NO: 138 is C. In some embodiments, N at position 63 of SEQ ID NO: 138 is C. In some embodiments, N at position 99 of SEQ ID NO: 138 is C. In some embodiments, N at position 120 of SEQ ID NO: 138 is C.

[0168] 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, N at position 84 of SEQ ID NO: 139 is C. In some embodiments, N at position 106 of SEQ ID NO: 139 is C.

[0169] 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, N at position 136 of SEQ ID NO: 140 is C. In some embodiments, N at position 138 of SEQ ID NO: 140 is C. In some embodiments, N at position 82 of SEQ ID NO: 118 is C. In some embodiments, N at position 85 of SEQ ID NO: 118 is C.

[0170] 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, N at position 51 of SEQ ID NO: 141 is C.

[0171] The MeCP2 trans-splicing molecule described herein features a binding domain (BD) configured to bind / anneal a target MeCP2 intron and / or exon. In some embodiments, the target MeCP2 intron is MeCP2 intron 3 (SEQ No. 4). In some embodiments, the binding domain is a nucleic acid sequence that is at least 80% complementary (e.g., at least 85% complementary, at least 90% complementary, at least 91% complementary, at least 92% complementary, at least 93% complementary, at least 94% complementary, at least 95% complementary, at least 96% complementary, at least 97% complementary, at least 98% complementary, at least 99% complementary, or 100% complementary) to the sequence of the target MeCP2 intron pre-mRNA (e.g., target MeCP2 intron). MeCP2 trans-splicing molecules containing such binding domains can promote trans-splicing between the trans-splicing molecule and the target MeCP2 pre-mRNA while inhibiting endogenous target cis-splicing (e.g., by creating a chimeric molecule having a coding domain with one or more functional MeCP2 exons encoding a wild-type MeCP2 amino acid sequence and a portion of the endogenous MeCP2 mRNA). In some embodiments relating to a trans-splicing molecule-encoding sequence (e.g., a vector encoding a trans-splicing molecule), the binding domain-encoding sequence encodes a nucleic acid sequence that is at least 80% complementary (e.g., at least 85% complementary, at least 86% complementary, at least 87% complementary, at least 88% complementary, at least 89% complementary, at least 90% complementary, at least 91% complementary, at least 92% complementary, at least 93% complementary, at least 94% complementary, at least 95% complementary, at least 96% complementary, at least 97% complementary, at least 98% complementary, at least 99% complementary, or 100% complementary) to the sequence of the target MeCP2 intron pre-mRNA.

[0172] In some embodiments, the binding domain is a DNA sequence having at least 80% identity with any one of SEQ ID NOs: 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).

[0173] As described in detail herein, the first step in the design of the MeCP2 exon editor began with the screening and selection of highly efficient BD sequences complementary to the targeted pre-mRNA intron. As shown herein, the BD targeting Intron 1 of MeCP2 exhibited the highest level of trans-splicing and was therefore identified as an effective component of an exemplary MeCP2-targeted exon editor for the treatment of the Rett syndrome patient population. See Figs. 6-9.

[0174] Any binding domain disclosed herein may include one or more nucleotide changes (e.g., nucleotide substitutions) relative to the natural pre-mRNA sequence, wherein one or more nucleotide changes disrupt the coding splice site within the binding domain sequence. The coding splice site-reducing nucleotide changes may cause a lack of complementarity between the binding domain and the binding site on the pre-mRNA at the location of the nucleotide changes. However, despite the lack of complementarity at the location(s) of one or more nucleotide changes, the binding domain can nevertheless bind to the binding site and support effective trans-splicing. This is illustrated in FIG. 19, where an exon editor comprising a 150-nucleotide binding domain having five coding splice site-reducing nucleotide substitutions (BD sequence: SEQ ID NO: 88) provided effective trans-splicing into MeCP2 intron 2.

[0175] In some embodiments, the RNA exon editor of the present disclosure comprises a binding domain that specifically binds to a binding site within MeCP2 intron 1, 2, or 3. In some embodiments, the RNA exon editor of the present disclosure comprises a binding domain that specifically binds to any one of the binding sites disclosed herein.

[0176] In some embodiments, the binding domain of the RNA exon editor of the present disclosure binds to the same sequence of MeCP2 pre-mRNA to which any one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 47, 55, 56, 57, 58, 59, 88, 89, 90, 91, or 92 binds. In some embodiments, this binding domain has 100% complementarity with the binding site.

[0177] 4. Hemi-intron

[0178] In some embodiments, the hemi-intron includes a 3' splice site. In some embodiments, the hemi-intron includes a polypyrimidine track (pPy), a branch (BP), and / or a 3' splice site to mediate trans-splicing. In some embodiments, the hemi-intron has a single splice site, which indicates that the splice site is designed for preferential trans-splicing but not for cis-splicing because the splice site is absent.

[0179] Alternative hemi-introns may be selected by those skilled in the art according to known methods and principles. In some embodiments, the 3' splice site common sequence is the nucleic acid sequence YAG / [wherein Y is a pyrimidine (cytosine or thymidine); and " / " indicates the location of the 3' splice site; e.g., TAG]. In some embodiments, the RNA exon editor includes the sequence TAG immediately next to the 5' for the first nucleotide of the coding domain sequence in the 5' to 3' direction (e.g., the coding domain sequence of MeCP2 exon 1, exon 2, or exon 3, or exon 4). In some embodiments, endogenous splice sites corresponding to exons and introns adjacent to the splice site may be used to maintain any splicing control signal. Thus, in some embodiments, the hemi-intron 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 at the 3'-terminus of MeCP2 intron 1, intron 2, or intron 3. In some embodiments, the hemi-intron comprises BP and pPy. In some embodiments, the hemi-intron comprises BP, pPy, and a 3' splice site (splice acceptor). In some embodiments, the hemi-intron comprises BP, pPy, and a 3' splice site operably connected 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; and A is the site of branching. In some embodiments, BP has the sequence YNYTRAC, where Y is a pyrimidine (C or U), N is any nucleotide, R is a purine; and A is the site of branching.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 the site of branching. In some embodiments, BP is at least 10 nucleotides upstream of the 3' splice site. In some embodiments, BP is 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%, and 90% of the nucleotides between the BP sequence and the 3' splice site (i.e., pPy) are pyrimidines. In some embodiments, all of the nucleotides between the BP sequence and the 3' splice site (i.e., pPy) are pyrimidines.

[0180] In some embodiments, the suitable 3' splice site includes a TAG. In some embodiments, the hemi-intron includes a nucleotide sequence presented in SEQ ID NO: 17 or a sequence having at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity with either SEQ ID NO: 17.

[0181] The hemi-intron can be operably linked to the coding domain sequence (CDS) at the 5' end (e.g., directly linked to the CDS or having an intervening sequence connecting the 3' end of the hemi-intron and the 5' end of the CDS).

[0182] 5. 3' Untranslated Section

[0183] In some embodiments, the nucleic acid trans-splicing molecule comprises a 3' untranslated region. In some embodiments, the 3' untranslated region comprises, is essentially composed of, or is composed of a heterologous 3' untranslated region. In some embodiments, the 3' UTR is an RDH1pA 3' UTR, which is a synthetic 3' UTR comprising a highly conserved 110 bp MeCP2 distal polyadenylation signal and an upstream miRNA-binding panel comprising 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 comprises SEQ ID NO: 19. In some embodiments, the 3' UTR is an mWPRE 3' UTR. The DNA sequence encoding the mWPRE 3' UTR comprises SEQ ID NO: 20. In some embodiments, the 3' UTR comprises a truncated WPRE sequence. A DNA sequence encoding a truncated WPRE 3' UTR comprises SEQ ID NO: 87. In some embodiments, the truncated WPRE sequence comprises, is essentially composed of, or consists of 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, is essentially composed of, or consists of one of SEQ ID NO: 19 or 20.In some embodiments, the 3' untranslated region comprises a sequence having at least 80% sequence identity with one of 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).

[0184] In some embodiments, the 3' untranslated region may be operably connected to the coding domain as 3' (e.g., directly connected to the coding domain or having an intervention sequence connecting the 5' end of the 3' untranslated region and the 3' end of the coding domain).

[0185] In addition to the above, the nucleic acid trans-splicing molecule may include a hemi-intron domain at one or more positions within the molecule. In some embodiments, the hemi-intron domain is operatively connected to the binding domain at the 3' end (e.g., directly connected to the binding domain). The hemi-intron domain may be of any suitable size. In some embodiments, the hemi-intron domain is greater than 20 nucleotide lengths (e.g., 20 to 100 nucleotide lengths or 20 to 85 nucleotide lengths). In some cases, the hemi-intron domain comprises, is essentially composed of, or is composed 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).

[0186] The inventors discovered that a truncated variant of the WPRE 3' UTR sequence can be used to enhance the in-target splicing of RNA exon editors. A comparison was conducted between two different identical RNA exon editors, one having the WPRE 3' UTR sequence of SEQ ID NO: 20 and the other having a truncated variant of this sequence ("WPRE3"; SEQ ID NO: 87) with 203 internal nucleotides deleted. The trans-splicing efficiency of the two RNA exon editors was tested. In two replicates of the experiment, the RNA exon editor with the truncated WPRE 3' UTR had 18 to 19% in-target splicing, whereas the exon editor with the full-length WPRE 3' UTR had 13 to 14% in-target splicing. It was surprising that the truncated WPRE 3' UTR sequence, which is 42.6% the length of the full-length WPRE sequence, worked as well as or better than the full-length sequence.

[0187] Some embodiments of the RNA exon editor comprise the following components operably connected in the 5' to 3' direction: a binding domain configured to bind a binding site within a target region of the pre-mRNA of the target gene, a hemi-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 is missing 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 is missing 50-200, 50-150, or 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, which are operably linked to the 3' of a truncated WPRE 3' UTR sequence.

[0188] 6.3' Warrior Terminator Domain

[0189] In some cases, the trans-splicing molecule comprises a 3' transcription terminator domain. In some embodiments, this 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 the human long non-coding RNA MALAT1 (e.g., wild-type MALAT1). In some embodiments, the 3' transcription terminator domain comprises a tRNA-like domain. A 3' transcription terminator domain useful as part of the MeCP2 trans-splicing molecule is described in International Patent Publication No. WO 2020 / 214973, the entirety of which is incorporated herein by reference. For example, in some embodiments, a region of the RNA exon editor operably connected to the 3' end of the CDS comprises a terminator domain that is essentially composed of, or composed of, a MALAT1+mascRNA domain such as SEQ ID NO: 77, or a variant thereof such as SEQ ID NO: 22, which is an anti-mut1 variant as described in PCT / US2023 / 066969.

[0190] In some embodiments, the exemplary RNA exon editor described herein comprises a binding domain that binds to intron 1 of MeCP2, wherein the exemplary intron 1-binding RNA exon editor may comprise a binding domain, a hemi-intron, a coding domain sequence (e.g., SEQ ID NO: 76 or SEQ ID NO: 9), and a 3' transcription terminator.

[0191] In some embodiments, the exemplary RNA exon editor described herein comprises a binding domain that binds to intron 2 of MeCP2, wherein the exemplary intron 2-binding RNA exon editor may comprise a binding domain, a hemi-intron, a coding sequence (e.g., SEQ ID NO: 76), and a 3' transcription terminator.

[0192] In some embodiments, the exemplary RNA exon editor described herein comprises a binding domain that binds to intron 3 of MeCP2, wherein the exemplary intron 3-binding RNA exon editor may comprise a binding domain, a hemi-intron, a coding sequence (e.g., SEQ ID NO: 14), and a 3' transcription terminator.

[0193] In some embodiments, the binding of the trans-splicing molecule to the target pre-mRNA is mediated by percent complementarity (i.e., based on the base-pair characteristics of the nucleic acid), triple helix formation, or protein-nucleic acid interactions (as described in the documents incorporated 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. Also included herein is an RNA or DNA capable of hybridizing with one of the aforementioned RNA or DNA, preferably under strict conditions, e.g., in 2.5x SSC buffer at 60°C and under multiple washes at a lower buffer concentration, e.g., in 0.5x SSC buffer at 37°C. The trans-splicing molecule In vitro When synthesized, these trans-splicing molecules are modified in their base moiety, sugar moiety, or phosphate backbone to improve, for example, molecular stability, hybridization with target mRNA, transport into the cell, and cellular stability against enzymatic cleavage. For example, modification of the trans-splicing molecule to reduce its total charge can enhance the molecule's cellular uptake. Additionally, modifications can reduce susceptibility to nucleases or chemical degradation. Nucleic acid molecules can be synthesized to be conjugated to other molecules, for example, peptides, hybridization-triggering crosslinkers, transporters, hybridization-triggering cleavages, etc.

[0194] Various other well-known modifications to nucleic acid molecules can be introduced as a means to increase intracellular stability and half-life (see also above for oligonucleotides). Possible modifications are known in the art. Modifications that can be performed on the structure of synthetic trans-splicing molecules include backbone modifications.

[0195] 7. Linker

[0196] In some embodiments, the trans-splicing molecule of the present disclosure comprises a nucleotide linker 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.

[0197] 8. Cell line assay

[0198] 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 may be acquired or engineered to express targeted MeCP2 pre-mRNA at sufficient levels.

[0199] 9. Animal models

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

[0201] FVB / N-Tg(MECP2 * Further details regarding R270X / GFP)AHzo / J are available on the Jackson Laboratory website. Briefly, as described herein, MeCP2-R270X transplant-genetic mice express human MeCP2 truncated at amino acid R270 and tagged with EGFP at the C-terminus. As measured by immunohistochemistry, the expression of the transplant gene replicates the expression of the human MECP2 gene in the cortex, hippocampus, cerebellum, hypothalamus, and brainstem. The MeCP2 protein, like the endogenous protein, is entirely localized in the nucleus and concentrates in heterochromatic foci. MeCP2-R270X transplant-genetic mice do not exhibit an apparent phenotype until combined with the mouse MeCP2 knockout allele. When combined with the mouse MeCP2 knockout (stock number 003890), the median lifespan of the mouse is 85 days, which is slightly longer than that of mice with MeCP2 knockout alone (76 days).

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

[0203] 10. RNA Exon Editor Screening Platform

[0204] As described herein, a 3' RNA exon editor comprises several functional sequence elements, such as a binding domain (BD) for pre-mRNA targeting and a hemi-intron providing a splice acceptor site. In some embodiments, the hemi-intron includes a fork and a polypyrimidine track in addition to the 3' splice acceptor site. When manipulating an RNA exon editor for a given gene target, various sequence options for each of these elements are tested for their ability to contribute to high trans-splicing (TS) efficiency. This testing can be achieved through the cloning and transfection of individual RNA exon editor variants and efficiency analysis via RT-qPCR / ddPCR and Western blot as described below.

[0205] 11. RNA Exon Editor Screening in Individual Formats:

[0206] This approach can be applied to test a small number of variable elements within RNA exon editor sequences before initiating a library-based multiplex screen, to validate the performance of RNA exon editors identified in the multiplex screen, or to improve the performance of lead candidates. Evaluation of TS efficiency occurs at the RNA and protein levels.

[0207] At the RNA level, TS activity is evaluated by isolating total RNA from cells followed by reverse transcription and real-time quantitative PCR (RT-qPCR), measuring, for example, the RNA copy numbers of the following targets: RNF20 (housekeeping gene for normalization); natural (MeCP2) mRNA; exon editor RNA; intratarget, exon-edit RNA (ONT) which is the product of positive TS; ONT + exon editor + OFT (off-target) - a single assay capturing all three of these targets. OFT represents an incorrect RNA molecule that the RNA exon editor can transsplice.

[0208] ONT TS efficiency, also referred to as percentage replacement, represents the portion of the total MeCP2 mRNA population that underwent successful TS and is calculated via the following equation: % ONT TS = 100 * (ONT Copy Count / (ONT Copy Count + Natural Copy Count)).

[0209] RNA exon editor TS efficiency is the portion of the RNA exon editor transcriptome population accurately trans-spliced ​​with MeCP2 RNA and is calculated through the following equation: % exon editor TS = 100 * (ONT copy count / (ONT copy count + ExxonEditor copy count + OFT copy count)).

[0210] At the protein level, TS activity is measured through Western blot analysis applied to proteins extracted from cell or tissue samples. The cytoskeletal protein beta-actin can be used as a loading control. Levels of ONT protein are measured using MeCP2-specific antibodies (Ab). For constructs containing a tag at the C-terminus, e.g., a Myc tag, an antibody specific to the tag (e.g., a Myc-specific Ab) can be used to evaluate ONT protein levels by probing a Western blot.

[0211] 12. Exemplary Combinations

[0212] Some embodiments of the RNA exon editor include a combination of the binding domain, hemi-intron, and 3' untranslated region and 3' transcription terminator domain disclosed above. In some embodiments, the RNA exon editor comprises a binding domain selected from any 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, a hemi-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 hemi-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 comprises a binding domain and a splice site 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. In some embodiments, the RNA exon editor comprises a binding domain and a hemi-intron selected from one of SEQ ID NOs: 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.In some embodiments, 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 SEQ ID NO: 17 or a functional variant thereof. In some embodiments, 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 SEQ ID NO: 19 or a functional variant thereof. In some embodiments, 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 SEQ ID NO: 19 or 20 or a functional variant thereof. In some embodiments, 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 SEQ ID NO: 22 or a functional variant thereof. In some embodiments, the RNA exon editor comprises a binding domain selected from one of SEQ ID NOs: 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 NOs: 7-14, 76, 85, or 86.

[0213] An exemplary configuration of the RNA exon editor of the present disclosure comprises the following (in the 5' to 3' direction):

[0214] Exon Editor A: [BD]-[Hemi-Intron]-[CDS];

[0215] Exon Editor B: [BD]-[Hemi-Intron]-[CDS]-[3' UTR];

[0216] Exon Editor C: [BD]-[Hemi-Intron]-[CDS]-[Terminator];

[0217] Exon Editor D: [BD]-[Hemi-Intron]-[CDS]-[3' UTR]-[Terminator];

[0218] Exon Editor E: [BD]-[BP]-[pPy]-[3' Splice Section]-[CDS];

[0219] Exon Editor F: [BD]-[BP]-[pPy]-[3' Splice Site]-[CDS]-[3' UTR];

[0220] Exon Editor G: [BD]-[BP]-[pPy]-[3' Splice Site]-[CDS]-[Terminator]; and

[0221] Exon Editor H: [BD]-[BP]-[pPy]-[3' Splice Site]-[CDS]-[3' UTR]-[Terminator];

[0222] Here, "BD" represents any BD disclosed in Section VI.C.3; "hemi-intron" represents any hemi-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 fork point sequence disclosed in Section VI.C.4; "pPy" represents any polypyrimidine track 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 composition encoding any exon editor 1 to 8 includes a promoter sequence operably connected upstream of the BD of any exon editor 1 to 8.

[0223] In some embodiments, the RNA exon editor of the present disclosure comprises 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, operably connected to the 3' end of a coding domain sequence in the 5' to 3' direction. In some embodiments, the RNA exon editor of the present disclosure comprises the WPRE sequence of SEQ ID NO: 87, a MALAT1 terminator anti-Mut1 variant of SEQ ID NO: 22, and a 3xUBS sequence of SEQ ID NO: 79, operably connected to the 3' end of a coding domain sequence in the 5' to 3' direction.

[0224] Exemplary combinations of additional RNA exon editor components are presented in embodiments 1 to 279 listed below.

[0225] D. Vector

[0226] Trans-splicing molecules can be delivered to target cells of an individual using various techniques, for example, using different vector modalities such as recombinant adeno-associated virus (AAV) vectors or non-viral vectors. Thus, a vector containing / encoding a trans-splicing molecule (e.g., a viral or non-viral vector containing / encoding a trans-splicing molecule, e.g., a DNA vector containing / encoding a trans-splicing molecule) is provided herein. Any suitable nucleic acid vector may be used in conjunction with the present composition and method to design and assemble the components of the trans-splicing molecule and the recombinant AAV. In one embodiment, the vector is a recombinant AAV carrying a trans-splicing molecule driven by a promoter that expresses the trans-splicing molecule in selected cells of an individual. Methods for assembling recombinant vectors are known in the art. For example, Ausubel et al., Current Protocols in Molecular Biology , John Wiley & Sons, New York, 1989; Kay, M.A. et al., Nat. Medic , 2001, 7(I):33-40; and Walther W. and Stein U.; Drugs See 2000, 60(2):249-71.

[0227] In certain embodiments described herein, the trans-splicing molecule is delivered via an AAV vector to selected cells requiring treatment, e.g., neuronal cells. Various naturally occurring AAV serotypes are available. Since many natural variants exist in AAV capsids, AAVs with characteristics specifically suited for neuronal cells are identified and used. Artificial AAV vectors can be manipulated by conventional molecular biology techniques and these particles can be optimized for cell-specific delivery of the trans-splicing molecule nucleic acid sequence, minimization of immunogenicity, tuning of stability and particle lifetime, efficient degradation, accurate delivery to the nucleus, etc. Such artificial capsids can be generated by any suitable technique using a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) in combination with a heterogeneous sequence that may be obtained from a different selected AAV, a non-contiguous portion of the same AAV, a non-AAV viral source, or a non-viral source. Artificial AAVs may be, without limitation, detyped AAVs, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids. Detyped vectors in which the capsid of one AAV is replaced with a heterologous capsid protein are useful for delivering the trans-splicing molecules described herein.

[0228] The expression of the trans-splicing molecule described herein can be achieved in selected cells through delivery by recombinantly engineered AAV or artificial AAV containing a sequence containing / encoding the desired trans-splicing molecule. The use of AAV is a common mode of exogenous DNA delivery because it is relatively non-toxic, provides efficient gene delivery, and can be optimized for specific purposes. Among the well-characterized serotypes of AAV isolated from human or non-human primates, human serotype 2 has been widely used in experiments for efficient gene delivery in different target tissues and animal models.

[0229] In some embodiments, 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 U.S. Patent No. 6,156,303), AAV3-3 or variants thereof (e.g., SEQ ID NOs. 200 and 217 of WO2005033321), AAV4 or variants thereof (e.g., SEQ ID NO. 63 of US20030138772), AAV4-4 or variants thereof (e.g., SEQ ID NOs. 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 its Variants (e.g., sequence number 1-3 of US20030138772), AAV7.2 or variants thereof, AAV8 or variants thereof (e.g., SEQ ID NOs. 4 and 95 of US20030138772 or AAV8(b) (having the amino acid sequence Pro-Glu-Arg-Thr-Ala-Met-Ser-Leu-Pro at amino acid positions 587 to 595 relative to wild-type AAV8, the whole as described in U.S. Patent No. 9,567,376 incorporated herein by reference)), AAV9 or variants thereof (e.g., SEQ ID NOs. 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 (e.g., N. Pulicherla et al., the entirety of which is incorporated herein by reference). Molecular TherapySee 19(6):1070-1078 (2011)), AAV10 or a variant thereof (e.g., Sequence No. 117 of US20030138772), AAV11 or a variant thereof (e.g., Sequence No. 118 of US20030138772), AAV12 or a variant thereof (e.g., Sequence No. 119 of US20030138772), 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 its Variants, AAV42-5a or its variants, AAV42-5b or its variants, AAV42-6b or its variants, AAV42-8 or its variants, AAV42-10 or its variants, AAV42-11 or its variants, AAV42-12 or its variants, AAV42-13 or its variants, AAV42-15 or its variants, AAV42-aa or its variants, AAV43-1 or its variants, AAV43-12 or its variants, AAV43-20 or its variants, AAV43-21 or its variants, AAV43-23 or its variants, AAV43-25 or its variants, AAV43-5 or its variants, AAV44.1 or Variants of this, 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, AAV2-3 / rh.61 or its variants, AAV2-4 / rh.50 or its variants, AAV2-5 / rh.51 or variants thereof, AAV3.1 / hu.6 or variants thereof, AAV3.1 / hu.9 or variants thereof, AAV3-9 / rh.52 or variants thereof, AAV3-11 / rh.53 or variants thereof, AAV4-8 / rh.64 or variants thereof, AAV4-9 / rh.54 or variants thereof (e.g., SEQ ID NO: 116 of WO2005033321), AAV4-19 / rh.55 or variants thereof (e.g., SEQ ID NO: 117 of WO2005033321), AAV5-3 / rh.57 or variants thereof, AAV5-22 / rh.58 or variants thereof, AAV7.3 / hu.7 or variants thereof, AAV16.8 / hu.10 or variants thereof, 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 / hu.61 or variants thereof, AAV33.12 / hu.17 or variants thereof, AAV33.4 / hu.15 or variants thereof, AAV33.8 / hu.16 or variants thereof, AAV52 / hu.19 or variants thereof, AAV52.1 / hu.20 or variants thereof, AAV58.2 / hu.25 or variants thereof, AAVA3.3 or variants thereof, AAVA3.4 or variants thereof, AAVA3.5 or variants 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 ID NO: 2 or 3 of US20140359799), 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 NOs. 8 and 24 of US20150159173), AAVCy.5R1 or a variant thereof, AAVCy.5R2 or a variant thereof, AAVCy.5R3 or a variant thereof, AAVCy.5R4 or a variant thereof, AAVcy.6 or a variant thereof, AAVhu.1 or a variant thereof (e.g., SEQ ID NO. 8 of WO2005033321 144), AAVhu.2 or a variant thereof (e.g., SEQ ID NO: 143 of WO2005033321), AAVhu.3 or a variant thereof (e.g., SEQ ID NO: 145 of WO2005033321), AAVhu.4 or a variant thereof (e.g., SEQ ID NO: 141 of WO2005033321), AAVhu.5 or a variant thereof, AAVhu.6 or a variant thereof (e.g., SEQ ID NO: 84 of WO2005033321), AAVhu.7 or a variant thereof (e.g., SEQ ID NO: 150 of WO2005033321), AAVhu.9 or a variant thereof (e.g., SEQ ID NO: 155 of WO2005033321), AAVhu.10 or a variant thereof (e.g., SEQ ID NO: 156 of WO2005033321), AAVhu.11 or a variant thereof (e.g., SEQ ID NO: 153 of WO2005033321), AAVhu.13 or a variant thereof (SEQ ID NOs: 16 and 32 of US20150159173), AAVhu.15 or a variant thereof (e.g., SEQ ID NO: 147 of WO2005033321), AAVhu.16 or a variant thereof (e.g., SEQ ID NO: 148 of WO2005033321), AAVhu.17 or a variant thereof (e.g., SEQ ID NO: 83), AAVhu.18 or a variant thereof (e.g., SEQ ID NO: 149 of WO2005033321), AAVhu.19 or a variant thereof (e.g., SEQ ID NO: 133 of WO2005033321), AAVhu.20 or a variant thereof (e.g., SEQ ID NO: 134 of WO2005033321), AAVhu.21 or a variant thereof (e.g., SEQ ID NO: 135 of WO2005033321), AAVhu.22 or a variant thereof (e.g., SEQ ID NO: 138 of WO2005033321), AAVhu.23.2 or a variant thereof (e.g., SEQ ID NO: 137 of WO2005033321), AAVhu.24 or a variant thereof (e.g., SEQ ID NO: 136 of WO2005033321), AAVhu.25 or a variant thereof (e.g., SEQ ID NO: 146 of WO2005033321), AAVhu.26 or a variant thereof (e.g., SEQ ID NO: 17 and 33 of US20150159173), AAVhu.27 or a variant thereof (e.g., SEQ ID NO: 140 of WO2005033321), AAVhu.28 or a variant thereof (e.g., SEQ ID NO: 42 of US20150159173), AAVhu.29 or a variant thereof (e.g., SEQ ID NO: 132 of WO2005033321), AAVhu.29R or a variant thereof, AAVhu.31 or a variant thereof (e.g., SEQ ID NO: 121 of WO2005033321), AAVhu.32 or a variant thereof (SEQ ID NO: 122 of WO2005033321), AAVhu.34 or a variant thereof (e.g., SEQ ID NO: 125 of WO2005033321), AAVhu.35 or a variant thereof (e.g., SEQ ID NO: 164 of WO2005033321), AAVhu.37 or a variant thereof (e.g., SEQ ID NOs: 18 and 34 of US20150159173), AAVhu.39 or a variant thereof (e.g., SEQ ID NO: 102 of WO2005033321), AAVhu.40 or a variant thereof (e.g., SEQ ID NO: 87 of WO2005033321), AAVhu.41 or a variant thereof (e.g., SEQ ID NO: 91 of WO2005033321), AAVhu.42 or a variant thereof (e.g., SEQ ID NO: 85 of WO2005033321), AAVhu.43 or a variant thereof (e.g., SEQ ID NO: 160 of WO2005033321), AAVhu.44 or a variant thereof (e.g., SEQ ID NO: 45 of US20150159173), AAVhu.44R1 or a variant thereof, AAVhu.44R2 or a variant thereof, AAVhu.44R3 or a variant thereof, AAVhu.45 or a variant thereof (e.g., SEQ ID NO: 127 of WO2005033321), AAVhu.46 or a variant thereof (e.g., SEQ ID NO: 159 of WO2005033321), AAVhu.47 or a variant thereof (e.g., SEQ ID NO: 128 of WO2005033321), AAVhu.48 or a variant thereof (e.g., SEQ ID NO: 38 of US20150159173), AAVhu.48R1 or a variant thereof, AAVhu.48R2 or a variant thereof, AAVhu.48R3 or a variant thereof, AAVhu.49 or a variant thereof (e.g., SEQ ID NO: 189 of WO2005033321), AAVhu.51 or a variant thereof (e.g., SEQ ID NO: 190 of WO2005033321), AAVhu.52 or a variant thereof (e.g., SEQ ID NO: 191 of WO2005033321), AAVhu.53 or a variant thereof (e.g., SEQ ID NOs: 19 and 35 of US20150159173), AAVhu.54 or a variant thereof (e.g., SEQ ID NO: 188 of WO2005033321), AAVhu.55 or a variant thereof (e.g., SEQ ID NO: 187 of WO2005033321), AAVhu.56 or a variant thereof (e.g., SEQ ID NO: 192 of WO2005033321), AAVhu.57 or a variant thereof (e.g., of WO2005033321 SEQ ID NO: 193), AAVhu.58 or a variant thereof (e.g., SEQ ID NO: 194 of WO2005033321), AAVhu.60 or a variant thereof (e.g., SEQ ID NO: 184 of WO2005033321), AAVhu.61 or a variant thereof (e.g., SEQ ID NO: 185 of WO2005033321), AAVhu.63 or a variant thereof (e.g., SEQ ID NO: 195 of WO2005033321), AAVhu.64 or a variant thereof (e.g., SEQ ID NO: 196 of WO2005033321), AAVhu.66 or a variant thereof (e.g., SEQ ID NO: 197 of WO2005033321), AAVhu.67 or a variant 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 NOs: 9 and 25 of US20150159173), AAVrh.12 or variants thereof, AAVrh.13 or variants thereof (e.g., SEQ ID NOs. 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. 1 of US20150159173), 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 NOs: 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 NOs: 21 and 37 of US20150159173), AAVrh.46 or variants thereof (e.g., SEQ ID NOs. 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. 44 of US20150159173), 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 a variant thereof (e.g., SEQ ID NO: 108 of WO2005033321), AAVrh.51 or a variant thereof (e.g., SEQ ID NO: 104 of WO2005033321), AAVrh.52 or a variant thereof (e.g., SEQ ID NO: 96 of WO2005033321), AAVrh.53 or a variant thereof (e.g., SEQ ID NO: 97 of WO2005033321), AAVrh.54 or a variant thereof (e.g., SEQ ID NO: 49 of US20150159173), AAVrh.56 or a variant thereof (e.g., SEQ ID NO: 152 of WO2005033321), AAVrh.57 or a variant thereof (e.g., SEQ ID NO: 108 of WO2005033321 105), AAVrh.58 or a variant thereof (e.g., SEQ ID NO: 48 of US20150159173), AAVrh.61 or a variant thereof (e.g., SEQ ID NO: 107 of WO2005033321), AAVrh.62 or a variant thereof (e.g., SEQ ID NO: 114 of WO2005033321), AAVrh.64 or a variant thereof (e.g., SEQ ID NO: 43 of US20150159173), AAVrh.64R1 or a variant thereof, AAVrh.64R2 or a variant thereof, AAVrh.67 or a variant thereof (e.g., SEQ ID NO: 47 of US20150159173), AAVrh.73 or a variant thereof (e.g., of US20150159173 SEQ ID NO: 5) or AAVrh.74 or a variant thereof (e.g., SEQ ID NO: 6 of US2015015917).Non-limiting examples of variants are sequence numbers of US20030138772, the contents of which are incorporated herein by reference in their entirety: 9, 27-45, 47-62, 66-69, 73-81, 84-94, 96, 97, 99, and 101-113, and of WO2005033321, the contents of which are incorporated herein by reference in their entirety: 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. Includes 204-212, 215, 219, and 224-236. 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 within the AAV capsid protein at one or more positions interacting with heparin sulfate proteoglycan or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, which are numberings based on the VP1 numbering of AAV2.

[0230] Unless otherwise specified, the AAV ITR and other selected AAV components described herein may be readily selected from any AAV serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or other known and unknown AAV serotypes, without limitation. In one embodiment, the ITR is derived from AAV2. These ITRs or other AAV components may be readily isolated from AAV serotypes using techniques available to those skilled in the art. Such AAV may be isolated or obtained from academic, commercial, or public sources (e.g., American Type Culture Collection, Manassas, Virginia, USA). Alternatively, AAV sequences may be obtained through synthesis or other suitable means by referring to publicly available sequences available in literature or databases such as GenBank, PubMed, or others.

[0231] AAV fragments preferred for assembly into a vector include a cap protein comprising vp1, vp2, vp3, and a hypervariable region, a rep protein comprising rep 78, rep 68, rep 52, and rep 40, and sequences encoding these proteins. These fragments can be readily utilized in various vector systems and host cells. These fragments may be used alone, in combination with other AAV serotype sequences or fragments, or in combination with elements from other AAV or non-AAV viral sequences. As used herein, artificial AAV serotypes include, without limitation, AAVs having non-naturally occurring capsid proteins. Such artificial capsids may be generated by any suitable technique by using a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) in combination with a heterogeneous sequence that may be obtained from a different selected AAV serotype, a non-adjacent portion of the same AAV serotype, a non-AAV viral source, or a non-viral source. Artificial AAV serotypes may be, without limitation, detyped AAVs, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids. Detyped vectors in which the capsid of one AAV is utilized together with an ITR from an AAV having a different capsid protein are useful as described herein. In one embodiment, the AAV is AAV2 / 5 (i.e., an AAV having an AAV2 ITR and an AAV5 capsid). In another embodiment, the AAV is AAV2 / 8 (i.e., an AAV having an AAV2 ITR and an AAV8 capsid). In one embodiment, the AAV comprises an AAV8 capsid. Such an AAV8 capsid comprises the amino acid sequence identified under NCBI reference sequence: YP_077180.1. In another embodiment, the AAV8 capsid comprises a capsid encoded by nt 2121 through 4337 of GenBank accession: AF513852.1.

[0232] In one embodiment, a vector useful in the composition and method described herein contains, at least, a sequence encoding a selected AAV serotype capsid, e.g., an AAV2 capsid, or a fragment thereof. In another embodiment, a useful vector contains, at least, a sequence encoding a selected AAV serotype rep protein, e.g., an AAV2 rep protein, or a fragment thereof. Optionally, such a vector may contain both AAV cap and rep proteins. In a vector in which both AAV rep and cap are provided, the AAV rep and AAV cap sequences may both be of a single serotype origin, e.g., an AAV2 origin.

[0233] Alternatively, a vector derived from an AAV serotype different from the one in which the rep sequence provides the cap sequence may be used. In one embodiment, the rep and cap sequences are expressed from separate sources (e.g., separate vectors, or host cells and vectors). In another embodiment, these rep sequences are fused within a frame to the cap sequence of a different AAV serotype to form a chimeric AAV vector such as that described in U.S. Patent No. 7,282,199, incorporated herein by reference.

[0234] Suitable recombinant AAV (rAAV) is produced by culturing a host cell comprising, as defined herein, a nucleic acid sequence encoding an AAV serotype capsid protein or a fragment thereof; a functional rep gene; a minigene consisting of, for example, an AAV ITR and a trans-splicing molecular nucleic acid sequence; and a helper function sufficient to allow the minigene to be packaged into the AAV capsid protein. The components required for culturing in a host cell to package the AAV minigene into the AAV capsid may be provided to the host cell trans-. Alternatively, any one or more of the required components (e.g., minigene, rep sequence, cap sequence, and / or helper function) may be provided by a stable host cell engineered to contain one or more of the required components using a method known to those skilled in the art.

[0235] In one embodiment, the AAV comprises a promoter (or a functional fragment of a promoter). The selection of the promoter to be used in the rAAV may be made from a number of constitutive or inducible promoters capable of expressing the selected transplant gene in the desired target cells. For example, see 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., in the brainstem). In some embodiments, the promoter is cell-specific. The term "cell-specific" means that a specific promoter selected for the recombinant vector can direct the expression of the selected transplant gene in a specific cell type. In some embodiments, the promoter is specific for the expression of the transplant gene in neuronal cells.

[0236] In another embodiment, the promoter is a natural promoter for the target gene to be expressed. Useful promoters include, without limitation, human synapsin 1 gene promoters, neuron-specific enolase (NSE) promoters, human synapsin 1 promoters, CaMK kinase promoters, or MeCP2 promoters, as well as promoters CAGGS and neuron-specific promoters. Other suitable promoters include inducible promoters, wherein these promoters initiate transcription only when the host cell is exposed to a stimulus that acts as a trigger for promoter activation.

[0237] Other conventional regulatory sequences contained in mini-genes or rAAVs are also disclosed in documents, such as WO 2014 / 124282, which is incorporated herein by reference and incorporated herein by reference. Those skilled in the art may select from these and other expression control sequences without departing from the scope described herein.

[0238] Selected genetic elements may be delivered by any suitable method including those described herein. Methods used to construct any embodiment described herein are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual , see Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for generating rAAV virions are widely known, and the selection of appropriate methods is not limited to the methods and compositions described herein. For example, K. Fisher et al., each incorporated herein by reference, J. Virol See ., 1993 70: 520-532 and U.S. Patent 5,478,745.

[0239] In some embodiments, the trans-splicing molecule comprises a proviral plasmid, e.g., that disclosed in International Patent Publication No. WO 2012 / 158757 incorporated herein by reference. Such a proviral plasmid comprises a wild-type 5' AAV2 ITR sequence adjacent to a unique restriction site allowing the immediate removal or replacement of said ITR by operable association; a promoter comprising a 49-nucleic acid cytomegalovirus sequence upstream of a cytomegalovirus (CMV)-chicken beta-actin sequence, or a neuron-specific promoter / enhancer, a promoter adjacent to a unique restriction site allowing the immediate removal or replacement of the entire promoter sequence, and an upstream sequence adjacent to a unique restriction site allowing the immediate removal or replacement of only the upstream CMV or enhancer sequence from the promoter sequence. The trans-splicing molecule described herein may be inserted into a site of a multi-cloning polylinker, wherein the trans-splicing molecule is operably linked to a promoter and is under the regulatory control of the promoter. A bovine growth hormone polyadenylation sequence adjacent to a unique restriction site allowing the immediate removal or replacement of the poly A sequence; and a wild-type 3' AAV2 ITR sequence adjacent to a unique restriction site allowing the immediate removal or replacement of the 3' ITR are also part of this plasmid. The plasmid backbone contains elements necessary for bacterial cell replication, e.g., a kanamycin resistance gene, and is adjacent to a transcription terminator / insulator sequence.

[0240] In some embodiments, the proviral plasmid comprises (a) a wild-type 5' AAV2 ITR sequence operatively associated with (i) a unique restriction site allowing the immediate removal or replacement of the ITR; and (ii) a modular recombinant AAV genome comprising (A) a 49-nucleic acid CMV sequence upstream of a CMV-chicken beta-actin sequence or (B) a promoter comprising a neuronal cell-specific promoter / enhancer. The promoter is operatively associated with a unique restriction site allowing the immediate removal or replacement of the entire promoter sequence, and the upstream sequence is operatively associated with a unique restriction site allowing the immediate removal or replacement of only the upstream CMV or enhancer sequence from the promoter sequence. A multi-cloning polylinker sequence allowing the insertion of a trans-splicing molecular sequence comprising any of those described herein, wherein the trans-splicing molecule is operatively linked to the promoter and is under its regulatory control; A bovine growth hormone polyadenylation sequence adjacent to a unique restriction site allowing immediate removal or replacement of the above poly-A sequence; and a wild-type 3' AAV2 ITR sequence adjacent to a unique restriction site allowing immediate removal or replacement of the 3' ITR are also part of this proviral plasmid. The proviral plasmid contains elements necessary for replication in bacterial cells and also contains a plasmid backbone further containing a kanamycin resistance gene, said plasmid backbone is adjacent to a transcription terminator / insulator sequence. The proviral plasmid described herein may also contain a non-coding lambda phage 5.1 kb stuffer sequence in the plasmid backbone to increase backbone length and prevent reverse packaging of non-functional AAV genomes.

[0241] In a further embodiment, the promoter of the provirus plasmid is modified to reduce the size of the promoter to allow a larger trans-splicing molecular sequence to be inserted into rAAV. In one embodiment, as described in International Patent Publication No. WO 2017 / 087900, the whole of which is incorporated herein by reference, a CMV / CBA hybrid promoter normally containing non-coding exons and introns totaling about 1,000 base pairs is replaced with a 130-base pair chimeric intron.

[0242] These proviral plasmids are then utilized in current conventional packaging methodologies to generate recombinant viruses expressing trans-splicing molecular transfer genes carried by the proviral plasmids. Suitable production cell lines are readily selected by those skilled in the art. For example, suitable host cells may be selected from any biological organism, including prokaryotic (e.g., bacterial) cells and eukaryotic cells, including insect cells, yeast cells, and mammalian cells. Briefly, the proviral plasmid is transfected into the selected packaging cell and may exist there transiently. Alternatively, a minigene or gene expression cassette to which the ITR is attached is stably integrated into the host cell genome as a chromosome or as an episome. Suitable transfection techniques are known and can be readily utilized to deliver the recombinant AAV genome into the host cell. Typically, the proviral plasmid is cultured in a host cell expressing cap and / or rep proteins. In host cells, a minigene consisting of a trans-splicing molecule to which an AAV ITR is attached is rescued and packaged into a capsid protein or envelope protein to form an infectious viral particle. Thus, recombinant AAV infectious particles are produced by culturing packaging cells containing a proviral plasmid in the presence of a viral sequence sufficient to allow the packaging of the gene-expressing cassette viral genome into the infectious AAV envelope or capsid.

[0243] Alternatively, trans-splicing molecules may be delivered using non-AAV vectors, e.g., non-viral vectors. Any suitable non-viral vector technology known in the art or described herein may be used. Such non-viral vectors suitable for the delivery of trans-splicing molecules include liposomes (e.g., cationic liposomes, monolayer liposomes, or multilayer liposomes), nanoparticles (e.g., polymeric nanoparticles, lipid nanoparticles (LNPs), PEGylated nanoparticles (e.g., PEGylated LNPs), peptide nanoparticles, metal nanoparticles, and others), dendrimers (e.g., cationic dendrimers, e.g., polypropyleneimine dendrimers), and exosomes (e.g., immunologically inactive and / or targeted exosomes, e.g., Alvarez-Erviti, et al., 2011). Nat. Biotechnol. It includes (prepared using the technique described in 29:341), and microvesicles. In some cases, the trans-splicing molecules described herein may be delivered using cell-penetrating peptides (CPPs), which can facilitate the delivery of trans-splicing molecules into the interior of the target cell by translocating the cell membrane of the target cell.

[0244] E. Pharmaceutical composition and kit

[0245] A pharmaceutical composition comprising rAAV comprising any of a nucleic acid trans-splicing molecule, a proviral plasmid, or the MeCP2 nucleic acid trans-splicing molecules described herein is provided herein. In some embodiments, the pharmaceutical composition comprises any of the 3' trans-splicing molecules described herein.

[0246] These pharmaceutical compositions are free from contamination and In vivoIt may be manufactured to be suitable for administration. The pharmaceutical compositions described herein may be evaluated for contamination by conventional methods and then formulated into pharmaceutical compositions intended for a suitable route of administration. Another composition containing trans-splicing molecules, e.g., naked DNA, may be formulated with a similarly suitable carrier. Such formulations involve the use of a pharmaceutically and / or physiologically acceptable vehicle or carrier, particularly one directed for administration to target cells (e.g., neurons). In one embodiment, a carrier suitable for administration to target cells comprises a buffered saline, an isotonic sodium chloride solution, or other buffers, e.g., HEPES, to maintain the pH at an appropriate physiological level, and optionally, other medicinal products, agents, stabilizers, buffers, carriers, ajuvants, diluents, etc.

[0247] 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. Various 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 comprises Tween. If the virus is to be stored for a long period, it may be frozen in the presence of glycerol or TWEEN®20.

[0248] In another embodiment, a composition containing the trans-splicing molecule described herein comprises a surfactant. A surfactant useful for preventing the AAV from adhering to an inert interface and thus ensuring the delivery of a desired dose, such as Pluronic F68 (also known as Poloxamer 188, LUTROL®F68), may be included. As an example, one exemplary composition designed for the treatment of Rett syndrome described herein comprises a recombinant adeno-associated vector having a nucleic acid sequence encoding the 3' trans-splicing molecule described herein, under the control of a regulatory sequence for expressing the trans-splicing molecule in the neuronal cells of a mammalian subject, and a pharmaceutically acceptable carrier. The carrier is an isotonic sodium chloride solution and comprises the surfactant Pluronic F68. In one embodiment, the trans-splicing molecule is any of those described herein.

[0249] In yet another exemplary embodiment, the composition comprises a nucleic acid sequence under the control of a promoter directing the expression of a trans-splicing molecule in a rAAV virus comprising any of the MeCP2 trans-splicing molecules described herein for MeCP2 gene editing, in neurons of the brain, neural stem cells, astrocytes, or oligodendrocytes, or any combination thereof (e.g., neurons of the brainstem), wherein the composition is formulated with a carrier and additional components suitable for intracerebral delivery (e.g., via slow delivery or convection-enhanced infusion) or intraventricular delivery. In yet another embodiment, the kit may include a composition or components for the production or assembly of the composition, comprising a carrier, rAAV particles, a surfactant, and / or components for generating rAAV, as well as suitable laboratory hardware for manufacturing the composition. Such a kit may further include instructions for administering the composition to an individual as a treatment for Rett syndrome, for example.

[0250] Additionally, a kit containing a pharmaceutical composition comprising a 3' trans-splicing molecule is provided herein (e.g., wherein the trans-splicing molecule is packaged in any AAV vector described herein). In some embodiments, the kit includes instructions for mixing the pharmaceutical composition before administration.

[0251] F. Methods and Uses

[0252] The nucleic acid trans-splicing molecules (e.g., nucleic acid trans-splicing molecules and nucleic acid trans-splicing molecule-encoding vectors) and compositions described above are useful for expressing functional MeCP2 in target cells of an individual (e.g., neurons, neural stem cells, astrocytes, or oligodendrocytes, or any combination thereof) and / or regulating the expression of MeCP2, in a method for treating a disease or disorder associated with a mutation in the MeCP2 gene, e.g., Rett syndrome, which includes delaying or improving symptoms associated with Rett syndrome.

[0253] In some embodiments, symptoms of Rett syndrome include, without limitation, developmental delay, cognitive problems, slowed brain and head growth, abnormal hand movements, hyperventilation, speech disorders, movement and coordination disorders (e.g., walking, toe-walking, or wide gait disorders), repetitive movements, unsettled crying or screaming, seizures, and impaired ability to socially interact.

[0254] The nucleic acid trans-splicing molecules (e.g., nucleic acid trans-splicing molecules and nucleic acid trans-splicing molecule-encoding vectors) and compositions described above are useful for their use in treating diseases or disorders associated with mutations in the MeCP2 gene, e.g., Rett syndrome, which include expressing functional MeCP2 in target cells of an individual to be further applied (e.g., neurons, neural stem cells, astrocytes, or oligodendrocytes, or any combination thereof) and / or regulating the expression of MeCP2, e.g., delaying or improving symptoms associated with Rett syndrome as described herein, or for their use in the manufacture of agents for treating diseases or disorders associated with mutations in the MeCP2 gene, e.g., Rett syndrome, which include delaying or improving symptoms associated with Rett syndrome as described herein. These methods and uses involve contacting a target MeCP2 gene (e.g., MeCP2 pre-mRNA) with a trans-splicing molecule [e.g., a 3' trans-splicing molecule as described herein, a composition containing the same (e.g., a pharmaceutical composition) or a drug containing the same] under conditions in which the coding domain of a trans-splicing molecule is spliced ​​to the target MeCP2 pre-mRNA to correct the expression of MeCP2 in target cells, thereby replacing a portion of the target pre-mRNA having one or more defects or mutations with the biologically functional (i.e., healthy), normal, wild-type, or corrected mRNA of the target gene. Thus, the methods and compositions are used to treat Rett syndrome pathologies and symptoms associated with specific mutations.

[0255] In some embodiments, a method for expressing functional MeCP2 in target cells is provided herein by contacting (e.g., transduction) the target cells with any of the nucleic acid trans-splicing molecules, vectors (e.g., AAV vectors), or compositions described herein. In one embodiment, contact involves direct administration of a composition (e.g., a pharmaceutical composition) to an affected individual. In another embodiment, contact involves cultured cells (e.g., neuronal cells or precursors thereof) and treated neuronal cells re-implanted in an individual and In vitro It may occur. In another embodiment, the present method comprises administering rAAV comprising any of the 3' MeCP2 trans-splicing molecules described herein. These methods comprise administering a composition of any of those described herein at an effective concentration to an individual requiring treatment.

[0256] In some embodiments, the present method comprises selecting one or more trans-splicing molecules to treat an individual having a disorder associated with MeCP2 mutations / s. In some embodiments, the use of one or more trans-splicing molecules to treat an individual having a disorder associated with MeCP2 mutations / s, or the use thereof in the manufacture of a drug to treat an individual having a disorder associated with MeCP2 mutations / s, is included herein. Such methods and uses include selecting one or more trans-splicing molecules to treat an individual having a disorder associated with MeCP2 mutations, or to use one or more of these selected trans-splicing molecules in the treatment of an individual having a disorder associated with MeCP2 mutations / s, or to use one or more of these selected trans-splicing molecules in the manufacture of a drug to treat an individual having a disorder associated with MeCP2 mutations / s. Such selection may be based on the genotype of the individual. In some embodiments, the disorder associated with MeCP2 may be an X-linked disorder. Methods for screening and identifying specific mutations in MeCP2 are known in the art.

[0257] The method of the present disclosure comprises 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, the causative mutations associated with Rett syndrome include those listed in Table 1, and most of the mutations identified to date have been found in exon 3 or 4 of MeCP2. Thus, in some embodiments, the method of the present disclosure comprises administering a single trans-splicing molecule to correct at least one of the pathological mutations in exon 3 or exon 4 of the MeCP2 gene, regardless of the location of any other mutation that may be present in other alleles, for example.

[0258] The nucleic acid trans-splicing molecules and vectors, proviral plasmids, and AAVs described herein, as well as compositions comprising such nucleic acid trans-splicing molecules and vectors, proviral plasmids, and AAVs, are intended for use in medical treatment, particularly for the treatment of Rett syndrome. In some embodiments, for example, when using an AAV vector (or other gene therapy vector), the AAV vector may be administered systemically for delivery throughout the brain. In some embodiments, for example, when using an AAV vector (or other gene therapy vector), the AAV vector may be administered systemically for delivery throughout the brain and the enteric nervous system. In some embodiments, for example, when using an AAV vector (or other gene therapy vector), the AAV vector may be administered via direct injection into the brain. In some embodiments, direct injection involves intrathecal injection of the AAV vector into the cerebrospinal fluid. Intrathecal injection provides an efficient delivery method to the CNS, where, for example, neurons may be targeted. In some embodiments, at least one of the brainstem, midbrain, or cortex, or any combination thereof, may be targeted via convective-enhanced diffusion (CED) delivery of the injection into and / or near the brainstem, midbrain, or cortex, or any combination thereof. In some embodiments, the injection may be directed to the brainstem, midbrain, or cortex, or any combination thereof, to provide greater coverage of the brain structures associated with Rett syndrome. Such injection may be performed using magnetic resonance imaging-guided injection. This treatment method is particularly useful for human subjects with Rett syndrome. This treatment includes human subjects with Rett syndrome, including those with a genetic predisposition to the development of Rett syndrome who do not exhibit symptoms of Rett syndrome.Accordingly, in some embodiments, treatment of a human subject with Rett syndrome may include treatment of any human subject having a pathological mutation associated with Rett syndrome.

[0259] In some embodiments, the effective concentration of a recombinant adeno-associated virus having a trans-splicing molecule as described herein is about 10 8 to 10 13 The vector genome is in the range of milliliters (vg / mL). The rAAV infectivity unit is McLaughlin et al., J. Virol It is measured as described in . 1988, 62: 1963. In another embodiment, the concentration is 10 9 to 10 13 It is in the vg / mL range. In another embodiment, the effective concentration is about 1.5 x 10⁻⁶ 11 It is vg / mL. In another embodiment, the effective concentration is about 5 x 10⁻⁶ 11 It is vg / mL. In one embodiment, the effective concentration is about 1.5 x 10⁻⁶ 10 It is vg / mL. In another embodiment, the effective concentration is about 2.8 x 10⁻⁶ 11 It is vg / mL. In another additional embodiment, the effective concentration is about 1.5 x 10⁻⁶ 12 It is vg / mL. In another embodiment, the effective concentration is about 1.5 x 10⁻⁶ 13 It is vg / mL.

[0260] It is desirable to utilize the lowest effective dose of the virus (total genome copy delivered) to reduce the risk of undesirable effects, such as toxicity, and other problems associated with administration to the brain. The effective dose of a recombinant adeno-associated virus having a trans-splicing molecule as described herein is approximately 10 per dose (i.e. per injection). 8 to 10 13 It is a vector genome (vg) range. In one embodiment, the dosage is 10 9 to 10 13It is in the vg range. In another embodiment, the effective dose is about 1.5 x 10⁻⁶ 11 It is vg. In another embodiment, the effective dose is about 5 x 10 11 It is vg. In one embodiment, the effective dose is about 1.5 x 10⁻⁶ 10 It is vg. In another embodiment, the effective dose is about 2.8 x 10⁻⁶ 11 It is vg. In another additional embodiment, the effective dose is about 1.5 x 10⁻⁶ 12 It is vg. In another embodiment, the effective concentration is about 1.5 x 10⁻⁶ 13 vg. The attending physician may select another dosage within these ranges or in other units, taking into account the physical condition of the individual to be treated, i.e., the individual's age; the composition to be administered; and the specific disorder; the targeted cells and, in the case of progressive disorders, the degree of development of the disorder.

[0261] In some embodiments, the composition may be delivered in volumes ranging from about 50 μL to about 1 mL, including all numbers 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 an additional other 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 about 750 μL. In another embodiment, the volume is about 850 μL. In another embodiment, the volume is about 1,000 μL.

[0262] In some embodiments, the treatments and uses described herein replace at least 10% of the target MeCP2 mRNA in target cells (e.g., at least 11% of the target MeCP2 mRNA in target cell(s), at least 12% of the target MeCP2 mRNA in target cell(s), at least 13% of the target MeCP2 mRNA in target cell(s), at least 14% of the target MeCP2 mRNA in target cell(s), at least 15% of the target MeCP2 mRNA in target cell(s), at least 16% of the target MeCP2 mRNA in target cell(s), at least 17% of the target MECP2 mRNA in target cell(s), at least 18% of the target MECP2 mRNA in target cell(s), at least 19% of the target MECP2 mRNA in target cell(s). In some embodiments, the treatments and uses described herein are at least 20% of the target MECP2 mRNA in target cells (e.g., at least 21% of the target MECP2 mRNA in target cell(s), at least 22% of the target MECP2 mRNA in target cell(s), at least 23% of the target MECP2 mRNA in target cell(s), at least 24% of the target MECP2 mRNA in target cell(s), at least 25% of the target MECP2 mRNA in target cell(s), at least 26% of the target MECP2 mRNA in target cell(s), at least 27% of the target MECP2 mRNA in target cell(s), at least 28% of the target MECP2 mRNA in target cell(s), at least 29% of the target MECP2 mRNA in target cell(s), at least 30% of the target MECP2 mRNA in target cell(s), at least 31% of the target MECP2 mRNA in target cell(s), at least 32% of the target MECP2 mRNA in target cell(s), target More than 33% of target MECP2 mRNA in cell(s), more than 34% of target MECP2 mRNA in target cell(s), more than 35% of target MECP2 mRNA in target cell(s), more than 36% of target MECP2 mRNA in target cell(s),More than 37% of target MECP2 mRNA in target cell(s), more than 38% of target MECP2 mRNA in target cell(s), more than 39% of target MECP2 mRNA in target cell(s), more than 40% of target MECP2 mRNA in target cell(s), more than 41% of target MECP2 mRNA in target cell(s), more than 42% of target MECP2 mRNA in target cell(s), more than 43% of target MECP2 mRNA in target cell(s), more than 44% of target MECP2 mRNA in target cell(s), more than 45% of target MECP2 mRNA in target cell(s), more than 46% of target MECP2 mRNA in target cell(s), more than 47% of target MECP2 mRNA in target cell(s), more than 48% of target MECP2 mRNA in target cell(s), more than 49% of target MECP2 mRNA in target cell(s), or target MECP2 in target cell(s) Replaces more than 50% of mRNA. In some embodiments, the treatments and uses described herein are at least 50% of the target MECP2 mRNA in target cells (e.g., at least 51% of the target MECP2 mRNA in target cell(s), at least 52% of the target MECP2 mRNA in target cell(s), at least 53% of the target MECP2 mRNA in target cell(s), at least 54% of the target MECP2 mRNA in target cell(s), at least 55% of the target MECP2 mRNA in target cell(s), at least 56% of the target MECP2 mRNA in target cell(s), at least 57% of the target MECP2 mRNA in target cell(s), at least 58% of the target MECP2 mRNA in target cell(s), at least 59% of the target MECP2 mRNA in target cell(s), at least 60% of the target MECP2 mRNA in target cell(s), at least 61% of the target MECP2 mRNA in target cell(s), at least 62% of the target MECP2 mRNA in target cell(s), target More than 63% of target MECP2 mRNA in cell(s),More than 64% of target MECP2 mRNA in target cell(s), more than 65% of target MECP2 mRNA in target cell(s), more than 66% of target MECP2 mRNA in target cell(s), more than 67% of target MECP2 mRNA in target cell(s), more than 68% of target MECP2 mRNA in target cell(s), more than 69% of target MECP2 mRNA in target cell(s), more than 70% of target MECP2 mRNA in target cell(s), more than 71% of target MECP2 mRNA in target cell(s), more than 72% of target MECP2 mRNA in target cell(s), more than 73% of target MECP2 mRNA in target cell(s), more than 74% of MECP2 mRNA in target cell(s), more than 75% of target MECP2 mRNA in target cell(s), more than 76% of target MECP2 mRNA in target cell(s), of target MECP2 mRNA in target cell(s). 77% or more, 78% or more of target MECP2 mRNA in target cell(s), 79% or more of target MECP2 mRNA in target cell(s), or 80% or more of target MECP2 mRNA in target cell(s) (e.g., 81% or more of target MECP2 mRNA in target cell(s), 82% or more of target MECP2 mRNA in target cell(s), 83% or more of target MECP2 mRNA in target cell(s), 84% or more of target MECP2 mRNA in target cell(s), 85% or more of target MECP2 mRNA in target cell(s), 86% or more of target MECP2 mRNA in target cell(s), 87% or more of target MECP2 mRNA in target cell(s), 88% or more of target MECP2 mRNA in target cell(s), 89% or more of target MECP2 mRNA in target cell(s), 90% or more of target MECP2 mRNA in target cell(s), target More than 91% of target MECP2 mRNA in cell(s), more than 92% of target MECP2 mRNA in target cell(s),Replaces more than 93% of target MECP2 mRNA in target cell(s), more than 94% of target MECP2 mRNA in target cell(s), more than 95% of target MECP2 mRNA in target cell(s), more than 96% of target MECP2 mRNA in target cell(s), more than 97% of target MECP2 mRNA in target cell(s), more than 98% of target MECP2 mRNA in target cell(s), more than 99% of target MECP2 mRNA in target cell(s), or more than 100% of target MECP2 mRNA in target cell(s).

[0263] For each of the described methods and uses, the treatment or use may be used to prevent the occurrence of further damage or to rescue tissues with mild, moderate, or advanced disease. As used herein, the term “rescue” means to prevent the progression of disease and / or to prevent the spread of damage to undamaged cells and / or to improve damage to damaged cells.

[0264] Thus, in one embodiment, the composition is administered before the onset of the disease. In another embodiment, the composition is administered before the onset of symptoms. In another embodiment, the composition is administered after the onset of symptoms. In a further embodiment, the composition is administered, for example, when less than 90% of the target cells remain functional compared to reference tissue. In a further embodiment, the composition is administered, for example, when more than 10% of the target cells remain functional compared to reference tissue. In a further embodiment, the composition is administered when more than 20% of the target cells remain functional. In a further embodiment, the composition is administered when more than 30% of the target cells remain functional. In a further embodiment, the composition is administered when more than 40% of the target cells remain functional. In a further embodiment, the composition is administered when more than 50% of the target cells remain functional. In a further embodiment, the composition is administered when more than 60% of the target cells remain functional. In another additional embodiment, the composition is administered when more than 70% of the target cells remain functional. In another additional embodiment, the composition is administered when more than 80% of the target cells remain functional. In another additional embodiment, the composition is administered when more than 90% of the target cells remain functional. In yet another additional embodiment, the composition is administered when more than 95% of the target cells remain functional.

[0265] In yet another embodiment, any of the methods or uses described above are performed in combination with another or secondary therapy. The therapy may be any currently known or yet unknown therapy that helps prevent, stop, or improve any of these mutations or defects or related effects. The secondary therapy may be administered before, simultaneously with, or after the administration of the pharmaceutical composition described above. In one embodiment, the secondary therapy comprises a non-specific approach to maintain the health of neuronal cells, such as the administration of neurotrophic factors, antioxidants, and / or anti-apoptotic agents. The non-specific approach is achieved through the injection of proteins, recombinant DNA, recombinant viral vectors, stem cells, fetal tissue, or genetically modified cells. The latter may comprise genetically modified cells that are encapsulated.

[0266] For use in these methods, the volume and viral titer of each injection are determined individually and may be the same or different from other injections performed in the brain, for example. The dosage, administration, and regimen may be determined by the attending physician to whom the teachings of this disclosure are given.

[0267] In some embodiments, the method of the present disclosure does not involve modifying the germline genetic identity of any human or other animal.

[0268] The following embodiments do not limit the scope of the embodiments described herein. Those skilled in the art will understand that modifications may be made to the following embodiments intended to be included within the spirit and scope of the invention.

[0269] G. Specific embodiments

[0270] The present disclosure is illustrated by the specific embodiments listed below:

[0271] 1. As an exon editor construct encoding an RNA exon editor, the exon editor construct is:

[0272] (a) A binding domain that binds to a target intron of MeCP2 pre-mRNA;

[0273] (b) hemi-intron; and

[0274] (c) An exon editor construct comprising a sequence encoding a coding domain containing one or more MeCP2 exons.

[0275] 2. An exon editor construct of Embodiment 1, wherein the target intron of the MeCP2 pre-mRNA is intron 1 or intron 2.

[0276] 3. In Embodiment 1 or Embodiment 2, the binding domain is:

[0277] Nucleotides -100 to 1500 or -50 to 1449 of intron 1 (Sequence No. 1);

[0278] Nucleotides 3950 to 4250 or 4020-4169 of intron 1; or

[0279] An exon editor construct that binds to a binding site containing nucleotides 1-300 of intron 2 (sequence number: 2 and 3).

[0280] 4. In any one of the aforementioned embodiments, the binding domain is:

[0281] Nucleotides of intron 1 (SEQ ID NO: 1) -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000; or

[0282] An exon editor construct that binds to a binding site comprising nucleotides 50-300, 50-250, 75-300, 75-250, 80-300, 80-250, 90-300, 90-250, 95-300, 95-250, or 99-248 of intron 2 (SEQ No. 2: 2).

[0283] 5. In any one of the aforementioned embodiments, the binding domain has a size of 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 an exon editor construct ranging from about 150 nucleotides.

[0284] 6. An exon editor composition in any one of the aforementioned embodiments, wherein the binding domain has a size in the range of 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.

[0285] 7. An exon editor composition in any one of the aforementioned embodiments, wherein the binding domain has a size of about 100-200 nucleotides; about 100-150 nucleotides; about 125-200 nucleotides; about 125-150 nucleotides; or about 150 nucleotides; or a size of 100-200 nucleotides; 100-150 nucleotides; 125-200 nucleotides; 125-150 nucleotides; or a size of 150 nucleotides.

[0286] 8. An Exon Editor composition in any one of the above 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 binds.

[0287] 9. In any one of the aforementioned embodiments, the binding domain comprises a stretch of adjacent nucleotides that is 100% complementary to a portion of the binding site to which it binds, wherein the stretch of adjacent nucleotides is at least 5 nucleotide lengths, at least 10 nucleotide lengths, at least 15 nucleotide lengths, at least 20 nucleotide lengths, at least 25 nucleotide lengths, at least 30 nucleotide lengths, at least 35 nucleotide lengths, at least 40 nucleotide lengths, at least 45 nucleotide lengths, at least 50 nucleotide lengths, at least 55 nucleotide lengths, at least 60 nucleotide lengths, at least 65 nucleotide lengths, at least 70 nucleotide lengths, at least 75 nucleotide lengths, at least 80 nucleotide lengths, at least 85 nucleotide lengths, at least 90 nucleotide lengths, at least 95 nucleotide lengths, and at least 100 An exon editor composition having a nucleotide length of at least 105 nucleotide lengths, at least 110 nucleotide lengths, at least 115 nucleotide lengths, at least 120 nucleotide lengths, at least 130 nucleotide lengths, at least 135 nucleotide lengths, at least 140 nucleotide lengths, at least 145 nucleotide lengths, or at least 150 nucleotide lengths.

[0288] 10. An exon editor composition in any one of the embodiments described above, wherein the sequence encoding the binding domain comprises, is essentially made of, or is made of any one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 47.

[0289] 11. An exon editor composition in any one of the embodiments described above, wherein the sequence encoding the binding domain comprises, is essentially composed of, or is composed of SEQ ID NO: 55.

[0290] 12. An exon editor composition in any one of the embodiments described above, wherein the sequence encoding the binding domain comprises, is essentially made of, or is made of any one of SEQ ID NOs: 24, 28, 29, or 30.

[0291] 13. In any one of the aforementioned embodiments, the coding domain is:

[0292] Sequence Nos. 7 and 8 or sequences 90% identical to Sequence Nos. 7 and 8;

[0293] Sequence No. 76 or a sequence that is at least 90% identical to Sequence No. 76;

[0294] Sequence number: 12;

[0295] Sequence number: 14; or

[0296] An exon editor composition comprising, essentially consisting of, or consisting of Sequence No. 9.

[0297] 14. An Exon Editor composition in any one of the aforementioned embodiments, wherein the coding domain comprises, is essentially made of, or is made of either SEQ ID NO: 76 or 14.

[0298] 15. An exon editor composition in any one of the embodiments described above, wherein the coupling domain, the hemi-intron, and the coding domain are operatively connected in the 5'-to-3' direction.

[0299] 16. An exon editor construct in any one of the embodiments described above, wherein the hemi-intron comprises a 3' splice site and optionally, a branching sequence and a polypyrimidine track, and the binding domain, the hemi-intron comprising the 3' splice site, and the coding domain are operatively connected in the 5'-to-3' direction.

[0300] 17. An exon editor composition of embodiment 16, wherein the hemi-intron comprises, is essentially composed of, or is composed of a polypyrimidine track, a branching point, and a 3' splice site.

[0301] 18. In either embodiment 16 or 17, the hemi-intron is:

[0302] An exon editor composition comprising, or essentially composed of, or composed of, sequence number: 17 or a sequence having at least 90% identity with sequence number: 17.

[0303] 19. An exon editor composition in any one of the embodiments described above, further comprising a 3' untranslated region (3' UTR), a binding domain; a hemi-intron; a coding domain; and the 3' UTR being operatively connected in the 5'-to-3' direction.

[0304] 20. An exon editor composition of embodiment 19, wherein the 3' UTR comprises an RDH1pA 3' UTR (SEQ ID: 19) or an mWPRE 3' UTR (SEQ ID: 20).

[0305] 21. In embodiment 20,

[0306] The RDH1pA 3' UTR comprises, is essentially composed of, or is composed of SEQ ID NO: 19 or a sequence having at least 90% identity with SEQ ID NO: 19; or

[0307] An exon editor composition in which the mWPRE 3' UTR comprises, is essentially composed of, or is composed of a sequence having at least 90% identity with SEQ ID NO: 20 or SEQ ID NO: 20.

[0308] 22. In any one of the aforementioned embodiments, the Exon Editor creation is:

[0309] Sequence number: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 47, or 55; a combined domain comprising, essentially consisting of, or consisting of;

[0310] A hemi-intron comprising, essentially consisting of, or consisting of SEQ ID NO: 17;

[0311] Sequence Number: 76, a coding domain that includes, is essentially composed of, or is composed of;

[0312] and includes, or is essentially composed of, or comprises a 3' UTR consisting of SEQ ID NO: 20, and

[0313] Here, the joining domain, hemi-intron, coding domain, and 3' UTR are operatively connected in the 5'-to-3' direction, exon editor construct.

[0314] 23. An exon editor composition of embodiment 22, wherein the exon editor composition comprises, is essentially made of, or is made of SEQ ID NO: 78, 80, 81, 82, 83, or 84.

[0315] 24. An exon editor composition of embodiment 22, wherein the exon editor composition comprises, is essentially made of, or is made of SEQ ID NO: 78 or 80.

[0316] 25. An exon editor construct in any one of embodiments 1 to 19, further comprising a triple helix terminator, a binding domain; a hemi-intron; a coding domain; if present, a 3' UTR; and the triple helix terminator operatively connected in the 5'-to-3' direction.

[0317] 26. An exon editor composition of embodiment 25, wherein the triple helix terminator comprises, is essentially composed of, or is composed of a sequence having at least 90% identity with SEQ ID NO: 77 or SEQ ID NO: 77.

[0318] 27. An exon editor composition of embodiment 25, wherein the triple helix terminator comprises, is essentially composed of, or is composed of SEQ ID NO: 22.

[0319] 28. An exon editor composition, wherein in any one of the embodiments described above, further comprises a sequence encoding a poly A sequence, e.g., SEQ ID NO: 21.

[0320] 29. An exon editor composition, wherein in any one of the embodiments described above, further comprises a sequence encoding an epitope tag, a binding domain; a hemi-intron; a coding domain; an epitope tag; if present, a 3' UTR; and if present, a triple helix terminator operably connected in the 5'-to-3' direction.

[0321] 30. An exon editor composition of embodiment 28, wherein the exon editor composition comprises, is essentially made of, or is made of SEQ ID NO: 78.

[0322] 31. RNA exon editor transcribed from an exon editor construct of any one of embodiments 1 to 30.

[0323] 32. An exon editor construct in any one of embodiments 1 to 30, wherein the MeCP2 pre-mRNA comprises at least one mutation associated with Rett syndrome.

[0324] 33. An exon editor composition in any one of embodiments 1 to 30, wherein 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.

[0325] 34. An exon editor construct in any one of embodiments 32 to 33, wherein at least one mutation associated with Rett syndrome is X-linked.

[0326] 35. An exon editor construct in any one of embodiments 32 to 34, wherein the MeCP2 protein containing at least one mutation associated with Rett syndrome is expressed in at least one of neural stem cells, neurons, astrocytes, or oligodendrocytes, or any combination thereof.

[0327] 36. A vector comprising an exon editor creation of any one of embodiments 1 to 30 or 32 to 35.

[0328] 37. In embodiment 36, the vector comprises a 5' control domain operably connected to a 5' bonding domain.

[0329] 38. A vector in any one of embodiments 36 to 37, wherein the 5' regulatory domain comprises a constitutive promoter or a tissue-specific promoter.

[0330] 39. Vector in embodiment 38, wherein the constitutive promoter is a CMV promoter.

[0331] 40. A provirus plasmid comprising an exon editor construct of any one of embodiments 1 to 30 or 32 to 35.

[0332] 41. An adeno-associated virus (AAV) comprising an exon editor composition of any one of embodiments 1 to 30 or 32 to 35, wherein the AAV optionally comprises a 5' control domain operatively linked to the exon editor composition at 5'.

[0333] 42. The AAV of embodiment 41, wherein the AAV comprises a 5' control domain operably connected to a binding domain at 5'.

[0334] 43. An AAV in any one of embodiments 41 to 42, wherein the 5' control domain comprises a constitutive promoter.

[0335] 44. An AAV in embodiment 43, wherein the constitutive promoter is a CMV promoter.

[0336] 45. In any one of embodiments 41 to 44, the AAV exhibits neuronal tendencies.

[0337] 46. ​​In any one of embodiments 41 to 45, the AAV is AAV9, AAV8, AAV5, or AAV2, or a variant of AAV9, AAV8, AAV5, or AAV2.

[0338] 47. A composition comprising an exon editor construct of any one of embodiments 1 to 30 or 32 to 35, a vector of any one of embodiments 36 to 39, a provirus plasmid of embodiment 40, or an AAV of any one of embodiments 41 to 46.

[0339] 48. A composition comprising a pharmaceutically acceptable excipient in embodiment 47.

[0340] 49. A method for restoring the functional level of MeCP2 protein in target cells by expressing biologically active MeCP2 in target cells, comprising transfecting the target cells with an exon editor construct of any one of embodiments 1 to 30 or 32 to 35, a vector of any one of embodiments 36 to 39, a provirus plasmid of embodiment 40, an AAV of any one of embodiments 41 to 46, or a composition of any one of embodiments 47 to 48.

[0341] 50. A method according to 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 MeCP2 pre-mRNA containing at least one mutation associated with Rett syndrome in target cells is modified to become a transcript encoding functional MeCP2 in which at least one mutation is corrected.

[0342] 51. A method according to 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 MeCP2 pre-mRNA containing at least one mutation associated with Rett syndrome in target cells is modified to become a transcript encoding functional MeCP2 in which at least one mutation is corrected.

[0343] 52. A method according to embodiment 51, wherein at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of MeCP2 pre-mRNA containing at least one mutation associated with Rett syndrome in target cells is modified to become a transcript encoding functional MeCP2 in which at least one mutation is corrected.

[0344] 53. A method in any one of embodiments 49 to 52, wherein the functional level of MeCP2 is restored in a target cell by expressing a biologically functional MeCP2 protein.

[0345] 54. A method for reducing the expression of MeCP2 containing at least one mutation associated with Rett syndrome in a subject, comprising transfecting or transducing a target cell, more particularly a neuron, in the subject with an exon editor construct of any one of embodiments 1 to 30 or 32 to 35, a vector of any one of embodiments 36 to 39, a provirus plasmid of embodiment 40, an AAV of any one of embodiments 41 to 46, or a composition of any one of embodiments 47 to 48.

[0346] 55. A method for correcting at least one mutation in the MeCP2 exon sequence of MeCP2 pre-mRNA in a target cell of a subject, 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 provirus plasmid of embodiment 40, an AAV of any one of embodiments 41-46, or a composition of any one of embodiments 47-48.

[0347] 56. A method for treating Rett syndrome in a subject requiring treatment, 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 provirus plasmid of embodiment 40, an AAV of any one of embodiments 41-46, or a composition of any one of embodiments 47-48 in a therapeutically effective amount.

[0348] 57. A method in any one of embodiments 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 MeCP2 pre-mRNA containing at least one mutation associated with Rett syndrome in target cells is modified to become a transcript encoding functional MeCP2 in which at least one mutation is corrected.

[0349] 58. A method according to 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 MeCP2 pre-mRNA containing at least one mutation associated with Rett syndrome in target cells is modified to become a transcript encoding functional MeCP2 in which at least one mutation is corrected.

[0350] 59. A method according to embodiment 58, wherein at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of MeCP2 pre-mRNA containing at least one mutation associated with Rett syndrome in target cells is modified to become a transcript encoding functional MeCP2 in which at least one mutation is corrected.

[0351] 60. A method in any one of embodiments 54 to 59, wherein the functional level of MeCP2 is restored in target cells by expressing a biologically functional MeCP2 protein.

[0352] 61. A method comprising administering to the brain of a subject any one of embodiments 54 to 60 an exon editor construct of any one of embodiments 1 to 30 or 32 to 35, a vector of any one of embodiments 36 to 39, a provirus plasmid of embodiment 40, an AAV of any one of embodiments 41 to 46, or a composition of any one of embodiments 47 to 48.

[0353] 62. A method in any one of embodiments 54 to 61, wherein the subject is a mammal, preferably a rodent, a non-human primate, or a human.

[0354] 63. A method in any one of embodiments 54 to 62, wherein the subject has a genetic predisposition to Rett syndrome or is diagnosed with Rett syndrome.

[0355] 64. An exon editor construct of any one of embodiments 1 to 30 or 32 to 35, a vector of any one of embodiments 36 to 39, a provirus plasmid of embodiment 40, an AAV of any one of embodiments 41 to 46, or a composition of any one of embodiments 47 to 48 for use in the prevention or treatment of Rett syndrome in subjects requiring prevention or treatment.

[0356] 65. An exon editor construct of any one of embodiments 1 to 30 or 32 to 35, a vector of any one of embodiments 36 to 39, a provirus plasmid of embodiment 40, an AAV of any one of embodiments 41 to 46, or a composition of any one of embodiments 47 to 48 for use in the manufacture of a drug for the treatment or prevention of Rett syndrome in subjects requiring treatment or prevention.

[0357] 66. Operablely connected in the 5' to 3' direction:

[0358] (a) A binding domain configured to bind to a binding site within the target region of MeCP2 pre-mRNA;

[0359] (b) hemi-intron; and

[0360] (c) An RNA exon editor comprising a coding domain comprising a sequence encoding one or more MeCP2 exons.

[0361] 67. An RNA exon editor according to embodiment 66, wherein the target region comprises or is composed of nucleotides -100 to 1500 of MeCP2 intron 1 (positions 15 to 1615 of sequence number 111).

[0362] 68. An RNA exon editor according to embodiment 66, wherein the target region comprises or is composed of nucleotides -100 to 100 of MeCP2 intron 1 (positions 15 to 215 of sequence number 111).

[0363] 69. An RNA exon editor according to embodiment 66, wherein the target region comprises or is composed of nucleotides -50 to 1449 of MeCP2 intron 1 (positions 65 to 1564 of sequence number 111).

[0364] 70. An RNA exon editor according to embodiment 66, wherein the target region comprises or is composed of nucleotides -50 to 99 of MeCP2 intron 1 (positions 65 to 214 of sequence number 111).

[0365] 71. An RNA exon editor according to embodiment 66, wherein the target region comprises or is composed of nucleotides 51 to 649 of MeCP2 intron 1 (positions 51 to 649 of SEQ ID NO: 1).

[0366] 72. An RNA exon editor according to embodiment 66, wherein the target region comprises or is composed of nucleotides 51 to 524 of MeCP2 intron 1 (positions 51 to 524 of SEQ ID NO: 1).

[0367] 73. An RNA exon editor according to embodiment 66, wherein the target region comprises or is composed of nucleotides 51 to 1449 of MeCP2 intron 1 (positions 51 to 1449 of SEQ ID NO: 1).

[0368] 74. An RNA exon editor according to embodiment 66, wherein the target region comprises or is composed of nucleotides 125 to 524 of MeCP2 intron 1 (positions 125 to 524 of SEQ ID NO: 1).

[0369] 75. An RNA exon editor according to embodiment 66, wherein the target region comprises or is composed of nucleotides 125 to 649 of MeCP2 intron 1 (positions 125 to 649 of sequence number 1).

[0370] 76. An RNA exon editor according to embodiment 66, wherein the target region comprises or is composed of nucleotides 820 to 1449 of MeCP2 intron 1 (positions 820 to 1449 of SEQ ID NO: 1).

[0371] 77. An RNA exon editor according to embodiment 66, wherein the target region comprises or is composed of nucleotides 3950 to 4250 of MeCP2 intron 1 (positions 3950 to 4250 of SEQ ID NO: 1).

[0372] 78. An RNA exon editor according to embodiment 66, wherein the target region comprises or is composed of nucleotides 4020 to 4169 of MeCP2 intron 1 (positions 4020 to 4169 of SEQ ID NO: 1).

[0373] 79. An RNA exon editor according to embodiment 66, wherein the target region comprises or is composed of nucleotides 1 to 300 of MeCP2 intron 2 (positions 1 to 300 of SEQ ID NO: 2).

[0374] 80. An RNA exon editor according to embodiment 66, wherein the target region comprises or is composed of nucleotides 99 to 248 of MeCP2 intron 2 (positions 99 to 248 of sequence number 2).

[0375] 81. An RNA exon editor in any one of embodiments 66 to 80, wherein the binding site comprises or is composed of at least 25 consecutive nucleotides of a target region.

[0376] 82. An RNA exon editor in any one of embodiments 66 to 80, wherein the binding site comprises or is composed of at least 50 consecutive nucleotides of a target region.

[0377] 83. An RNA exon editor in any one of embodiments 66 to 80, wherein the binding site comprises or is composed of 25 to 100 consecutive nucleotides of a target region.

[0378] 84. An RNA exon editor in any one of embodiments 66 to 80, wherein the binding site comprises or is composed of 50 to 100 consecutive nucleotides of a target region.

[0379] 85. An RNA exon editor in any one of embodiments 66 to 80, wherein the binding site comprises or is composed of 50 to 150 consecutive nucleotides of a target region.

[0380] 86. An RNA exon editor in any one of embodiments 66 to 80, wherein the binding site comprises or is composed of 100 to 200 consecutive nucleotides of a target region.

[0381] 87. An RNA exon editor in any one of embodiments 66 to 80, wherein the binding site comprises or is composed of 100 to 150 consecutive nucleotides of a target region.

[0382] 88. An RNA exon editor in any one of embodiments 66 to 80, wherein the binding site comprises or is composed of 150 consecutive nucleotides of a target region.

[0383] 89. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain comprises or is composed of a nucleotide sequence that is at least 80% complementary to the entire binding site.

[0384] 90. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain comprises or is composed of a nucleotide sequence that is at least 90% complementary to the entire binding site.

[0385] 91. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain comprises or is composed of a nucleotide sequence that is 100% complementary to the entire binding site.

[0386] 92. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain comprises or is composed of a nucleotide sequence that is at least 80% complementary to 25 consecutive nucleotides within the binding site.

[0387] 93. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain comprises or is composed of a nucleotide sequence that is at least 90% complementary to 25 consecutive nucleotides within the binding site.

[0388] 94. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain comprises or is composed of a nucleotide sequence that is 100% complementary to 25 consecutive nucleotides within the binding site.

[0389] 95. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain comprises or is composed of a nucleotide sequence that is at least 80% complementary to 50 consecutive nucleotides within the binding site.

[0390] 96. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain comprises or is composed of a nucleotide sequence that is at least 90% complementary to 50 consecutive nucleotides within the binding site.

[0391] 97. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain comprises or is composed of a nucleotide sequence that is 100% complementary to 50 consecutive nucleotides within the binding site.

[0392] 98. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain comprises or is composed of a nucleotide sequence that is at least 80% complementary to 75 consecutive nucleotides within the binding site.

[0393] 99. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain comprises or is composed of a nucleotide sequence that is at least 90% complementary to 75 consecutive nucleotides within the binding site.

[0394] 100. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain comprises or is composed of a nucleotide sequence that is 100% complementary to 75 consecutive nucleotides within the binding site.

[0395] 101. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain comprises or is composed of a nucleotide sequence that is at least 80% complementary to 100 consecutive nucleotides within the binding site.

[0396] 102. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain comprises or is composed of a nucleotide sequence that is at least 90% complementary to 100 consecutive nucleotides within the binding site.

[0397] 103. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain comprises or is composed of a nucleotide sequence that is 100% complementary to 100 consecutive nucleotides within the binding site.

[0398] 104. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain comprises or is composed of a nucleotide sequence that is at least 80% complementary to 150 consecutive nucleotides within the binding site.

[0399] 105. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain comprises or is composed of a nucleotide sequence that is at least 90% complementary to 150 consecutive nucleotides within the binding site.

[0400] 106. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain comprises or is composed of a nucleotide sequence that is 100% complementary to 150 consecutive nucleotides within the binding site.

[0401] 107. An RNA exon editor in any one of embodiments 66 to 106, wherein the binding domain is at least 25 nucleotides long.

[0402] 108. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain is at least 50 nucleotides long.

[0403] 109. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain is at least 100 nucleotides long.

[0404] 110. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain is at least 150 nucleotides long.

[0405] 111. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain is 25 to 150 nucleotides long.

[0406] 112. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain is 50 to 100 nucleotides long.

[0407] 113. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain is 50 to 150 nucleotides long.

[0408] 114. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain is 100 to 150 nucleotides long.

[0409] 115. An RNA exon editor in any one of embodiments 66 to 88, wherein the binding domain is 100 to 200 nucleotides long.

[0410] 116. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 23 (where U is substituted for each T).

[0411] 117. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 23 (where U is substituted for each T).

[0412] 118. An RNA exon editor according to Embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 23 (where U is substituted for each T).

[0413] 119. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 24 (where U is substituted for each T).

[0414] 120. An RNA exon editor according to embodiment 119, wherein the binding domain comprises nucleotides 23C and / or 72C numbered according to SEQ ID NO: 24 (where U is substituted for each T).

[0415] 121. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 24 (where U is substituted for each T).

[0416] 122. An RNA exon editor according to Embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 24 (where U is substituted for each T).

[0417] 123. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 25 (where U is substituted for each T).

[0418] 124. An RNA exon editor according to embodiment 123, wherein the binding domain comprises nucleotides 97C and / or 146C numbered according to SEQ ID NO: 25 (where U is substituted for each T).

[0419] 125. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 25 (where U is substituted for each T).

[0420] 126. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 25 (where U is substituted for each T).

[0421] 127. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 26 (where U is substituted for each T).

[0422] 128. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 26 (where U is substituted for each T).

[0423] 129. An RNA exon editor according to Embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 26 (where U is substituted for each T).

[0424] 130. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 27 (where U is substituted for each T).

[0425] 131. An RNA exon editor according to 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 U is substituted for each T).

[0426] 132. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 27 (where U is substituted for each T).

[0427] 133. An RNA exon editor according to Embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 27 (where U is substituted for each T).

[0428] 134. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 28 (where U is substituted for each T).

[0429] 135. An RNA exon editor according to embodiment 134, wherein the binding domain comprises nucleotide 133C numbered according to SEQ ID NO: 28 (where U is substituted for each T).

[0430] 136. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 28 (where U is substituted for each T).

[0431] 137. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 28 (where U is substituted for each T).

[0432] 138. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 29 (where U is substituted for each T).

[0433] 139. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 29 (where U is substituted for each T).

[0434] 140. An RNA exon editor according to Embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 29 (where U is substituted for each T).

[0435] 141. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 30 (where U is substituted for each T).

[0436] 142. An RNA exon editor according to embodiment 141, wherein the binding domain comprises nucleotide 4C, 71C, or 101C, or any combination thereof, numbered according to SEQ ID NO: 30 (where U is substituted for each T).

[0437] 143. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 30 (where U is substituted for each T).

[0438] 144. An RNA exon editor according to Embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 30 (where U is substituted for each T).

[0439] 145. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 31 (where U is substituted for each T).

[0440] 146. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 31 (where U is substituted for each T).

[0441] 147. An RNA exon editor according to Embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 31 (where U is substituted for each T).

[0442] 148. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 32 (where U is substituted for each T).

[0443] 149. An RNA exon editor according to embodiment 148, wherein the binding domain comprises nucleotide 119A numbered according to SEQ ID NO: 32 (where U is substituted for each T).

[0444] 150. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 32 (where U is substituted for each T).

[0445] 151. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 32 (where U is substituted for each T).

[0446] 152. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 33 (where U is substituted for each T).

[0447] 153. An RNA exon editor according to embodiment 152, wherein the binding domain comprises nucleotide 133C numbered according to SEQ ID NO: 33 (where U is substituted for each T).

[0448] 154. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 33 (where U is substituted for each T).

[0449] 155. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 33 (where U is substituted for each T).

[0450] 156. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 47 (where U is substituted for each T).

[0451] 157. An RNA exon editor according to embodiment 156, wherein the binding domain comprises nucleotide 149C numbered according to SEQ ID NO: 47 (where U is substituted for each T).

[0452] 158. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 47 (where U is substituted for each T).

[0453] 159. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 47 (where U is substituted for each T).

[0454] 160. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 55 (where U is substituted for each T).

[0455] 161. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 55 (where U is substituted for each T).

[0456] 162. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 55 (where U is substituted for each T).

[0457] 163. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 88, and the binding domain has a C at one or more of positions 25, 59, 63, 99, or 120 numbered according to SEQ ID NO: 88 (where U is substituted for each T).

[0458] 164. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 88, and the binding domain has C at positions 25, 59, 63, 99, or 120 numbered according to SEQ ID NO: 88 (where U is substituted for each T).

[0459] 165. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 88, and the binding domain has a C at one or more of positions 25, 59, 63, 99, or 120 numbered according to SEQ ID NO: 88 (where U is substituted for each T).

[0460] 166. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 88, and the binding domain has C at positions 25, 59, 63, 99, or 120 numbered according to SEQ ID NO: 88 (where U is substituted for each T).

[0461] 167. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 88 (where U is substituted for each T).

[0462] 168. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 89 (where U is substituted for each T).

[0463] 169. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 89 (where U is substituted for each T).

[0464] 170. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 89 (where U is substituted for each T).

[0465] 171. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 90, and the binding domain has an A at position 84 or a C at position 106 numbered according to SEQ ID NO: 90 (where U is substituted for each T).

[0466] 172. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 90, and the binding domain has A at position 84 and C at position 106, numbered according to SEQ ID NO: 90 (where U is substituted for each T).

[0467] 173. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 90, and the binding domain has an A at position 84 or a C at position 106 numbered according to SEQ ID NO: 90 (where U is substituted for each T).

[0468] 174. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 90, and the binding domain has an A at position 84 or a C at position 106 numbered according to SEQ ID NO: 90 (where U is substituted for each T).

[0469] 175. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 90 (where U is substituted for each T).

[0470] 176. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 91, and the binding domain has a C at position 136 or 138 numbered according to SEQ ID NO: 91 (where U is substituted for each T).

[0471] 177. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 91, and the binding domain has C at positions 136 and 138 numbered according to SEQ ID NO: 91 (where U is substituted for each T).

[0472] 178. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 91, and the binding domain has a C at position 136 or 138 numbered according to SEQ ID NO: 91 (where U is substituted for each T).

[0473] 179. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 91, and the binding domain has C at positions 136 and 138 numbered according to SEQ ID NO: 91 (where U is substituted for each T).

[0474] 180. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 91 (where U is substituted for each T).

[0475] 181. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 92, and the binding domain has a C at position 51 numbered according to SEQ ID NO: 92 (where U is substituted for each T).

[0476] 182. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 92, and the binding domain has a C at position 51 numbered according to SEQ ID NO: 92 (where U is substituted for each T).

[0477] 183. An RNA exon editor according to embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity with SEQ ID NO: 92 (where U is substituted for each T).

[0478] 184. An RNA exon editor in any one of embodiments 66 to 183, wherein the binding domain comprises one or more nucleotide substitutions relative to an endogenous MeCP2 mRNA sequence, and the one or more nucleotide substitutions destroy coding splice sites within the binding domain sequence.

[0479] 185. An RNA exon editor 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.

[0480] 186. An RNA exon editor in any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence encoding a wild-type MeCP2 exon 4 amino acid sequence.

[0481] 187. An RNA exon editor 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.

[0482] 188. An RNA exon editor in any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence encoding a wild-type MeCP2 exon 3 amino acid sequence and a wild-type MeCP2 exon 4 amino acid sequence.

[0483] 189. An RNA exon editor 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.

[0484] 190. An RNA exon editor in any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence encoding a wild-type MeCP2 exon 2 amino acid sequence, a wild-type MeCP2 exon 3 amino acid sequence, and a wild-type MeCP2 exon 4 amino acid sequence.

[0485] 191. An RNA exon editor, wherein in any one of embodiments 66 to 184, the coding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 7 (where U is substituted for each T) in the 5' to 3' direction and a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 8 (where U is substituted for each T).

[0486] 192. An RNA exon editor, wherein in any one of embodiments 66 to 184, the coding domain comprises the nucleotide sequence of SEQ ID NO: 7 (where U is substituted for each T) and the nucleotide sequence of SEQ ID NO: 8 (where U is substituted for each T) in the 5' to 3' direction.

[0487] 193. An RNA exon editor, wherein in any one of embodiments 66 to 184, the coding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 11 (where U is substituted for each T) in the 5' to 3' direction and a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 12 (where U is substituted for each T).

[0488] 194. An RNA exon editor, wherein in any one of embodiments 66 to 184, the coding domain comprises the nucleotide sequence of SEQ ID NO: 11 (where U is substituted for each T) and the nucleotide sequence of SEQ ID NO: 12 (where U is substituted for each T) in the 5' to 3' direction.

[0489] 195. An RNA exon editor, wherein in any one of embodiments 66 to 184, the coding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 10 (where U is substituted for each T) in the 5' to 3' direction, a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 11 (where U is substituted for each T), and a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 12 (where U is substituted for each T).

[0490] 196. An RNA exon editor, wherein in any one of embodiments 66 to 184, the coding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 12 (where U is substituted for each T).

[0491] 197. An RNA exon editor in any one of embodiments 66 to 184, wherein the coding domain comprises the nucleotide sequence of SEQ ID NO: 12 (where U is substituted for each T).

[0492] 198. An RNA exon editor, wherein in any one of embodiments 66 to 184, the coding domain comprises the nucleotide sequence of SEQ ID NO: 10 (where U is substituted for each T), the nucleotide sequence of SEQ ID NO: 11 (where U is substituted for each T), and the nucleotide sequence of SEQ ID NO: 12 (where U is substituted for each T) in the 5' to 3' direction.

[0493] 199. An RNA exon editor, wherein in any one of embodiments 66 to 184, the coding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 9 (where U is substituted for each T).

[0494] 200. An RNA exon editor, wherein in any one of embodiments 66 to 184, the coding domain comprises the nucleotide sequence of SEQ ID NO: 9 (where U is substituted for each T).

[0495] 201. An RNA exon editor, wherein in any one of embodiments 66 to 184, the coding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 13 (where U is substituted for each T) in the 5' to 3' direction and a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 14 (where U is substituted for each T).

[0496] 202. An RNA exon editor, wherein in any one of embodiments 66 to 184, the coding domain comprises the nucleotide sequence of SEQ ID NO: 13 (where U is substituted for each T) and the nucleotide sequence of SEQ ID NO: 14 (where U is substituted for each T) in the 5' to 3' direction.

[0497] 203. An RNA exon editor, wherein in any one of embodiments 66 to 184, the coding domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 14 (where U is substituted for each T).

[0498] 204. An RNA exon editor in any one of embodiments 66 to 184, wherein the coding domain comprises the nucleotide sequence of SEQ ID NO: 14 (where U is substituted for each T).

[0499] 205. An RNA exon editor in any one of embodiments 66 to 204, wherein the coding domain comprises at least one nucleotide substitution relative to the endogenous RNA molecular sequence, and the at least one nucleotide substitution destroys a coding splice site within the coding domain sequence.

[0500] 206. An RNA exon editor according to claim 205, wherein at least one nucleotide substitution is a synonymous nucleotide substitution.

[0501] 207. In any one of embodiments 66 to 184, the coding domain comprises a MeCP2 exon 3 nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 85 (where U is substituted for each T) in the 5' to 3' direction and a MeCP2 exon 4 nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 86 (where U is substituted for each T), wherein the MeCP2 exon 3 nucleotide sequence is numbered according to SEQ ID NO: 7, and the following nucleotides: 43T, 49G, 55A, 67C, 85G, 91G, 94A, 97A, 103C, 106A, 109G, 115A, 118T, 119T, 120C, 130T, 133G, 176T, The following nucleotides, comprising one or more of 177C, 182T, 183C, 184G, 206T, 207C, 223A, 239C, 241T, 280A, 290C, 317C, 320T, 321C, or 331A, wherein the MeCP2 exon 4 nucleotide sequence is numbered according to SEQ NO: 8: 49T, 55A, 61T, 67G, 94T, 106A, 107C, 130A, 286T, 304A, 595T, 683C, 740T, 741C, 806T, 807C, 808T, 809C, 824T, 825C, 851T, RNA exon editor comprising one or more of 852C, 854T, 855C, 892G, 980C, 982C, 1034C, 1055C, 1072A, 1073C, or 1075T.

[0502] 208. An RNA exon editor, wherein in any one of embodiments 66 to 184, the coding domain comprises the nucleotide sequence of SEQ ID NO: 85 (where U is substituted for each T) and the nucleotide sequence of SEQ ID NO: 86 (where U is substituted for each T) in the 5' to 3' direction.

[0503] 209. In any one of embodiments 66 to 184, the coding domain comprises a nucleotide sequence including a MeCP2 exon 3 sequence and a MeCP2 exon 4 sequence, the nucleotide sequence has at least 90% sequence identity with SEQ ID NO: 76 (where U is substituted for each T), and the MeCP2 exon 3 nucleotide sequence is numbered according to SEQ ID NO: 7, 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, The following nucleotides, comprising one or more of 207C, 223A, 239C, 241T, 280A, 290C, 317C, 320T, 321C, or 331A, wherein the MeCP2 exon 4 nucleotide sequence is numbered according to SEQ NO: 8: 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, RNA exon editor containing one or more of 982C, 1034C, 1055C, 1072A, 1073C, or 1075T.

[0504] 210. An RNA exon editor comprising, in any one of embodiments 66 to 209, a branching point, a polypyrimidine track, and a 3' splice site, wherein the hemi-intron is operably connected in the 5' to 3' direction.

[0505] 211. An RNA exon editor according to embodiment 210, wherein the branching point is at least 15 to 25 nucleotides upstream of the 3' splice site.

[0506] 212. An RNA exon editor according to embodiment 210 or 211, wherein the 3' splice site comprises the nucleotide sequence YAG, wherein Y is a pyrimidine.

[0507] 213. An RNA exon editor in any one of embodiments 210 to 212, wherein the polypyrimidine track comprises at least 10 consecutive pyrimidine nucleotides.

[0508] 214. An RNA exon editor, wherein in any one of embodiments 210 to 213, the hemi-intron comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 17 (where U is substituted for each T).

[0509] 215. An RNA exon editor in any one of embodiments 210 to 214, wherein the hemi-intron comprises the nucleotide sequence of SEQ ID NO: 17 (where U is substituted for each T).

[0510] 216. An RNA exon editor, wherein in any one of embodiments 66 to 215, the RNA exon editor further comprises a 3' untranslated region (3'UTR) operably connected to the coding domain as 3'.

[0511] 217. An RNA exon editor comprising, in any one of embodiments 66 to 215, a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 19 (where U is substituted for each T) (RHD1pA 3' UTR).

[0512] 218. An RNA exon editor according to embodiment 216 or 217, wherein the 3' UTR comprises the nucleotide sequence of SEQ ID NO: 19 (where U is substituted for each T).

[0513] 219. An RNA exon editor according to embodiment 216, wherein the 3' UTR comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 20 (where U is substituted for each T) (mWPRE 3' UTR).

[0514] 220. An RNA exon editor according to embodiment 219, wherein the 3' UTR comprises the nucleotide sequence of SEQ ID NO: 20 (where U is substituted for each T).

[0515] 221. An RNA exon editor according to embodiment 216, wherein the 3' UTR comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 87 (where U is substituted for each T) (mWPRE 3' UTR is truncated).

[0516] 222. An RNA exon editor according to embodiment 221, wherein the 3' UTR comprises the nucleotide sequence of SEQ ID NO: 87 (where U is substituted for each T).

[0517] 223. An RNA exon editor comprising, in any one of embodiments 66 to 222, a polyadenylation sequence having at least 90% sequence identity with SEQ ID NO: 21 (where U is substituted for each T) (Sv40pA element).

[0518] 224. An RNA exon editor according to embodiment 223, wherein the polyadenylation sequence comprises the nucleotide sequence of SEQ ID NO: 21 (where U is substituted for each T).

[0519] 225. An RNA exon editor, wherein in any one of embodiments 66 to 222, a transcription terminator domain operably linked to the 3' UTR to the 3' UTR is further included.

[0520] 226. An RNA exon editor according to embodiment 225, wherein the transcription terminator domain comprises a triple helix terminator.

[0521] 227. An RNA exon editor according to embodiment 225 or 226, wherein the transcription terminator domain comprises a MALAT1 terminator.

[0522] 228. An RNA exon editor 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 U is substituted for each T).

[0523] 229. An RNA exon editor in any one of embodiments 225 to 228, wherein the transcription terminator domain comprises the nucleotide sequence of SEQ ID NO: 77 (where U is substituted for each T).

[0524] 230. An RNA exon editor, wherein in any one of embodiments 225 to 227, the transcription terminator domain comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 22 (where U is substituted for each T).

[0525] 231. An RNA exon editor 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 U is substituted for each T).

[0526] 232. An RNA exon editor comprising, in any one of embodiments 225 to 231, a 3xUBS sequence operably linked to a transcription terminator domain at 3'.

[0527] 233. An RNA exon editor according to embodiment 232, wherein the 3xUBS sequence comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 79 (where U is substituted for each T).

[0528] 234. An RNA exon editor according to embodiment 232 or 233, wherein the 3xUBS sequence comprises the nucleotide sequence of SEQ ID NO: 79 (where U is substituted for each T).

[0529] 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 U is substituted for each T).

[0530] 236. RNA exon editor comprising the nucleotide sequence of nucleotides 601–2522 of SEQ ID NO: 78 (where U is substituted for each T).

[0531] 237. An RNA exon editor comprising a nucleotide sequence having at least 90% sequence identity with nucleotide 601-2483 of SEQ ID NO: 80 (where U is substituted for each T).

[0532] 238. RNA exon editor comprising a neurotide sequence of nucleotides 601–2483 of SEQ ID NO: 80 (where U is substituted for each T).

[0533] 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 U is substituted for each T).

[0534] 240. RNA exon editor comprising the nucleotide sequence of nucleotides 601–2522 of SEQ ID NO: 81 (where U is substituted for each T).

[0535] 241. An RNA exon editor comprising a neurotide sequence having at least 90% sequence identity with nucleotides 601-3011 of SEQ ID NO: 82 (where U is substituted for each T).

[0536] 242. RNA exon editor comprising the nucleotide sequence of nucleotides 601-3011 of SEQ ID NO: 82 (where U is substituted for each T).

[0537] 243. An RNA exon editor comprising a nucleotide sequence having at least 90% sequence identity with nucleotide 601-3250 of SEQ ID NO: 83 (where U is substituted for each T).

[0538] 244. RNA exon editor comprising the nucleotide sequence of nucleotides 601–3250 of SEQ ID NO: 83 (where U is substituted for each T).

[0539] 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 U is substituted for each T).

[0540] 246. RNA exon editor comprising the nucleotide sequence of nucleotides 601–3128 of SEQ ID NO: 84 (where U is substituted for each T).

[0541] 247. Operablely connected in the 5' to 3' direction:

[0542] (a) A binding domain configured to bind to a binding site within the target region of MeCP2 pre-mRNA;

[0543] (b) Hemi-intron;

[0544] (c) a coding domain comprising a sequence encoding one or more MeCP2 exons; and

[0545] (d) An RNA exon editor comprising a truncated WPRE 3' UTR having at least 90% sequence identity with sequence number: 87 (where U is substituted for each T).

[0546] 248. An RNA exon editor according to embodiment 247, wherein the truncated WPRE 3' UTR comprises or is composed of SEQ ID NO: 87.

[0547] 249. An exon editor composition encoding any one of the RNA exon editors of embodiments 66 to 248.

[0548] 250. An Exon Editor composition of embodiment 249, further comprising a promoter operably connected to a 5' in a combination domain.

[0549] 251. An exon editor composition of embodiment 249, further comprising a sequence having at least 90% sequence identity with SEQ ID NO: 15, which is operably linked to the binding domain at 5'.

[0550] 252. An exon editor composition of embodiment 249, further comprising the sequence of SEQ ID NO: 15, which is operably connected to the binding domain at 5'.

[0551] 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.

[0552] 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.

[0553] 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.

[0554] 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.

[0555] 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.

[0556] 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.

[0557] 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.

[0558] 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.

[0559] 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.

[0560] 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.

[0561] 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.

[0562] 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.

[0563] 265. A vector comprising an Exon Editor creation of any one of embodiments 249 to 264.

[0564] 266. In embodiment 265, the vector comprises a 5' control domain operably connected to a 5' bonding domain.

[0565] 267. In embodiment 266, a vector in which the 5' control domain includes a constructive promoter.

[0566] 268. A vector according to embodiment 266, wherein the 5' regulatory domain comprises a tissue-specific promoter.

[0567] 269. A provirus plasmid comprising an exon editor construct of any one of embodiments 249 to 264.

[0568] 270. An adeno-associated virus (AAV) comprising an exon editor composition of any one of embodiments 249 to 264.

[0569] 271. In embodiment 270, the AAV exhibits neuronal tendencies.

[0570] 272. A composition comprising an exon editor construct of any one of embodiments 249 to 264, a vector of any one of embodiments 265 to 268, a provirus plasmid of embodiment 269, or an AAV of embodiments 270 to 271, and a pharmaceutically acceptable excipient.

[0571] 273. A method for expressing functional MeCP2 in target cells, comprising transfecting target cells with an exon editor construct of any one of embodiments 249 to 264, a vector of any one of embodiments 265 to 268, a provirus plasmid of embodiment 269, or an AAV of embodiments 270 to 271.

[0572] 274. The method of embodiment 273, wherein the MeCP2 pre-mRNA comprises one or more mutations associated with Rett syndrome.

[0573] 275. The method of embodiment 274, wherein at least one mutation associated with Rett syndrome is in MeCP2 exon 3 or MeCP2 exon 4.

[0574] 276. A method for reducing the expression of MeCP2 containing at least one mutation associated with Rett syndrome in a subject, comprising transfecting or transducing a neuron in the subject with an exon editor construct of any one of embodiments 249 to 264, a vector of any one of embodiments 265 to 268, a provirus plasmid of embodiment 269, an AAV of embodiments 270 to 271, or a composition of embodiment 272.

[0575] 277. A method for correcting at least one mutation in the MeCP2 exon sequence of MeCP2 pre-mRNA in a target cell of a subject, comprising administering to the subject an exon editor construct of any one of embodiments 249 to 264, a vector of any one of embodiments 255 to 268, a provirus plasmid of embodiment 269, an AAV of embodiments 270 to 271, or a composition of embodiment 272.

[0576] 278. A method for treating Rett syndrome in a subject requiring treatment, comprising administering to the subject an exon editor construct of any one of embodiments 249 to 264, a vector of any one of embodiments 255 to 268, a provirus plasmid of embodiment 269, an AAV of embodiments 270 to 271, or a composition of embodiment 272 in a therapeutically effective amount.

[0577] 279. A method in any one of embodiments 273 to 278, 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 MeCP2 pre-mRNA containing at least one mutation associated with Rett syndrome in target cells is modified to become a transcript encoding functional MeCP2 in which at least one mutation is corrected.

[0578] H. Examples

[0579] As described herein, the inventors designed and tested a MeCP2 RNA exon editor for their ability to replace mutant MeCP2 exons 3 and 4 with wild-type and modified / engineered MeCP2 sequences via RNA trans-splicing. This MeCP2 RNA exon editor has significant therapeutic potential because the correction of mutations observed ...

Claims

Claim 1 An exon editor construct encoding an RNA exon editor, wherein the exon editor construct comprises: (a) a binding domain that binds to a target intron of MeCP2 pre-mRNA, wherein the target intron of MeCP2 pre-mRNA is intron 1 or intron 2; (b) a hemi-intron; and (c) a sequence encoding a coding domain comprising one or more MeCP2 exons. Claim 2 An exon editor construct according to claim 1, wherein the binding domain binds to a site comprising: nucleotides -100 to 1500 or -50 to 1449 of intron 1; nucleotides 3950 to 4250 or 4020 to 4169 of intron 1. Claim 3 An exon editor construct according to claim 1, wherein the binding domain binds to a binding site comprising nucleotide 1-300 of intron 2. Claim 4 An exon editor composition according to 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 of intron 1. Claim 5 An exon editor construct according to claim 3, wherein the binding domain binds to a binding site comprising nucleotides 50-300, 50-250, 75-300, 75-250, 80-300, 80-250, 90-300, 90-250, 95-300, 95-250, or 99-248 of intron 2. Claim 6 An exon editor composition according to any one of claims 1 to 5, wherein the binding domain has a size in the range of 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. Claim 7 An exon editor composition according to any one of claims 1 to 5, wherein the binding domain has a size of about 100-200 nucleotides; about 100-150 nucleotides; about 125-200 nucleotides; about 125-150 nucleotides; or about 150 nucleotides; or a size of 100-200 nucleotides; 100-150 nucleotides; 125-200 nucleotides; 125-150 nucleotides; or a size of 150 nucleotides. Claim 8 An Exon Editor composition 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 binds. Claim 9 In any one of claims 1 to 8, the binding domain comprises a stretch of adjacent nucleotides that is 100% complementary to a portion of the binding site to which it binds, wherein the stretch of adjacent nucleotides is at least 5 nucleotide lengths, at least 10 nucleotide lengths, at least 15 nucleotide lengths, at least 20 nucleotide lengths, at least 25 nucleotide lengths, at least 30 nucleotide lengths, at least 35 nucleotide lengths, at least 40 nucleotide lengths, at least 45 nucleotide lengths, at least 50 nucleotide lengths, at least 55 nucleotide lengths, at least 60 nucleotide lengths, at least 65 nucleotide lengths, at least 70 nucleotide lengths, at least 75 nucleotide lengths, at least 80 nucleotide lengths, at least 85 nucleotide lengths, at least 90 nucleotide lengths, at least 95 nucleotide lengths, and at least 100 An exon editor composition having a nucleotide length of at least 105 nucleotide lengths, at least 110 nucleotide lengths, at least 115 nucleotide lengths, at least 120 nucleotide lengths, at least 130 nucleotide lengths, at least 135 nucleotide lengths, at least 140 nucleotide lengths, at least 145 nucleotide lengths, or at least 150 nucleotide lengths. Claim 10 An exon editor composition according to any one of claims 1 to 9, wherein the sequence encoding the binding domain comprises, is essentially made of, or is made of any one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 47, 116, 117, 118, 119, 120, or 121. Claim 11 An exon editor composition according to any one of claims 1 to 9, wherein the sequence encoding the binding domain comprises, is essentially composed of, or is composed of SEQ ID NO: 55, 56, 57, 58, 59, 88, or 138. Claim 12 An exon editor composition according to any one of claims 1 to 9, wherein the sequence encoding the binding domain comprises, is essentially composed of, or is composed of any one of SEQ ID NOs: 24, 28, 29, 30, 116, 119, or 120. Claim 13 An exon editor composition according to any one of claims 1 to 12, wherein the coding domain comprises: a sequence identical to SEQ ID NO: 7 and 8 or SEQ ID NO: 7 and 8 by 90%; a sequence identical to SEQ ID NO: 76 or SEQ ID NO: 76 by at least 90%; SEQ ID NO: 13 and 14, or a sequence identical to SEQ ID NO: 13 and 14 by 90%; SEQ ID NO: 12; SEQ ID NO: 14; or SEQ ID NO: 9, comprising, being substantially composed of, or being composed of. Claim 14 An exon editor composition according to any one of claims 1 to 12, wherein the coding domain comprises, is essentially composed of, or is composed of either SEQ ID NO: 76 or 14. Claim 15 An exon editor composition according to any one of claims 1 to 14, wherein the binding domain, hemi-intron, and coding domain are operatively connected in the 5'-to-3' direction. Claim 16 An exon editor construct according to any one of claims 1 to 15, wherein the hemi-intron comprises a 3' splice site and optionally a fork sequence and a polypyrimidine track, and the binding domain, the hemi-intron comprising the 3' splice site, and the coding domain are operatively connected in the 5'-to-3' direction. Claim 17 In paragraph 16, the exon editor composition wherein the hemi-intron comprises, is essentially composed of, or is composed of a polypyrimidine track, a branching point, and a 3' splice site. Claim 18 An exon editor composition according to either claim 16 or 17, wherein the hemi-intron comprises, is essentially composed of, or is composed of a sequence having at least 90% identity with SEQ ID NO: 17 or SEQ ID NO:

17. Claim 19 An exon editor composition according to any one of claims 1 through 18, further comprising a 3' untranslated region (3' UTR), wherein the combining domain; hemi-intron; coding domain; and the 3' UTR are operatively connected in the 5'-to-3' direction. Claim 20 An exon editor composition according to claim 19, wherein the 3' UTR comprises an RDH1pA 3' UTR (SEQ ID: 19) or an mWPRE 3' UTR (SEQ ID: 20). Claim 21 An exon editor composition according to claim 20, wherein the RDH1pA 3' UTR comprises, is essentially composed of, or is composed of a sequence having at least 90% identity with SEQ ID NO: 19 or SEQ ID NO: 19; and the mWPRE 3' UTR comprises, is essentially composed of, or is composed of a sequence having at least 90% identity with SEQ ID NO: 20 or SEQ ID NO:

20. Claim 22 An exon editor construct according to any one of claims 1 to 21, wherein the exon editor construct comprises: a binding domain comprising, essentially consisting of, or consisting of 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 hemi-intron comprising, essentially consisting of, or consisting of SEQ ID NO: 17; a coding domain comprising, essentially consisting of, or consisting of SEQ ID NO: 76; and a 3' UTR comprising, essentially consisting of, or consisting of SEQ ID NO: 20, wherein the binding domain, the hemi-intron, the coding domain, and the 3' UTR are operatively connected in a 5'-to-3' direction. Claim 23 In claim 22, the exon editor composition comprises, is essentially made of, or is made of SEQ ID NO: 78, 80, 81, 82, 83, or 84. Claim 24 In claim 22, the exon editor composition comprises, is essentially composed of, or is composed of SEQ ID NO: 78, 80, or 83. Claim 25 An exon editor construct according to any one of claims 1 to 24, further comprising a triple helix terminator, wherein the binding domain; hemi-intron; coding domain; if present, 3' UTR; and the triple helix terminator is operatively connected in the 5'-to-3' direction. Claim 26 In claim 25, the exon editor composition wherein the triple helix terminator comprises, is essentially composed of, or is composed of a sequence having at least 90% identity with SEQ ID NO: 77 or SEQ ID NO:

77. Claim 27 In claim 25, the exon editor composition wherein the triple helix terminator comprises, is essentially composed of, or is composed of SEQ ID NO:

22. Claim 28 An exon editor composition according to any one of claims 1 to 27, further comprising a sequence encoding a poly A sequence, e.g., sequence number 21. Claim 29 An exon editor construct according to any one of claims 1 to 28, further comprising a sequence encoding an epitope tag, said binding domain; hemi-intron; coding domain; epitope tag; if present, a 3' UTR; and if present, a triple helix terminator operably connected in the 5'-to-3' direction. Claim 30 In claim 28, the exon editor composition comprising, essentially consisting of, or consisting of SEQ ID NO:

78. Claim 31 An RNA exon editor transcribed from an exon editor product of any one of claims 1 to 30. Claim 32 An exon editor construct according to any one of claims 1 to 30, wherein the MeCP2 pre-mRNA comprises at least one mutation associated with Rett syndrome. Claim 33 An exon editor composition according to any one of claims 1 to 30, wherein 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. Claim 34 An exon editor construct according to paragraph 32 or 33, wherein at least one mutation associated with Rett syndrome is X-linked. Claim 35 An exon editor construct according to any one of claims 32 to 34, wherein the MeCP2 protein containing at least one mutation associated with Rett syndrome is expressed in at least one of neural stem cells, neurons, astrocytes, or oligodendrocytes, or any combination thereof. Claim 36 A vector comprising an Exon Editor creation of any one of paragraphs 1 through 30 or paragraphs 32 through 35. Claim 37 In claim 36, the vector comprises a 5' control domain operably connected to a bonding domain at 5'. Claim 38 A vector according to claim 36 or 37, wherein the 5' regulatory domain comprises a constitutive promoter or a tissue-specific promoter. Claim 39 In paragraph 38, the above-mentioned constitutive promoter is a CMV promoter, vector. Claim 40 A provirus plasmid comprising an exon editor construct of any one of claims 1 to 30 or claims 32 to 35. Claim 41 An adeno-associated virus (AAV) comprising an exon editor composition of any one of claims 1 through 30 or 32 through 35, wherein the AAV optionally comprises a 5' control domain operatively linked to the exon editor composition at 5'. Claim 42 In paragraph 41, the AAV comprises a 5' control domain operatively connected to the 5' of the binding domain. Claim 43 AAV according to claim 41 or 42, wherein the 5' control domain includes a constitutive promoter. Claim 44 In paragraph 43, the above-mentioned constitutive promoter is a CMV promoter, AAV. Claim 45 In any one of paragraphs 41 to 44, the AAV exhibits neuronal orientation. Claim 46 In any one of paragraphs 41 to 45, the AAV is AAV9, AAV8, AAV5, or AAV2, or a variant of AAV9, AAV8, AAV5, or AAV2. Claim 47 A composition comprising 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 provirus plasmid of claim 40, or an AAV of any one of claims 41 to 46. Claim 48 A composition comprising a pharmaceutically acceptable excipient in paragraph 47. Claim 49 A method for restoring the functional level of MeCP2 protein in target cells by expressing biologically active MeCP2 in target cells, comprising transfecting or transducing 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 provirus plasmid of claim 40, an AAV of any one of claims 41 to 46, or a composition of claims 47 to 48. Claim 50 In claim 49, a method 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 MeCP2 pre-mRNA containing at least one mutation associated with Rett syndrome in target cells is modified to become a transcript encoding functional MeCP2 in which at least one mutation is corrected. Claim 51 A method according to 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 MeCP2 pre-mRNA containing at least one mutation associated with Rett syndrome in target cells is modified to become a transcript encoding functional MeCP2 in which at least one mutation is corrected. Claim 52 A method according to claim 51, wherein at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of MeCP2 pre-mRNA containing at least one mutation associated with Rett syndrome in target cells is modified to become a transcript encoding functional MeCP2 in which at least one mutation is corrected. Claim 53 A method according to any one of claims 49 to 52, wherein the functional level of MeCP2 is restored in a target cell by expressing a biologically functional MeCP2 protein. Claim 54 A method for reducing the expression of MeCP2 containing at least one mutation associated with Rett syndrome in a subject, 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 provirus plasmid of claim 40, an AAV of any one of claims 41 to 46, or a composition of claim 47 or 48. Claim 55 A method for correcting at least one mutation in the MeCP2 exon sequence of MeCP2 pre-mRNA in a target cell of a subject, 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 provirus plasmid of claim 40, an AAV of any one of claims 41 to 46, or a composition of claims 47 to 48. Claim 56 A method for treating Rett syndrome in a subject requiring treatment, 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 provirus plasmid of claim 40, an AAV of any one of claims 41 to 46, or a composition of claim 47 or 48 in a therapeutically effective amount. Claim 57 A method according to 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 MeCP2 pre-mRNA containing at least one mutation associated with Rett syndrome in target cells is modified to become a transcript encoding functional MeCP2 in which at least one mutation is corrected. Claim 58 In claim 57, a method 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 MeCP2 pre-mRNA containing at least one mutation associated with Rett syndrome in target cells is modified to become a transcript encoding functional MeCP2 in which at least one mutation is corrected. Claim 59 In claim 58, a method wherein at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of MeCP2 pre-mRNA containing at least one mutation associated with Rett syndrome in target cells is modified to become a transcript encoding functional MeCP2 in which at least one mutation is corrected. Claim 60 A method according to any one of claims 54 to 59, wherein the functional level of MeCP2 is restored in a target cell by expressing a biologically functional MeCP2 protein. Claim 61 A method according to any one of claims 54 to 60, wherein the method comprises administering to the brain of a 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 provirus plasmid of claim 40, an AAV of any one of claims 41 to 46, or a composition of any one of claims 47 to 48. Claim 62 A method according to any one of claims 54 to 61, wherein the subject is a mammal, preferably a rodent, a non-human primate, or a human. Claim 63 A method according to any one of paragraphs 54 to 62, wherein the subject has a genetic predisposition to Rett syndrome or is diagnosed with Rett syndrome. Claim 64 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 provirus plasmid of claim 40, an AAV of any one of claims 41 to 46, or a composition of any one of claims 47 to 48 for use in preventing or treating Rett syndrome in subjects requiring treatment. Claim 65 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 provirus plasmid of claim 40, an AAV of any one of claims 41 to 46, or a composition of any one of claims 47 to 48 for use in the manufacture of a drug for the treatment or prevention of Rett syndrome in subjects requiring treatment. Claim 66 An RNA exon editor operably connected in the 5' to 3' direction, comprising: (a) a binding domain configured to bind to a binding site within a target region of MeCP2 pre-mRNA; (b) a hemi-intron; and (c) a coding domain comprising a sequence encoding one or more MeCP2 exons; wherein the coding domain comprises at least one nucleotide variation relative to a wild-type MeCP2 sequence at a coding splice site. Claim 67 An RNA exon editor operably connected in the 5' to 3' direction, comprising: (a) a binding domain configured to bind to a binding site within a target region of MeCP2 pre-mRNA; (b) a hemi-intron; and (c) a coding domain comprising a sequence encoding one or more MeCP2 exons; wherein the binding domain comprises at least one nucleotide variation relative to the corresponding wild-type MeCP2 pre-mRNA sequence at a coding splice site. Claim 68 An RNA exon editor operably connected in the 5' to 3' direction, comprising: (a) a binding domain configured to bind to a binding site within a target region of the pre-mRNA of a target gene; (b) a hemi-intron; (c) a coding domain comprising a sequence encoding one or more exons of a target gene; and (d) a 3'-truncated WPRE 3' UTR sequence comprising a sequence having at least 90% sequence identity with SEQ ID NO: 87 or composed thereof. Claim 69 In claim 68, an RNA exon editor in which the 3'-truncated WPRE 3' UTR sequence comprises or consists of SEQ ID NO: 87.

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