Mecp2 trans-splicing molecules

Abstract

Exon editor constructs are provided, which include a binding domain that binds a target intron of a MeCP2 pre-mRNA, a hemi-intron, and a coding domain comprising one or more MeCP2 exons. Also provided are methods of expressing functional MeCP2 in a target cell that include contacting a target cell with exon editor constructs provided herein.

Description

MECP2 TRANS-SPLICING MOLECULESI. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of United States provisional application no. 63 / 598,693, filed November 14, 2023, the contents of which are incorporated herein in their entirety by reference thereto.II. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on November 13, 2024, is named ASC-003WO_SL.xml and is 329,544 bytes in size.III. BACKGROUND

[0003] Rett Syndrome is a devastating neurodevelopmental disorder that predominantly afflicts girls, for which there is no effective treatment. Rett patients typically say their first few words but shortly thereafter lose their ability to talk, walk, or purposefully use their hands. There is no “disease-modifying” treatment available for these patients. Even with the best current standard of care, they often suffer daily from vomiting, seizures, and inconsolable crying.IV. SUMMARY

[0004] Gene therapy has great potential to treat Rett Syndrome (RTT), which is caused by mutations in the methyl CpG-binding protein 2 (MECP2) gene. Conventional gene therapy approaches such as gene replacement, however, could lead to overproduction of MeCP2, which causes a disease called MeCP2 Duplication Syndrome. Moreover, with thousands of different mutations that can potentially cause Rett Syndrome, individual base editor drugs would not address the needs of a significant number of patients. Gene editing approaches like CRISPR / Cas9-based editing would also require introducing Cas9 into the brain, increasing the risk of immune rejection. Therefore, there is a need in the field for alternative strategies for correcting mutations in the MeCP2 gene.

[0005] To address these unmet needs, an RNA exon editing solution is described herein which comprises delivering an exon editor construct that encodes a therapeutic RNA exon editor designed to trans-splice into endogenous MeCP2 pre-mRNA comprising at least one mutation. Trans-splicing replaces one or more exons of the MeCP2 pre-mRNA with exons from the therapeutic RNA exon editor that encode functional amino acid sequences present in biologically active MeCP2 protein, thereby correcting one or more mutations in the endogenous MeCP2 pre-mRNA. Rett Syndrome is a promising candidate for an exon editing approach. Exon-editing can correct -95% of Rett patient mutations with a single therapeutic RNA exon editor. Exemplary MeCP2 RNA exon editors are described herein.

[0006] In some embodiments, an exon editor construct is described herein that comprises: (a) a binding domain that binds a target intron of a 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 VLC.3 below. In some embodiments, the hemi-intron comprises any of the hemi-introns disclosed in Section VLC.4. In some embodiments, the coding domain sequence comprises any of the CDSs disclosed in Section VLC.2. In some embodiments, the exon editor construct further comprises, operably linked 3’ to the CDS, a 3’ UTR sequence. In some embodiments, the 3’ UTR sequence comprises any of the 3’ UTR sequences disclosed in Section VLC.5. In some embodiments, the exon editor construct further comprises, operably linked 3’ to the CDS, a polyA tail disclosed herein. In some embodiments, the exon editor construct further comprises, operably linked 3’ to the CDS, a transcription terminator sequence. In some embodiments, the transcription terminator sequence comprises any of the transcription terminator sequences disclosed in Section VI. C.6.

[0007] Embodiments disclosed herein include an exon editor construct that encodes an RNA exon editor, wherein the exon editor construct comprises sequences encoding: (a) a binding domain that binds a target intron of a MeCP2 pre-mRNA; (b) a hemi-intron; and (c) a coding domain comprising one or more MeCP2 exons. In some embodiments, the target intron of the 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 (SEQ ID NO: 1); nucleotides 3950 to 4250 or 4020-4169 of intron 1 ; or nucleotides 1-300 of intron 2 (SEQ ID NOs: 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 (SEQ ID NO: 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 (SEQ ID NOs: 2 and 3). In some embodiments, the binding domain ranges in size from about 50-300 nucleotides; about 50-250 nucleotides; about 50-150 nucleotides; about 50-100 nucleotides; about 75-300 nucleotides; about 75-250 nucleotides; about 75-200 nucleotides; about 75-150 nucleotides; about 100-300 nucleotides; about 100-250 nucleotides; about 100-200 nucleotides; about 100-150 nucleotides; about 125-300 nucleotides; about 125-250 nucleotides; about 125-200 nucleotides; about 125-150 nucleotides; or about 150 nucleotides. In some embodiments, the binding domain ranges in size from 50-300 nucleotides; 50-250 nucleotides; 50-150 nucleotides; 50-100 nucleotides; 75-300 nucleotides; 75-250 nucleotides; 75-200 nucleotides; 75-150 nucleotides; 100-300 nucleotides; 100-250 nucleotides; 100-200 nucleotides; 100-150 nucleotides; 125-300 nucleotides; 125-250nucleotides; 125-200 nucleotides; 125-150 nucleotides; or 150 nucleotides. In some embodiments, the exon editor construct of any one of the preceding embodiments, wherein the binding domain ranges in size from about 100-200 nucleotides; about 100-150 nucleotides; about 125-200 nucleotides; about 125-150 nucleotides; or about 150 nucleotides; or ranges in size from 100-200 nucleotides; 100-150 nucleotides; 125-200 nucleotides; 125-150 nucleotides; or 150 nucleotides. In some embodiments, the binding domain is at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the binding site to which it binds. In some embodiments, the binding domain comprises stretches of contiguous nucleotides that are 100% complementary to a portion of the binding site to which it binds, wherein the stretches of contiguous nucleotides are at least 5 nucleotides long, at least 10 nucleotides long, at least 15 nucleotides long, at least 20 nucleotides long, at least 25 nucleotides long, at least 30 nucleotides long, at least 35 nucleotides long, at least 40 nucleotides long, at least 45 nucleotides long, at least 50 nucleotides long, at least 55 nucleotides long, at least 60 nucleotides long, at least 65 nucleotides long, at least 70 nucleotides long, at least 75 nucleotides long, at least 80 nucleotides long, at least 85 nucleotides long, at least 90 nucleotides long, at least 95 nucleotides long, at least 100 nucleotides long, at least 105 nucleotides long, at least 110 nucleotides long, at least 115 nucleotides long, at least 120 nucleotides long, at least 130 nucleotides long, at least 135 nucleotides long, at least 140 nucleotides long, at least 145 nucleotides long, or at least 150 nucleotides long. In some embodiments, the sequence encoding the binding domain comprises, consists essentially of, or consists of any one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, or 47. In some embodiments, the sequence encoding the binding domain comprises, consists essentially of, or consists of SEQ ID NO: 55. In some embodiments, the sequence encoding the binding domain comprises, consists essentially of, or consists of any one of SEQ ID NOs: 24, 28, 29, or 30. In some embodiments, the coding domain comprises, consists essentially of, or consists of: SEQ ID NOs: 7 and 8 or a sequence 90% identical to SEQ ID NOs: 7 and 8; SEQ ID NO: 76 or a sequence 90% identical to SEQ ID NO: 76; SEQ ID NO: 12; SEQ ID NO: 14; or SEQ ID NO: 9.

[0008] In some embodiments, the coding domain comprises, consists essentially of, or consists of any one of SEQ ID NOs: 76 or 14.

[0009] In some embodiments, the binding domain, the hemi-intron, and the coding domain are operatively linked in a 5’-to-3’ direction.

[0010] In some embodiments, the hemi-intron comprises a 3’ splice site and optionally, a branchpoint sequence and a polypyrimidine tract, wherein the binding domain, the hemi-intron comprising the 3’ splice site, and the coding domain are operatively linked in a 5’-to-3’ direction.In some embodiments, the hemi-intron comprises, consists essentially of, or consists of: polypyrimidine tract, a branchpoint, and a 3’ splice site. In some embodiments, the hemi-intron comprises, consists essentially of, or consists of: SEQ ID NO: 17 or a sequence having at least 90% identity to SEQ ID NO: 17.

[0011] In some embodiments, the exon editor construct further comprises a 3’ untranslated region (3’ UTR), wherein the binding domain; the hemi-intron; the coding domain; and the 3’ UTR; are operatively linked in a 5’-to-3’ direction. In some embodiments, the 3’ UTR comprises an RDH1 pA 3’ UTR (SEQ ID NO: 19), a mWPRE 3’ UTR (SEQ ID NO: 20), or a truncated version thereof such as, for example, a WPRE3 3’ UTR (SEQ ID NO: 87). In some embodiments, the RDHI pA 3’ UTR comprises, consists essentially of, or consists of SEQ ID NO: 19 or a sequence having at least 90% identity to SEQ ID NO: 19; or the mWPRE 3’ UTR comprises, consists essentially of, or consists of SEQ ID NO: 20 or a sequence having at least 90% identity to SEQ ID NO: 20. In some embodiments, the 3’ UTR comprises a truncated WPRE sequence. In some embodiments, the truncated WPRE sequence comprises, consists essentially of, or consists of SEQ ID NO: 87 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 87.

[0012] In some embodiments, the exon editor construct comprises: a binding domain comprising, consisting essentially 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, consisting essentially of, or consisting of SEQ ID NO: 17; a coding domain comprising, consisting essentially of, or consisting of SEQ ID NO: 76; and a 3’ UTR comprising, consisting essentially of, or consisting of SEQ ID NO: 20, wherein the binding domain, the hemi-intron, the coding domain, and the 3’ UTR are operatively linked in a 5’-to-3’ direction.

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

[0014] In some embodiments, the exon editor construct further comprises a triple helix terminator, wherein the binding domain; the hemi-intron; the coding domain; the 3’ UTR, when present; and the triple helix terminator are operatively linked in a 5’-to-3’ direction. In some embodiments, the triple helix terminator comprises, consists essentially of, or consists of SEQ ID NO: 77 or a sequence having at least 90% identity to SEQ ID NO: 77. In some embodiments, the triple helix terminator comprises, consists essentially of, or consists of SEQ ID NO: 22.

[0015] In some embodiments, the exon editor further comprises a sequence encoding a poly A sequence, for example, SEQ ID NO: 21 .

[0016] In some embodiments, the exon editor construct further comprises a sequence encoding an epitope tag, wherein the binding domain; the hemi-intron; the coding domain; theepitope tag; the 3’ UTR, when present; and the triple helix terminator, when present; are operatively linked in a 5’-to-3’ direction. In some embodiments, the exon editor construct comprises, consists essentially of, or consists of SEQ ID NO: 78.

[0017] Also disclosed herein is an RNA exon editor transcribed from the exon editor construct of any of the embodiments described above.

[0018] In some embodiments, the MeCP2 pre-mRNA comprises 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 an MeCP2 gene allele or at least one mutation in exon 4 of an 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 comprising the 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.

[0019] Also disclosed herein is a vector comprising the exon editor construct of any of the embodiments described above. In some embodiments, the vector comprises a 5’ regulatory domain operatively linked 5’ to the 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.

[0020] Also disclosed herein is a proviral plasmid comprising the exon editor construct of any of the embodiments described above.

[0021] Also disclosed herein is an adeno-associated virus (AAV) comprising the exon editor construct of any of the embodiments described above, wherein the AAV optionally comprises a 5’ regulatory domain operatively linked 5’ to the exon editor construct. In some embodiments, the AAV comprises a 5’ regulatory domain operatively linked 5’ to the binding domain. 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 tropism. In some embodiments, the AAV is AAV9, AAV8, AAV5, or AAV2.

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

[0023] Also disclosed is a method of expressing biologically active MeCP2 in a target cell to restore functional levels of MeCP2 protein in the target cell, the method comprising transducing the target cell with the exon editor construct of any of the embodiments described above, the vector of any of the embodiments described above, the proviral plasmid of any of theembodiments described above, the AAV of any of the embodiments described above, or the 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 the MeCP2 pre-mRNAs comprising at least one mutation associated with Rett Syndrome in the target cell are modified to become transcripts that encode functional MeCP2 wherein the 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-mRNAs comprising at least one mutation associated with Rett Syndrome in the target cell are modified to become transcripts that encode functional MeCP2 wherein the 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-mRNAs comprising at least one mutation associated with Rett Syndrome in the target cell are modified to become transcripts that encode functional MeCP2 wherein the at least one mutation is corrected. In some embodiments, functional levels of MeCP2 are restored in the target cell by expressing biologically functional MeCP2 protein.

[0024] Also disclosed is a method of reducing expression of MeCP2 comprising at least one mutation associated with Rett Syndrome in a subject, the method comprising transfecting or transducing a target cell, more particularly a neuron, in the subject with the exon editor construct of any of the embodiments described above, the vector of any of the embodiments described above, the proviral plasmid of any of the embodiments described above, the AAV of any of the embodiments described above, or the composition of any of the embodiments described above.

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

[0026] Also disclosed is a method of treating Rett Syndrome in a subject in need thereof, the method comprising administering to the subject the exon editor construct of any of the embodiments described above, the vector of any of the embodiments described above, the proviral plasmid of any of the embodiments described above, the AAV of any of the embodiments described above, or the composition of any of the embodiments described above.

[0027] In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of the MeCP2 pre-mRNAs comprising at least one mutation associated with Rett Syndrome in the target cell are modifiedto become transcripts that encode functional MeCP2 wherein the 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-mRNAs comprising at least one mutation associated with Rett Syndrome in the target cell are modified to become transcripts that encode functional MeCP2 wherein the 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-mRNAs comprising at least one mutation associated with Rett Syndrome in the target cell are modified to become transcripts that encode functional MeCP2 wherein the at least one mutation is corrected. In some embodiments, functional levels of MeCP2 are restored in the target cell by expressing biologically functional MeCP2 protein. In some embodiments, the method comprising administration of the exon editor construct of any one of embodiments 1 -29 or 31-34, the vector of any one of embodiments 35-38, the proviral plasmid of embodiment 39, the AAV of any one of embodiments 40-45, or the composition of any one of embodiments 46-47 to the subject’s brain. In some embodiments, the subject is a mammal, preferentially a rodent, non-human primate, or a human. In some embodiments, the subject is genetically predisposed to have Rett Syndrome or has been diagnosed with Rett Syndrome.

[0028] Also disclosed is the exon editor construct of any of the embodiments described above, the vector of any of the embodiments described above, the proviral plasmid of any of the embodiments described above, the AAV of any of the embodiments described above, or the composition of any of the embodiments described above for use in preventing or treating Rett Syndrome in a subject in need thereof.

[0029] Also disclosed is the exon editor construct of any of any of the embodiments described above, the vector of any of the embodiments described above, the proviral plasmid of any of the embodiments described above, the AAV of any of the embodiments described above, or the composition of any of the embodiments described above for use in the preparation of a medicament for the treatment or prevention of Rett Syndrome in a subject in need thereof.

[0030] Exemplary RNA exon editors disclosed herein include the exon editors of enumerated embodiments 66 to 248. Exemplary constructs encoding RNA exon editors disclosed herein include the exon editor constructs of enumerated embodiments 1 to 35 and 249 to 264.Exemplary vectors disclosed herein include the vectors of enumerated embodiments 36 to 39 and 265 to 268. Exemplary proviral plasmids disclosed herein include the proviral plasmids of enumerated embodiments 40 and 269. Exemplary AAVs disclosed herein include the AAVs of enumerated embodiments 41 to 46, 270, and 271. Exemplary compositions disclosed herein include the compositions of enumerated embodiments 47, 48, and 272. Exemplary methods of expressing biologically active MeCP2 include the methods of enumerated embodiments 49 to53 and 273 to 275. Exemplary methods of reducing expression of MeCP2 comprising at least one mutation associated with Rett Syndrome include the methods of enumerated embodiments54 and 276. Exemplary methods of correcting at least one mutation in an MeCP2 exon sequence include the methods of enumerated embodiments 55 and 277. Exemplary methods of treating Rett Syndrome in a subject in need thereof include the methods of enumerated embodiments 56 to 63, 278 and 279.V. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 is a schematic representation that depicts the frequency and position of missense mutations in MeCP2 associated with Rett Syndrome (adapted from Ehrhart, F., et. aL, Scientific Data, 2021). MeCP2 protein structural / functional domains include: the N-terminal domain (NTD); methyl binding domain (MBD); intervening domain (ID); transcription repression domain (TRD); and the C-terminal domain (CTD). Exon editors described herein would correct mutations in exons 3 and 4, treating roughly 95% of RTT patients.

[0032] 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 exon editor can correct mutations in exons 3 and 4.

[0033] FIG. 3 depicts the gene structure of human MeCP2. The gene structure and sequence length of MeCP2 is depicted with exons 1-4 and introns 1-3. The 3’ UTR (which is contained in exon 4) differs between MeCP2 isoforms, thus only the length of the coding sequence of exon 4 is indicated.

[0034] FIG. 4 presents an illustration of MeCP2 E1 and E2 mRNA isoforms. The E1 isoform mRNA consists of exons 1 , 3, and 4. The translation start codon for translation of the E1 isoform protein is in exon 1, as indicated. The E2 mRNA isoform consists of exons 1 , 2, 3, and 4. The translation start codon for translation of the E2 isoform protein is in exon 2, as indicated.

[0035] FIG. 5 depicts domains of an exemplary 3’ MeCP2 exon editor. Each domain of the 3’ exon editor is indicated. Promoter: sequence necessary for driving expression of the exon editor. Binding domain: sequence anti-sense to the target pre-mRNA, which recruits the exon editor to the target via base pairing. Hemi-intron: sequence with branch point, poly-pyrimidine tract, and splice acceptor site. MeCP2 exons 3 and 4 coding domain sequence (CDS): the coding sequence of MeCP2 exons 3 and 4. Myc tag: an epitope tag used to detect protein produced by exon editing. 3’ untranslated region (UTR): regulatory sequence. Terminator: sequence for termination of transcription of the exon editor. In some embodiments, the expression of an RNA exon editor is driven by a CMV / CMV enhancer / promoter. In some embodiments, the RNA exon editor comprises a binding domain targeting MeCP2 intron 1, ahemi-intron, a splice site sequence, replacement wild-type MeCP2 exons, 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, replacement wild-type MeCP2 exons, 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, modified replacement wild-type MeCP2 exons, and a 3’ UTR. In some embodiments, the 3’ UTR is the native MeCP2 3’ UTR or a truncated version thereof. In some embodiments, the 3’ UTR is a heterologous 3’ UTR (e.g., an artificial 3’ UTR assembled via genetic engineering).

[0036] FIG. 6 depicts a MeCP2 intron 1 binding domain scan. Relative position of binding domains (depicted as shapes above the bar indicating Intron 1) across the length of intron 1 of MeCP2.

[0037] FIG. 7 presents a plot that shows MeCP2 intron 1 hot spot identification. HEK293 cells were transfected with MeCP2 intron 1 -targeting RNA Exon Editors that target various regions of intron 1. Cells were harvested 48 hours post-transfection and assayed for trans-splicing efficiencies by RT-qPCR. The nomenclature for the binding domain is: (nucleotide base position in the intron of the 5’ end start position of the binding domain)_(length (nt) of the binding domain). For example, 51_150 indicates that the binding domain is the reverse complement sequence of bases 51-200 in intron 1 , which correspond to positions 51-200 of SEQ ID NO: 1. Position -50 represents the position 50 nucleotides upstream of the exon 1 - intron junction.Sequence identifiers of tested binding domains are set forth in Table 4. Various binding domains tested included cryptic splice site-mitigating nucleotide substitutions, as indicated in Table 4.

[0038] FIG. 8 demonstrates validation of intron 1 binding domains. Select exon editors from Figure 7 were retested by two individual operators. HEK293 cells were transfected with MeCP2 intron 1 -targeting RNA Exon Editors that target various regions of intron 1. Cells were harvested 48 hours post-transfection and assayed for trans-splicing efficiencies by RT-qPCR. The nomenclature for the binding domain is: (nucleotide base position in the intron of the 5’ end start position of the binding domain)_(length (nt) of the binding domain). For example, 51 _150 indicates that the binding domain is the reverse complement sequence of bases 51-200 in intron 1. The % replacement is calculated as the copy number of edited MeCP2 mRNA / total MeCP2 mRNA x 100. Sequences of tested binding domains are set forth in Table 4.

[0039] FIG. 9 Summary of binding domain scanning profile across MeCP2 Intron 1. The percentages represent the trans-splicing efficiency of the RNA Exon Editor having a binding domain in the indicated position.

[0040] FIG. 10 depicts a schematic of an exemplary binding domain scanning profile across MeCP2 Intron 2. Relative positions of the binding domains (depicted in green) are shown at the 5’ and 3’ termini of intron 2.

[0041] FIG. 11 presents a plot showing activity of exemplary MeCP2 intron 2 binding domains tested. HEK293 cells were transfected with MeCP2 intron 2-targeting RNA Exon Editors that target various regions of intron 2. Cells were harvested 48 hours post-transfection and assayed for trans-splicing efficiencies by RT-qPCR. The nomenclature for the binding domain is: (nucleotide base position in the intron of the 5’ end start position of the binding domain)_(length (nt) of the binding domain). For example, 99_150 indicates that the binding domain is the reverse complement sequence of bases 99-248 in intron 2, which correspond to positions 99- 248 in SEQ ID NO: 2. The % replacement is calculated as the copy number of edited MeCP2 mRNA (E2 isoform) I total MeCP2 RNA (E2 isoform) x 100. Sequence identifiers of tested binding domains are set forth in Table 5.

[0042] FIG. 12 presents a schematic of a binding domain scanning profile across MeCP2 Intron 2. The percentages represent the trans-splicing efficiency of the RNA Exon Editor having a binding domain in the indicated position.

[0043] FIG. 13 depicts an exemplary binding domain scanning profile across MeCP2 Intron 3 Relative positions of the binding domains (depicted in green) are shown across the length of intron 3.

[0044] FIG. 14 presents a plot showing activity of exemplary MeCP2 intron 3 binding domains tested. HEK293 cells were transfected with MeCP2 intron 3-targeting RNA Exon Editors that target various regions of intron 3. Cells were harvested 48 hours post-transfection and assayed for trans-splicing efficiencies by RT-qPCR. The nomenclature for the binding domain is: (nucleotide base position in the intron of the 5’ end start position of the binding domain)_(length (nt) of the binding domain). For example, 26_150 indicates that the binding domain is the reverse complement sequence of bases 26-175 in intron 3, which correspond to positions 26- 175 of SEQ ID NO: 4. The % replacement is calculated as the copy number of edited MeCP2 mRNA / total MeCP2 RNA x 100. Sequence identifiers of tested binding domains are set forth in Table 7.

[0045] FIG. 15 presents a summary of intron 3 binding domain performance. The percentages represent the trans-splicing efficiency of the RNA Exon Editor having a binding domain in the indicated position.

[0046] FIG. 16 shows that certain 3’ termini modifications improve exon editor performance. Protein from edited mRNA (left) and Exon editor expression (right) are plotted for editors with the indicated elements at the 3’ termini. Plots represent performance of editors relative to Editor 1. HEK293 cells were transfected with MeCP2 Exon Editors. Cells were harvested 48 hours post-transfection and assayed for (1) protein produced from edited mRNA by western blot, and (2) exon editor expression by RT-qPCR.

[0047] FIG. 17 shows that relative MeCP2 exon editing is recapitulated in different cell types. HEK293 and U-251 MG cells were transfected with MeCP2 Exon Editors as indicated. Cells were harvested 48 hours post-transfection and assayed fortrans-splicing efficiencies by RT-qPCR. The % replacement is calculated as the copy number of edited MeCP2 mRNA / total MeCP2 RNA x 100.

[0048] FIG. 18 presents an immunoblot of protein produced from MeCP2 edited mRNA. LI- 251 cells were transfected with plasmids encoding either full-length chimeric (edited) mRNA mimics, or exon editors. Cells were harvested 48 hours post-transfection and assayed by western blot. Only exon editors with active splice sites produce protein of the expected size.

[0049] FIG. 19 presents a plot showing activity of exemplary MeCP2 intron 2 binding domains tested by two different operators. HEK293 cells were transfected with MeCP2 intron 2- targeting RNA Exon Editors that target various regions of intron 2. Cells were harvested 48 hours post-transfection and assayed for trans-splicing efficiencies by RT-qPCR. The nomenclature for the binding domain is: (nucleotide base position in the intron of the 5’ end start position of the binding domain)_(length (nt) of the binding domain). For example, 99_150 indicates that the binding domain is the reverse complement sequence of bases 99-248 in intron 2, which correspond to positions 99-248 in SEQ ID NO: 2. Various binding domains tested included cryptic splice site-mitigating nucleotide substitutions, as indicated in Table 6. FIG. 19 shows a comparison between a binding domain with cryptic splice site-mitigating nucleotide substitutions (MeCP2_intron2_99_150SM (SEQ ID NO: 88)) and a binding domain lacking the substitutions (MeCP2_intron2_99_150 (SEQ ID NO: 55)). The % replacement is calculated as the copy number of edited MeCP2 mRNA (E2 isoform) / total MeCP2 RNA (E2 isoform) x 100.VI. DETAILED DESCRIPTION A. Introduction

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

[0051] Rett syndrome (RTT) is a rare neurological disorder that occurs at a frequency of 1 out of every 10,000 female births worldwide. It is even rarer in boys. A diagnosis is typically made when a child is between 6 to 18 months old and begins to miss developmental milestones or lose abilities already mastered. Rett syndrome is characterized by severe impairments with respect to speaking, walking, eating, and even breathing. A hallmark of Rett syndrome is near constant repetitive hand movements. Symptoms may include: seizures or Rett episodes, loss of speech, loss of purposeful use of hands, involuntary hand movements, loss of mobility, gaitdisturbances, loss of muscle tone, scoliosis, breathing issues, sleep disturbances, inconsolable crying, and / or slowed growth rate for the head, feet and / or hands.

[0052] 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 transcriptional regulator that is highly expressed in neurons. Inactivating mutations in MeCP2 alter the expression of many genes and ultimately lead to aberrations in neuronal morphology and circuitry. There are more than 900 different mutations found in the MeCP2 gene, most of which are found in eight different “hot spots.” See FIG 1. Rett syndrome presents with a wide range of disability ranging from mild to severe impairment. The course and severity of Rett syndrome is determined by the location, type and severity of the mutation and X-inactivation.

[0053] In addition to Rett Syndrome, mutations in the MeCP2 gene are also associated with other diseases such as MECP2 duplication syndrome, MECP2-re\ated severe neonatal encephalopathy, and pyramidal signs, parkinsonism, and macroorchidism (PPM-X) syndrome. There are no approved therapies to treat any of the diseases associated with mutations in the MeCP2 gene.

[0054] Gene therapy holds great promise for treating Rett Syndrome and other diseases associated with mutations in the MeCP2 gene. However, a major barrier is that 2-fold increases in MECP2 gene copy number and corresponding gene expression levels cause disease phenotypes, creating a narrow therapeutic dosing window for traditional gene replacement. This dosing window may be even narrower, since half of the cells in, e.g., Rett syndrome patients express fully functional MeCP2 protein, due to random X chromosome inactivation. It may not, therefore, be possible for traditional gene replacement to deliver adequate MeCP2 to cells that need it while avoiding over-expression phenotypes in the cells that do not need it.

[0055] Exon-editing molecules utilize RNA trans-splicing to excise and replace diseased exons. As described herein, a single exon-editor can correct a 3’ portion of an mRNA by substituting the usual intra-molecular “cis” splicing reaction with an inter-molecular “trans” splicing reaction See FIG. 2. Exon-editing for treatment of Rett syndrome offers at least two advantages. First, a single exon-editor that targets either MeCP2 intron 1 or intron 2 can correct ~95% of MECP2 patient mutations, whereas a single exon-editor that targets MeCP2 intron 3 can correct ~75% of MECP2 patient mutations. Second, exon-editing therapy inherently limits the maximum expression of corrected mRNAs in each and every cell to normal endogenous levels, regardless of promoter or vector dose. This inherent control arises because exon-editors act on expressed pre-mRNA, thus correcting a target pre-mRNA transcript only if it is expressed. This degree of control may be essential for the half of Rett patient cells that already express the correct amount of fully functional MECP2. In these cells, exon-editing replaces onefunctional mRNA with another, whereas gene replacement therapy would lead to undesirable, disease-causing overexpression of MECP2 mRNA.

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

[0057] Compositions and methods described herein involve trans-splicing molecules (e.g., pre-mRNA trans-splicing molecules) for treating diseases or disorders caused by a mutation in the MeCP2 gene. Such mutations comprise point mutations and deletions, the vast majority of which have been identified in exon 3 or exon 4 of the MeCP2 gene. See FIG. 1. Thecompositions and methods described herein employ nucleic acid trans-splicing molecules encoding RNA trans-splicing molecules that target pre-mRNA for gene therapy (e.g., in vivo gene therapy, as, e.g., delivered by adeno-associated virus) to treat diseases caused by at least one mutation in MeCP2, such as Rett Syndrome. The compositions and methods described herein also employ nucleic acid trans-splicing molecules encoding RNA trans-splicing molecules that target pre-mRNA for gene therapy (e.g., in vivo gene therapy) in combination with other therapeutic agents described herein to treat Rett Syndrome.

[0058] As described herein, Rett Syndrome refers to a neurodevelopmental disorder associated with mutations in the methyl CpG binding protein 2 (MECP2) gene. Rett Syndrome is a progressive neurologic developmental disorder and is one of the most common causes of cognitive disability in females.

[0059] MECP2 is a chromatin-associated protein that can both activate and repress transcription. It is required for maturation of neurons and is developmentally regulated. The MeCP2 gene is a member of a family of nuclear proteins that comprise a methyl-CpG binding domain (MBD). MeCP2 can specifically bind to methylated CpGs and may also interact with other co-regulator complexes. MeCP2 is X-linked and subject to X inactivation.Exon replacement by MeCP2 pre-mRNA trans-splicing

[0060] Therapeutic RNA exon editors can potentially correct ~95% of Rett patient mutations with a single MeCP2 RNA exon editor that replaces all of the exons in which -95% of mutations occur. The inventors have developed exemplary MeCP2 RNA exon editors that are capable of replacing a substantial portion of mutated MeCP2 protein (e.g., at least about 15%) in cultured cells with biologically active, functional MeCP2 protein. MeCP2 RNA exon editors described herein comprise a coding domain sequence that includes the sequence of multiple MeCP2 exons that encode functional (e.g., wild-type) MeCP2 amino acid sequences, a splice domain sequence that splices to native MeCP2 pre-mRNA, and a binding domain sequence that anneals to an intron of native MeCP2 pre-mRNA. Exemplary such MeCP2 RNA exon editors will be administered to animal subjects in vivo, and it is expected that 15% or more of mutated MeCP2 protein will be replaced with functional MeCP2 protein and that the RNA exon editor will provide sufficient replacement of mutated MeCP2 to achieve a therapeutic effect in vivo. Although not wishing to be bound be theory, exemplary MeCP2 RNA exon editors will confer therapeutic effect by achieving at least one of restoring functional levels of transcriptional regulation, restoring functional levels of binding to methylated DNA, or restoring functional levels of interactions with co-regulator complexes, or any combination thereof.B. Definitions

[0061] As used herein, “trans-splicing” refers to joining a first RNA molecule comprising one or more exons (e.g., exogenous exons or exons that are part of a coding domain of an RNA exon editor) to a second RNA molecule (e.g., an endogenous pre-mRNA molecule) and replacing a portion of the second RNA molecule with a portion of the first RNA molecule through a spliceosome-mediated mechanism. The general mechanism for an RNA trans-splicing reaction is illustrated in, e.g., FIG. 2.

[0062] 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, RNA exon editors comprise 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 comprising a splice site; and (c) a coding domain configured to be trans-spliced to the target nucleic acid, which can replace one or more exons in the target nucleic acid (e.g., exons comprising at least one mutation). RNA exon editors are effector molecules that physically bind to target pre-mRNA. The terms “DNA encoding an RNA exon editor,” “exon editor construct that encodes an RNA exon editor,” or “DNA sequence encoding an RNA exon editor” may be used to refer to a DNA sequence, construct, or vector that encodes the RNA exon editor. In some embodiments, a vector may be an AAV vector that comprises a DNA sequence encoding an RNA exon editor. In some embodiments, a DNA sequence encoding an RNA exon editor comprises cDNA, e.g., as part of a functional exon (e.g., a functional MeCP2 exon) for replacement of at least one MeCP2 exon comprising a mutation in a target gene (e.g., pre-mRNA). In some instances, sequences of RNA exon editor components are disclosed herein as DNA sequences. For any sequence disclosed herein as a DNA sequence, an RNA sequence with U substituted for each T in the sequence is also contemplated. Thus, if a given SEQ ID NO is identified as having a sequence that may be included in an RNA exon editor, a version of the SEQ ID NO with U substituted for each T is also contemplated.

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

[0064] As used herein, “% RNA replacement” refers to the portion of the total target mRNA population that has undergone successful trans splicing (TS), and is calculated via the followingequation: % on-target (ONT) TS = 100*(ONT copy number / (ONT copy number + Native copy number)).

[0065] As used herein, “relative trans-splicing efficiency” refers to a ratio of a test transsplicing efficiency to a reference trans-splicing efficiency, wherein the test trans-splicing efficiency is the trans-splicing efficiency of an RNA exon editor (a first RNA exon editor; test) described herein and the reference trans-splicing efficiency is the trans-splicing efficiency of a reference RNA exon editor (e.g., an RNA exon editor comprising the same elements as the first RNA exon editor except that the binding domain is replaced with a scrambled binding domain or non-targeting binding domain). Relative trans-splicing efficiency of an RNA exon editor may be given as a ratio (a.k.a. fold increase) of the first trans-splicing RNA (test) efficiency over the reference trans-splicing efficiency tested under similar conditions.

[0066] As used herein, the term “operably linked” or “operatively linked” refers to an arrangement of elements, wherein the components so described are configured so as to perform their usual function. A nucleic acid is “operably linked” to another nucleic acid sequence when it is placed into a functional relationship with the other nucleic acid sequence. Elements need not be contiguous to be operably linked. Thus, for example, intervening sequences can be present between operably linked sequences (e.g., a binding domain and a coding sequence can be separated by intervening sequences and the binding domain is still considered to be “operably linked” to the coding sequence).

[0067] As used herein, the term “coding domain” refers to a nucleic acid sequence (e.g., an RNA sequence, a DNA sequence, or combination of RNA and DNA) that encodes a portion of a protein (e.g., a target protein in which a mutation is being corrected). Thus, a coding domain may include one or more functional exons (e.g., a sequence of functional exons). In some instances, one or more functional exons of a coding domain are not separated by introns (e.g., as in endogenous pre-mRNA) but adjacent to one another (e.g., as cDNA). In some instances, a coding domain can include one or more introns (e.g., native introns) or untranslated regions (UTRs, e.g., native UTRs) between or otherwise adjacent to (e.g., upstream or downstream of) exons.

[0068] As used herein, a “native 3’ MeCP2 untranslated region” or “native 3’ MeCP2 UTR” refers to a sequence greater than 20 nucleotides in length that has at least 90% sequence identity with a region of a native MeCP2 gene (e.g., a human MeCP2 gene) that is 3’ to the translation termination codon. In some embodiments, RNA exon editors comprise a native 3’ MeCP2 untranslated region. The native 3’ MeCP2 untranslated region comprises alternative polyA signals spanning from 0.1 kb to 8.5 kb of the MeCP2 3’ UTR that correspond to various lengths of native MeCP2 3’ UTRs that are differentially expressed in different tissues. The 8kb 3’ UTR is enriched in brain tissue compared to shorter MeCP2 3’ UTRs. In some embodiments, thenative 3’ MeCP2 UTR is replaced by a 3’ UTR of a shorter length. In some embodiments, the 3’ UTR is a RDHI pA 3’ UTR, which is a synthetic 3’ UTR comprising 110 bp of the highly conserved MeCP2 distal polyadenylation signal and an upstream miRNA-binding panel containing sites for three additional miRNAs endogenous to the MECP2 3 ' UTR: miR-19, miR- 22, and miR-132. The DNA sequence encoding RDHIpA 3’ UTR comprises SEQ ID NO: 19.

[0069] As used herein, a “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 a functional (biologically active) portion of MeCP2 protein. In some embodiments, a “functional sequence of 3’ MeCP2 exons” refers to a nucleic acid sequence comprising exons 3-4 of MeCP2 that encodes a functional (biologically active) portion of MeCP2 protein. In some embodiments, a “functional sequence of 3’ MeCP2 exons” refers to a nucleic acid sequence comprising exons 2-4 of MeCP2 that encodes a functional (biologically active) portion of MeCP2 protein. In some embodiments, a “functional sequence of 3’ MeCP2 exons” refers to a nucleic acid sequence comprising exon 4 of MeCP2 that encodes a functional (biologically active) portion of MeCP2 protein. When trans-spliced to an endogenous MeCP2 exon 5’ to the binding site, a functional sequence of 3’ MeCP2 exons provides expression of functional MeCP2 protein (e.g., non-mutated MeCP2 protein). In some instances, the functional sequence of 3’ MeCP2 exons includes a sequence of exons abutting the exon to which the RNA exon editor is being trans-spliced (e.g., an RNA exon editor that binds MeCP2 intron 1 and trans-splices with endogenous MeCP2 exon 1 can include a functional sequence of 3’ MeCP2 exons that includes exons 3 and 4).

[0070] As used herein, the term “functional”, when used in the context of a protein, refers to a biologically active protein. The term “functional” may also be used to refer to the amount of activity of a protein that is necessary to support normal cellular functions. With respect to MeCP2, the term “functional” may be used to refer to the amount of MeCP2 protein activity that is necessary to restore MeCP2 activity levels to support normal cellular functions within the context of, for example, neurons in the brain. Such levels are sufficient to reduce or prevent disruption of gene expression, neuronal development, synaptic maturation, plasticity, and / or other disease manifestations associated with reduced levels of MeCP2 activity. More particularly, since defective (non-functional) MeCP2 protein comprising at least one mutation leads to defects in synaptic and circuit-level defects in brain function, restoring functional levels of MeCP2 refers, at least in part, to an increase in biologically active MeCP2 protein so as to reduce the amount or degree of gene expression defects, synaptic defects, circuit-level defects and / or other defects in brain function associated with reduced levels of MeCP2 activity. Although not wishing to be bound be theory, the term “functional” may be used to refer to the amount of MeCP2 protein activity necessary to achieve at least one of restoring functional levelsof transcriptional regulation, restoring functional levels of binding to methylated DNA, or restoring functional levels of interactions with co-regulator complexes, or any combination thereof.

[0071] In the context of treating a condition associated with pathogenic MeCP2 activity (e.g., Rett syndrome) or use of a therapeutic agent comprising an RNA exon editor or a DNA sequence encoding an 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 condition associated with defective (non-functional) MeCP2 protein comprising at least one mutation and / or reduced levels of MeCP2 activity (e.g., Rett syndrome). In some embodiments, such methods or uses lead to an increase in MeCP2 protein activity. In some embodiments, such an increase in MeCP2 protein activity increases functional (biologically active) MeCP2 activity levels 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% of MeCP2 activity (e.g., 96%, 97%, 98%, 99%, or 100%) relative to that of a control (normal / wildtype) cell in which functional (biologically active) MeCP2 is expressed at levels that support normal cellular functions. In some embodiments, such an increase in MeCP2 protein activity restores MeCP2 activity levels 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% of MeCP2 activity (e.g., 96%, 97%, 98%, 99%, or 100%) relative to that of a cell in which MeCP2 is present at normal, wildtype levels such as those present in cells in which non-mutated MeCP2 is expressed.

[0072] As used herein, “cryptic splice site corrected”, “cryptic splice site mitigated”, or “cryptic splice site resistant” refers to an RNA exon editor or a nucleic acid molecule encoding an RNA exon editor or a portion of either thereof (e.g., a coding domain sequence therein) that has been modified to change individual nucleotides therein to reduce the frequency of splicing that occurs at a cryptic splice site identified in the context of the RNA exon editor. In some embodiments, the modifications do not result in any changes in amino acid sequences encoded thereby. In some embodiments, the cryptic splice site resistant nucleic acid sequence within an RNA exon editor is a coding domain sequence (CDS). In some embodiments, the cryptic splice site resistant MeCP2 CDS comprises, consists essentially of, or consists of exons 3 and 4 or exon 4 of the MeCP2 gene, wherein cryptic splice sites have been identified in the context of an MeCP2 RNA exon editor and wherein at least one of the cryptic splice sites has been modified to reduce the frequency of splicing at the at least one site, while not altering amino acids encoded thereby.

[0073] A “hemi-intron” as used herein, refers to a nucleic acid sequence comprising motifs that are recognized by the spliceosome and mediate trans-splicing. In some embodiments, ahemi-intron comprises a polypyrimidine tract (pPy), a branchpoint (BP), and / or a 3’ splice site, or any combination thereof. See, e.g., SEQ ID NO: 17 as an exemplary hemi-intron. The 3’ splice site may be a consensus 3’ splice site. In some embodiments, the hemi-intron comprises other regulatory elements that have one or more of the following effects: increase recruitment of splicing factors to the RNA exon editor, increase transcription / expression of functional MeCP2, increase on target (ONT) trans-splicing efficiency, decrease off target (OFT) trans-splicing, or decrease cis-splicing on the target pre-mRNA, or any combination thereof.

[0074] As used herein, the “binding domain” of an RNA exon editor or a nucleic acid molecule encoding same is a polynucleotide sequence that binds a target gene (e.g., an endogenous pre-mRNA) at a binding site via hybridization (i.e., full or partial complementarity to the binding site).

[0075] As used herein, the term “binding site” refers to an endogenous polynucleotide sequence in the target pre-mRNA (e.g., a 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’-most nucleotide bound by the binding domain to the 3’-most 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 within 1-10 nucleotides longer or shorter than the binding domain (i.e., some of the nucleotides of either the binding site or the binding domain are unhybridized). In embodiments involving binding domains having at least two nonoverlapping sequences with at least 80% complementarity to the binding site, the binding site may be substantially shorter than the binding domain.

[0076] As used herein, “complementarity,” and grammatical variations thereof, refers to the percentage of nucleotide bases of a given sequence that pairs through hydrogen bonding with a reference sequence. In the absence of a given percentage of complementarity, the terms “complement” and “complementary” refer to 100% complementarity.

[0077] As used herein, a given sequence (e.g., a binding domain sequence) is “100% complementary to,” or has “100% complementarity” with a reference sequence (e.g., an endogenous pre-mRNA binding site) if each of the nucleotide bases of the given sequence pairs through hydrogen bonding with the reference sequence, thereby hybridizing to form a double stranded sequence (e.g., through Watson-Crick base-pairing, e.g., each A pairs with a T or U, and each C pairs with a G). For instance, a binding domain that is in an anti-sense orientation to a binding site is complementary to the binding site. RNA pairing includes G pairing with U; therefore, an RNA binding domain having G-U pairing with its binding site can be 100% complementary with the binding site. Accordingly, a binding domain that is exactly the reverse complement of its binding site (i.e., A's of the binding domain are paired with U’s of the bindingsite) can be modified to replace any one or more of the A's with G’s without substantially affecting binding.

[0078] As used herein, a given sequence (e.g., a binding domain sequence) is “at least X% complementary to,” or has “X% complementarity” with a reference sequence (e.g., an endogenous pre-mRNA binding site) if X% of the nucleotide bases of the given sequence pairs through hydrogen bonding with the reference sequence, e.g., hybridizing to form a double stranded sequence (e.g., through Watson-Crick base-pairing, e.g., A pairs with T or U, and C pairs with G). For instance, 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 if at least 135 of its 150 residues pair through hydrogen bonding with the binding site through Watson-Crick base pairing, leaving 15 or fewer mismatched nucleotides.

[0079] “ Binding” between a binding domain and an intron, as used herein, refers to hydrogen bonding (e.g., double helix formation, or Watson Crick pairing) between the binding domain and, e.g., the target intron in a degree sufficient to mediate trans-splicing by bringing the RNA exon editor into association with the target (e.g., pre-mRNA). In some embodiments, the hydrogen bonds between the binding domain and the target intron are between nucleotide bases that are complementary to and in anti-sense orientation from one another (e.g., hybridized to one another).

[0080] As used herein, the term “mutation” may be used to refer to any aberrant nucleic acid sequence that encodes a defective protein product (e.g., a non-functional protein product, a non-biologically active protein, a protein product having reduced function, a protein product having pathogenic or aberrant function, and / or a protein product that is produced in less than normal or greater than normal quantities). Mutations include base pair mutations (e.g., single nucleotide polymorphisms), duplications, missense mutations, frameshift mutations, deletions, insertions, and splice mutations. In some embodiments, a mutation refers to a nucleic acid sequence that is different in one or more portions of its sequence than a corresponding wildtype nucleic acid sequence or functional variant thereof. In some embodiments, a mutation refers to a nucleic acid sequence that encodes a protein having an amino acid sequence that is different from a corresponding wildtype protein or functional variant thereof. A “mutated exon” (e.g., a mutated MeCP2 exon) refers to an exon comprising a mutation or an exon sequence that reflects a mutation in a different region, such as a cryptic exon resulting from a mutation in an intron.

[0081] The term “MeCP2” (Methyl CpG binding protein 2) refers to any native MeCP2 from any vertebrate source, including mammals such as primates (e.g., human, African green monkeys, and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated, as well as functionally equivalent or improved variants (e.g., natural or syntheticvariants), mutants, muteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof. Functionally equivalent and improved variants can be determined on the basis of known MeCP2 signaling. MeCP2 encompasses full-length, unprocessed MeCP2, as well as any form of MeCP2 that results from native processing in the cell. An exemplary human MeCP2 sequence is provided as National Center for Biotechnology Information (NCBI) Reference Sequence: NM_001110792.2 (specific for the E1 isoform, which lacks exon 2) or NM_004992.4 (specific for the E2 isoform, which includes exons 1-4). In some instances, an MeCP2 fragment is encoded by a therapeutic agent comprising a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 10, 11, or 12, or any combination thereof (e.g., a cassette of exon 3 and exon 4 sequences or of exon 2, 3, and 4 sequences) or SEQ ID NO: 9 or a fragment thereof (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 10, 11, or 12, or any combination thereof (e.g., a cassette of exon 3 and exon 4 sequences) or SEQ ID NO: 9, or a functional portion thereof, and / or a codon-modified variant thereof. Exemplary such codon-modified variants comprise 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, functional MeCP2 exons comprise codon-modified variants of exon 4 (e.g., SEQ ID NO: 14 or 86).

[0082] As used herein, a “variant” refers to a polynucleotide that differs in at least one nucleic acid residue from the reference polynucleotide sequence, such as a naturally occurring polynucleotide sequence, or a polypeptide (e.g., an AAV capsid sequence) that differs in at least one amino acid residue from the reference polypeptide sequence, such as a naturally occurring polypeptide sequence or, e.g., any of the rAAV sequences described herein. In this context, the difference in at least one residue may include, for example, a substitution of a nucleic acid residue to another nucleic acid, a deletion, or an insertion, or a substitution of an amino acid residue to another amino acid. A variant may be a homolog, isoform, or transcript variant of a polynucleotide as defined herein, wherein the homolog, isoform or transcript variant is characterized by a degree of identity or homology, respectively, as defined herein. In some instances, a variant of a polynucleotide or polypeptide includes 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 acidsubstitutions, 9 nucleic acid or amino acid substitutions, or 10 nucleic acid or amino acid substitutions). Nucleic acid substitutions that result in the expressed polypeptide having an exchanged amino acid from the same class are referred to herein as conservative substitutions. In particular, these are amino acids having aliphatic side chains, positively or negatively charged side chains, or aromatic groups in the side chains, the side chains of which can form hydrogen bridges, e.g., side chains which have a hydroxyl function. By conservative substitution, e.g., 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 substituted by another amino acid having a corresponding hydrophobic side chain (e.g., serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine)).

[0083] In some instances, insertions, deletions, and / or non-conservative substitutions are also encompassed by the term variant, e.g., at those positions that do not cause a substantial modification of the three-dimensional structure of the protein. Modifications to a three- dimensional structure by insertion(s) or deletion(s) can readily be determined by a person of skill in the art, e.g., using CD spectra (circular dichroism spectra).

[0084] The term “homologous” refers to the degree of identity between sequences of two nucleic acid sequences. The homology of sequences is determined by comparing two sequences aligned under standard conditions over the sequence length to be compared. The sequences to be compared herein may have an addition or deletion (for example, gap and the like) in the optimum alignment of the two sequences. In some embodiments, sequence homology is calculated by creating an alignment 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.

[0085] As used herein, the term “heterologous,” when used to describe a first element in reference to a second element indicates that the first element and second element do not exist in nature disposed as described. For example, a heterologous nucleic acid sequence (e.g., a heterologous 3’ UTR), can refer to a nucleic acid sequence that is included in a construct of the present disclosure operably linked to nucleic acid sequences with which it is not found operably linked in nature.

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

[0087] As used herein, relating to AAV, the term variant means any AAV sequence which is derived from a known AAV sequence, including those 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 greater sequence identity over the amino acid or nucleic acid sequence. In another embodiment, the AAV capsid includes variants which may include up to about 10% variation from any described or known AAV capsid sequence. That is, the AAV capsid shares about 90% identity to about 99.9% identity, about 95% to about 99% identity or about 97% to about 98% identity to an AAV capsid provided herein and / or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with an AAV capsid. When determining the percent identity of an AAV capsid, the comparison may be made over any of the variable proteins (e.g., vp1, vp2, or vp3).

[0088] The ITRs or other AAV components may be readily isolated or engineered using techniques available to those of skill in the art from an AAV. Such AAV may be isolated, engineered, or obtained from academic, commercial, or public sources (e.g., the American Type Culture Collection, Manassas, Va.). Alternatively, the AAV sequences may be engineered through synthetic or other suitable means by reference to published sequences such as are available in the literature or in databases such as, e.g., GenBank, PubMed, or the like. AAV viruses may be engineered by conventional molecular biology techniques, making it possible to optimize these particles for cell specific delivery of nucleic acid sequences, for minimizing immunogenicity, for tuning stability and particle lifetime, for efficient degradation, for accurate delivery to the nucleus, etc.

[0089] As used herein, the term “subject,” “individual,” or “patient” includes any mammal in need of these methods of treatment or prophylaxis, including primates, such as humans. Other mammals in need of such treatment or prophylaxis include non-human primates (NHP; 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 for developing a disease or disorder caused by a mutation in the MeCP2 gene. In another embodiment, the individual has shown clinical signs of a disease or disorder caused by a mutation in the MeCP2 gene, such as Rett syndrome. The individual may be any age during which treatment or prophylactic therapy may be beneficial. For example, in some embodiments, the individual is 0-5 years of age, 5-10 years of age, 10-20 years of age, 20-30 years of age, 30-40 years of age, 30-50 years of age, 40-50 years of age, 50-60 years of age, 60-70 years of age, or more than 70 years of age.

[0090] As used herein, the terms “disorder associated with a mutation” or “mutation associated with a disorder” refer to a correlation between a disorder and a mutation. In some embodiments, a disorder associated with a mutation is known or suspected to be wholly orpartially, or directly or indirectly, caused by the mutation. For example, an individual having the mutation may be at risk of developing the disorder, and the risk may additionally depend on other factors, such as other (e.g., independent) mutations (e.g., in the same or a different gene), or environmental factors.

[0091] As used herein, the term “treatment,” or a grammatical derivation thereof, is defined as reducing the progression of a disease, reducing the severity of a disease symptom, retarding progression of a disease symptom, removing a disease symptom, or delaying onset of a disease. In some embodiments, the term “treatment” is used to refer to a persistent or durable effect of a therapeutic agent such as an RNA exon editor described herein.

[0092] As used herein, the term “prevention” of a disorder, or a grammatical derivation thereof, is defined as reducing the risk of onset of a disease, e.g., as a prophylactic therapy for an individual who is at risk for developing a disorder associated with a mutation. An individual can be characterized as “at risk” for 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 who is at risk for developing a disorder has one or more MeCP2 mutations associated with the disorder. Additionally, or alternatively, an individual can be characterized as “at risk” for developing a disorder if the individual has a family history of the disorder.

[0093] MeCP2 disorders are inherited in an X-linked manner. Greater than 99% of MeCP2 disorders are simplex cases (i.e., a single occurrence in a family), resulting from a de novo pathogenic variant or possibly from inheritance of the pathogenic variant from a parent who has germline mosaicism. In rare circumstances, an MeCP2 variant may be inherited from a heterozygous mother wherein X-chromosome inactivation results in minimal to no clinical findings. For known heterozygote mothers, the risk of child inheriting the MeCP2 variant is 50%.

[0094] Treating or preventing a disorder in an individual can be performed by directly administering DNA encoding an RNA exon editor (e.g., via a vector comprising a DNA sequence encoding an RNA exon editor) or RNA exon editor to the individual. In some embodiments, the vector comprising a DNA sequence encoding an RNA exon editor comprises an AAV vector. In some embodiments, the AAV vector is administered via an AAV viral particle. Alternatively, host cells containing the RNA exon editor may be administered to the individual.

[0095] The term “administering” or a grammatical derivation thereof, as used in the methods described herein, refers to delivering an RNA exon editor or a DNA sequence encoding same (e.g., within a vector, e.g., an AAV vector or AAV particle) or a composition thereof, or an ex vivo-treated cell, to the individual in need thereof, e.g., an individual having a mutation or defect in MeCP2. In some embodiments, the brain is targeted for delivery of an RNA exon editor or a DNA sequence encoding same (e.g., within a vector, e.g., an AAV vector or AAV particle) or acomposition thereof. In some embodiments, brain cells for which delivery is targeted comprise e.g., neurons, neural stem cells, astrocytes, or oligodendrocytes, or any combination thereof).

[0096] In some embodiments thereof, the method involves delivering an RNA exon editor or a DNA sequence encoding same (e.g., within a vector, e.g., an AAV vector or AAV particle) or a composition thereof to the individual by intravenous (IV) delivery, intracerebral (IC) delivery (e.g., slow delivery injection or convection-enhanced diffusion injection), intracerebroventricular (ICV) delivery, intrathecal delivery, or intracisternal magna delivery. In some embodiments thereof, neurons, neural stem cells, astrocytes, or oligodendrocytes, or any combination thereof are targeted. In some embodiments, IC injections involve stereotaxic implantation of microinjection guide sleeves to improve delivery to a specific locus in the brain. In some embodiments, the composition is administered systemically (e.g., intravenously). Still other methods of administration may be selected by one of skill in the art, in view of this disclosure.

[0097] As used herein, “modulating expression of MeCP2” refers to increasing the expression of functional MeCP2 protein generated following trans-splicing. Increasing expression of functional MeCP2 protein generated following trans-splicing is associated with a decrease in expression of endogenous mutated (non-functional) MeCP2. Modulating expression of MeCP2 may be used to refer, e.g., to increasing the expression of MeCP2 protein generated following trans-splicing (e.g., MeCP2 transcript or protein product that has an RNA exon editor- mediated corrected mutation site relative to its endogenous mutated transcript or protein product). Modulating expression of MeCP2 may also be used to refer, e.g., to decreasing the expression of endogenous (e.g., mutated) MeCP2. Upon replacement of the endogenous MeCP2 exon / s comprising the at least one mutation via trans-splicing, a functional MeCP2 protein is expressed.

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

[0099] The term “pharmaceutically acceptable” means safe for administration to a mammal, such as a human. In some embodiments, a pharmaceutically acceptable composition is approved by a regulatory agency of the Federal or a state government or listed in the U. S.Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0100] The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic molecule (e.g., a trans-splicing molecule or a trans-splicing molecule including 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.

[0101] The terms “a” and “an” mean “one or more of.” For example, “a gene” is understood to represent one or more such genes. As such, the terms “a” and “an,” “one or more of a (or an),” and “at least one of a (or an)” are used interchangeably herein.

[0102] As used herein, the term “about” refers to a value within ± 10% variability from the reference value, unless otherwise specified.C. RNA Exon Editors

[0103] Provided herein are RNA exon editors and DNA sequences encoding same (e.g., vectors comprising DNA sequences encoding RNA exon editors or AAV viral particles comprising DNA sequences encoding RNA exon editors) useful for correcting mutations in MeCP2 by replacing at least one MeCP2 exon comprising at least one mutation with a functional MeCP2 exon (e.g., an MeCP2 exon 3’ to the binding site, e.g., exons 3-4, exons 2-4, or exon 4 of MeCP2). 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 permits replacement of the defective or mutated portion of the pre-mRNA exon(s) with a nucleic acid sequence, e.g., the exon(s) having a functional (e.g., normal) sequence without the mutation. The functional sequence can be a wildtype, naturally occurring sequence or a corrected sequence with some other modification, e.g., codon optimization.

[0104] In some embodiments, RNA exon editors comprise a binding domain, a hemi-intron, and a coding domain. In some embodiments, RNA exon editors comprise a binding domain, a splice site, and a coding domain. In some embodiments, the RNA exon editor has a 3’ regulatory domain comprising a native 3’ MeCP2 untranslated region (UTR) or a heterologous 3’ UTR (e.g., a sequence having at least 80% sequence identity with SEQ ID NO: 19 or SEQ ID NO: 20). In some embodiments, the RNA exon editor has a 3’ splice site of YAG / [wherein Y is a pyrimidine (cytosine or thymidine (which may be uracil in RNA));denotes the location of the 3’ splice site.; e.g., TAG]. In some embodiments, the RNA exon editor comprises a hemi-intron comprising, consisting essentially of, or consisting of 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% identityto SEQ ID NO: 17. 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 terminator sequence (e.g., SEQ ID NO: 22), or a combination thereof. See, e.g., FIGs. 5 and 16.

[0105] In some embodiments, an RNA exon editor described herein includes, operatively linked in a 5’ to 3’ direction: a binding domain, a hemi-intron, a coding domain sequence (e.g., a CDS, e.g., a sequence encoding a functional sequence of MeCP2 exons, e.g., a functional sequence of MeCP2 exons 3’ to the binding site), and a 3’ untranslated region. In some embodiments, an RNA exon editor described herein includes, operatively linked in a 5’ to 3’ direction: a binding domain, a hemi-intron, a coding domain sequence (e.g., a CDS, e.g., a sequence encoding a functional sequence of MeCP2 exons, e.g., a functional sequence of MeCP2 exons 3’ to the binding site), a 3’ untranslated region, a termination and poly A sequence, and a triple helix terminator.

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

[0107] An MeCP2 gene targeted by a trans-splicing molecule described herein can contain one or multiple mutations that are associated with Rett syndrome. An exemplary human MeCP2 sequence is provided as National Center for Biotechnology Information (NCBI) Reference Sequence: NM_001110792.2 (specific for the E1 isoform) or NM_004992.4 (specific for the E2 isoform). In addition to published sequences, all corrections later obtained or naturally occurring conservative and non-disease-causing variant sequences that occur in the human or other mammalian population are also included. Additional conservative nucleotide replacements or those causing codon optimizations are also included. The sequences as provided by the database accession numbers may also be used to search for homologous sequences in the same or another mammalian organism.

[0108] MeCP2 protein comprises several well-defined structural / functional domains as follows: N-terminal domain (NTD); methyl binding domain (MBD); intervening domain (ID); transcription repression domain (TRD); NCoR interaction domain (NID); and the C-terminaldomain (CTD) (see FIG. 1). The TRD includes the nuclear localization sequence (NLS). Genetic mutations in the coding region of the X-chromosome-linked MeCP2 gene alter the ability with which its encoded protein MeCP2 binds DNA within the context of chromatin. Mutations affecting the MBD of MeCP2 affect the stability and affinity of its DNA binding.

[0109] It is anticipated that the MeCP2 nucleic acid sequences and resulting proteins expressed may tolerate certain minor modifications at the nucleic acid level to include, for example, modifications to the nucleotide bases which are silent, e.g., preference codons. In other embodiments, nucleic acid base modifications which change the amino acids, e.g., to improve expression of the resulting peptide / protein are envisioned. In some embodiments, modification of allelic variations, caused by the natural degeneracy of the genetic code are envisioned.

[0110] Also included as modifications of MeCP2 genes are analogs or modified versions of the encoded amino acid sequences. Typically, such analogs differ from the specifically identified proteins by only one to four codon changes. Conservative replacements are those that take place within a family of amino acids that are related in their side chains and chemical properties.

[0111] The nucleic acid sequence of a functional MeCP2 gene may be derived from any mammal which natively expresses functional MeCP2 or a homolog thereof. In other embodiments, certain modifications are made to the MeCP2 gene sequence in order to enhance expression in the target cell. Such modifications include codon optimization.

[0112] As described herein above, Rett syndrome is caused by mutations in the MeCP2 gene. Compositions comprising RNA exon editors described herein can correct about 95% of the mutations associated with Rett syndrome because the RNA exon editors replace the entirety of exons 3 and 4 of the MeCP2 gene, wherein about 95% of the identified mutations are found.

[0113] The attached sequence listing provides various sequences of human MeCP2. SEQ ID NO: 1 is a partial nucleotide sequence of wild type MeCP2 intron 1 , starting at position 1 of intron 1. SEQ ID NO: 113 is the complete nucleotide sequence of wild type MeCP2 intron 1.SEQ ID NO: 2 is a partial nucleotide sequence of wild type MeCP2 intron 2, starting at position 1 of intron 2. SEQ ID NO: 112 is the complete nucleotide sequence of wild type MeCP2 intron 2. SEQ ID NO: 3 is a partial nucleotide sequence of wild type MeCP2 intron 2, ending with the final nucleotide of intron 2. SEQ ID NO: 4 is the complete nucleotide sequence of wild type MeCP2 intron 3. SEQ ID NO: 5 is the nucleotide sequence of the open reading frame of wild type MeCP2 isoform E1. SEQ ID NO: 6 is the sequence of the open reading frame of wild type MeCP2 E1 exon 1. SEQ ID NO: 7 is the sequence of wild type MeCP2 E1 exon 3. SEQ ID NO: 7 is the sequence of wild type MeCP2 E1 exon 4. SEQ ID NO: 9 is the nucleotide sequence of the open reading frame of wild type MeCP2 isoform E2. SEQ ID NO: 10 is the sequence of the openreading frame of wild type MeCP2 E2 exon 2. SEQ ID NO: 11 is the sequence of wild type MeCP2 E2 exon 3. SEQ ID NO: 7 is the sequence of wild type MeCP2 E2 exon 4.2. Coding Domains

[0114] In some embodiments, the coding domain of a 3’ trans-splicing molecule includes all MeCP2 exons (e.g., functional MeCP2 exons) that are 3’ to the target MeCP2 intron (e.g., MeCP2 intron 1). In some embodiments in which a 3’ trans-splicing molecule targets MeCP2 intron 1, the coding domain may include functional MeCP2 exons 3-4. In some embodiments, functional MeCP2 exons 3-4 are encoded by a sequence comprising SEQ ID NOs: 11 and 12 or a sequence having at least 95% sequence identity to a sequence comprising SEQ ID NOs: 11 and 12. In some embodiments in which a 3’ trans-splicing molecule targets MeCP2 intron 1 , the coding domain may include functional MeCP2 exons 2-4. In some embodiments, functional MeCP2 exons 2-4 are encoded by a sequence comprising SEQ ID NO: 9 or a sequence having at least 95% sequence identity to 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’s may be substituted for T’s in the sequence. Thus, for example, in some embodiments, functional MeCP2 exons 2-4 include an RNA version of SEQ ID NO: 9 having U substituted for each T in SEQ ID NO: 9 or a sequence having at least 95% sequence identity to SEQ ID NO: 9 having U substituted for each T in SEQ ID NO: 9. In some embodiments, the binding domain binds to intron 2, and the coding domain includes 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 to a sequence comprising SEQ ID NOs: 11 and 12. In some embodiments, the binding domain binds to intron 3, and the coding domain includes functional MeCP2 exon 4. In some embodiments, functional MeCP2 exon 4 is encoded by a sequence comprising SEQ ID NO: 12 or a sequence having at least 95% sequence identity to SEQ ID NO: 12. In some embodiments, functional MeCP2 exons comprise codon-modified variants of exon 3 (e.g., SEQ ID NO: 13 or 85) and codon-modified variants of exon 4 (e.g., SEQ ID NO: 14 or 86) 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, functional MeCP2 exons comprise codon-modified variants of exon 4 (e.g., SEQ ID NO: 14).

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

[0116] In some embodiments, all or a portion of the coding domain or coding domainencoding sequence, or one or more functional MeCP2 exons thereof, is a codon-optimized sequence in which a nucleic acid sequence has been modified, e.g., to enhance expression or stability, without resulting in a change in the encoded amino acid. Codon optimization may be performed in a manner such as that described in, e.g., U.S. Patent Nos. 7,561 ,972, 7,561 ,973, and 7,888,112, each of which is incorporated herein by reference in its entirety. A codon- optimized version of MeCP2 Exon 3 is set forth in SEQ ID NO: 13. A codon-optimized version of MeCP2 Exon 4 is set forth in SEQ ID NO: 14. For delivery via a recombinant AAV, as described herein, in one embodiment, the coding domain can be a nucleic acid sequence of up to 4,000 nucleotide bases in length (e.g., from 3,000 to 4,000 nucleotide bases in length, from 3,100 to 3,800 nucleotide bases in length, from 3,200 to 3,700 nucleotide bases in length, or from 3,300 to 3,500 nucleotide bases in length, e.g., from 3,000 to 3,100 nucleotide bases in length, from 3,100 to 3,200 nucleotide bases in length, from 3,200 to 3,300 nucleotide bases in length, from3,300 to 3,400 nucleotide bases in length, from 3,400 to 3,500 nucleotide bases in length, from3,500 to 3,600 nucleotide bases in length, from 3,600 to 3,700 nucleotide bases in length, from3,700 to 3,800 nucleotide bases in length, from 3,800 to 3,900 nucleotide bases in length, or from 3,900 to 4,000 nucleotide bases in length, e.g., about 3,108 nucleotide bases in length, about 3,285 nucleotide bases in length, about 3,375 nucleotide bases in length, about 3,503 nucleotide bases in length, about 3,630 nucleotide bases in length, about 3,540 nucleotide bases in length, about 3,363 nucleotide bases in length, about 3,273 nucleotide bases in length, about 3,145 nucleotide bases in length, or about 3,018 nucleotide bases in length).

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

[0118] Also encompassed herein is a method for modifying an RNA molecule in a cell, the method comprising providing to the cell an exogenous RNA molecule (via, e.g., a DNA vector encoding the exogenous RNA molecule or viral particle comprising such a DNA vector) comprising, in a 5’ to 3’ direction: (a) a binding domain sequence configured to bind to an intronof the endogenous target RNA molecule; (b) a splice acceptor sequence configured to splice to a splice donor sequence of the endogenous target RNA molecule; and (c) a cDNA coding domain sequence comprising one or more nucleotide changes that differ from that of an endogenous target RNA molecule in the cell; wherein the one or more nucleotide changes disrupt one or more cryptic splice sites within the coding domain sequence of the exogenous RNA molecule. In some embodiments, the nucleotide change is a synonymous nucleotide change. In some embodiments, the cryptic splice site is identified experimentally. In some embodiments, the cryptic splice site is predicted based on in silico analysis.

[0119] Also encompassed herein is a method of increasing trans-splicing efficiency of an RNA exon editor or improving therapeutic performance of an RNA exon editor comprising introducing a nucleotide change into a coding domain sequence of the RNA exon editor, wherein the nucleotide change disrupts a cryptic 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 cryptic splice site is identified experimentally. In some embodiments, the cryptic splice site is predicted based on in silico analysis.

[0120] Cryptic splice site-mitigating nucleotide changes can include changes that eliminate or reduce the ability of a cryptic splice site (e.g., a cryptic splice site within a coding domain or binding domain sequence of an RNA exon editor) to be used in a splicing reaction. For example, a cryptic splice site identified in the context of an RNA exon editor typically comprises a splice site, a polypyrimidine tract, and a branchpoint. In some embodiments, one or more nucleotide changes may be introduced into at least one of a splice site, a polypyrimidine tract, or a branchpoint, or any combination thereof of a cryptic splice site identified in the context of an RNA exon editor. In some embodiments, the nucleotide change is designed so as to minimize the potential impact on a protein encoded thereby. A person of skill in the art would appreciate that if a nucleotide change made to reduce the frequency of cryptic splice site usage also altered the amino acid encoded by a trans-spliced RNA, conservative amino acid changes would be preferred over non-conservative amino acid changes. Moreover, such a skilled person could readily analyze the protein sequence and structure with an eye toward functional domains and significant sequences therein to evaluate whether such changes could reasonably be expected to alter function of a protein encoded by a trans-spliced protein. A skilled person could also test a protein comprising such an amino acid change to determine if biological activity is altered using assays known in the art. In some embodiments, more than one nucleotide is changed within a cryptic splice site identified. Under some circumstances, a determination of how many nucleotides should be changed is made empirically based on in silico predictions and / or experimental results. In some embodiments, one or more (also referred to herein as at least one) synonymous nucleotide changes may be introduced into at least one of a splice site, apolypyrimidine tract, or a branchpoint, or any combination thereof of a cryptic splice site identified in the context of an RNA exon editor. Synonymous nucleotide changes do not alter the amino acid sequence of a protein encoded by a trans-spliced RNA. In some embodiments, more than one synonymous nucleotide change may be introduced into at least one of a splice site, a polypyrimidine tract, or a branchpoint, or any combination thereof of a cryptic splice site identified in the context of an RNA exon editor.

[0121] Further to the above, the experimental results and sequence information are analyzed generally as follows. Changes to remove cryptic splice sites identified experimentally are made by searching for and replacing certain elements of splice donor sites. AG sites (and more strongly CAG sites) at the end of a splice donor site are prioritized for introduction of nucleotide changes. If an AG site is not found or could not be changed without introducing a non- synonymous nucleotide change, then the sequence 42 - 4 base pairs upstream of the splice site is scanned for branch points (e.g., sequences matching YNAH, wherein Y is a pyrimidine, N represents any nucleotide, H is adenine, cytosine, or thymine (or uracil in RNA), and A is the branch point adenine). Any such branch point sequences identified are then analyzed and considered for introduction of one or more nucleotide changes to reduce cryptic splice site usage at the experimentally identified cryptic splice site. In addition, the sequences are also scanned for the presence of polypyrimidine tracts (multiple Ys (pyrimidines) immediately upstream of the terminal AG). Typically, such polypyrimidine tracts comprise at least 5 pyrimidines within 10 base pairs upstream of the splice site. Once identified, such polypyrimidine tracts are then analyzed and considered for introduction of one or more nucleotide changes to reduce cryptic splice site usage at the experimentally identified cryptic splice site.

[0122] In some embodiments, the cryptic splice site, or off-target splice site, that is changed to mitigate off-target splicing is a site that has been identified empirically as a site of off-target splicing. Such sites can be identified, for example, using techniques described in Example 7 of PCT / US23 / 66969 (published as WO2023 / 220742), which is hereby incorporated by reference in its entirety. In some embodiments, all cryptic splice sites that have a frequency of usage above a predetermined threshold are changed by cryptic splice site-mitigating nucleotide changes.

[0123] In some embodiments, the cryptic splice site, or off-target splice site, that is changed to mitigate off-target splicing is a site that has been predicted to be a site of off-target splicing. Such predictions can be made based on sequence analysis to identify a canonical splice site, polypyrimidine tract, and / or branchpoint of a putative cryptic splice site therein.

[0124] The present inventors used a proprietary combination of proprietary software and know how to identify cryptic splice sites having high probability of impact on RNA exon editor activity. The proprietary combination also relies on an order of operation regarding the belowlisted cycles that has been determined by the present inventors to impact therapeutic performance of RNA exon editors generated thereby. Briefly, three iterative cycles of analysis and modification were performed on the codon domain sequences whereby in the first cycle, high ranked cryptic splice sites were identified using proprietary software and modified to reduce / eliminate splice site usage; whereby in the second cycle, sequences generated following the first cycle were further analyzed using a combination of proprietary software and know how to identify and rank additional cryptic splice sites and alter these sites to reduce / eliminate cryptic splice site usage at these sites; and whereby in the third cycle, sequences generated following the second cycle were further analyzed using a combination of proprietary software and know how to identify sequences requiring further modification to improve and / or enhance therapeutic performance of RNA exon editors generated thereby.

[0125] A modified version of the above process was implemented with respect to analysis and modification of the binding domain sequences. Briefly, two iterative cycles of analysis and modification of the binding domain sequences were performed using a combination of proprietary software and know how to identify and rank cryptic splice sites and alter these sites to reduce / eliminate cryptic splice site usage at these sites in a first cycle, followed by a second cycle, wherein sequences generated following the first cycle were further analyzed using a combination of proprietary software and know how to identify sequences requiring further modification to improve or enhance therapeutic performance of RNA exon editors generated thereby.

[0126] By performing the above analysis, the inventors identified cryptic splice sites in the sequences of codon-optimized versions of MeCP2 exon 3 (SEQ ID NO: 13) and exon 4 (SEQ ID NO: 14), along with a probability score which gives the relative likelihood of the cryptic splice site being involved in an off-target splicing reaction. Nucleotides that contribute to cryptic splice sites present in codon-optimized exon 3 (SEQ ID NO: 13) and relative probability scores are set forth in Table 2. A relative probability score of 1 indicates that such sites have the highest relative probability of cryptic site use in the scoring system presented herein.Table 2: Exon 3 Cryptic Splice Sites

[0127] Nucleotides that contribute to cryptic splice sites present in codon-optimized exon 4 (SEQ ID NO: 14) and relative probability scores are set forth in Table 3.Table 3: Exon 4 Cryptic Splice Sites

[0128] In some embodiments, a coding domain sequence comprises an MeCP2 exon 3 nucleotide sequence comprising a cryptic splice site mitigating nucleotide change relative to SEQ ID NO:13 at one or more of the positions identified in Table 2. In some embodiments, the coding domain sequence has a nucleotide change relative to SEQ ID NO: 13 at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13 of the positions set forth in Table 2. In some embodiments, the coding domain sequence has a nucleotide change relative to SEQ ID NO: 13 at one or more of the positions set forth in Table 2 having a relative probability of 1. In some embodiments, the coding domain sequence has a nucleotide change relative to SEQ ID NO: 13 at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 of the positions set forth in Table 2 having a relative probability of 1.

[0129] In some embodiments, a coding domain sequence comprises an MeCP2 exon 4 nucleotide sequence comprising a cryptic splice site mitigating nucleotide change relative to SEQ ID NO:14 at one or more of the positions identified in Table 3. In some embodiments, the coding domain sequence has a nucleotide change relative to SEQ ID 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 set forth in Table 3. In some embodiments, the coding domain sequence has a nucleotide change relative to SEQ ID NO: 14 at one or more of the positions set forth in Table 3 having a relative probability of 1. In some embodiments, the coding domain sequence has a nucleotide change relative to SEQ ID NO: 14 at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the positions set forth in Table 2 having a relative probability of 1.

[0130] In some embodiments, a cryptic splice site-mitigating nucleotide change causes a nucleotide sequence that matches a canonical splice site consensus sequence to no longer match the canonical sequence. In some embodiments, a cryptic splice site-mitigating nucleotide change eliminates a potential splice site nucleotide. In some embodiments, a cryptic splice sitemitigating nucleotide change eliminates a potential polypyrimidine tract nucleotide. In some embodiments, a cryptic splice site-mitigating nucleotide change eliminates a potential branch point nucleotide. In some embodiments, a cryptic splice site-mitigating nucleotide change is a synonymous nucleotide change. In some embodiments, a cryptic splice site-mitigating nucleotide change causes a change in an amino acid encoded by the exon editor. In some embodiments, the amino acid change is a conservative amino acid substitution. A sequence of MeCP2 exon 3 having cryptic splice site-mitigating nucleotide changes is set forth in SEQ ID NO: 85. A sequence of MeCP2 exon 4 having cryptic splice site-mitigating nucleotide changes is set forth in SEQ ID NO: 86. A sequence of MeCP2 exons 3 and 4 having cryptic splice sitemitigating nucleotide changes is set forth in SEQ ID NO: 76. The sequences in SEQ ID NOs: 76, 85, and 86 also have nucleotide changes done for the purpose of codon optimization.

[0131] In some embodiments, the coding domain sequence of a MeCP2 trans-splicing molecule has nucleotide changes at one or more of the following nucleotides in MeCP2 exon 3, numbering according to SEQ ID NO: 7: 4A, 7A, 1 1 T, 12C, 13A, 16A, 22G, 28C, 37C, 43T, 49G, 55A, 67C, 85G, 91 G, 94A, 97A, 103C, 106A, 109G, 1 15A, 119T, 120C, 127T, 130T, 133G, 176T, 177C, 182T, 183C, 184G, 206T, 207C, 223A, 239C, 241 T, 280A, 290C, 317C, 320T, 3210, or 331 A. In some embodiments, the CDS has nucleotide changes at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 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, a U is substituted in place of T in 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, numbering according to SEQ ID NO: 8: 7G, 10A, 13A, 19T, 20C, 22C, 23T, 24C, 25T, 28A, 35T, 40T, 49T, 55A, 61 T, 67G, 94T, 106A, 107C, 130A, 286T, 304A, 595T, 683C, 740T, 741C, 806T, 807C, 809T, 810C, 824T, 825C, 851 T, 8520, 854T, 855C, 892G, 980C, 9820, 9840, 10340, 1055C, 1072A, 10730, 1075T, 1 117T, 1 1180, 1123T, 1 1240, 1 125G, 1147T, 1 1480, 1164A, 11800, 1182T, 1221A, 12310, 12580, 1261T, 12620, or 1272A. In some embodiments, the CDS has nucleotide changes at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 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. In some embodiments, a U is substituted in place of T in the disclosed positions.

[0132] In some embodiments, the coding domain sequence of a MeCP2 trans-splicing molecule comprises an exon 3 nucleotide sequence having one or more of, or any combination of, the following nucleotides at the indicated positions: 43T, 49G, 55A, 670, 85G, 91 G, 94A, 97A, 1030, 106A, 109G, 115A, 1 18T, 1 19T, 120C, 130T, 133G, 176T, 177C, 182T, 183C, 184G, 206T, 207C, 223A, 2390, 241T, 280A, 290C, 317C, 320T, 321 C, or 331A (numbering according to SEQ ID NO: 7). In some embodiments, the coding domain sequence comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 85, wherein the coding domain sequence comprises one or more of, or any combination of, the following nucleotides at the indicated positions: 43T, 49G, 55A, 67C, 85G, 91G, 94A, 97A, 103C, 106A, 109G, 1 15A, 118T, 1 19T, 1200, 130T, 133G, 176T, 177C, 182T, 183C, 184G, 206T, 207C, 223A, 239C, 241T, 280A, 2900, 317C, 320T, 3210, or 331A (numbering according to SEQ ID NO: 85).

[0133] In some embodiments, the coding domain sequence of an MeCP2 trans-splicing molecule comprises an exon 4 nucleotide sequence having one or more of, or any combination of, the following nucleotides at the indicated positions: 49T, 55A, 61 T, 67G, 94T, 106A, 107C, 130A, 286T, 304A, 595T, 683C, 740T, 7410, 806T, 8070, 808T, 8090, 824T, 825C, 851 T, 8520, 854T, 8550, 892G, 9800, 9820, 1034C, 10550, 1072A, 1073C, or 1075T (numbering accordingto SEQ ID NO: 8). In some embodiments, the coding domain sequence comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 86, wherein the coding domain sequence comprises one or more of, or any combination of, the following nucleotides at the indicated positions: 49T, 55A, 61T, 67G, 94T, 106A, 107C, 130A, 286T, 304A, 595T, 683C, 740T, 741 C, 806T, 807C, 808T, 809C, 824T, 825C, 851 T, 852C, 854T, 855C, 892G, 980C, 982C, 1034C, 1055C, 1072A, 1073C, or 1075T (numbering according to SEQ ID NO: 86).3. Binding Domains

[0134] MeCP2 trans-splicing molecules described herein feature a binding domain (BD) configured to bind / anneal to a target MeCP2 intron and / or exon. In some embodiments, a BD is configured to bind / anneal to 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 a sequence of the target MeCP2 intron pre-mRNA (e.g., a target MeCP2 intron; 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, at least 76% complementary to, at least 77% complementary to, at least 78% complementary to, at least 79% complementary to, at least 80% complementary to, at least 81% complementary to, at least 82% complementary to, at least 83% complementary to, at least 84% complementary to, at least 85% complementary to, at least 86% complementary to, at least 87% complementary to, at least 88% complementary to, at least 89% complementary to, at least 90% complementary to, at least 91% complementary to, at least 92% complementary to, at least 93% complementary to, at least 94% complementary to, at least 95% complementary to, at least 96% complementary to, at least 97% complementary to, at least 98% complementary to, at least 99% complementary to, or 100% complementary to a sequence of the target MeCP2 intron and / or exon pre-mRNA (e.g., a target MeCP2 intron; intron 1 , intron 2, or intron 3).

[0135] Further to the above, the present inventors tested mouse MeCP2 exon editors comprising BDs that bind to analogous positions of mouse MeCP2 intron 1 pre-mRNA relative to human MeCP2 pre-mRNA. Exemplary such mouse MeCP2 exon editors comprising BDs that are at least 80% identical (e.g., 82% identical) to analogous human binding domain sequences target human MeCP2 pre-mRNA and were shown to achieve 10% ONT trans-splicing into a human MeCP2 pre-mRNA target. Such exemplary mouse MeCP2 exon editors, therefore, achieve significant levels of cross species on-target trans-splicing. By way of comparison, positionally analogous human MeCP2 exon editors achieve 15% ONT into the human MeCP2 pre-mRNA target. In some embodiments, exemplary mouse MeCP2 exon editors comprise stretches of up to 35-40 nucleotides that are fully complementary to the human MeCP2 pre-mRNA. In some embodiments, most of the mismatches between the mouse MeCP2 exon editor and the human MeCP2 pre-mRNA target are located towards the 5' end of the binding domain.

[0136] MeCP2 trans-splicing molecules described herein feature 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 to (e.g., at least 85% complementary to, at least 90% complementary to, at least 91% complementary to, at least 92% complementary to, at least93% complementary to, at least 94% complementary to, at least 95% complementary to, at least96% complementary to, at least 97% complementary to, at least 98% complementary to, at least99% complementary to, or 100% complementary to) a sequence of the target MeCP2 intron pre-mRNA (e.g., a target MeCP2 intron). An MeCP2 trans-splicing molecule comprising such a binding domain may suppress endogenous target cis-splicing while enhancing trans-splicing between the trans-splicing molecule and the target MeCP2 pre-mRNA (e.g., by creating a chimeric molecule having a portion of endogenous MeCP2 mRNA and a coding domain having one or more functional MeCP2 exons that encode wildtype MeCP2 amino acid sequences). In some embodiments involving trans-splicing molecule-encoding sequences (e.g., vectors encoding trans-splicing molecules), the binding domain-encoding sequence encodes a nucleic acid sequence that is at least 80% complementary to (e.g., at least 85% complementary to, at least 86% complementary to, at least 87% complementary to, at least 88% complementary to, at least 89% complementary to, at least 90% complementary to, at least 91% complementary to, at least 92% complementary to, at least 93% complementary to, at least 94% complementary to, at least 95% complementary to, at least 96% complementary to, at least 97% complementary to, at least 98% complementary to, at least 99% complementary to, or 100% complementary to) a sequence of the target MeCP2 intron pre-mRNA.

[0137] In some embodiments, the present disclosure provides trans-splicing molecules (or vectors encoding same) that bind MeCP2 at intron 1 , e.g., wherein the nucleic acid trans- splicing molecule is configured to trans-splice a coding domain to endogenous MeCP2 exon 1. In some embodiments, the trans-splicing molecule binds MeCP2 pre-mRNA at a position spanning the exon1-intron1 junction. In some embodiments, trans-splicing molecules described herein include those in which the binding domain binds a binding site having any one or more (e.g., six or more, eight or more, ten or more, or twelve or more, twenty-five or more, fifty or more, 75 or more, 100 or more, 150 or more, 200 or more, or 250 or more, or a range of 6 to 12, 12 to 25, 25 to 50, 25 to 75, 25 to 100, 25 to 150, 25 to 200, 50 to 75, 50 to 100, 50 to 150, 50 to 200, 50 to 250, 100 to 150, 100 to 200, or 100 to 250) of nucleotides -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of intron 1 or 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 defined as the first nucleotide of MeCP2 intron 1 (position 115 of SEQ ID NO: 111 or position 1 of SEQ ID NO: 1 ), with negative numbers increasing proceeding in a 5’ direction from position -1 and positive numbers increasing proceeding in a 3' direction from position 1. In some embodiments, transsplicing molecules described herein include those in which the binding domain binds a binding site within 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, 125-649, 125-809, 125-969, 125-1129, 125-1289, 125-1449, 250-399, 250-524, 250-649, 250-809, 250-969, 250-1129, 250-1289, 250- 1449, 375-399, 375-524, 375-649, 375-809, 375-969, 375-1129, 375-1289, 375-1449, 500-524, 500-649, 500-809, 500-969, 500-1129, 500-1289, 500-1449, 660-809, 660-969, 660-1129, 660- 1289, 660-1449, 820-969, 820-1129, 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 of SEQ ID NO:1. In some embodiments, trans-splicing molecules described herein include those in which the binding domain binds a binding site comprising at least 100 consecutive nucleotides within 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, 125-649, 125-809, 125-969, 125-1129, 125-1289, 125-1449, 250-399, 250-524, 250-649, 250-809, 250-969, 250-1129, 250-1289, 250- 1449, 375-399, 375-524, 375-649, 375-809, 375-969, 375-1129, 375-1289, 375-1449, 500-524, 500-649, 500-809, 500-969, 500-1129, 500-1289, 500-1449, 660-809, 660-969, 660-1129, 660- 1289, 660-1449, 820-969, 820-1129, 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 of SEQ ID NO:1. In some embodiments, trans-splicing molecules described herein include those in which the binding domain comprises a length of about 50-300 nucleotides; about 50-250 nucleotides; about 50-200 nucleotides; about 50-150 nucleotides; about 50-100 nucleotides; about 75-300 nucleotides; about 75-250 nucleotides; about 75-200 nucleotides; about 75-150 nucleotides; about 100-300 nucleotides; about 100-250 nucleotides; about 100-200 nucleotides; about 100- 150 nucleotides; about 125-300 nucleotides; about 125-250 nucleotides; about 125-200 nucleotides; about 125-150 nucleotides; about 50 nucleotides; about 100 nucleotides; about 125 nucleotides; about 150 nucleotides; about 200 nucleotides; or about 250 nucleotides. In some embodiments, trans-splicing molecules described herein include those in which the binding domain comprises 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; 125-300 nucleotides; 125-250 nucleotides; 125-200nucleotides; 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 ranging from 100 to 200 nucleotides in length and binds MeCP2 at a binding site within nucleotides 1-1449 of SEQ ID NO:1 , wherein the binding domain comprises 50 consecutive nucleotides that are 100% complementary to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and binds MeCP2 at a binding site within nucleotides 1-1449 of SEQ ID NO:1 , wherein the binding domain comprises 50 consecutive nucleotides that are 90% to 100% complementary to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 100% complementary to a binding site comprising at least 100 consecutive nucleotides within nucleotides 1-1449 of SEQ ID NO:1. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 100% complementary to a binding site comprising at least 75 consecutive nucleotides within nucleotides 1-1449 of SEQ ID NO:1. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 90% to 100% complementary to a binding site comprising at least 100 consecutive nucleotides within nucleotides 1-1449 of SEQ ID NO:1. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and binds MeCP2 at 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 ranging from 100 to 200 nucleotides in length and binds MeCP2 at a binding site within nucleotides 3950-4250 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 ranging from 100 to 200 nucleotides in length and is 100% complementary to a binding site comprising at least 75 consecutive nucleotides within nucleotides 3950-4250 of SEQ ID NO:1. In someembodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 100% complementary to a binding site comprising at least 100 consecutive nucleotides within nucleotides 3950-4250 of SEQ ID NO:1. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 90% to 100% complementary to a binding site comprising at least 100 consecutive nucleotides within nucleotides 3950-4250 of SEQ ID NO:1.

[0138] In some instances, the binding domain includes any six or more consecutive nucleotides within nucleotides -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of MeCP2 intron 1 (e.g., any eight or more consecutive nucleic acids within nucleotides MeCP2 intron 1 , any ten or more consecutive nucleic acids within nucleotides -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of MeCP2 intron 1 , any 12 or more consecutive nucleic acids within nucleotides -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of MeCP2 intron 2, any 20 or more consecutive nucleic acids within nucleotides -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of MeCP2 intron 1 , any 30 or more consecutive nucleic acids within nucleotides -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of MeCP2 intron 1 , any 40 or more consecutive nucleic acids within nucleotides -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of MeCP2 intron 1 , any 50 or more consecutive nucleic acids within nucleotides -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of MeCP2 intron 1 , any 75 or more consecutive nucleic acids within nucleotides -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of MeCP2 intron 1 , any 100 or more consecutive nucleic acids within nucleotides -100 to 1500, -50 to 1450, 3950 to 4250, 4020- 4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of MeCP2 intron 1 , any 150 or more consecutive nucleic acids within nucleotides -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of MeCP2 intron 1 , any 200 or more consecutive nucleic acids within nucleotides -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of MeCP2 intron 1 , or any 250 or more consecutive nucleic acids within nucleotides -100 to 1500, -50 to 1450, 3950 to 4250, 4020-4170, -100 to 1300, -100 to 1000, -50 to 1300, or -50 to 1000 of MeCP2 intron 1 ) , wherein nucleotide position -1 is defined as the last nucleotide of MeCP2 exon 1 (position 114 of SEQ ID NO: 111) and position 1 is defined as the first nucleotide of MeCP2 intron 1 (position 115 of SEQ ID NO: 111), with negative numbers increasing proceeding in a 5’ direction from position -1 and positive numbers increasing proceeding in a 3’ direction from position 1.

[0139] In some instances, the binding site includes 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 nucleic acids 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, any ten or more consecutive nucleic acids 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 , any 12 or more consecutive nucleic acids 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 , any 20 or more consecutive nucleic acids 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 , any 30 or more consecutive nucleic acids 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 , any 40 or more consecutive nucleic acids 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 , any 50 or more consecutive nucleic acids 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 , any 75 or more consecutive nucleic acids 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 , any 100 or more consecutive nucleic acids 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, any 150 or more consecutive nucleic acids 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 , any 200 or more consecutive nucleic acids 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 , or any 250 or more consecutive nucleic acids 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) wherein nucleotide position -1 is defined as the last nucleotide of MeCP2 exon 1 (position 114 of SEQ ID NO: 111) and position 1 is defined as the first nucleotide of MeCP2 intron 1 (position 115 of SEQ ID NO: 111), with negative numbers increasing proceeding in a 5’ direction from position -1 and positive numbers increasing proceeding in a 3' direction from position 1.

[0140] In some embodiments, a binding domain has at least two non-overlapping sequences with at least 80% complementarity to the binding site.

[0141] In some embodiments, the binding domain includes a nucleic acid sequence having at least 80% identity (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) to any one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, or 47. In any binding domainsequences disclosed herein, U’s may be substituted for all the T’s in the sequence. Thus, for example, in some embodiments, the binding domain includes a nucleic acid sequence having at least 80% identity (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) to any one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, or 47 having U substituted for each T in the sequences.

[0142] In some embodiments, the binding domain is a DNA sequence having at least 80% identity (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) to 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 or consists of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, or 47.

[0143] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 24, wherein the binding domain comprises nucleotide 23C and / or 72C, numbering according to SEQ ID NO: 24.

[0144] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 25, wherein the binding domain comprises nucleotide 97C and / or 146C, numbering according to SEQ ID NO: 25.

[0145] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 27, wherein the binding domain comprises nucleotide 8C, 56C, 72C, or 85C, or any combination thereof, numbering according to SEQ ID NO: 27.

[0146] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 28, wherein the binding domain comprises nucleotide 133C, numbering according to SEQ ID NO: 28.

[0147] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 30, wherein the binding domain comprises nucleotide 4C, 71 C, or 101 C, or any combination thereof, numbering according to SEQ ID NO: 30.

[0148] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 32, wherein the binding domain comprises nucleotide 1 19A, numbering according to SEQ ID NO: 32.

[0149] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 33, wherein the binding domain comprises nucleotide 133C, numbering according to SEQ ID NO: 33.

[0150] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 47, wherein the binding domain comprises nucleotide 149C, numbering according to SEQ ID NO: 47.

[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 to any one of SEQ ID NOs: 116 to 137, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, N is a nucleotide other than G. In some embodiments, the binding domain comprises or consists of any one of SEQ ID NOs: 116 to 137, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, the nucleotide at any of the positions designated as N is non-complementary to the corresponding position of a native MeCP2 pre-mRNA.

[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 to SEQ ID NO: 116, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, the N at position 23 of SEQ ID NO: 116 is C. In some embodiments, the N at position 72 of SEQ ID NO:116 is C.

[0153] In some embodiments, the binding domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 117, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, the N at position 97 of SEQ ID NO: 117 is C. In some embodiments, the N at position 146 of SEQ ID NO:117 is C.

[0154] In some embodiments, the binding domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 118, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, the N at position 8 of SEQ ID NO: 118 is C. In some embodiments, the N at position 56 of SEQ ID NO:118 is C. In some embodiments, the N at position 82 of SEQ ID NO: 118 is C. In some embodiments, the N at position 85 of SEQ ID NO: 118 is C.

[0155] In some embodiments, the binding domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 119, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, the N at position 133 of SEQ ID NO: 119 is C.

[0156] In some embodiments, the binding domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 120, whereineach N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, the N at position 4 of SEQ ID NO: 120 is C. In some embodiments, the N at position 71 of SEQ ID NO: 120 is C. In some embodiments, the N at position 101 of SEQ ID NO: 120 is C.

[0157] In some embodiments, the binding domain comprises a nucleotide sequence having at least 50 consecutive nucleotides that have 100% sequence identity to a consecutive stretch of nucleotides 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 that have 100% sequence identity to a consecutive stretch of nucleotides 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 that have 100% sequence identity to a consecutive stretch of nucleotides of any one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, or 47.

[0158] Some embodiments of MeCP2 trans-splicing molecules described herein feature 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 2. In some embodiments, the binding domain is a nucleic acid sequence that is at least 80% complementary to (e.g., at least 85% complementary to, at least 90% complementary to, at least 91% complementary to, at least 92% complementary to, at least 93% complementary to, at least 94% complementary to, at least 95% complementary to, at least 96% complementary to, at least 97% complementary to, at least 98% complementary to, at least 99% complementary to, or 100% complementary to) a sequence of the target MeCP2 intron pre-mRNA (e.g., a target MeCP2 intron). An MeCP2 trans-splicing molecule comprising such a binding domain may suppress endogenous target cis-splicing while enhancing trans-splicing between the trans-splicing molecule and the target MeCP2 pre-mRNA (e.g., by creating a chimeric molecule having a portion of endogenous MeCP2 mRNA and a coding domain having one or more functional MeCP2 exons that encode wildtype MeCP2 amino acid sequences). In some embodiments involving trans-splicing molecule-encoding sequences (e.g., vectors encoding trans-splicing molecules), the binding domain-encoding sequence encodes a nucleic acid sequence that is at least 80% complementary to (e.g., at least 85% complementary to, at least 86% complementary to, at least 87% complementary to, at least 88% complementary to, at least 89% complementary to, at least 90% complementary to, at least 91% complementary to, at least 92% complementary to, at least 93% complementary to, at least 94% complementary to, at least 95% complementary to, at least 96% complementary to, at least 97% complementary to, at least 98% complementary to, at least 99% complementary to, or 100% complementary to) a sequence of the target MeCP2 intron pre-mRNA.

[0159] In some embodiments, the present disclosure provides trans-splicing molecules (or vectors encoding same) that bind MeCP2 at intron 2, e.g., wherein the nucleic acid trans-splicing molecule is configured to trans-splice a coding domain to endogenous MeCP2 exon 1 or exon 2. In some embodiments, trans-splicing molecules described herein include those in which the binding domain binds a binding site having any one or more (e.g., six or more, eight or more, ten or more, or twelve or more, twenty-five or more, fifty or more, 75 or more, 100 or more, 150 or more, 200 or more, or 250 or more) of 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. In some embodiments, trans-splicing molecules described herein include those in which the binding domain comprises a length of about 50-300 nucleotides; about 50-250 nucleotides; about 50- 200 nucleotides; about 50-150 nucleotides; about 50-100 nucleotides; about 75-300 nucleotides; about 75-250 nucleotides; about 75-200 nucleotides; about 75-150 nucleotides; about 100-300 nucleotides; about 100-250 nucleotides; about 100-200 nucleotides; about 100- 150 nucleotides; about 125-300 nucleotides; about 125-250 nucleotides; about 125-200 nucleotides; about 125-150 nucleotides; about 50 nucleotides; about 100 nucleotides; about 125 nucleotides; about 150 nucleotides; about 200 nucleotides; or about 250 nucleotides. In some embodiments, trans-splicing molecules described herein include those in which the binding domain comprises 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; 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 ranging from 100 to 200 nucleotides in length and binds MeCP2 at a binding site within nucleotides 1-450 of SEQ ID NO: 2, wherein the binding domain comprises 50 consecutive nucleotides that are 100% complementary to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and binds MeCP2 at a binding site within nucleotides 1-450 of SEQ ID NO: 2, wherein the binding domain comprises 50 consecutive nucleotides that are 90% to 100% complementary to 50 consecutivenucleotides of the binding site. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and binds MeCP2 at a binding site within nucleotides 1-450 of SEQ ID NO: 2, wherein the binding domain comprises 75 consecutive nucleotides that are 90% to 100% complementary to 75 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 100% complementary to a binding site comprising at least 100 consecutive nucleotides within nucleotides 1-450 of SEQ ID NO: 2. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 90% to 100% complementary to a binding site comprising at least 100 consecutive nucleotides within nucleotides 1-450 of SEQ ID NO:2. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and binds MeCP2 at a binding site within nucleotides 99-248 of SEQ ID NO: 2, wherein the binding domain comprises 50 consecutive nucleotides that are 100% complementary to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and binds MeCP2 at a binding site within nucleotides 99-248 of SEQ ID NO: 2, wherein the binding domain comprises 50 consecutive nucleotides that are 90% to 100% complementary to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 100% complementary to a binding site comprising at least 100 consecutive nucleotides within nucleotides 99-248 of SEQ ID NO: 2. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 90% to 100% complementary to a binding site comprising at least 100 consecutive nucleotides within nucleotides 99-248 of SEQ ID NO: 2. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and binds MeCP2 at a binding site within nucleotides 149-300, 199-350, 249-400, or 299-450 of SEQ ID NO: 2, wherein the binding domain comprises 50 consecutive nucleotides that are 100% complementary to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and binds MeCP2 at a binding site within nucleotides 149-300, 199-350, 249-400, or 299-450 of SEQ ID NO: 2, wherein the binding domain comprises 50 consecutive nucleotides that are 90% to 100% complementary to 50 consecutive nucleotides of the binding site. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is 100% complementary to a binding site comprising at least 100 consecutive nucleotides within nucleotides 149-300, 199-350, 249-400, or 299-450 of SEQ ID NO: 2. In some embodiments, the binding domain comprises a sequence ranging from 100 to 200 nucleotides in length and is90% 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.

[0160] In some instances, the binding domain includes any six or more consecutive nucleotides 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 (e.g., any eight or more consecutive nucleic acids within nucleotides MeCP2 intron 2, any ten 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, any 12 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, any 20 or more consecutive nucleic acids within nucleotides 1-300, 50-300, 50-250, 75-300, 75-250, SO- SOO, 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, any 30 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, 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 of MeCP2 intron 2, any 50 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, any 75 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, any 100 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, 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 MeCP2 intron 2, any 200 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, or any 250 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).

[0161] In some instances, the binding site includes any six or more consecutive nucleotides 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 of MeCP2 intron 2 (e.g., any eight or more consecutive nucleic acids within nucleotides 90-300, 90-250, 95-300, 95-250, or 99-248 of MeCP2 intron 2, any ten 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 of MeCP2 intron 2, any 12 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 of MeCP2 intron 2, any 20 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 of MeCP2 intron 2, any 30 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 of MeCP2 intron 2, any 40 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 of MeCP2 intron 2, any 50 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 of MeCP2 intron 2, any 75 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 of MeCP2 intron 2, any 100 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 of MeCP2 intron 2, any 150 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 of MeCP2 intron 2, any 200 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 of MeCP2 intron 2, or any 250 or more consecutive nucleic acids withinnucleotides 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).

[0162] In some embodiments, a binding domain has at least two non-overlapping sequences with at least 80% complementarity to the binding site.In some embodiments, the binding domain includes a nucleic acid sequence having at least 80% identity (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) to SEQ ID NO: 55, 56, 57, 58, 59, 88, 89, 90, 91 , or 92, including versions of SEQ ID NO: 55, 56, 57, 58, 59, 88, 89, 90, 91 , or 92 with U substituted for each T. 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.

[0163] In some embodiments, the binding domain comprises a nucleotide sequence having at least 50 consecutive nucleotides that have 100% sequence identity to a consecutive stretch of nucleotides 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 that have 100% sequence identity to a consecutive stretch of nucleotides 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 that have 100% sequence identity to a consecutive stretch of nucleotides of any one of SEQ ID NOs: 55, 56, 57, 58, 59, 88, 89, 90, 91 , or 92.

[0164] In some embodiments, the binding domain comprises one or more nucleotide substitutions relative to an endogenous MeCP2 mRNA sequence, wherein the one or more nucleotide substitutions disrupt a cryptic splice site within the binding domain sequence. In some embodiments, the binding domain comprises a nucleic acid sequence having one or more of the following changes 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 (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) to SEQ ID NO: 88,wherein the binding domain comprises the following nucleotides, numbering according to SEQ ID NO: 88: 25C, 59C, 63C, 99C, or 120C.

[0165] In some embodiments, the binding domain is a DNA sequence having at least 80% identity (e.g., at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at leanst 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) to SEQ ID NO: 55.

[0166] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 90, wherein the binding domain comprises nucleotide 84A and / or 106C, numbering according to SEQ ID NO: 90.

[0167] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 91 , wherein the binding domain comprises nucleotide 136C and / or 138C, numbering according to SEQ ID NO: 91.

[0168] In some embodiments, the binding domain is a DNA sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 92, wherein the binding domain comprises nucleotide 51 C or any combination thereof, numbering according to SEQ ID NO: 92.

[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 to any one of SEQ ID NOs: 138 to 152, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, N is a nucleotide other than G. In some embodiments, the binding domain comprises or consists of any one of SEQ ID NOs: 139 to 152, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, the nucleotide at any of the positions designated as N is non-complementary to the corresponding position of a native MeCP2 pre-mRNA.

[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 to SEQ ID NO: 138, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, the N at position 25 of SEQ ID NO: 138 is C. In some embodiments, the N at position 59 of SEQ ID NO:138 is C. In some embodiments, the N at position 63 of SEQ ID NO: 138 is C. In some embodiments, the N at position 99 of SEQ ID NO: 138 is C. In some embodiments, the N at position 120 of SEQ ID NO: 138 is C.

[0171] In some embodiments, the binding domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 139, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, the N at position 84 of SEQ ID NO: 139 is A. In some embodiments, the N at position 106 of SEQ ID NO:139 is C.

[0172] In some embodiments, the binding domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 140, wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, the N at position 136 of SEQ ID NO: 140 is C. In some embodiments, the N at position 138 of SEQ ID NO: 140 is C. In some embodiments, the N at position 82 of SEQ ID NO: 1 18 is C. In some embodiments, the N at position 85 of SEQ ID NO: 1 18 is C.

[0173] In some embodiments, the binding domain comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 141 , wherein each N is independently A, T (or U in the case of RNA), C, or G. In some embodiments, the N at position 51 of SEQ ID NO: 141 is C.

[0174] MeCP2 trans-splicing molecules described herein feature 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 ID NO: 4). In some embodiments, the binding domain is a nucleic acid sequence that is at least 80% complementary to (e.g., at least 85% complementary to, at least 90% complementary to, at least 91 % complementary to, at least 92% complementary to, at least 93% complementary to, at least 94% complementary to, at least 95% complementary to, at least 96% complementary to, at least 97% complementary to, at least 98% complementary to, at least 99% complementary to, or 100% complementary to) a sequence of the target MeCP2 intron pre-mRNA (e.g., a target MeCP2 intron). An MeCP2 trans-splicing molecule comprising such a binding domain may suppress endogenous target cis-splicing while enhancing trans- splicing between the trans-splicing molecule and the target MeCP2 pre-mRNA (e.g., by creating a chimeric molecule having a portion of endogenous MeCP2 mRNA and a coding domain having one or more functional MeCP2 exons that encode wildtype MeCP2 amino acid sequences). In some embodiments involving trans-splicing molecule-encoding sequences (e.g., vectors encoding trans-splicing molecules), the binding domain-encoding sequence encodes a nucleic acid sequence that is at least 80% complementary to (e.g., at least 85% complementary to, at least 86% complementary to, at least 87% complementary to, at least 88% complementary to, at least 89% complementary to, at least 90% complementary to, at least 91 % complementary to, at least 92% complementary to, at least 93% complementary to, at least 94% complementary to, at least 95% complementary to, at least 96% complementary to, at least 97% complementary to, at least 98% complementary to, at least 99% complementary to, or 100% complementary to) a sequence of the target MeCP2 intron pre-mRNA.

[0175] In some embodiments, the binding domain is a DNA sequence having at least 80% identity (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) to any one of SEQ ID NOs: 67, 68, 69, 70, 71, 72, 73, 74, or 75.

[0176] As detailed herein, the first stage of MeCP2 Exon Editor design began with the screening and selection of a highly efficient BD sequence, which is complementary to the targeted pre-mRNA intron. As shown herein, BDs that target intron 1 of MeCP2 exhibited the highest levels of trans-splicing and therefore, have been identified as effective elements of exemplary MeCP2-targeting Exon Editors for treatment of the Rett Syndrome patient population. See FIGs. 6-9.

[0177] Any of the binding domains disclosed herein may comprise one or more nucleotide changes (e.g., a nucleotide substitution) relative to a native pre-mRNA sequence, wherein the one or more nucleotide changes disrupt a cryptic splice site within the binding domain sequence. A cryptic splice site-mitigating nucleotide change may cause a lack of complementarity between the binding domain and the binding site on the pre-mRNA at the position of the nucleotide change. However, despite a lack of complementarity at the position(s) of the one or more nucleotide changes, the binding domain may nevertheless bind to the binding site and support effective trans-splicing. This is shown in FIG. 19, in which an exon editor comprising a 150-nucleotide binding domain that has five cryptic splice site-mitigating nucleic acid substitutions (BD sequence: SEQ ID NO: 88) provided effective trans-splicing into MeCP2 intron 2.

[0178] In some embodiments, an 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, an RNA exon editor of the present disclosure comprises a binding domain that specifically binds to any one of the binding sites disclosed herein.

[0179] In some embodiments, a binding domain of an 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, such a binding domain has 100% complementarity to the binding site.4. Hemi-lntrons

[0180] In some embodiments, a hemi-intron comprises a 3’ splice site. In some embodiments, a hemi-intron comprises a polypyrimidine tract (pPy) tract, a branch point (BP), and / or a 3’ splice site to mediate trans-splicing. In some embodiments, a hemi-intron has a single splice site, which denotes that the splice site is designed for preferential trans-splicing, but not cis-splicing, due to the lack of a corresponding splice site.

[0181] Alternative hemi-introns may be selected by one of skill in the art according to known methods and principles. In some embodiments, the 3’ splice site consensus sequence is thenucleic acid sequence YAG / [wherein Y is a pyrimidine (cytosine or thymidine);denotes the location of the 3’ splice site.; e.g., TAG]. In some embodiments, an RNA exon editor comprises, in a 5’ to 3’ direction, the sequence TAG immediately 5’ to the first nucleotide of the coding domain sequence (e.g., the coding domain sequence of MeCP2 exon 1, exon 2, or exon 3, or exon 4). In some embodiments, the endogenous splice sites that correspond to the exon and intron proximal to the splice site can be employed to maintain any splicing regulatory signals. Thus, in some embodiments, a hemi-intron may comprise the sequence of the 3’-most 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides of MeCP2 intron 1 , intron 2, or intron 3. In some embodiments, the hemi-intron comprises a BP and a pPy. In some embodiments, the hemi-intron comprises a BP, a pPy, and a 3’ splice site (splice acceptor). In some embodiments, a hemi-intron comprises, operably linked in a 5’ to 3’ direction, a BP, a pPy, and a 3’ splice site. In some embodiments, the BP has a sequence YUNAY, where Y is a pyrimidine (C or U) and N is any nucleotide; the A is the site of branch formation. In some embodiments, the BP has the sequence YNYTRAC where Y is a pyrimidine (C or U), N is any nucleotide, and R is a purine; the A is the site of branch formation. In some embodiments, the BP has the sequence YNAH, wherein Y is a pyrimidine, N represents any nucleotide, H is adenine, cytosine, or thymine; the A is the site of branch formation. In some embodiments, the BP is at least 10 nucleotides upstream of the 3’ splice site. In some embodiments, the BP is IQ- 12, 10-15, 10-17, or 10-20 nucleotides upstream of the 3’ splice site. In some embodiments, the BP is separated from the 3’ splice site by a pPy of 9, 10, 11 , 12, 13, 14, 15, or 16 nucleotides (i.e., the BP and 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%, 90%, of the nucleotides between the BP sequence and the 3’ splice site (i.e., the pPy) are pyrimidines. In some embodiments, all of the nucleotides between the BP sequence and the 3’ splice site (i.e., the pPy) are pyrimidines.

[0182] In some embodiments, a suitable 3’ splice site comprises TAG. In some embodiments, a hemi-intron comprises the nucleotide sequence set forth 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 to any one of SEQ ID NO: 17.

[0183] A hemi-intron can be operably linked 5’ to a coding domain sequence (CDS) (e.g., directly connected to a CDS or have intervening sequences connecting the 3’ end of the hemi- intron and the 5’ end of the CDS).5. 3’ Untranslated Region

[0184] In some embodiments, the nucleic acid trans-splicing molecule includes a 3’ untranslated region. In some embodiments, the 3’ untranslated region comprises, consistsessentially of, or consists of a heterologous 3’ untranslated region. In some embodiments, the 3’ UTR is a RDHI pA 3’ UTR, which is a synthetic 3' UTR comprising 110 bp of the highly conserved MeCP2 distal polyadenylation signal and an upstream miRNA-binding panel containing sites for three additional miRNAs endogenous to the MeCP2 3' UTR: miR-19, miR-22, and miR-132. A DNA sequence encoding RDHIpA 3’ UTR comprises SEQ ID NO: 19. In some embodiments, the 3’ UTR is a mWPRE 3’ UTR. A DNA sequence encoding 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, consists essentially of, or consists of SEQ ID NO: 87 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 87. In some embodiments, the 3’ untranslated region comprises, consists essentially of, or consists of either of SEQ ID NOs: 19 or 20. In some embodiments, the 3’ untranslated region comprises a sequence having at least 80% sequence identity (e.g., at least 85% sequence identity, at least 86% sequence identity, at least 87% sequence identity, at least88% sequence identity, at least 89% sequence identity, at least 90% sequence identity, at least91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to either of SEQ ID NOs: 19 or 20.

[0185] In some embodiments, a 3’ untranslated region can be operably linked 3’ to a coding domain (e.g., directly connected to a coding domain or have intervening sequences connecting the 5’ end of the 3’ untranslated region and the 3’ end of the coding domain).

[0186] Further to the above, nucleic acid trans-splicing molecules may include a hemi-intron domain at one or more positions within the molecule. In some embodiments, the hemi-intron domain is operatively linked 3’ to the binding domain (e.g., directly connected to the binding domain). The hemi-intron domain may be any suitable size. In some embodiments, the hemi- intron domain is longer than 20 nucleotides in length (e.g., between 20 and 100 nucleotides in length or between 20 and 85 nucleotides in length). In some instances, the hemi-intron domain comprises, consists essentially of, or consists of a nucleic acid sequence having at least 80% identity (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) with SEQ ID NO: 17.

[0187] The inventors discovered that a truncated variant of the WPRE 3’ UTR sequence can be used to enhance on-target splicing of RNA exon editors. A comparison was made between two otherwise 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 in which 203 internal nucleotides were deleted (“WPRE3”; SEQ ID NO: 87). 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 18-19% on-target splicing, whereas the exon editor with the full- length WPRE 3’ UTR had 13-14% on-target splicing. It was surprising that a truncated WPRE 3’ UTR sequence that was 42.6% of the length of the full-length WPRE sequence worked as well as or better than the full-length sequence.

[0188] Some embodiments of RNA exon editors comprise the following components, operably linked in a 5’ to 3' direction: a binding domain configured to bind a binding site within a target region of a pre-mRNA of a 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 lacks at least 50, 75, 100, 125, 150, 175, or 200 consecutive nucleotides of SEQ ID NO: 20. In some embodiments, the truncated WPRE 3’ UTR sequence lacks 50-200, 50-150, 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 to 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, operably linked 3’ to the truncated WPRE 3’ UTR sequence, one or more of a poly A sequence, a triple helix terminator sequence, or a 3x UBS sequence, or any combination thereof.6. 3’ Transcription Terminator Domains

[0189] In some instances, the trans-splicing molecule includes a 3’ transcription terminator domain. In some embodiments, such 3’ transcription terminator domains form a triple helical structure that effectively caps the 3’ end of the trans-splicing molecule. In some instances, the 3’ transcription terminator domain is from the human long non-coding RNA MALAT1 (e.g., wildtype MALAT1). In some embodiments, the 3’ transcription terminator domain includes a tRNA-like domain. 3’ transcription terminator domains useful as part of the present MeCP2 trans-splicing molecules are described in International Patent Publication No. WO 2020 / 214973, which is herein incorporated by reference in its entirety. For example, in some embodiments, the region of an RNA exon editor operably linked to the 3’ end of the CDS includes a terminator domainthat comprises, consists essentially of, or consists of a MALATI+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, exemplary RNA exon editors described herein include those that comprise binding domains that bind to intron 1 of MeCP2, wherein such exemplary intron 1- binding RNA exon editors 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, exemplary RNA exon editors described herein include those that comprise binding domains that bind to intron 2 of MeCP2, wherein such exemplary intron 2- binding RNA exon editors may comprise a binding domain, a hemi-intron, the coding sequence (e.g., SEQ ID NO: 76), and a 3’ transcription terminator.

[0192] In some embodiments, exemplary RNA exon editors described herein include those that comprise binding domains that bind to intron 3 of MeCP2, wherein such exemplary intron 3- binding RNA exon editors may comprise a binding domain, a hemi-intron, the coding sequence (e.g., SEQ ID NO: 14), and a 3’ transcription terminator.

[0193] In some embodiments, binding of a trans-splicing molecule to the target pre-mRNA is mediated by percent complementarity (i.e., based on base-pairing characteristics of nucleic acids), triple helix formation, or protein-nucleic acid interaction (as described in documents cited herein) or any combination thereof. In one embodiment, the nucleic acid trans-splicing molecule includes DNA, RNA, or DNA / RNA hybrid molecules, wherein the DNA or RNA is either single or double stranded. Also included herein are RNAs or DNAs, which can hybridize to one of the aforementioned RNAs or DNAs, preferably under stringent conditions, for example, at 60°C in 2.5x SSC buffer and several washes at 37°C at a lower buffer concentration, for example, 0.5x SSC buffer. When trans-splicing molecules are synthesized in vitro, such trans-splicing molecules can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization to the target mRNA, transport into the cell, stability in the cells to enzymatic cleavage, etc. For example, modification of a trans- splicing molecule to reduce the overall charge can enhance the cellular uptake of the molecule. In addition, modifications can be made to reduce susceptibility to nuclease or chemical degradation. The nucleic acid molecules may be synthesized in such a way as to be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

[0194] Various other well-known modifications to the nucleic acid molecules can be introduced as a means of increasing intracellular stability and half-life (see also above for oligonucleotides). Possible modifications are known to the art. Modifications, which may be made to the structure of synthetic trans-splicing molecules include backbone modifications.7. Linkers

[0195] In some embodiments, trans-splicing molecules of the present disclosure comprise nucleotide linkers between one or more of the components thereof. 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.8. Cell Line Assays

[0196] In some instances, trans-splicing molecules described herein are tested in cultured cell lines. To screen, select, and improve functionality of RNA exon editors, cultured cell lines may be acquired or engineered to express the targeted MeCP2 pre-mRNA at a sufficient level.9. Animal Models

[0197] Exemplary exon editors described herein target human intron 1 or intron 2. To test their efficacy in vivo, the present inventors will use a mouse with a human MeCP2 gene knocked into an autosome: FVB / N-Tg(MECP2*R270X / GFP)AHzo / J. The mouse mutant MeCP2(tm1.1) generated recapitulates many of the phenotypes seen in humans and is a standard model used in the field. Accordingly, this mouse model will be used to evaluate the ability of RNA exon editors described herein to rescue MeCP2 mutant defects in an in vivo context.

[0198] Additional details regarding FVB / N-Tg(MECP2*R270X / GFP)AHzo / J are available via The Jackson Laboratory website. Briefly, as described therein, MeCP2-R270X transgenic mice express human MeCP2 that is truncated at amino acid R270 and tagged with EGFP at the C terminal. Expression of the transgene, as determined using immunohistochemistry, recapitulates that of the human MECP2 gene in the cortex, hippocampus, cerebellum, hypothalamus, and brainstem. MeCP2 protein localizes entirely in the nucleus and concentrates at heterochromatic foci, like that of the endogenous protein. The MeCP2-R270X transgenic mice demonstrate no overt phenotype until combined with a mouse Mecp2 knock-out allele. When combined with a mouse Mecp2 knock-out (Stock No. 003890), median lifespan of the mice is 85 days, slightly longer than mice carrying the Mecp2 knock-out alone (76 days).

[0199] In some embodiments, AAV9 comprising an exemplary RNA exon editor described herein is administered via intracerebroventricular injection to P0-P1 neonate FVB / N- Tg(MECP2*R270X / GFP)AHzo / J mice.10. RNA Exon Editor Screening Platform

[0200] As described herein, 3’ RNA exon editors comprise several functional sequence elements, such as a binding domain (BD) for pre-mRNA targeting and a hemi-intron that provides a splice acceptor site. In some embodiments, a hemi-intron comprises a branchpoint and a polypyrimidine tract in addition to a 3’ splice acceptor site. When engineering an RNA exon editor for a given gene target, a variety of sequence options for each of these elements is tested for its ability to contribute to high trans-splicing (TS) efficiency. Such testing may be accomplished via cloning and transfecting individual RNA exon editor variants and analyzing efficiency via RT-qPCR / ddPCR and Western blot as are described below.11. RNA exon editor screening in individual format:

[0201] This approach can be applied to test a small-scale number of variable elements within an RNA exon editor sequence prior to initiating a library-based multiplexed screen, to validate the performance of RNA exon editors identified in a multiplexed screen, and / or to improve performance of lead candidates. Evaluation of TS efficiency occurs at the RNA and protein levels.

[0202] At the RNA level, TS activity is evaluated via isolation of total RNA from cells followed by reverse transcription and real-time quantitative PCR (RT-qPCR) measuring RNA copy numbers of, e.g., the following targets: RNF20 (housekeeping gene for normalization); Native (MeCP2) mRNA; Exon Editor RNA; On-target, exon-edited RNA (ONT), which is the product of positive TS; ONT+Exon Editor+OFT (off-target) - a single assay that captures all three of these targets. OFT represents incorrect RNA molecules to which the RNA exon editor may transsplice.

[0203] ONT TS efficiency, also referred to as percent replacement, represents the portion of the total MeCP2 mRNA population that has undergone successful TS, and is calculated via the following equation: % ONT TS = 100*(ONT copy number / (ONT copy number + Native copy number)).

[0204] RNA exon editor TS efficiency is the portion of the RNA exon editor transcript population that has been correctly trans-spliced into the MeCP2 RNA and is calculated via the following equation: % Exon Editor TS = 100*(ONT copy number / (ONT copy number + Exon Editor copy number + OFT copy number)).

[0205] At the protein level, TS activity is measured via Western blot analysis applied to protein extracted from cell or tissue samples. Beta-actin, which is a cytoskeletal protein, can be used as a loading control. The level of ONT protein is measured using MeCP2-specific antibodies (Ab). For those constructs that include a tag, e.g., a Myc tag at the C-terminus, an Abspecific for the tag (e.g., a Myc-specific Ab) can be used to probe Western blots to assess ONT protein levels.12. Exemplary Combinations

[0206] Some embodiments of RNA exon editors include combinations of binding domains, hemi-introns, and 3’ untranslated regions, and 3’ transcriptional terminator domains disclosed above. In some embodiments, the RNA exon editor comprises a binding domain selected from 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 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 NOs: 7-14, 76, 85, or 86, or combinations or functional variants 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, a 3’ UTR of SEQ ID NO: 19, 20, or 87 or a MALAT1 terminator of SEQ ID NO: 22 or 77. 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 splice site. 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 hemi-intron. 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 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 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 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 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 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 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 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 that comprises one or more of SEQ ID NOs: 7-14, 76, 85, or 86.

[0207] Exemplary configurations of RNA exon editors of the present disclosure include the following (in 5’ to 3’ direction):Exon Editor A: [BD]-[hemi-intron]-[CDS];Exon Editor B: [BD]-[hemi-intron]-[CDS]-[3’ UTR];Exon Editor C: [BD]-[hemi-intron]-[CDS]-[terminator];Exon Editor D: [BD]-[hemi-intron]-[CDS]-[3' UTR]-[terminator];Exon Editor E: [BD]-[BP]-[pPy]-[3' splice site]-[CDS];Exon Editor F: [BD]-[BP]-[pPy]-[3’ splice site]-[CDS]-[3’ UTR];Exon Editor G: [BD]-[BP]-[pPy]-[3’ splice site]-[CDS]-[terminator]; andExon Editor H: [BD]-[BP]-[pPy]-[3’ splice site]-[CDS]-[3’ UTR]-[terminator]; wherein “BD” represents any of the BDs disclosed in Section VI. C.3; “hemi-intron” represents any of the hemi-introns disclosed in Section VI.C.4; “CDS” represents any of the CDSs disclosed in Section VI.C.2; “3’ UTR” represents any of the 3’ UTRs disclosed in Section VI. C.5; “terminator” represents any of the terminators disclosed in Section VLC.6; “BP” represents any of the branch point sequences disclosed in Section VI.C.4; “pPy” represents any of the polypyrimidine tract sequences disclosed in Section VI.C.4; “3’ splice site” represents the nucleotide sequence YAG, wherein in Y is a pyrimidine; andrepresents a phosphodiester bond or a linker. In some embodiments, an exon editor construct encoding any of Exon Editors 1 to 8 comprises a promoter sequence operably linked upstream of the BD of any of Exon Editors 1 to 8.

[0208] In some embodiments, an RNA exon editor of the present disclosure comprises, operably linked to the 3’ end of the coding domain sequence, in a 5’ to 3’ direction: a WPRE sequence having at least 90% identity to SEQ ID NO: 87, a MALAT1 terminator anti-Mut1 variant having at least 90% identity to SEQ ID NO: 22, and a 3xUBS sequence having at least 90% identity to SEQ ID NO: 79. In some embodiments, an RNA exon editor of the present disclosure comprises, operably linked to the 3’ end of the coding domain sequence, in a 5’ to 3' direction: the WPRE sequence of SEQ ID NO: 87, the MALAT 1 terminator anti-Mut1 variant of SEQ ID NO: 22, and the 3xUBS sequence of SEQ ID NO: 79.

[0209] Additional exemplary combinations of RNA exon editor components are set forth in Enumerated embodiments 1 to 279 below.D. Vectors

[0210] Trans-splicing molecules can be delivered to target cells of an individual using various techniques, e.g., using recombinant adeno-associated virus (AAV) vectors or other vector modalities, such as non-viral vectors. Thus, provided herein are vectors comprising / encoding trans-splicing molecules (e.g., viral or non-viral vectors comprising / encoding trans-splicingmolecules, e.g., DNA vectors comprising / encoding trans-splicing molecules). Any suitable nucleic acid vector may be used in conjunction with the present compositions and methods to design and assemble the components of the trans-splicing molecule and a recombinant AAV. In one embodiment, the vector is a recombinant AAV carrying the trans-splicing molecule driven by a promoter that expresses a trans-splicing molecule in selected cells of an individual.Methods for assembly of the recombinant vectors are known in the art. See, e.g., Ausubel et aL, Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A. et al., Nat. Medic, 2001, 7(l):33-40; and Walther W. and Stein U„ Drugs 2000, 60(2):249-71.

[0211] In certain embodiments described herein, the trans-splicing molecule is delivered to the selected cells, e.g., neuronal cells, in need of treatment by means of an AAV vector. A variety of naturally occurring serotypes of AAV are available. Many natural variants in the AAV capsid exist, allowing identification and use of an AAV with properties specifically suited for neuronal cells. Artificial AAV vectors may be engineered by conventional molecular biology techniques, making it possible to optimize these particles for cell specific delivery of the trans- splicing molecule nucleic acid sequences, for minimizing immunogenicity, for tuning stability and particle lifetime, for efficient degradation, for accurate delivery to the nucleus, etc. Such artificial capsids may, e.g., be generated by any suitable technique, using a selected AAV sequence (e.g., a fragment of a vp1 capsid protein) in combination with heterologous sequences which may be obtained from a different selected AAV, non-contiguous portions of the same AAV, from a non-AAV viral source, or from a non-viral source. An artificial AAV may be, without limitation, a pseudotyped AAV, chimeric AAV capsid, recombinant AAV capsid, or “humanized” AAV capsid. Pseudotyped vectors, wherein the capsid of one AAV is replaced with a heterologous capsid protein, are useful for delivering trans-splicing molecules described herein.

[0212] The expression of trans-splicing molecules described herein can be achieved in the selected cells through delivery by recombinantly engineered AAVs or artificial AAVs that contain sequences comprising / encoding the desired trans-splicing molecule. The use of AAVs is a common mode of exogenous delivery of DNA as it is relatively non-toxic, provides efficient gene transfer, and can be optimized for specific purposes. Among the well-characterized serotypes of AAVs isolated from human or non-human primates, human serotype 2 has been widely used for efficient gene transfer experiments in different target tissues and animal models.

[0213] In some embodiments, the AAV is AAV1 or a variant thereof (e.g., SEQ ID NO: 6 or 64 of US20030138772 or SEQ ID NO: 11 or 27 of US20150159173), AAV2 or a variant thereof (e.g., SEQ ID NO: 7 or 70 of US20030138772, SEQ ID NO: 7 or 23 of US20150159173, or SEQ ID NO: 7 of US20150159173), AAV2G9 or a variant thereof, AAV3 or a variant thereof (e.g., SEQ ID NO: 8 or 71 of US20030138772), AAV3a or a variant thereof, AAV3b or a variant thereof (e.g.,SEQ ID NO: 1 and 10 of U.S. Pat. No. 6,156,303), AAV3-3 or a variant thereof (e.g., SEQ ID NO: 200 and 217 of W02005033321), AAV4 or a variant thereof (e.g., SEQ ID NO: 63 of US20030138772), AAV4-4 or a variant thereof (e.g., SEQ ID NO: 201 or 218 of W02005033321), AAV5 or a variant thereof (e.g., SEQ ID NO: 114 of US20030138772), AAV6 or a variant thereof (e.g., SEQ ID NO: 65 of US20030138772), AAV6.1 or a variant thereof (e.g., SEQ ID NO: 29 of US20150159173), AAV6.2 or a variant thereof, AAV6.1.2 or a variant thereof, AAV7 or a variant thereof (e.g., SEQ ID NO: 1-3 of US20030138772), AAV7.2 or a variant thereof, AAV8 or a variant thereof (e.g., SEQ ID NO: 4 and 95 of US20030138772 or AAV8(b) (having the amino acid sequence of Pro-Glu-Arg-Thr-Ala-Met-Ser-Leu-Pro at amino acid positions 587-595 as compared to wildtype AAV8, as described in U.S. Patent No. 9,567,376, which is incorporated herein by reference in its entirety), AAV9 or a variant thereof (e.g., SEQ ID NO: 5 and 100 of US20030138772), AAV9.9 or a variant thereof, AAV9.11 or a variant thereof, AAV9.13 or a variant thereof, AAV9.16 or a variant thereof, AAV9.24 or a variant thereof, AAV9.45 or a variant thereof, AAV9.47 or a variant thereof, AAV9.61 or a variant thereof, AAV9.68 or a variant thereof, AAV9.84 or a variant thereof (see, e.g., N. Pulicherla et al. Molecular Therapy 19(6):1070-1078 (2011), herein incorporated by reference in its entirety), AAV10 or a variant thereof (e.g., SEQ ID NO: 117 of US20030138772), AAV11 or a variant thereof (e.g., SEQ ID NO: 118 of US20030138772), AAV12 or a variant thereof (e.g., SEQ ID 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, AAV42-1 b or a variant thereof, AAV42-2 or a variant thereof, AAV42-3a or a variant thereof, AAV42-3b or a variant thereof, AAV42-4 or a variant thereof, AAV42-5a or a variant thereof, AAV42-5b or a variant thereof, AAV42-6b or a variant thereof, AAV42-8 or a variant thereof, AAV42-10 or a variant thereof, AAV42-11 or a variant thereof, AAV42-12 or a variant thereof, AAV42-13 or a variant thereof, AAV42-15 or a variant thereof, AAV42-aa or a variant thereof, AAV43-1 or a variant thereof, AAV43-12 or a variant thereof, AAV43-20 or a variant thereof, AAV43-21 or a variant thereof, AAV43-23 or a variant thereof, AAV43-25 or a variant thereof, AAV43-5 or a variant thereof, AAV44.1 or a variant thereof, AAV44.2 or a variant thereof, AAV44.5 or a variant thereof, AAV223.1 or a variant thereof, AAV223.2 or a variant thereof, AAV223.4 or a variant thereof, AAV223.5 or a variant thereof, AAV223.6 or a variant thereof, AAV223.7 or a variant thereof, AAV1-7 / rh.48 or a variant thereof, AAV1-8 / rh.49 or a variant thereof, AAV2-15 / rh.62 or a variant thereof, AAV2-3 / rh.61 or a variant thereof, AAV2-4 / rh.5O or a variant thereof, AAV2-5 / rh.51 or a variant thereof, AAV3.1 / hu.6 or a variant thereof, AAV3.1 / hu.9 or a variant thereof, AAV3-9 / rh.52 or a variant thereof, AAV3-11 / rh.53 or a variant thereof, AAV4-8 / rh.64 or a variant thereof, AAV4-9 / rh.54 or a variant thereof (e.g., SEQ ID NO: 116 of WQ2005033321), AAV4-19 / rh.55 or a variant thereof (e.g., SEQ ID NO: 117 of WQ2005033321), AAV5-3 / rh.57 or a variantthereof, AAV5-22 / rh.58 or a variant thereof, AAV7.3 / hu.7 or a variant thereof, AAV16.8 / hu.1 O or a variant thereof, AAV16.12 / hu.11 or a variant thereof, AAV29.3 / bb.1 or a variant thereof, AAV29.5 / bb.2 or a variant thereof, AAV106.1 / hu.37 or a variant thereof, AAV1 14.3 / hu.4O or a variant thereof, AAV127.2 / hu.41 or a variant thereof, AAV127.5 / hu.42 or a variant thereof, AAV128.3 / hu.44 or a variant thereof, AAV130.4 / hu.48 or a variant thereof, AAV145.1 / hu.53 or a variant thereof, AAV145.5 / hu.54 or a variant thereof, AAV145.6 / hu.55 or a variant thereof, AAV161.1 O / hu.6O or a variant thereof, AAV161.6 / hu.61 or a variant thereof, AAV33.12 / hu.17 or a variant thereof, AAV33.4 / hu.15 or a variant thereof, AAV33.8 / hu.16 or a variant thereof, AAV52 / hu.19 or a variant thereof, AAV52.1 / hu.2O or a variant thereof, AAV58.2 / hu.25 or a variant thereof, AAVA3.3 or a variant thereof, AAVA3.4 or a variant thereof, AAV A3.5 or a variant thereof, AAVA3.7 or a variant thereof, AAVC1 or a variant thereof, AAVC2 or a variant thereof, AAVC5 or a variant thereof, AAV-DJ or a variant thereof (e.g., SEQ ID NO: 2 or 3 ofUS20140359799), 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-1 O / rh.4O 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 ofUS20150159173), AAVCh.5R1 or a variant thereof, AAVcy.2 or a variant thereof, AAVcy.3 or a variant thereof, AAVcy.4 or a variant thereof, AAVcy.5 or a variant thereof (e.g., SEQ ID NO: 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: 144 of WQ2005033321), AAVhu.2 or a variant thereof (e.g., SEQ ID NO: 143 of WQ2005033321), AAVhu.3 or a variant thereof (e.g., SEQ ID NO: 145 of W02005033321), AAVhu.4 or a variant thereof (e.g., SEQ ID NO: 141 of WQ2005033321), AAVhu.5 or a variant thereof, AAVhu.6 or a variant thereof (e.g., SEQ ID NO: 84 of W02005033321), AAVhu.7 or a variant thereof (e.g., SEQ ID NO: 150 of WQ2005033321), AAVhu.9 or a variant thereof (e.g., SEQ ID NO: 155 of WQ2005033321), AAVhu.10 or a variant thereof (e.g., SEQ ID NO: 156 of W02005033321), AAVhu.1 1 or a variant thereof (e.g., SEQ ID NO: 153 of WQ2005033321), AAVhu.13 or a variant thereof (SEQ ID NO: 16 and 32 of US20150159173), AAVhu.15 or a variant thereof (e.g., SEQ ID NO: 147 of WQ2005033321), AAVhu.16 or a variant thereof (e.g., SEQ ID NO: 148 of W02005033321), AAVhu.17 or a variant thereof (e.g., SEQ ID NO: 83 of W02005033321), AAVhu.18 or a variant thereof (e.g., SEQ ID NO: 149 of W02005033321), AAVhu.19 or a variant thereof (e.g., SEQ ID NO: 133 of WQ2005033321), AAVhu.20 or a variant thereof (e.g., SEQ ID NO: 134 of WQ2005033321), AAVhu.21 or a variant thereof (e.g., SEQ ID NO: 135 of W02005033321), AAVhu.22 or a variant thereof (e.g., SEQ ID NO: 138 of W02005033321), AAVhu.23.2 or a variant thereof (e.g., SEQ IDNO: 137 of W02005033321), AAVhu.24 or a variant thereof (e.g., SEQ ID NO: 136 of W02005033321), AAVhu.25 or a variant thereof (e.g., SEQ ID NO: 146 of W02005033321), 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 W02005033321), 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 W02005033321), AAVhu.29R or a variant thereof, AAVhu.31 or a variant thereof (e.g., SEQ ID NO: 121 of W02005033321), AAVhu.32 or a variant thereof (SEQ ID NO: 122 of W02005033321), AAVhu.34 or a variant thereof (e.g., SEQ ID NO: 125 of W02005033321), AAVhu.35 or a variant thereof (e.g., SEQ ID NO: 164 of W02005033321), AAVhu.37 or a variant thereof (e.g., SEQ ID NO: 18 and 34 of US20150159173), AAVhu.39 or a variant thereof (e.g., SEQ ID NO: 102 of W02005033321), AAVhu.40 or a variant thereof (e.g., SEQ ID NO: 87 of W02005033321), AAVhu.41 or a variant thereof (e.g., SEQ ID NO: 91 of W02005033321), AAVhu.42 or a variant thereof (e.g., SEQ ID NO: 85 of W02005033321), AAVhu.43 or a variant thereof (e.g., SEQ ID NO: 160 of W02005033321), 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 W02005033321), AAVhu.46 or a variant thereof (e.g., SEQ ID NO: 159 of W02005033321), AAVhu.47 or a variant thereof (e.g., SEQ ID NO: 128 of W02005033321), 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 W02005033321), AAVhu.51 or a variant thereof (e.g., SEQ ID NO: 190 of W02005033321), AAVhu.52 or a variant thereof (e.g., SEQ ID NO: 191 of W02005033321), AAVhu.53 or a variant thereof (e.g., SEQ ID NO: 19 and 35 of US20150159173), AAVhu.54 or a variant thereof (e.g., SEQ ID NO: 188 of W02005033321), AAVhu.55 or a variant thereof (e.g., SEQ ID NO: 187 of W02005033321), AAVhu.56 or a variant thereof (e.g., SEQ ID NO: 192 of W02005033321), AAVhu.57 or a variant thereof (e.g., SEQ ID NO: 193 of W02005033321), AAVhu.58 or a variant thereof (e.g., SEQ ID NO: 194 of W02005033321), AAVhu.60 or a variant thereof (e.g., SEQ ID NO: 184 of W02005033321), AAVhu.61 or a variant thereof (e.g., SEQ ID NO: 185 of W02005033321), AAVhu.63 or a variant thereof (e.g., SEQ ID NO: 195 of W02005033321), AAVhu.64 or a variant thereof (e.g., SEQ ID NO: 196 of W02005033321), AAVhu.66 or a variant thereof (e.g., SEQ ID NO: 197 of W02005033321), AAVhu.67 or a variant thereof (e.g., SEQ ID NO: 198 of W02005033321), AAVhu.14 / 9 or a variant thereof, AAVhu.t 19 or a variant thereof, AAVrh.2 or a variant thereof (e.g., SEQ ID NO: 39 of US20150159173), AAVrh.2R or a variant thereof, AAVrh.8 or a variant thereof (e.g., SEQ ID NO: 41 of US20150159173), AAVrh.8R or a variant thereof, AAVrh.10 or a variant thereof (e.g., SEQ ID NO: 9 and 25 of US20150159173), AAVrh.12 or a variant thereof,AAVrh.13 or a variant thereof (e.g., SEQ ID NO: 10 and 26 of US20150159173), AAVrh.13R or a variant thereof, AAVrh.14 or a variant thereof, AAVrh.17 or a variant thereof, AAVrh.18 or a variant thereof, AAVrh.19 or a variant thereof, AAVrh.20 or a variant thereof (e.g., SEQ ID NO: 1 of US20150159173), AAVrh.21 or a variant thereof, AAVrh.22 or a variant thereof, AAVrh.23 or a variant thereof, AAVrh.24 or a variant thereof, AAVrh.25 or a variant thereof, AAVrh.31 or a variant thereof, AAVrh.32 or a variant thereof, AAVrh.33 or a variant thereof, AAVrh.34 or a variant thereof, AAVrh.35 or a variant thereof, AAVrh.36 or a variant thereof, AAVrh.37 or a variant thereof (e.g., SEQ ID NO: 40 of US20150159173), AAVrh.37R2 or a variant thereof, AAVrh.38 or a variant thereof (e.g., SEQ ID NO: 86 of W02005033321), AAVrh.39 or a variant thereof (e.g., SEQ ID NO: 3, 20, or 36 of US20150159173), AAVrh.40 or a variant thereof (e.g., SEQ ID NO: 92 of W02005033321), AAVrh.43 or a variant thereof (e.g., SEQ ID NO: 21 and 37 of US20150159173), AAVrh.46 or a variant thereof (e.g., SEQ ID NO: 4 and 22 ofUS20150159173), AAVrh.48 or a variant thereof (e.g., SEQ ID NO: 44 of US20150159173), AAVrh.48.1 or a variant thereof (e.g., SEQ ID NO: 44 of US20150159173), AAVrh.48.1 .2 or a variant thereof, AAVrh.48.2 or a variant thereof, AAVrh.49 or a variant thereof (e.g., SEQ ID NO: 103 of W02005033321), AAVrh.50 or a variant thereof (e.g., SEQ ID NO: 108 of W02005033321), AAVrh.51 or a variant thereof (e.g., SEQ ID NO: 104 of W02005033321), AAVrh.52 or a variant thereof (e.g., SEQ ID NO: 96 of W02005033321), AAVrh.53 or a variant thereof (e.g., SEQ ID NO: 97 of W02005033321), 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 W02005033321), AAVrh.57 or a variant thereof (e.g., SEQ ID NO: 105 of W02005033321), 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 W02005033321), AAVrh.62 or a variant thereof (e.g., SEQ ID NO: 114 of W02005033321), 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., SEQ ID NO: 5 of US20150159173), or AAVrh.74 or a variant thereof (e.g., SEQ ID NO: 6 of US2015015917). Non-limiting examples of variants include SEQ ID Nos: 9, 27-45, 47-62, 66-69, 73-81 , 84-94, 96, 97, 99, and 101-1 13 of US20030138772, the contents of which are herein incorporated by reference in its entirety, and SEQ ID Nos: 1 , 2, 4-82, 89, 90, 93-95, 98, 100, 101 , 109-113, 1 18-120, 124, 126, 131 , 139, 142, 151 , 154, 158, 161 , 162, 165-183, 202, 204-212, 215, 219, and 224-236 of W02005033321 , the contents of which are herein incorporated by reference in its entirety. In one embodiment, the AAV serotype is any of those described in U.S. 2021 / 0189430, the contents of which is herein incorporated by reference in its entirety. The amino acid sequence of the AAV may include one or more amino acid substitutions in an AAV capsid protein at one or more positions that interacts with a heparan sulfate proteoglycan or atone or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588, numbering based on VP1 numbering of AAV2.

[0214] Unless otherwise specified, the AAV ITRs, and other selected AAV components described herein, may be readily selected from among any AAV serotype, including, without limitation, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or other known and unknown AAV serotypes. In one embodiment, the ITRs are from AAV2. These ITRs or other AAV components may be readily isolated using techniques available to those of skill in the art from an AAV serotype. Such AAV may be isolated or obtained from academic, commercial, or public sources (e.g., the American Type Culture Collection, Manassas, VA). Alternatively, the AAV sequences may be obtained through synthetic or other suitable means by reference to published sequences such as are available in the literature or in databases such as, e.g., GenBank, PubMed, or the like.

[0215] Desirable AAV fragments for assembly into vectors include the cap proteins, including the vp1, vp2, vp3, and hypervariable regions, the rep proteins, including rep 78, rep 68, rep 52, and rep 40, and the sequences encoding these proteins. These fragments may be readily utilized in a variety of vector systems and host cells. Such 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, AAV with a non-naturally occurring capsid protein. Such an artificial capsid may be generated by any suitable technique, using a selected AAV sequence (e.g., a fragment of a vp1 capsid protein) in combination with heterologous sequences which may be obtained from a different selected AAV serotype, non-contiguous portions of the same AAV serotype, from a non-AAV viral source, or from a non-viral source. An artificial AAV serotype may be, without limitation, a pseudotyped AAV, a chimeric AAV capsid, a recombinant AAV capsid, or a “humanized” AAV capsid. Pseudotyped vectors, wherein the capsid of one AAV is utilized with the ITRs 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 AAV2 ITRs and an AAV5 capsid). In another embodiment, the AAV is AAV2 / 8 (i.e., an AAV having AAV2 ITRs and an AAV8 capsid). In one embodiment, the AAV includes an AAV8 capsid. Such AAV8 capsid includes the amino acid sequence found under NCBI Reference Sequence: YP_077180.1. In another embodiment, the AAV8 capsid includes a capsid encoded by nt 2121 to 4337 of GenBank accession: AF513852.1.

[0216] In one embodiment, the vectors useful in compositions and methods described herein contain, at a minimum, sequences encoding a selected AAV serotype capsid, e.g., an AAV2 capsid, or a fragment thereof. In another embodiment, useful vectors contain, at a minimum, sequences encoding a selected AAV serotype rep protein, e.g., AAV2 rep protein, or a fragmentthereof. Optionally, such vectors may contain both AAV cap and rep proteins. In vectors in which both AAV rep and cap are provided, the AAV rep and AAV cap sequences can both be of one serotype origin, e.g., an AAV2 origin.

[0217] Alternatively, vectors may be used in which the rep sequences are from an AAV serotype which differs from that which is providing the cap sequences. In one embodiment, the rep and cap sequences are expressed from separate sources (e.g., separate vectors, or a host cell and a vector). In another embodiment, these rep sequences are fused in frame to cap sequences of a different AAV serotype to form a chimeric AAV vector, such as those described in U.S. Patent No. 7,282,199, which is incorporated by reference herein.

[0218] A suitable recombinant AAV (rAAV) is generated by culturing a host cell which contains a nucleic acid sequence encoding an AAV serotype capsid protein, or fragment thereof, as defined herein; a functional rep gene; a minigene composed of, e.g., AAV ITRs and a trans-splicing molecule nucleic acid sequence; and sufficient helper functions to permit packaging of the minigene into the AAV capsid protein. The components required to be cultured in the host cell to package an AAV minigene in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., minigene, rep sequences, cap sequences, and / or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art.

[0219] In one embodiment, the AAV includes a promoter (or a functional fragment of a promoter). The selection of the promoter to be employed in the rAAV may be made from among a wide number of constitutive or inducible promoters that can express the selected transgene in the desired target cell. See, e.g., the list of promoters identified in International Patent Publication No. WO 2014 / 012482, incorporated by reference herein. 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 the particular promoter selected for the recombinant vector can direct expression of the selected transgene in a particular cell type. In some embodiments, the promoter is specific for expression of the transgene in neuronal cells.

[0220] In another embodiment, the promoter is the native promoter for the target gene to be expressed. Useful promoters include, without limitation, the promoter CAGGS and neuronal specific promoters, including, without limitation, a human synapsin 1 gene promoter, a neuronspecific enolase (NSE) promoter, human synapsin 1 promoter, a CaMK kinase promoter, or an MeCP2 promoter. Other suitable promoters comprise inducible promoters, wherein such promoters initiate transcription only when the host cell is exposed to a stimulus which acts as a trigger for activating the promoter.

[0221] Other conventional regulatory sequences contained in the mini-gene or rAAV are also disclosed in documents such as WO 2014 / 124282 and others cited and incorporated by reference herein. One of skill in the art may make a selection among these, and other, expression control sequences without departing from the scope described herein.

[0222] The selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment described herein are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods of generating rAAV virions are well known and the selection of a suitable method is not a limitation on methods and constructs described herein. See, e.g., K. Fisher et aL, J. Virol., 1993 70: 520-532 and U.S. Patent 5,478,745, each of which is incorporated by reference herein.

[0223] In some embodiments, the trans-splicing molecule is included in a proviral plasmid, such as those disclosed in International Patent Publication No. WO 2012 / 158757, incorporated herein by reference. Such a proviral plasmid contains a modular recombinant AAV genome comprising in operative association: a wildtype 5’ AAV2 ITR sequence flanked by unique restriction sites that permit ready removal or replacement of said ITR; a promoter comprising a 49-nucleic acid cytomegalovirus sequence upstream of a cytomegalovirus (CMV)-chicken beta actin sequence, or a neuron-specific promoter / enhancer, the promoter flanked by unique restriction sites that permit ready removal or replacement of the entire promoter sequence, and the upstream sequence flanked by unique restriction sites that permit ready removal or replacement of only the upstream CMV or enhancer sequence, from the promoter sequence. The trans-splicing molecule described herein can be inserted into the site of a multi-cloning polylinker, wherein the trans-splicing molecule is operably linked to, and under the regulatory control of, the promoter. A bovine growth hormone polyadenylation sequence flanked by unique restriction sites that permit ready removal or replacement of said poly A sequence; and a wildtype 3’ AAV2 ITR sequence flanked by unique restriction sites that permit ready removal or replacement of the 3’ ITR; are also part of such a plasmid. The plasmid backbone comprises the elements necessary for replication in bacterial cells, e.g., a kanamycin resistance gene, and is itself flanked by transcriptional terminator / insulator sequences.

[0224] In some embodiments, a proviral plasmid comprises: (a) a modular recombinant AAV genome comprising in operative association: (i) a wildtype 5’ AAV2 ITR sequence flanked by unique restriction sites that permit ready removal or replacement of said ITR; (ii) a promoter comprising (A) a 49-nucleic acid CMV sequence upstream of a CMV-chicken beta actin sequence or (B) a neuronal cell-specific promoter / enhancer. The promoter is flanked by unique restriction sites that permit ready removal or replacement of the entire promoter sequence, andthe upstream sequence flanked by unique restriction sites that permit ready removal or replacement of only the upstream CMV or enhancer sequence, from the promoter sequence. Also part of this proviral plasmid is a multi-cloning polylinker sequence that permits insertion of a trans-splicing molecule sequence including any of those described herein, wherein the transsplicing molecule is operably linked to, and under the regulatory control of, the promoter; a bovine growth hormone polyadenylation sequence flanked by unique restriction sites that permit ready removal or replacement of said poly A sequence; and a wildtype 3’ AAV2 ITR sequence flanked by unique restriction sites that permit ready removal or replacement of the 3’ ITR. The proviral plasmid also contains a plasmid backbone comprising the elements necessary for replication in bacterial cells, and further comprising a kanamycin resistance gene, said plasmid backbone flanked by transcriptional terminator / insulator sequences. The proviral plasmid described herein may also contain in the plasmid backbone a non-coding lambda phage 5.1 kb stuffer sequence to increase backbone length and prevent reverse packaging of non-functional AAV genomes.

[0225] In yet a further aspect, the promoter of the proviral plasmid is modified to reduce the size of the promoter to permit larger trans-splicing molecule sequences to be inserted in the rAAV. In one embodiment, the CMV / CBA hybrid promoter, which normally includes a noncoding exon and intron totaling about 1 ,000 base pairs, is replaced with a 130-base pair chimeric intron, as described in International Patent Publication No. WO 2017 / 087900, which is incorporated herein by reference in its entirety.

[0226] These proviral plasmids are then employed in currently conventional packaging methodologies to generate a recombinant virus expressing the trans-splicing molecule transgene carried by the proviral plasmids. Suitable production cell lines are readily selected by one of skill in the art. For example, a suitable host cell can 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 a selected packaging cell, where it may exist transiently. Alternatively, the minigene or gene expression cassette with its flanking ITRs is stably integrated into the genome of the host cell, either chromosomally or as an episome. Suitable transfection techniques are known and may readily be utilized to deliver the recombinant AAV genome to the host cell. Typically, the proviral plasmids are cultured in the host cells which express the cap and / or rep proteins. In the host cells, the minigene consisting of the trans-splicing molecule with flanking AAV ITRs is rescued and packaged into the capsid protein or envelope protein to form an infectious viral particle. Thus, a recombinant AAV infectious particle is produced by culturing a packaging cell carrying the proviral plasmid in the presence of sufficient viral sequences to permit packaging of the gene expression cassette viral genome into an infectious AAV envelope or capsid.

[0227] Alternatively, trans-splicing molecules can be delivered using a non-AAV vector, e.g., a non-viral vector. Any suitable non-viral vector technology known in the art or described herein may be used. Such non-viral vectors amenable for delivery of trans-splicing molecules include liposomes (e.g., cationic liposomes, unilamellar liposomes, or multilamellar liposomes), nanoparticles (e.g., polymeric nanoparticles, lipid nanoparticles (LNPs), PEGylated nanoparticles (e.g., PEGylated LNPs), peptide nanoparticles, metal nanoparticles, and the like), dendrimers (e.g., cationic dendrimers, e.g., polypropylenimine dendrimers), exosomes (e.g., immunologically inert and / or targeted exosomes, e.g., made using techniques described in Alvarez-Erviti, et aL, 2011 , Nat. Biotechnol. 29:341), and microvesicles. In some instances, the trans-splicing molecules described herein may be delivered using cell penetrating peptides (CPPs), which can translocate the plasma membrane of a target cell and facilitate the delivery of a trans-splicing molecule to the interior of the target cell.E. Pharmaceutical Compositions and Kits

[0228] Provided herein are pharmaceutical compositions including a nucleic acid trans- splicing molecule, a proviral plasmid, or a rAAV comprising any of the MeCP2 nucleic acid trans-splicing molecules described herein. In some embodiments, the pharmaceutical composition includes any of the 3’ trans-splicing molecules described herein.

[0229] Such pharmaceutical compositions may be prepared so as to be free / pure of contamination and suitable for in vivo administration. The pharmaceutical compositions described herein may be assessed for contamination by conventional methods and then formulated into a pharmaceutical composition intended for a suitable route of administration. Still other compositions containing the trans-splicing molecule, e.g., naked DNA, may be formulated similarly with a suitable carrier. Such formulation involves the use of a pharmaceutically and / or physiologically acceptable vehicle or carrier, particularly directed for administration to the target cell (e.g., a neuron). In one embodiment, carriers suitable for administration to the target cells include buffered saline, an isotonic sodium chloride solution, or other buffers, e.g., HEPES, to maintain pH at appropriate physiological levels, and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc.

[0230] In some embodiments, the carrier is a liquid for injection. Exemplary physiologically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline. A variety of such known carriers are provided in U.S. Patent No. 7,629,322, incorporated herein by reference. In one embodiment, the carrier is an isotonic sodium chloride solution. In another embodiment, the carrier is a balanced salt solution. In one embodiment, thecarrier includes tween. If the virus is to be stored long-term, it may be frozen in the presence of glycerol or TWEEN®20.

[0231] In other embodiments, compositions containing trans-splicing molecules described herein include a surfactant. Useful surfactants, such as Pluronic F68 (Poloxamer 188, also known as LUTROL® F68) may be included as they prevent AAV from sticking to inert surfaces and thus ensure delivery of the desired dose. As an example, one illustrative composition designed for the treatment of Rett Syndrome described herein comprises a recombinant adeno- associated vector carrying a nucleic acid sequence encoding a 3’ trans-splicing molecule as described herein, under the control of regulatory sequences which express the trans-splicing molecule in an neuronal cell of a mammalian subject, and a pharmaceutically acceptable carrier. The carrier is isotonic sodium chloride solution and includes a surfactant Pluronic F68. In one embodiment, the trans-splicing molecule is any of those described herein.

[0232] In yet another exemplary embodiment, the composition comprises a rAAV virus comprising any of the MeCP2 trans-splicing molecules described herein for MeCP2 gene correction, the nucleic acid sequence under the control of a promoter which directs expression of the trans-splicing molecule in neurons, neural stem cells, astrocytes, or oligodendrocytes, or any combination thereof of the brain (e.g., neurons in 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 intracerebroventricular delivery. In still another embodiment, the composition or components for production or assembly of this composition, including carriers, rAAV particles, surfactants, and / or the components for generating the rAAV, as well as suitable laboratory hardware to prepare the composition, may be incorporated into a kit. Such kits may further include instructions for administering the composition to an individual, e.g., as a treatment for Rett Syndrome.

[0233] Additionally provided herein are kits containing a pharmaceutical composition comprising a 3’ trans-splicing molecule (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 prior to administration.F. Methods and Uses

[0234] 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, and / or modulating expression of MeCP2, in a target cell (e.g., neurons, neural stem cells, astrocytes, or oligodendrocytes, or any combination thereof) of an individual in, e.g., methods for treating diseases or disorders associated withmutations in the MeCP2 gene, such as Rett Syndrome, including delaying or ameliorating symptoms associated with Rett Syndrome.

[0235] In some embodiments, symptoms of Rett Syndrome include, without limitation, developmental delays, cognitive problems, slowed brain and head growth, abnormal hand movements, hyperventilating, impairments in language, impairments in movement and coordination (e.g., problems walking, walking on the toes, or a wide-based gait), repetitive movements, inconsolable crying or screaming, seizures, and impaired ability to interact socially.

[0236] 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 furthermore useful for expressing functional MeCP2, and / or modulating expression of MeCP2, in a target cell (e.g., neurons, neural stem cells, astrocytes, or oligodendrocytes, or any combination thereof) of an individual as applied, e.g., to their use for treating diseases or disorders associated with mutations in the MeCP2 gene, such as Rett Syndrome, including delaying or ameliorating symptoms associated with Rett Syndrome as described herein or to their use in the preparation of a medicament for the treatment of diseases or disorders associated with mutations in the MeCP2 gene, such as Rett Syndrome, including delaying or ameliorating symptoms associated with Rett Syndrome as described herein. Such methods and uses involve contacting a target MeCP2 molecule (e.g., MeCP2 pre-mRNA) with a trans-splicing molecule as described herein [e.g., a 3’ trans-splicing molecule as described herein, a composition (e.g., a pharmaceutical composition) comprising same or a medicament comprising same], under conditions in which a coding domain of the trans-splicing molecule is spliced to the target MeCP2 pre-mRNA to replace a part of the targeted pre-mRNA carrying one or more defects or mutations, with a biologically functional (i.e., healthy), or normal or wildtype or corrected mRNA of the targeted gene, in order to correct expression of MeCP2 in the target cell. Thus, the methods and compositions are used to treat the Rett Syndrome pathologies and symptoms associated with the specific mutations.

[0237] In some embodiments, provided herein are methods of expressing functional MeCP2 in a target cell, by contacting (e.g., transducing) the target cell with any of the nucleic acid trans- splicing molecules, vectors (e.g., AAV vectors), or compositions described herein. In one embodiment, the contacting involves direct administration of the composition (e.g., pharmaceutical composition) to the affected individual. In another embodiment, the contacting may occur ex vivo with a cultured cell (e.g., a neuronal cell or precursor thereof) and the treated neuronal cell reimplanted in the individual. In another embodiment, the method involves administering an rAAV comprising any of the 3’ MeCP2 trans-splicing molecules described herein. These methods comprise administering to an individual in need thereof an effective concentration of a composition of any of those described herein.

[0238] In some embodiments, the methods include selecting one or more trans-splicing molecules for treating an individual having a disorder associated with mutation / s in MeCP2. In some embodiments, use of one or more trans-splicing molecules for treating an individual having a disorder associated with mutation / s in MeCP2 or use of same in the preparation of a medicament for the treatment of an individual having a disorder associated with mutation / s in MeCP2 is encompassed herein. Such methods and uses include selecting one or more trans- splicing molecules for treating an individual having a disorder associated with a mutation in MeCP2 or for use of such selected one or more trans-splicing molecules in treating an individual having a disorder associated with a mutation in MeCP2 or for use of such selected one or more trans-splicing molecules in the preparation of a medicament for the treatment of an individual having a disorder associated with mutation / s in MeCP2. Such selection can be based on the genotype of the individual. In some embodiments, a disorder associated with MeCP2 may be an X-linked disorder. Methods of screening for and identifying particular mutations in MeCP2 are known in the art.

[0239] Methods of the present disclosure include selecting a single trans-splicing molecule based on the location of a single mutation in MeCP2 (e.g., a mutation of one allele of the individual). As described herein, the causative mutations associated with Rett syndrome comprise those listed in Table 1 and the majority of mutations identified to date have been found in exons 3 or 4 of MeCP2. Thus, in some embodiments, methods of the present disclosure include 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, e.g., without regard to the location of any other mutations that may exist in the other allele.

[0240] Nucleic acid trans-splicing molecules described herein and vectors, proviral plasmids, and AAV comprising same, as well as compositions comprising such nucleic acid trans-splicing molecules and vectors, proviral plasmids, and AAV comprising same are for use in medical treatment, in particular for use in the treatment of Rett Syndrome. In some embodiments, when using, e.g., an AAV vector (or other gene therapy vector) the AAV vector may be administered systemically for delivery to the whole brain. In some embodiments, when using, e.g., an AAV vector (or other gene therapy vector) the AAV vector may be administered systemically for delivery to the whole brain and the enteric nervous system. In some embodiments, when using, e.g., an AAV vector (or other gene therapy vector) the AAV vector may be administered via direct infusion into the brain. In some embodiments, direct infusion comprises an intrathecal infusion of the AAV vector into the cerebrospinal fluid. Intrathecal infusion offers an efficient delivery mode into the CNS, wherein, e.g., neurons can be targeted. In some embodiments, at least one of the brainstem, midbrain, or cortex, or any combination thereof may be targeted via convection enhanced diffusion (CED) delivery of injections into and / or in the vicinity of thebrainstem, midbrain, or cortex, or any combination thereof. In some embodiments, injections may be directed to the brainstem, midbrain, or cortex, or any combination thereof to provide greater coverage of the structures of the brain implicated in Rett Syndrome. Such injections may be performed using magnetic resonance imaging-guided injections. Such methods for treatment are particularly useful for human subjects having Rett Syndrome. Such treatment involves human subjects having Rett Syndrome, including those having a genetic predisposition for developing Rett Syndrome that do not exhibit symptoms of Rett Syndrome. Accordingly, in some embodiments, treatment of human subjects with Rett Syndrome may include the treatment of any human subject carrying a pathological mutation associated with Rett Syndrome.

[0241] In some embodiments, an effective concentration of a recombinant adeno-associated virus carrying a trans-splicing molecule as described herein ranges between about 108and 1013vector genomes per milliliter (vg / mL). The rAAV infectious units are measured as described in McLaughlin et al., J. Virol. 1988, 62: 1963. In another embodiment, the concentration ranges between 109and 1013vg / mL. In another embodiment, the effective concentration is about 1.5 x 1011vg / mL. In another embodiment, the effective concentration is about 5 x 1011vg / mL. In one embodiment, the effective concentration is about 1 .5 x 1010vg / mL. In another embodiment, the effective concentration is about 2.8 x 1011vg / mL. In yet another embodiment, the effective concentration is about 1.5 x 1012vg / mL. In another embodiment, the effective concentration is about 1 .5 x 1013vg / mL.

[0242] It is desirable that the lowest effective dosage (total genome copies delivered) of virus be utilized in order to reduce the risk of undesirable effects, such as toxicity, and other issues related to administration to the brain. An effective dosage of a recombinant adeno-associated virus carrying a trans-splicing molecule as described herein ranges between about 108and 1013vector genomes (vg) per dose (i.e., per injection). In one embodiment, the dosage ranges between 109and 1013vg. In another embodiment, the effective dosage is about 1.5 x 1011vg. In another embodiment, the effective dosage is about 5 x 1011vg. In one embodiment, the effective dosage is about 1.5 x 1010vg. In another embodiment, the effective dosage is about 2.8 x 1011vg. In yet another embodiment, the effective dosage is about 1.5 x 1012vg. In another embodiment, the effective concentration is about 1.5 x 1013vg. Still other dosages in these ranges or in other units may be selected by the attending physician, taking into account the physical state of the individual being treated, including the age of the individual; the composition being administered, and the particular disorder; the targeted cell and the degree to which the disorder, if progressive, has developed.

[0243] In some embodiments, the composition may be delivered in a volume of from about 50 pL to about 1 mL, including all numbers within the range, depending on the size of the areato 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 pL. In another embodiment, the volume is about 70 pL. In another embodiment, the volume is about 100 pL. In another embodiment, the volume is about 125 pL. In another embodiment, the volume is about 150 pL. In another embodiment, the volume is about 175 pL. In yet another embodiment, the volume is about 200 pL. In another embodiment, the volume is about 250 pL. In another embodiment, the volume is about 300 pL. In another embodiment, the volume is about 350 pL. In another embodiment, the volume is about 400 pL. In another embodiment, the volume is about 450 pL. In another embodiment, the volume is about 500 pL. In another embodiment, the volume is about 600 pL. In another embodiment, the volume is about 750 pL. In another embodiment, the volume is about 850 pL. In another embodiment, the volume is about 1 ,000 pL.

[0244] In some embodiments, treatments and uses described herein replace 10% or more of the target MeCP2 mRNA in the target cell (e.g., 11% or more of the target MeCP2 mRNA in the target cell(s), 12% or more of the target MeCP2 mRNA in the target cell(s), 13% or more of the target MeCP2 mRNA in the target cell(s), 14% or more of the target MeCP2 mRNA in the target cell(s), 15% or more of the target MeCP2 mRNA in the target cell(s), 16% or more of the target MeCP2 mRNA in the target cell(s), 17% or more of the target MECP2 mRNA in the target cell(s), 18% or more of the target MECP2 mRNA in the target cell(s), 19% or more of the target MECP2 mRNA in the target cell(s). In some embodiments, treatments and uses described herein replace 20% or more of the target MECP2 mRNA in the target cell (e.g., 21% or more of the target MECP2 mRNA in the target cell(s), 22% or more of the target MECP2 mRNA in the target cell(s), 23% or more of the target MECP2 mRNA in the target cell(s), 24% or more of the target MECP2 mRNA in the target cell(s), 25% or more of the target MECP2 mRNA in the target cell(s), 26% or more of the target MECP2 mRNA in the target cell(s), 27% or more of the target MECP2 mRNA in the target cell(s), 28% or more of the target MECP2 mRNA in the target cell(s), 29% or more of the target MECP2 mRNA in the target cell(s), 30% or more of the target MECP2 mRNA in the target cell(s), 31% or more of the target MECP2 mRNA in the target cell(s), 32% or more of the target MECP2 mRNA in the target cell(s), 33% or more of the target MECP2 mRNA in the target cell(s), 34% or more of the target MECP2 mRNA in the target cell(s), 35% or more of the target MECP2 mRNA in the target cell(s), 36% or more of the target MECP2 mRNA in the target cell(s), 37% or more of the target MECP2 mRNA in the target cell(s), 38% or more of the target MECP2 mRNA in the target cell(s), 39% or more of the target MECP2 mRNA in the target cell(s), 40% or more of the target MECP2 mRNA in the target cell(s), 41% or more of the target MECP2 mRNA in the target cell(s), 42% or more of the target MECP2 mRNA in the target cell(s), 43% or more of the target MECP2 mRNA in the target cell(s), 44% or more of the MECP2 mRNA in the target cell(s), 45% or more of the target MECP2 mRNA in the target cell(s), 46% or more of the targetMECP2 mRNA in the target cell(s), 47% or more of the target MECP2 mRNA in the target cell(s), 48% or more of the target MECP2 mRNA in the target cell(s), 49% or more of the target MECP2 mRNA in the target cell(s), or 50% or more of the target MECP2 mRNA in the target cell(s)). In some embodiments, treatments and uses described herein replace 50% or more of the target MECP2 mRNA in the target cell (e.g., 51% or more of the target MECP2 mRNA in the target cell(s), 52% or more of the target MECP2 mRNA in the target cell(s), 53% or more of the target MECP2 mRNA in the target cell(s), 54% or more of the target MECP2 mRNA in the target cell(s), 55% or more of the target MECP2 mRNA in the target cell(s), 56% or more of the target MECP2 mRNA in the target cell(s), 57% or more of the target MECP2 mRNA in the target cell(s), 58% or more of the target MECP2 mRNA in the target cell(s), 59% or more of the target MECP2 mRNA in the target cell(s), 60% or more of the target MECP2 mRNA in the target cell(s), 61 % or more of the target MECP2 mRNA in the target cell(s), 62% or more of the target MECP2 mRNA in the target cell(s), 63% or more of the target MECP2 mRNA in the target cell(s), 64% or more of the target MECP2 mRNA in the target cell(s), 65% or more of the target MECP2 mRNA in the target cell(s), 66% or more of the target MECP2 mRNA in the target cell(s), 67% or more of the target MECP2 mRNA in the target cell(s), 68% or more of the target MECP2 mRNA in the target cell(s), 69% or more of the target MECP2 mRNA in the target cell(s), 70% or more of the target MECP2 mRNA in the target cell(s), 71 % or more of the target MECP2 mRNA in the target cell(s), 72% or more of the target MECP2 mRNA in the target cell(s), 73% or more of the target MECP2 mRNA in the target cell(s), 74% or more of the MECP2 mRNA in the target cell(s), 75% or more of the target MECP2 mRNA in the target cell(s), 76% or more of the target MECP2 mRNA in the target cell(s), 77% or more of the target MECP2 mRNA in the target cell(s), 78% or more of the target MECP2 mRNA in the target cell(s), 79% or more of the target MECP2 mRNA in the target cell(s), or 80% or more of the target MECP2 mRNA in the target cell(s) (e.g., 81 % or more of the target MECP2 mRNA in the target cell(s), 82% or more of the target MECP2 mRNA in the target cell(s), 83% or more of the target MECP2 mRNA in the target cell(s), 84% or more of the target MECP2 mRNA in the target cell(s), 85% or more of the target MECP2 mRNA in the target cell(s), 86% or more of the target MECP2 mRNA in the target cell(s), 87% or more of the target MECP2 mRNA in the target cell(s), 88% or more of the target MECP2 mRNA in the target cell(s), 89% or more of the target MECP2 mRNA in the target cell(s), 90% or more of the target MECP2 mRNA in the target cell(s), 91 % or more of the target MECP2 mRNA in the target cell(s), 92% or more of the target MECP2 mRNA in the target cell(s), 93% or more of the target MECP2 mRNA in the target cell(s), 94% or more of the target MECP2 mRNA in the target cell(s), 95% or more of the target MECP2 mRNA in the target cell(s), 96% or more of the target MECP2 mRNA in the target cell(s), 97% or more of the target MECP2 mRNA in the target cell(s), 98% or more of the target MECP2mRNA in the target cell(s), 99% or more of the target MECP2 mRNA in the target cell(s), or 100% or more of the target MECP2 mRNA in the target cell(s).

[0245] 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 tissue having mild, moderate, or advanced disease. As used herein, the term “rescue” means to prevent progression of the disease, prevent spread of damage to uninjured cells, and / or to improve damage in injured cells.

[0246] Thus, in one embodiment, the composition is administered before disease onset. In another embodiment, the composition is administered prior to the development of symptoms. In another embodiment, the composition is administered after development of symptoms. In yet another embodiment, the composition is administered when less than 90% of the target cells are functioning or remaining, e.g., as compared to a reference tissue. In yet another embodiment, the composition is administered when more than 10% of the target cells are functioning or remaining, e.g., as compared to a reference tissue. In yet another embodiment, the composition is administered when more than 20% of the target cells are functioning or remaining. In yet another embodiment, the composition is administered when more than 30% of the target cells are functioning or remaining. In yet another embodiment, the composition is administered when more than 40% of the target cells are functioning or remaining. In yet another embodiment, the composition is administered when more than 50% of the target cells are functioning or remaining. In yet another embodiment, the composition is administered when more than 60% of the target cells are functioning or remaining. In yet another embodiment, the composition is administered when more than 70% of the target cells are functioning or remaining. In yet another embodiment, the composition is administered when more than 80% of the target cells are functioning or remaining. In yet another embodiment, the composition is administered when more than 90% of the target cells are functioning or remaining. In yet another embodiment, the composition is administered when more than 95% of the target cells are functioning or remaining.

[0247] In yet another embodiment, any of the above-described methods or uses is performed in combination with another, or secondary, therapy. The therapy may be any now known, or as yet unknown, therapy which helps prevent, arrest or ameliorate these mutations or defects or any of the effects associated therewith. The secondary therapy can be administered before, concurrent with, or after administration of a pharmaceutical composition described above. In one embodiment, a secondary therapy involves non-specific approaches for maintaining the health of the neuronal cells, such as administration of neurotrophic factors, antioxidants, and / or anti-apoptotic agents. The non-specific approaches are achieved through injection of proteins, recombinant DNA, recombinant viral vectors, stem cells, fetal tissue, orgenetically modified cells. The latter could include genetically modified cells that are encapsulated.

[0248] For use in these methods, the volume and viral titer of each injection is determined individually and may be the same or different from other injections performed in, e.g., the brain. The dosages, administrations, and regimens may be determined by the attending physician given the teachings of this disclosure.

[0249] In some embodiments, methods of the disclosure do not involve modifying the germ line genetic identity of any human or other animal.

[0250] The examples that follow do not limit the scope of the embodiments described herein. One skilled in the art will appreciate that modifications can be made in the following examples which are intended to be encompassed by the spirit and scope of the invention.G. Specific Embodiments

[0251] The present disclosure is exemplified by the specific enumerated embodiments below:1. An exon editor construct that encodes an RNA exon editor, wherein the exon editor construct comprises sequences encoding:(a) a binding domain that binds a target intron of a MeCP2 pre-mRNA;(b) a hemi-intron; and(c) a coding domain comprising one or more MeCP2 exons.2. The exon editor construct of embodiment 1 , wherein the target intron of the MeCP2 pre- mRNA is intron 1 or intron 2.3. The exon editor construct of embodiment 1 or embodiment 2, wherein the binding domain binds to a binding site comprising: nucleotides -100 to 1500 or -50 to 1449 of intron 1 (SEQ ID NO: 1); nucleotides 3950 to 4250 or 4020-4169 of intron 1 ; or nucleotides 1-300 of intron 2 (SEQ ID NOs: 2 and 3).4. The exon editor construct of any one of the preceding embodiments, 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 (SEQ ID NO: 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 (SEQ ID NO: 2).5. The exon editor construct of any one of the preceding embodiments, wherein the binding domain ranges in size from about 50-300 nucleotides; about 50-250 nucleotides; about 50-150 nucleotides; about 50-100 nucleotides; about 75-300 nucleotides; about 75-250 nucleotides; about 75-200 nucleotides; about 75-150 nucleotides; about 100-300 nucleotides; about 100-250 nucleotides; about 100-200 nucleotides; about 100-150 nucleotides; about 125-300 nucleotides; about 125-250 nucleotides; about 125-200 nucleotides; about 125-150 nucleotides; or about 150 nucleotides.6. The exon editor construct of any one of the preceding embodiments, wherein the binding domain ranges in size from 50-300 nucleotides; 50-250 nucleotides; 50-150 nucleotides; 50-100 nucleotides; 75-300 nucleotides; 75-250 nucleotides; 75-200 nucleotides; 75-150 nucleotides; 100-300 nucleotides; 100-250 nucleotides; 100-200 nucleotides; 100-150 nucleotides; 125-300 nucleotides; 125-250 nucleotides; 125-200 nucleotides; 125-150 nucleotides; or 150 nucleotides.7. The exon editor construct of any one of the preceding embodiments, wherein the binding domain ranges in size from about 100-200 nucleotides; about 100-150 nucleotides; about 125- 200 nucleotides; about 125-150 nucleotides; or about 150 nucleotides; or ranges in size from 100-200 nucleotides; 100-150 nucleotides; 125-200 nucleotides; 125-150 nucleotides; or 150 nucleotides.8. The exon editor construct of any one of the preceding embodiments, wherein the binding domain is at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the binding site to which it binds.9. The exon editor construct of any one of the preceding embodiments, wherein the binding domain comprises stretches of contiguous nucleotides that are 100% complementary to a portion of the binding site to which it binds, wherein the stretches of contiguous nucleotides are at least 5 nucleotides long, at least 10 nucleotides long, at least 15 nucleotides long, at least 20 nucleotides long, at least 25 nucleotides long, at least 30 nucleotides long, at least 35 nucleotides long, at least 40 nucleotides long, at least 45 nucleotides long, at least 50 nucleotides long, at least 55 nucleotides long, at least 60 nucleotides long, at least 65 nucleotides long, at least 70 nucleotides long, at least 75 nucleotides long, at least 80nucleotides long, at least 85 nucleotides long, at least 90 nucleotides long, at least 95 nucleotides long, at least 100 nucleotides long, at least 105 nucleotides long, at least 110 nucleotides long, at least 115 nucleotides long, at least 120 nucleotides long, at least 130 nucleotides long, at least 135 nucleotides long, at least 140 nucleotides long, at least 145 nucleotides long, or at least 150 nucleotides long.10. The exon editor construct of any one of the preceding embodiments, wherein the sequence encoding the binding domain comprises, consists essentially of, or consists of any one of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, or 47.11. The exon editor construct of any one of the preceding embodiments, wherein the sequence encoding the binding domain comprises, consists essentially of, or consists of SEQ ID NO: 55.12. The exon editor construct of any one of the preceding embodiments, wherein the sequence encoding the binding domain comprises, consists essentially of, or consists of any one of SEQ ID NOs: 24, 28, 29, or 30.13. The exon editor construct of any one of the preceding embodiments, wherein the coding domain comprises, consists essentially of, or consists of :SEQ ID NOs: 7 and 8 or a sequence 90% identical to SEQ ID NOs: 7 and 8;SEQ ID NO: 76 or a sequence 90% identical to SEQ ID NO: 76;SEQ ID NO: 12;SEQ ID NO: 14; orSEQ ID NO: 9.14. The exon editor construct of any one of the preceding embodiments, wherein the coding domain comprises, consists essentially of, or consists of any one of SEQ ID NOs: 76 or 14.15. The exon editor construct of any one of the preceding embodiments, wherein the binding domain, the hemi-intron, and the coding domain are operatively linked in a 5’-to-3’ direction.16. The exon editor construct of any one of the preceding embodiments, wherein the hemi- intron comprises a 3’ splice site and optionally, a branchpoint sequence and a polypyrimidine tract, wherein the binding domain, the hemi-intron comprising the 3’ splice site, and the coding domain are operatively linked in a 5’-to-3’ direction.17. The exon editor construct of embodiment 16, wherein the hemi-intron comprises, consists essentially of, or consists of: polypyrimidine tract, a branchpoint, and a 3’ splice site.18. The exon editor construct of any one of embodiments 16 or 17, wherein the hemi-intron comprises, consists essentially of, or consists of:SEQ ID NO: 17 or a sequence having at least 90% identity to SEQ ID NO: 17.19. The exon editor construct of any one of the preceding embodiments, further comprising a 3’ untranslated region (3’ UTR), wherein the binding domain; the hemi-intron; the coding domain; and the 3’ UTR; are operatively linked in a 5'-to-3’ direction.20. The exon editor construct of embodiment 19, wherein the 3’ UTR comprises an RDHI pA 3’ UTR (SEQ ID NO: 19) or a mWPRE 3’ UTR (SEQ ID NO: 20).21. The exon editor construct of embodiment 20, wherein the RDHIpA 3’ UTR comprises, consists essentially of, or consists of SEQ ID NO: 19 or a sequence having at least 90% identity to SEQ ID NO: 19; or the mWPRE 3’ UTR comprises, consists essentially of, or consists of SEQ ID NO: 20 or a sequence having at least 90% identity to SEQ ID NO: 20.22. The exon editor construct of any one of the preceding embodiments, wherein the exon editor construct comprises: a binding domain comprising, consisting essentially 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, consisting essentially of, or consisting of SEQ ID NO: 17; a coding domain comprising, consisting essentially of, or consisting of SEQ ID NO: 76; and a 3’ UTR comprising, consisting essentially of, or consisting of SEQ ID NO: 20, wherein the binding domain, the hemi-intron, the coding domain, and the 3’ UTR are operatively linked in a 5’-to-3’ direction.23. The exon editor construct of embodiment 22, wherein the exon editor construct comprises, consists essentially of, or consists of SEQ ID NO: 78, 80, 81, 82, 83, or 84.24. The exon editor construct of embodiment 22, wherein the exon editor construct comprises, consists essentially of, or consists of SEQ ID NO: 78 or 80.25. The exon editor construct of any one of embodiments 1-19, further comprising a triple helix terminator, wherein the binding domain; the hemi-intron; the coding domain; the 3’ UTR, when present; and the triple helix terminator are operatively linked in a 5’-to-3’ direction.26. The exon editor construct of embodiment 25, wherein the triple helix terminator comprises, consists essentially of, or consists of SEQ ID NO: 77 or a sequence having at least 90% identity to SEQ ID NO: 77.27. The exon editor construct of embodiment 25, wherein the triple helix terminator comprises, consists essentially of, or consists of SEQ ID NO: 22.28. The exon editor construct of any one of the preceding embodiments, further comprising a sequence encoding a poly A sequence, for example, SEQ ID NO: 21.29. The exon editor construct of any one of the preceding embodiments, further comprising a sequence encoding an epitope tag, wherein the binding domain; the hemi-intron; the coding domain; the epitope tag; the 3’ UTR, when present; and the triple helix terminator, when present; are operatively linked in a 5’-to-3’ direction.30. The exon editor construct of embodiment 28, wherein the exon editor construct comprises, consists essentially of, or consists of SEQ ID NO: 78.31. An RNA exon editor transcribed from the exon editor construct of any one of embodiments 1-30.32. The exon editor construct of any one of embodiments 1 -30, wherein the MeCP2 pre-mRNA comprises at least one mutation associated with Rett Syndrome.33. The exon editor construct of any one of embodiments 1 -30, wherein the at least one mutation associated with Rett Syndrome comprises at least one mutation in exon 3 of an MeCP2 gene allele or at least one mutation in exon 4 of an MeCP2 gene allele, or any combination thereof.34. The exon editor construct of any one of embodiments 32-33, wherein the at least one mutation associated with Rett Syndrome is X-linked.35. The exon editor construct of any one of embodiments 32-34, wherein MeCP2 protein comprising the 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.36. A vector comprising the exon editor construct of any one of embodiments 1-30 or 32-35.37. The vector of embodiment 36, wherein the vector comprises a 5’ regulatory domain operatively linked 5’ to the binding domain.38. The vector of any one of embodiments 36-37, wherein the 5’ regulatory domain comprises a constitutive promoter or a tissue specific promoter.39. The vector of embodiment 38, wherein the constitutive promoter is a CMV promoter.40. A proviral plasmid comprising the exon editor construct of any one of embodiments 1-30 or 32-35.41. An adeno-associated virus (AAV) comprising the exon editor construct of any one of embodiments 1-30 or 32-35, wherein the AAV optionally comprises a 5’ regulatory domain operatively linked 5’ to the exon editor construct.42. The AAV of embodiment 41 , wherein the AAV comprises a 5’ regulatory domain operatively linked 5’ to the binding domain.43. The AAV of any one of embodiments 41-42, wherein the 5’ regulatory domain comprises a constitutive promoter.44. The AAV of embodiment 43, wherein the constitutive promoter is a CMV promoter.45. The AAV of any one of embodiments 41-44, wherein the AAV exhibits neuronal tropism.46. The AAV of any one of embodiments 41-45, wherein the AAV is AAV9, AAV8, AAV5, or AAV2 or a variant of AAV9, AAV8, AAV5, or AAV2.47. A composition comprising the exon editor construct of any one of embodiments 1 -30 or 32- 35, the vector of any one of embodiments 36-39, the proviral plasmid of embodiment 40, or the AAV of any one of embodiments 41-46.48. The composition of embodiment 47, comprising a pharmaceutically acceptable excipient.49. A method of expressing biologically active MeCP2 in a target cell to restore functional levels of MeCP2 protein in the target cell, the method comprising transducing the target cell with the exon editor construct of any one of embodiments 1-30 or 32-35, the vector of any one of embodiments 36-39, the proviral plasmid of embodiment 40, the AAV of any one of embodiments 41-46, or the composition of any one of embodiments 47-48.50. The method of embodiment 49, wherein at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of the MeCP2 pre- mRNAs comprising at least one mutation associated with Rett Syndrome in the target cell are modified to become transcripts that encode functional MeCP2 wherein the at least one mutation is corrected.51. The method of embodiment 50, wherein at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of the MeCP2 pre-mRNAs comprising at least one mutation associated with Rett Syndrome in the target cell are modified to become transcripts that encode functional MeCP2 wherein the at least one mutation is corrected.52. The method of embodiment 51 , wherein at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the MeCP2 pre-mRNAs comprising at least one mutation associated with Rett Syndrome in the target cell are modified to become transcripts that encode functional MeCP2 wherein the at least one mutation is corrected.53. The method of any one of embodiments 49-52, wherein functional levels of MeCP2 are restored in the target cell by expressing biologically functional MeCP2 protein.54. A method of reducing expression of MeCP2 comprising at least one mutation associated with Rett Syndrome in a subject, the method comprising transfecting or transducing a target cell, more particularly a neuron, in the subject with the exon editor construct of any one of embodiments 1-30 or 32-35, the vector of any one of embodiments 36-39, the proviral plasmidof embodiment 40, the AAV of any one of embodiments 41-46, or the composition of any one of embodiments 47-48.55. A method of correcting at least one mutation in an MeCP2 exon sequence in an MeCP2 pre-mRNA in a target cell of a subject, the method comprising administering to the subject the exon editor construct of any one of embodiments 1-30 or 32-35, the vector of any one of embodiments 36-39, the proviral plasmid of embodiment 40, the AAV of any one of embodiments 41-46, or the composition of any one of embodiments 47-48.56. A method of treating Rett Syndrome in a subject in need thereof, the method comprising administering to the subject the exon editor construct of any one of embodiments 1-30 or 32-35, the vector of any one of embodiments 36-39, the proviral plasmid of embodiment 40, the AAV of any one of embodiments 41-46, or the composition of any one of embodiments 47-48 in a therapeutically effective amount.57. The method of any one of embodiments 54-56, wherein at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of the MeCP2 pre-mRNAs comprising at least one mutation associated with Rett Syndrome in the target cell are modified to become transcripts that encode functional MeCP2 wherein the at least one mutation is corrected.58. The method of embodiment 57, wherein at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of the MeCP2 pre-mRNAs comprising at least one mutation associated with Rett Syndrome in the target cell are modified to become transcripts that encode functional MeCP2 wherein the at least one mutation is corrected.59. The method of embodiment 58, wherein at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the MeCP2 pre-mRNAs comprising at least one mutation associated with Rett Syndrome in the target cell are modified to become transcripts that encode functional MeCP2 wherein the at least one mutation is corrected.60. The method of any one of embodiments 54-59, wherein functional levels of MeCP2 are restored in the target cell by expressing biologically functional MeCP2 protein.61. The method of any one of embodiments 54-60, the method comprising administration of the exon editor construct of any one of embodiments 1-30 or 32-35, the vector of any one of embodiments 36-39, the proviral plasmid of embodiment 40, the AAV of any one of embodiments 41-46, or the composition of any one of embodiments 47-48 to the subject’s brain.62. The method of any one of embodiments 54-61 , wherein the subject is a mammal, preferentially a rodent, non-human primate, or a human.63. The method of any one of embodiments 54-62, wherein the subject is genetically predisposed to have Rett Syndrome or has been diagnosed with Rett Syndrome.64. The exon editor construct of any one of embodiments 1-30 or 32-35, the vector of any one of embodiments 36-39, the proviral plasmid of embodiment 40, the AAV of any one of embodiments 41-46, or the composition of any one of embodiments 47-48 for use in preventing or treating Rett Syndrome in a subject in need thereof.65. The exon editor construct of any one of embodiments 1-30 or 32-35, the vector of any one of embodiments 36-39, the proviral plasmid of embodiment 40, the AAV of any one of embodiments 41-46, or the composition of any one of embodiments 47-48 for use in the preparation of a medicament for the treatment or prevention of Rett Syndrome in a subject in need thereof.66. An RNA exon editor comprising, operably linked in a 5’ to 3’ direction:(a) a binding domain configured to bind to a binding site within a target region of a MeCP2 pre-mRNA;(b) a hemi-intron; and(c) a coding domain comprising a sequence encoding one or more MeCP2 exons.67. The RNA exon editor of embodiment 66, wherein the target region comprises or consists of nucleotides -100 to 1500 of MeCP2 intron 1 (positions 15 to 1615 of SEQ ID NO: 111).68. The RNA exon editor of embodiment 66, wherein the target region comprises or consists of nucleotides -100 to 100 of MeCP2 intron 1 (positions 15 to 215 of SEQ ID NO: 111).69. The RNA exon editor of embodiment 66, wherein the target region comprises or consists of nucleotides -50 to 1449 of MeCP2 intron 1 (positions 65 to 1564 of SEQ ID NO: 111).70. The RNA exon editor of embodiment 66, wherein the target region comprises or consists of nucleotides -50 to 99 of MeCP2 intron 1 (positions 65 to 214 of SEQ ID NO: 111).71. The RNA exon editor of embodiment 66, wherein the target region comprises or consists of nucleotides 51 to 649 of MeCP2 intron 1 (positions 51 to 649 of SEQ ID NO: 1).72. The RNA exon editor of embodiment 66, wherein the target region comprises or consists of nucleotides 51 to 524 of MeCP2 intron 1 (positions 51 to 524 of SEQ ID NO: 1).73. The RNA exon editor of embodiment 66, wherein the target region comprises or consists of nucleotides 51 to 1449 of MeCP2 intron 1 (positions 51 to 1449 of SEQ ID NO: 1).74. The RNA exon editor of embodiment 66, wherein the target region comprises or consists of nucleotides 125 to 524 of MeCP2 intron 1 (positions 125 to 524 of SEQ ID NO: 1).75. The RNA exon editor of embodiment 66, wherein the target region comprises or consists of nucleotides 125 to 649 of MeCP2 intron 1 (positions 125 to 649 of SEQ ID NO: 1).76. The RNA exon editor of embodiment 66, wherein the target region comprises or consists of nucleotides 820 to 1449 of MeCP2 intron 1 (positions 820 to 1449 of SEQ ID NO: 1).77. The RNA exon editor of embodiment 66, wherein the target region comprises or consists of nucleotides 3950 to 4250 of MeCP2 intron 1 (positions 3950 to 4250 of SEQ ID NO: 1 ).78. The RNA exon editor of embodiment 66, wherein the target region comprises or consists of nucleotides 4020 to 4169 of MeCP2 intron 1 (positions 4020 to 4169 of SEQ ID NO: 1).79. The RNA exon editor of embodiment 66, wherein the target region comprises or consists of nucleotides 1 to 300 of MeCP2 intron 2 (positions 1 to 300 of SEQ ID NO: 2).80. The RNA exon editor of embodiment 66, wherein the target region comprises or consists of nucleotides 99 to 248 of MeCP2 intron 2 (positions 99 to 248 of SEQ ID NO: 2).81. The RNA exon editor of any one of embodiments 66 to 80, wherein the binding site comprises or consists of at least 25 consecutive nucleotides of the target region.82. The RNA exon editor of any one of embodiments 66 to 80, wherein the binding site comprises or consists of at least 50 consecutive nucleotides of the target region.83. The RNA exon editor of any one of embodiments 66 to 80, wherein the binding site comprises or consists of 25 to 100 consecutive nucleotides of the target region.84. The RNA exon editor of any one of embodiments 66 to 80, wherein the binding site comprises or consists of 50 to 100 consecutive nucleotides of the target region.85. The RNA exon editor of any one of embodiments 66 to 80, wherein the binding site comprises or consists of 50 to 150 consecutive nucleotides of the target region.86. The RNA exon editor of any one of embodiments 66 to 80, wherein the binding site comprises or consists of 100 to 200 consecutive nucleotides of the target region.87. The RNA exon editor of any one of embodiments 66 to 80, wherein the binding site comprises or consists of 100 to 150 consecutive nucleotides of the target region.88. The RNA exon editor of any one of embodiments 66 to 80, wherein the binding site comprises or consists of 150 consecutive nucleotides of the target region.89. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 80% complementary to the entirety of the binding site.90. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 90% complementary to the entirety of the binding site.91. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at 100% complementary to the entirety of the binding site.92. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 80% complementary to 25 consecutive nucleotides within the binding site.93. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 90% complementary to 25 consecutive nucleotides within the binding site.94. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is 100% complementary to 25 consecutive nucleotides within the binding site.95. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 80% complementary to 50 consecutive nucleotides within the binding site.96. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 90% complementary to 50 consecutive nucleotides within the binding site.97. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is 100% complementary to 50 consecutive nucleotides within the binding site.98. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 80% complementary to 75 consecutive nucleotides within the binding site.99. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 90% complementary to 75 consecutive nucleotides within the binding site.100. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is 100% complementary to 75 consecutive nucleotides within the binding site.101. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 80% complementary to 100 consecutive nucleotides within the binding site.102. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 90% complementary to 100 consecutive nucleotides within the binding site.103. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is 100% complementary to 100 consecutive nucleotides within the binding site.104. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 80% complementary to 150 consecutive nucleotides within the binding site.105. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is at least 90% complementary to 150 consecutive nucleotides within the binding site.106. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain comprises or consists of a nucleotide sequence that is 100% complementary to 150 consecutive nucleotides within the binding site.107. The RNA exon editor of any one of embodiments 66 to 106, wherein the binding domain is at least 25 nucleotides in length.108. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain is at least 50 nucleotides in length.109. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain is at least 100 nucleotides in length.110. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain is at least 150 nucleotides in length.111. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain is 25 to 150 nucleotides in length.112. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain is 50 to 100 nucleotides in length.113. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain is 50 to 150 nucleotides in length.114. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain is 100 to 150 nucleotides in length.115. The RNA exon editor of any one of embodiments 66 to 88, wherein the binding domain is 100 to 200 nucleotides in length.116. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 23 (with U substituted for each T).117. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 23 (with U substituted for each T).118. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 23 (with U substituted for each T).119. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 24 (with U substituted for each T).120. The RNA exon editor of embodiment 119, wherein the binding domain comprises nucleotides 23C and / or 72C, numbering according to SEQ ID NO: 24 (with U substituted for each T).121. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 24 (with U substituted for each T).122. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 24 (with U substituted for each T).123. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 25 (with U substituted for each T).124. The RNA exon editor of embodiment 123, wherein the binding domain comprises nucleotides 97C and / or 146C, numbering according to SEQ ID NO: 25 (with U substituted for each T).125. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 25 (with U substituted for each T).126. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 25 (with U substituted for each T).127. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 26 (with U substituted for each T).128. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 26 (with U substituted for each T).129. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 26 (with U substituted for each T).130. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 27 (with U substituted for each T).131. The RNA exon editor of embodiment 130, wherein the binding domain comprises nucleotides 8C, 56C, 72C, or 85C, or any combination thereof, numbering according to SEQ ID NO: 27 (with U substituted for each T).132. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 27 (with U substituted for each T).133. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 27 (with U substituted for each T).134. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 28 (with U substituted for each T).135. The RNA exon editor of embodiment 134, wherein the binding domain comprises nucleotide 133C, numbering according to SEQ ID NO: 28 (with U substituted for each T).136. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 28 (with U substituted for each T).137. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 28 (with U substituted for each T).138. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 29 (with U substituted for each T).139. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 29 (with U substituted for each T).140. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 29 (with U substituted for each T).141. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 30 (with U substituted for each T).142. The RNA exon editor of embodiment 141 , wherein the binding domain comprises nucleotides 4C, 71 C, or 101C, or any combination thereof, numbering according to SEQ ID NO: 30 (with U substituted for each T).143. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 30 (with U substituted for each T).144. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 30 (with U substituted for each T).145. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 31 (with U substituted for each T).146. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 31 (with U substituted for each T).147. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 31 (with U substituted for each T).148. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 32 (with U substituted for each T).149. The RNA exon editor of embodiment 148, wherein the binding domain comprises nucleotide 119A, numbering according to SEQ ID NO: 32 (with U substituted for each T).150. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 32 (with U substituted for each T).151. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 32 (with U substituted for each T).152. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 33 (with U substituted for each T).153. The RNA exon editor of embodiment 152, wherein the binding domain comprises nucleotide 133C, numbering according to SEQ ID NO: 33 (with U substituted for each T).154. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 33 (with U substituted for each T).155. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 33 (with U substituted for each T).156. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 47 (with U substituted for each T).157. The RNA exon editor of embodiment 156, wherein the binding domain comprises nucleotide 149C, numbering according to SEQ ID NO: 47 (with U substituted for each T).158. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 47 (with U substituted for each T).159. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 47 (with U substituted for each T).160. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 55 (with U substituted for each T).161. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 55 (with U substituted for each T).162. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 55 (with U substituted for each T).163. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 88, wherein the binding domain has a C at one or more of positions 25, 59, 63, 99, or 120, numbering according to SEQ ID NO: 88 (with U substituted for each T).164. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 88, wherein the binding domain has a C at positions 25, 59, 63, 99, and 120, numbering according to SEQ ID NO: 88 (with U substituted for each T).165. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 88, wherein the binding domain has a C at one or more of positions 25, 59, 63, 99, or 120, numbering according to SEQ ID NO: 88 (with U substituted for each T).166. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 88, wherein the binding domain has a C at positions 25, 59, 63, 99, and 120, numbering according to SEQ ID NO: 88 (with U substituted for each T).167. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 88 (with U substituted for each T).168. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 89 (with U substituted for each T).169. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 89 (with U substituted for each T).170. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 89 (with U substituted for each T).171. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 90, wherein the binding domain has an A at position 84 or a C at position 106, numbering according to SEQ ID NO: 90 (with U substituted for each T).172. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 90, wherein the binding domain has an A at position 84 and a C at position 106, numbering according to SEQ ID NO: 90 (with U substituted for each T).173. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 90, wherein the binding domain has an A at position 84 or a C at position 106, numbering according to SEQ ID NO: 90 (with U substituted for each T).174. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 90, wherein the binding domain has an A at position 84 or a C at position 106, numbering according to SEQ ID NO: 90 (with U substituted for each T).175. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 90 (with U substituted for each T).176. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 91 , wherein the binding domain has C at position 136 or 138, numbering according to SEQ ID NO: 91 (with U substituted for each T).177. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 91 , wherein the binding domain has C at position 136 and 138, numbering according to SEQ ID NO: 91 (with U substituted for each T).178. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 91 , wherein the binding domain has C at position 136 or 138, numbering according to SEQ ID NO: 91 (with U substituted for each T).179. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 91 , wherein the binding domain has C at position 136 and 138, numbering according to SEQ ID NO: 91 (with U substituted for each T).180. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 91 (with U substituted for each T).181. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 92, wherein the binding domain has C at position 51 , numbering according to SEQ ID NO: 92 (with U substituted for each T).182. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 92, wherein the binding domain has C at position 51 , numbering according to SEQ ID NO: 92 (with U substituted for each T).183. The RNA exon editor of embodiment 66, wherein the binding domain comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 92 (with U substituted for each T).184. The RNA exon editor of any one of embodiments 66 to 183, wherein the binding domain comprises one or more nucleotide substitutions relative to an endogenous MeCP2 mRNAsequence, wherein the one or more nucleotide substitutions disrupt a cryptic splice site within the binding domain sequence.185. The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence encoding a functional MeCP2 exon 4 amino acid sequence.186. The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence encoding a wildtype MeCP2 exon 4 amino acid sequence.187. The RNA exon editor of 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.188. The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence encoding a wildtype MeCP2 exon 3 amino acid sequence and a wildtype MeCP2 exon 4 amino acid sequence.189. The RNA exon editor of 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.190. The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence encoding a wildtype MeCP2 exon 2 amino acid sequence, a wildtype MeCP2 exon 3 amino acid sequence, and a wildtype MeCP2 exon 4 amino acid sequence.191. The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises, in a 5’ to 3’ direction: a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 7 (with U substituted for each T) and a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 8 (with U substituted for each T).192. The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises, in a 5’ to 3’ direction: the nucleotide sequence of SEQ ID NO: 7 (with U substituted for each T) and the nucleotide sequence of SEQ ID NO: 8 (with U substituted for each T).193. The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises, in a 5’ to 3’ direction: a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1 1 (with U substituted for each T) and a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 12 (with U substituted for each T).194. The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises, in a 5’ to 3’ direction: the nucleotide sequence of SEQ ID NO: 11 (with U substituted for each T) and the nucleotide sequence of SEQ ID NO: 12 (with U substituted for each T).195. The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises, in a 5’ to 3’ direction: a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 10 (with U substituted for each T), a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 11 (with U substituted for each T), and a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 12 (with U substituted for each T).196. The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 12 (with U substituted for each T).197. The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises the nucleotide sequence of SEQ ID NO: 12 (with U substituted for each T).198. The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises, in a 5’ to 3’ direction: the nucleotide sequence of SEQ ID NO: 10 (with U substituted for each T), the nucleotide sequence of SEQ ID NO: 11 (with U substituted for each T), and the nucleotide sequence of SEQ ID NO: 12 (with U substituted for each T).199. The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 9 (with U substituted for each T).200. The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises the nucleotide sequence of SEQ ID NO: 9 (with U substituted for each T).201 . The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises, in a 5’ to 3’ direction: a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 13 (with U substituted for each T) and a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 14 (with U substituted for each T).202. The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises, in a 5’ to 3’ direction: the nucleotide sequence of SEQ ID NO: 13 (with U substituted for each T) and the nucleotide sequence of SEQ ID NO: 14 (with U substituted for each T).203. The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 14 (with U substituted for each T).204. The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises the nucleotide sequence of SEQ ID NO: 14 (with U substituted for each T).205. The RNA exon editor of any one of embodiments 66 to 204, wherein the coding domain comprises at least one nucleotide substitution relative to the endogenous RNA molecule sequence, wherein the at least one nucleotide substitution disrupts a cryptic splice site within the coding domain sequence.206. The RNA exon editor of claim 205, wherein the at least one nucleotide substitution is a synonymous nucleotide substitution.207. The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises, in a 5’ to 3’ direction: an MeCP2 exon 3 nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 85 (with U substituted for each T) and an MeCP2 exon 4 nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 86 (with U substituted for each T), wherein the MeCP2 exon 3 nucleotide sequence comprises one or more of the following nucleotides: 43T, 49G, 55A, 67C, 85G, 91 G, 94A, 97A, 103C, 106A, 109G, 115A, 1 18T, 119T, 120C, 130T, 133G, 176T, 177C, 182T, 183C, 184G, 206T, 207C, 223A, 239C, 241 T, 280A, 290C, 317C, 320T, 321 C, or 331 A, numbering according to SEQ ID NO: 7, and wherein the MeCP2 exon 4 nucleotide sequence comprises one or more of the following nucleotides: 49T, 55A, 61T, 67G, 94T, 106A, 107C, 130A, 286T, 304A, 595T, 683C, 740T, 741 C, 806T, 807C, 808T, 809C, 824T, 825C, 851 T, 852C, 854T, 855C, 892G, 980C, 982C, 1034C, 1055C, 1072A, 1073C, or 1075T, numbering according to SEQ ID NO: 8.208. The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises, in a 5’ to 3’ direction: the nucleotide sequence of SEQ ID NO: 85 (with U substituted for each T) and the nucleotide sequence of SEQ ID NO: 86 (with U substituted for each T).209. The RNA exon editor of any one of embodiments 66 to 184, wherein the coding domain comprises, a nucleotide sequence comprising an MeCP2 exon 3 sequence and an MeCP2 exon 4 sequence, wherein the nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 76 (with U substituted for each T), and wherein the MeCP2 exon 3 nucleotide sequence comprises one or more of the following nucleotides: 43T, 49G, 55A, 67C, 85G, 91 G, 94A, 97A, 103C, 106A, 109G, 115A, 1 18T, 1 19T, 120C, 130T, 133G, 176T, 177C, 182T, 183C, 184G, 206T, 207C, 223A, 239C, 241T, 280A, 290C, 317C, 320T, 321 C, or 331A, numbering accordingto SEQ ID NO: 7 , and wherein the MeCP2 exon 4 nucleotide sequence comprises one or more of the following nucleotides: 49T, 55A, 61 T, 67G, 94T, 106A, 107C, 130A, 286T, 304A, 595T, 683C, 740T, 741 C, 806T, 807C, 808T, 809C, 824T, 825C, 851 T, 852C, 854T, 855C, 892G, 980C, 982C, 1034C, 1055C, 1072A, 1073C, or 1075T, numbering according to SEQ ID NO: 8.210. The RNA exon editor of any one of embodiments 66 to 209, wherein the hemi-intron comprises, operably linked in a 5’ to 3’ direction, a branch point, a polypyrimidine tract, and a 3’ splice site.211. The RNA exon editor of embodiment 210, wherein the branch point is at least 15 to 25 nucleotides upstream of the 3’ splice site.212. The RNA exon editor of embodiment 210 or 211, wherein the 3’ splice site comprises the nucleotide sequence YAG, wherein Y is a pyrimidine.213. The RNA exon editor of any one of embodiments 210 to 212, wherein the polypyrimidine tract comprises at least 10 consecutive pyrimidine nucleotides.214. The RNA exon editor of any one of embodiments 210 to 213, wherein the hemi-intron comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 17(with U substituted for each T).215. The RNA exon editor of any one of embodiments 210 to 214, wherein the hemi-intron comprises the nucleotide sequence of SEQ ID NO: 17(with U substituted for each T).216. The RNA exon editor of any one of embodiments 66 to 215, further comprising a 3’ untranslated region (3’ UTR) operably linked 3’ to the coding domain.217. The RNA exon editor of any one of embodiments 66 to 215, wherein the 3’ UTR comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 19 (with U substituted for each T) (RHDI pA 3’ UTR).218. The RNA exon editor of embodiment 216 or 217, wherein the 3’ UTR comprises the nucleotide sequence of SEQ ID NO: 19 (with U substituted for each T).219. The RNA exon editor of embodiment 216, wherein the 3’ UTR comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 20 (with U substituted for each T) (mWPRE 3’ UTR).220. The RNA exon editor of embodiment 219, wherein the 3’ UTR comprises the nucleotide sequence of SEQ ID NO: 20 (with U substituted for each T).221. The RNA exon editor of embodiment 216, wherein the 3’ UTR comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 87 (with U substituted for each T) (mWPRE 3’ UTR truncated).222. The RNA exon editor of embodiment 221, wherein the 3’ UTR comprises the nucleotide sequence of SEQ ID NO: 87 (with U substituted for each T).223. The RNA exon editor of any one of embodiments 66 to 222, further comprising a polyadenylation sequence having at least 90% sequence identity to SEQ ID NO: 21 (with U substituted for each T) (Sv40pA element).224. The RNA exon editor of embodiment 223, wherein the polyadenylation sequence comprises the nucleotide sequence of SEQ ID NO: 21 (with U substituted for each T).225. The RNA exon editor of any one of embodiments 66 to 222, further comprising a transcription terminator domain operably linked 3’ to the 3’ UTR.226. The RNA exon editor of embodiment 225, wherein the transcription terminator domain comprises a triple helix terminator.227. The RNA exon editor of embodiment 225 or 226, wherein the transcription terminator domain comprises a MALAT1 terminator.228. The RNA exon editor of any one of embodiments 225 to 227, wherein the transcription terminator domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 77 (with U substituted for each T).229. The RNA exon editor of any one of embodiments 225 to 228, wherein the transcription terminator domain comprises the nucleotide sequence of SEQ ID NO: 77 (with U substituted for each T).230. The RNA exon editor of any one of embodiments 225 to 227, wherein the transcription terminator domain comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 22 (with U substituted for each T).231. The RNA exon editor of any one of embodiments 225 to 227 or 230, wherein the transcription terminator domain comprises the nucleotide sequence of SEQ ID NO: 22 (with U substituted for each T).232. The RNA exon editor of any one of embodiments 225 to 231 , further comprising a 3xUBS sequence operably linked 3’ to the transcription terminator domain.233. The RNA exon editor of embodiment 232, wherein the 3xllBS sequence comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 79 (with U substituted for each T).234. The RNA exon editor of embodiment 232 or 233, wherein the 3xUBS sequence comprises the nucleotide sequence of SEQ ID NO:79 (with U substituted for each T).235. An RNA exon editor comprising a nucleotide sequence having at least 90% sequence identity to nucleotides 601-2522 of SEQ ID NO: 78 (with U substituted for each T).236. An RNA exon editor comprising the nucleotide sequence of nucleotides 601-2522 of SEQ ID NO: 78 (with U substituted for each T).237. An RNA exon editor comprising a nucleotide sequence having at least 90% sequence identity to nucleotides 601-2483 of SEQ ID NO: 80 (with U substituted for each T).238. An RNA exon editor comprising the nucleotide sequence of nucleotides 601-2483 of SEQ ID NO: 80 (with U substituted for each T).239. An RNA exon editor comprising a nucleotide sequence having at least 90% sequence identity to nucleotides 601-2522 of SEQ ID NO: 81 (with U substituted for each T).240. An RNA exon editor comprising the nucleotide sequence of nucleotides 601-2522 of SEQ ID NO: 81 (with U substituted for each T).241. An RNA exon editor comprising a nucleotide sequence having at least 90% sequence identity to nucleotides 601-3011 of SEQ ID NO: 82 (with U substituted for each T).242. An RNA exon editor comprising the nucleotide sequence of nucleotides 601-3011 of SEQ ID NO: 82 (with U substituted for each T).243. An RNA exon editor comprising a nucleotide sequence having at least 90% sequence identity to nucleotides 601-3250 of SEQ ID NO: 83 (with U substituted for each T).244. An RNA exon editor comprising the nucleotide sequence of nucleotides 601-3250 of SEQ ID NO: 83 (with U substituted for each T).245. An RNA exon editor comprising a nucleotide sequence having at least 90% sequence identity to nucleotides 601-3128 of SEQ ID NO: 84 (with U substituted for each T).246. An RNA exon editor comprising the nucleotide sequence of nucleotides 601-3128 of SEQ ID NO: 84 (with U substituted for each T).247. An RNA exon editor comprising, operably linked in a 5’ to 3’ direction:(a) a binding domain configured to bind to a binding site within a target region of a MeCP2 pre-mRNA;(b) a hemi-intron;(c) a coding domain comprising a sequence encoding one or more MeCP2 exons; and(d) a truncated WPRE 3’ UTR comprising a sequence having at least 90% sequence identity to SEQ ID NO: 87 (with U substituted for each T).248. The RNA exon editor of embodiment 247, wherein the truncated WPRE 3' UTR comprises or consists of SEQ ID NO: 87.249. An exon editor construct encoding the RNA exon editor of any one of embodiments 66 to 248.250. The exon editor construct of embodiment 249, further comprising, operably linked 5’ to the binding domain, a promoter.251. The exon editor construct of embodiment 249, further comprising, operably linked 5’ to the binding domain, a sequence having at least 90% sequence identity to SEQ ID NO: 15.252. The exon editor construct of embodiment 249, further comprising, operably linked 5’ to the binding domain, the sequence of SEQ ID NO: 15.253. 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 to SEQ ID NO: 78.254. An exon editor construct encoding the RNA exon editor of embodiment 236, wherein the exon editor construct comprises the sequence of SEQ ID NO: 78.255. 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 to SEQ ID NO: 80.256. An exon editor construct encoding the RNA exon editor of embodiment 238, wherein the exon editor construct comprises the sequence of SEQ ID NO: 80.257. 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 to SEQ ID NO: 81.258. An exon editor construct encoding the RNA exon editor of embodiment 240, wherein the exon editor construct comprises the sequence of SEQ ID NO: 81.259. 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 to SEQ ID NO: 82.260. An exon editor construct encoding the RNA exon editor of embodiment 242, wherein the exon editor construct comprises the sequence of SEQ ID NO: 82.261. 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 to SEQ ID NO: 83.262. An exon editor construct encoding the RNA exon editor of embodiment 244, wherein the exon editor construct comprises the sequence of SEQ ID NO: 83.263. 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 to SEQ ID NO: 84.264. An exon editor construct encoding the RNA exon editor of embodiment 246, wherein the exon editor construct comprises the sequence of SEQ ID NO: 84.265. A vector comprising the exon editor construct of any one of embodiments 249 to 264.266. The vector of embodiment 265, wherein the vector comprises a 5’ regulatory domain operatively linked 5’ to the binding domain.267. The vector of embodiment 266, wherein the 5’ regulatory domain comprises a constitutive promoter.268. The vector of embodiment 266, wherein the 5’ regulatory domain comprises a tissue specific promoter.269. A proviral plasmid comprising the exon editor construct of any one of embodiments 249 to 264.270. An adeno-associated virus (AAV) comprising the exon editor construct of any one of embodiments 249 to 264.271. The AAV of embodiment 270, wherein the AAV exhibits neuronal tropism.272. A composition comprising the exon editor construct of any one of embodiments 249 to 264, the vector of any one of embodiments 265 to 268, the proviral plasmid of embodiment 269, or the AAV of embodiments 270 to 271 , and a pharmaceutically acceptable excipient.273. A method of expressing functional MeCP2 in a target cell, the method comprising transducing the target cell with the exon editor construct of any one of embodiments 249 to 264, the vector of any one of embodiments 265to 268, the proviral plasmid of embodiment 269, or the AAV of embodiments 270 to 271 .274. The method of embodiment 273, wherein the MeCP2 pre-mRNA comprises one or more mutations associated with Rett Syndrome.275. The method of embodiment 274, wherein the at least one mutation associated with Rett Syndrome is within MeCP2 exon 3 or MeCP2 exon 4.276. A method of reducing expression of MeCP2 comprising at least one mutation associated with Rett Syndrome in a subject, the method comprising transfecting or transducing a neuron in the subject with the exon editor construct of any one of embodiments 249 to 264, the vector of any one of embodiments 265 to 268, the proviral plasmid of embodiment 269, the AAV of embodiments 270 to 271 , or the composition of embodiment 272.277. A method of correcting at least one mutation in an MeCP2 exon sequence in an MeCP2 pre-mRNA in a target cell of a subject, the method comprising administering to the subject the exon editor construct of any one of embodiments 249 to 264, the vector of any one of embodiments 255 to 268, the proviral plasmid of embodiment 269, the AAV of embodiments 270 to 271 , or the composition of embodiment 272.278. A method of treating Rett Syndrome in a subject in need thereof, the method comprising administering to the subject the exon editor construct of any one of embodiments 249 to 264, the vector of any one of embodiments 255 to 268, the proviral plasmid of embodiment 269, the AAV of embodiments 270 to 271 , or the composition of embodiment 272, in a therapeutically effective amount.279. The method of 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 the MeCP2 pre-mRNAs comprising at least one mutation associated with Rett Syndrome in the target cell are modified to become transcripts that encode functional MeCP2 wherein the at least one mutation is corrected.H. Examples

[0252] As described herein, the present inventors designed and tested MeCP2 RNA exoneditors for their ability to replace mutant MeCP2 exon 3 and exon 4 with wild-type and modified / engineered MeCP2 sequences via RNA trans-splicing. Such MeCP2 RNA exon editorshave significant therapeutic potential since correction of mutations observed in MeCP2 exons 3 and 4 would treat approximately 95% of the Rett Syndrome patient population.

[0253] As described herein, HEK293 cells were transfected with RNA exon editors comprising different binding domains that bind to binding sites spanning MeCP2 introns 1 , 2, or 3 to identify editors exhibiting a high efficiency of RNA trans-splicing as reflected by percent replacement of MeCP2 RNA. See, for example, FIGs. 6-15. In some embodiments, MeCP2 RNA exon-editors comprise a CMV enhancer and CMV promoter, a binding domain complementary to the target sequence of MeCP2 intron 1 , a hemi-intron comprising a 3’ splice site, native and modified MeCP2 wild-type sequences, a Myc-tag, and a 3’ UTR. The Myc-tag is used for detection of on-target protein of edited RNA sequences and may be removed for any therapeutic editor.

[0254] Intron 1 binding domain scan. As described herein, RNA exon editors comprising 31 different binding domains across the length of intron 1 were tested. See FIG. 6. These RNA exon editors were initially tested in an exemplary screening format, results from which are presented in FIG. 7. Sequence identifiers of the binding domains tested are set forth in Table 4. Various binding domains tested included nucleotide substitutions at cryptic splice sites identified as having a significant threshold for usage based on combinatorial analytics as indicated in Table 4 (numbering for nucleotide changes starts at the beginning (5’ end) of the BD sequence). The nucleotide changes are indicated relative to a binding domain sequence that would be 100% complementary to a MeCP2 pre-mRNA binding site. For instance, the G23C nucleotide change of MeCP2_intron1_51_150 (SEQ ID NO: 24) in Table 4 indicates that a binding domain 100% complementary to the corresponding binding site of MeCP2 intron 1 would have a G at position 23 of SEQ ID NO: 24, but after the nucleotide change the binding domain has a C at position 23 of SEQ ID NO: 24. Based on results presented in, for example FIG. 7, the present inventors identified a hot spot from -50 nucleotides (nt) to 1450 nt (based on the first nucleotide of the intron as position 1. See, e.g., FIG. 7. To confirm the results of the exemplary screening format assay, select exon editors were retested and the % replacement of MeCP2 was calculated. See FIG. 8. FIG. 9 shows a summary of the activity of RNA exon editors comprising exemplary intron 1 binding domains. The top performing binding domains have 25-30% replacement of MeCP2 mRNA.TABLE 4 - Intron 1 Binding Domain Sequence Identifiers

[0255] Intron 2 binding domain scan. Intron 2 is nearly 60kb in length. Accordingly, the present inventors designed and tested RNA exon editors comprising binding domains near the 5’ and 3’ termini of intron 2 in an initial assay. RNA exon editors comprising ten different binding domains were tested as shown in FIG. 10. Sequence identifiers of the binding domains tested are set forth in Table 5. As shown in FIG. 1 1 , an exemplary RNA exon editor comprising a specific binding domain (mecp2_intron2_99_150) was identified that exhibited 10% replacement. Other RNA exon editors comprising different binding domains exhibited activity similar to that of a scrambled binding domain (negative control). FIG. 12 presents a summary of data pertaining to RNA exon editors comprising binding domains that bind to intron 2.TABLE 5 - Intron 2 Binding Domain Sequence Identifiers

[0256] Additional Intron 2 binding domain scan. MeCP2 intron 2 is 59,626 bp in length.The present inventors designed and tested RNA exon editors comprising 18 additional binding domains along intron 2, including variant binding domains in which nucleotide substitutions were made at cryptic splice sites identified as having a significant threshold for usage based on combinatorial analytics. Results are shown in FIG. 19. The additional binding domains tested did not perform better than the binding domains identified in FIG. 11.

[0257] Sequence identifiers of the binding domains tested are set forth in Table 6, along with any nucleotide substitutions in the tested binding domain sequences (numbering for nucleotide changes starts at the beginning (5’ end) of the BD sequence). The nucleotide chang...

Claims

CLAIMS1. An exon editor construct that encodes an RNA exon editor, wherein the exon editor construct comprises sequences encoding:(a) a binding domain that binds a target intron of a MeCP2 pre-mRNA, wherein the target intron of the MeCP2 pre-mRNA is intron 1 or intron 2;(b) a hemi-intron; and(c) a coding domain comprising one or more MeCP2 exons.

2. The exon editor construct of 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 4020-4169 of intron 1.

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

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

5. The exon editor construct of 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.

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

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

8. The exon editor construct of 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.

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

10. The exon editor construct of any one of claims 1 to 9, wherein the sequence encoding the binding domain comprises, consists essentially of, or consists 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.

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

12. The exon editor construct of any one of claims 1 to 9, wherein the sequence encoding the binding domain comprises, consists essentially of, or consists of any one of SEQ ID NOs: 24, 28, 29, 30, 116, 119, or 120.

13. The exon editor construct of any one of claims 1 to 12, wherein the coding domain comprises, consists essentially of, or consists of :SEQ ID NOs: 7 and 8 or a sequence 90% identical to SEQ ID NOs: 7 and 8;SEQ ID NO: 76 or a sequence 90% identical to SEQ ID NO: 76;SEQ ID NOs: 13 and 14 or a sequence 90% identical to SEQ ID NOs: 13 and 14;SEQ ID NO: 12;SEQ ID NO: 14; orSEQ ID NO: 9.

14. The exon editor construct of any one of claims 1 to 12, wherein the coding domain comprises, consists essentially of, or consists of any one of SEQ ID NOs: 76 or 14.

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

16. The exon editor construct of any one of claims 1 to 15, wherein the hemi-intron comprises a 3’ splice site and optionally, a branchpoint sequence and a polypyrimidine tract, wherein the binding domain, the hemi-intron comprising the 3’ splice site, and the coding domain are operatively linked in a 5’-to-3’ direction.

17. The exon editor construct of claim 16, wherein the hemi-intron comprises, consists essentially of, or consists of: polypyrimidine tract, a branchpoint, and a 3’ splice site.

18. The exon editor construct of any one of claims 16 or 17, wherein the hemi-intron comprises, consists essentially of, or consists of:SEQ ID NO: 17 or a sequence having at least 90% identity to SEQ ID NO: 17.

19. The exon editor construct of any one of claims 1 to 18, further comprising a 3’ untranslated region (3’ UTR), wherein the binding domain; the hemi-intron; the coding domain; and the 3’ UTR; are operatively linked in a 5’-to-3’ direction.

20. The exon editor construct of claim 19, wherein the 3’ UTR comprises an RDHI pA 3’ UTR (SEQ ID NO: 19) or a mWPRE 3’ UTR (SEQ ID NO: 20).

21. The exon editor construct of claim 20, wherein the RDHIpA 3’ UTR comprises, consists essentially of, or consists of SEQ ID NO: 19 or a sequence having at least 90% identity to SEQ ID NO: 19; or the mWPRE 3’ UTR comprises, consists essentially of, or consists of SEQ ID NO: 20 or a sequence having at least 90% identity to SEQ ID NO: 20.

22. The exon editor construct of any one of claims 1 to 21 , wherein the exon editor construct comprises: a binding domain comprising, consisting essentially 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, consisting essentially of, or consisting of SEQ ID NO: 17; a coding domain comprising, consisting essentially of, or consisting of SEQ ID NO: 76; and a 3’ UTR comprising, consisting essentially of, or consisting of SEQ ID NO: 20, wherein the binding domain, the hemi-intron, the coding domain, and the 3’ UTR are operatively linked in a 5’-to-3’ direction.

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

24. The exon editor construct of claim 22, wherein the exon editor construct comprises, consists essentially of, or consists of SEQ ID NO: 78, 80, or 83.

25. The exon editor construct of any one of claims 1 to 24, further comprising a triple helix terminator, wherein the binding domain; the hemi-intron; the coding domain; the 3’ UTR, when present; and the triple helix terminator are operatively linked in a 5’-to-3’ direction.

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

27. The exon editor construct of claim 25, wherein the triple helix terminator comprises, consists essentially of, or consists of SEQ ID NO: 22.

28. The exon editor construct of any one claims 1 to 27, further comprising a sequence encoding a poly A sequence, for example, SEQ ID NO: 21.

29. The exon editor construct of any one of claims 1 to 28, further comprising a sequence encoding an epitope tag, wherein the binding domain; the hemi-intron; the coding domain; the epitope tag; the 3’ UTR, when present; and the triple helix terminator, when present; are operatively linked in a 5’-to-3’ direction.

30. The exon editor construct of claim 28, wherein the exon editor construct comprises, consists essentially of, or consists of SEQ ID NO: 78.

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

32. The exon editor construct of any one of claims 1 to 30, wherein the MeCP2 pre-mRNA comprises at least one mutation associated with Rett Syndrome.

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

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

35. The exon editor construct of any one of claims 32 to 34, wherein MeCP2 protein comprising the 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.

36. A vector comprising the exon editor construct of any one of claims 1 to 30 or 32 to 35.

37. The vector of claim 36, wherein the vector comprises a 5’ regulatory domain operatively linked 5’ to the binding domain.

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

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

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

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

42. The AAV of claim 41, wherein the AAV comprises a 5’ regulatory domain operatively linked 5’ to the binding domain.

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

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

45. The AAV of any one of claims 41 to 44, wherein the AAV exhibits neuronal tropism.

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

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

48. The composition of claim 47, comprising a pharmaceutically acceptable excipient.

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

50. The method of claim 49, wherein at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of the MeCP2 pre-mRNAs comprising at least one mutation associated with Rett Syndrome in the target cell are modified to become transcripts that encode functional MeCP2 wherein the at least one mutation is corrected.

51. The method of claim 50, wherein at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of the MeCP2 pre-mRNAs comprising at least one mutation associated with Rett Syndrome in the target cell are modified to become transcripts that encode functional MeCP2 wherein the at least one mutation is corrected.

52. The method of claim 51 , wherein at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the MeCP2 pre-mRNAs comprising at least one mutation associated with Rett Syndrome in the target cell are modified to become transcripts that encode functional MeCP2 wherein the at least one mutation is corrected.

53. The method of any one of claims 49 to 52, wherein functional levels of MeCP2 are restored in the target cell by expressing biologically functional MeCP2 protein.

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

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

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

57. The method of any one of claims 54 to 56, wherein at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of the MeCP2 pre-mRNAs comprising at least one mutation associated with Rett Syndrome in the target cell are modified to become transcripts that encode functional MeCP2 wherein the at least one mutation is corrected.

58. The method of claim 57, wherein at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of the MeCP2 pre-mRNAs comprising at least one mutation associated with Rett Syndrome in the target cell are modified to become transcripts that encode functional MeCP2 wherein the at least one mutation is corrected.

59. The method of claim 58, wherein at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the MeCP2 pre-mRNAs comprising at least one mutation associated with Rett Syndrome in the target cell are modified to become transcripts that encode functional MeCP2 wherein the at least one mutation is corrected.

60. The method of any one of claims 54 to 59, wherein functional levels of MeCP2 are restored in the target cell by expressing biologically functional MeCP2 protein.

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

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

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

64. The exon editor construct of any one of claims 1 to 30 or 32 to 35, the vector of any one of claims 36 to 39, the proviral plasmid of claim 40, the AAV of any one of claims 41 to 46, or the composition of any one of claims 47 to 48 for use in preventing or treating Rett Syndrome in a subject in need thereof.

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

66. An RNA exon editor comprising, operably linked in a 5’ to 3' direction:(a) a binding domain configured to bind to a binding site within a target region of a 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 cryptic splice site.

67. An RNA exon editor comprising, operably linked in a 5’ to 3' direction:(a) a binding domain configured to bind to a binding site within a target region of a 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 a corresponding wild type MeCP2 pre-mRNA sequence at a cryptic splice site.

68. An RNA exon editor comprising, operably linked in a 5’ to 3' direction:(a) a binding domain configured to bind to a binding site within a target region of a pre- mRNA of a target gene;(b) a hemi-intron;(c) a coding domain comprising a sequence encoding one or more exons of the target gene; and(d) a 3’-truncated WPRE 3’ UTR sequence comprising or consisting of a sequence having at least 90% sequence identity to SEQ ID NO: 87.

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