WO2025166360A1

WO2025166360A1 - Compositions for and methods of engineering the transcriptome

Info

Publication number: WO2025166360A1
Application number: PCT/US2025/014341
Authority: WO
Inventors: Aravind Asokan; David FIFLIS
Original assignee: Duke University
Current assignee: Duke University
Priority date: 2024-02-03
Filing date: 2025-02-03
Publication date: 2025-08-07
Anticipated expiration: 2026-08-03

Abstract

Disclosed herein are compositions for and methods of generating chimeric RNA molecules via trans-splicing and methods for determining the efficacy of guide RNA and RNA targeting motifs in trans-splicing processes including, for example, SMaRT, CRAFT, and GRAFT.

Description

COMPOSITIONS FOR AND METHODS OF ENGINEERING THE TRANSCRIPTOME

I. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63/549,494 filed 3 February 2024 and U.S. Provisional Application No. 63/571,441 filed 29 March 2024, each of which is incorporated herein in its entirety.

II. REFERENCE TO THE SEQUENCE LISTING

[0002] The Sequence Listing submitted 3 February 2025 as an XML file named “24-3010-WO- Sequence-Listing”, created on 3 February 2025 and having a size of 204,800 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

III. BACKGROUND

[0003] The cellular RNA processing machinery has many attributes that can be exploited to manipulate the transcriptome. Specifically, RNA splicing is well conserved in higher eukaryotes and executed by a large ribonucleoprotein complex called the spliceosome. The canonical function of the spliceosome is to catalyze a dual trans-esterification reaction, that joins adjacent exons on the same transcript and removes the intervening intronic sequence; a process referred to as cis-splicing. This splicing machinery has been previously exploited to achieve mRNA transsplicing, which involves targeted incorporation of recombinant exon or exons into a pre-mRNA transcript. This approach features an RNA molecule comprised of an antisense sequence linked to a hemi-intron and one or more exons. Following delivery to the nucleus, the hybridization of the antisense binding domain to the target pre-mRNA enables the intronic sequence to incorporate the recombinant exons in trans by co-opting the splicing machinery, resulting in a chimeric mRNA product. This process has been enhanced through combination with next generation RNA targeting systems such as CRISPR/Casl3.

[0004] One bottleneck to the translation of technologies is optimizing the editing efficiency at a given target. Testing out individual variations of trans-splicing platforms is laborious and can become unwieldy quickly due to the vast parameter space that exists for such technologies (i.e., guide position, guide length, guide number, intron composition, untranslated regions, RNA binding protein recruitment domains, etc.). These efforts are further complicated by the transient nature of RNA editing, and that the region of the trans-splicing RNA responsible for catalyzing editing is not preserved in the final edited product (i.e., the guide is in the intron that undergoes splicing and as such is not a part of the mature RNA). Thus, a pooled approach to screen guide candidates is used, it is not possible to discern from the mature RNA which guide was responsible for trans-splicing. [0005] Thus, there remains an urgent need for a means to ascertain the efficacy of RNA guides and targeting motifs in a trans-splicing event. Consequently, the present disclosure provides compositions for and methods of identifying effective RNA guides and targeting motifs.

IV. BRIEF DESCRIPTION OF THE FIGURES

[0006] FIG. 1 is a schematic showing the generation of a 5’ replacement construct to be used in a disclosed method.

[0007] FIG. l is a schematic showing the replacement strategy for replacing in trans of exons in a 5’ segment of a pre-mRNA using a 5’ replacement construct.

[0008] FIG. 3 is a schematic showing the generation of internal replacement constructs to be used in a disclosed method.

[0009] FIG. 4 is a schematic showing the replacement strategy for replacing in trans of an internal exon of pre-mRNA using an internal replacement construct.

[0010] FIG. 5 is a schematic showing the generation of a 3’ replacement construct to be used in a disclosed method.

[0011] FIG. 6 is a schematic showing the replacement strategy for replacing in trans of exons in a 3’ segment of a pre-mRNA using a 3’ replacement construct.

[0012] FIG. 7 show the validation of trans-splicing in the DP71 transcript using disclosed composition and methods. Briefly, HEK293 cells were transfected with the following DNA constructs: Lane 1 : (SEQ ID NO:01), Lane 2 (SEQ ID NO:07), Lane 3 (SEQ ID NO:01 and SEQ ID NO:04), and Lane 4 (SEQ ID NO:01 and SEQ ID NO:07). 72 hours post-transfection, RNA was harvested with TriZOL reagent using the manufacturer’s directions. Purified RNA was converted to cDNA with Applied Biosciences’ High Capacity RNA-to-cDNA kit. Trans-splicing was then detected via PCR amplification using primers annealing to exon 72 (SEQ ID NO:34) and the mScarlet ORF (SEQ ID NO:35) of cDNA of cells.

[0013] FIG. 8A - FIG. 8B show the 3’ DMD Sanger sequencing confirmation of the transspliced product. FIG. 8A shows a schematic of cis (top) and trans (bottom) spliced RNA products while FIG. 8B shows the alignment of Sanger sequencing traces of cis (top) and trans (bottom) spliced RNA. Notable in the trans-spliced PCR product a silent A > G mutation was observed and highlighted. Briefly, cis-spliced RNA sample corresponds to the same transfection and harvest conditions as Lane 1 of FIG. 7 and trans-spliced sample was gel extracted from the band observed in Lane 4 of FIG. 7. These samples were amplified via PCR with primers comprising the sequence of SEQ ID NO:36 and SEQ ID NO:37.

[0014] FIG. 9 A - FIG. 9B shows the HTS data for DMD editing using the RNA editing efficiency. FIG. 9A shows the RNA editing strategy with no editing (top) and editing (bottom). FIG. 9B shows that editing efficiency was based on amplicon sequencing of total cDNA from cells amplified with sequencing primer set (SEQ ID NO:36 and SEQ ID NO:37). Efficiency was quantified as the percent of transcripts containing the silent A > G (E3580) mutation. Briefly, HEK293 cells were transfected with the following DNA constructs: Lane 1 (SEQ ID NO:01), Lane 2 (SEQ ID NO: 07), Lane 3 (SEQ ID NO:01 and SEQ ID NO: 04), Lane 4 (SEQ ID NO:01 and SEQ ID NO: 07). 72 hours post-transfection RNA was harvested with TriZOL reagent following manufacturer’s directions. Purified RNA was converted to cDNA with Applied Biosciences’ High-Capacity RNA-to-cDNA kit and was amplified via PCR amplification using primers SEQ ID NO:36 and SEQ ID NO:37 of cDNA of cells. Amplicons were then processed on an Illumina Hi-Seq and were analyzed using CRISPRESSO2 software.

[0015] FIG. 10 shows a comparison of the SMaRT technology vs. Protein Mediated TransSplicing HTS. Here, a comparison of RNA trans-splicing via anti-sense targeting based approach in comparison to the proposed RNP-mediated approach via HTS in accordance with one embodiment of the present disclosure. A direct comparison of editing efficiency at the DMD locus was compared between the two approaches to demonstrate the improvement over existing technology. Briefly, 3 separate guides targeting intron 74 of the DMD locus were chosen to compare the system (i.e., Guides A, B, and C). Editing efficiency was based on amplicon sequencing of total cDNA from cells amplified with sequencing primer set (SEQ ID NO:34 and SEQ ID NO:35). Efficiency was quantified as the percent of transcripts containing the silent A > G (E3580) mutation. HEK293 cells were transfected with the following DNA constructs, Guide A/SMaRT (SEQ ID NO: 10), Guide A/CRAFT (SEQ ID NO:01 and SEQ ID NO:05), Guide B/SMaRT (SEQ ID NO: 11), Guide B/CRAFT (SEQ ID NO:01 and SEQ ID NO:06), Guide C/SMaRT (SEQ ID NO: 12), Guide A/CRAFT (SEQ ID NO:01 and SEQ ID NO:07). Purified RNA was converted to cDNA with Applied Biosciences’ High-Capacity RNA-to-cDNA kit and amplified via PCR amplification using primers SEQ ID NO:36 and SEQ ID NO:37 of cDNA of cells. Amplicons were then processed on an Illumina Hi-Seq and analyzed using CRISPRESSO2 software.

[0016] FIG. HA - FIG. 11B shows a strategy for 3 ’ DMPK editing and the subsequent validation of 3 ’ trans-splicing in the DMPK transcript via binary PCR based readout. Briefly, HEK293 cells were transfected with the following DNA constructs: Lane 1 (SEQ ID NO:01), Lane 2 (SEQ ID NO: 09), Lane 3 (SEQ ID NO:01 and SEQ ID NO: 08), Lane 4 (SEQ ID NO:01 and SEQ ID NO: 09). 72 hours post-transfection RNA was harvested with TriZOL reagent following manufacturer’s directions. Purified RNA was converted to cDNA with Applied Biosciences’ High-Capacity RNA-to-cDNA kit. Trans-splicing was then detected via PCR amplification using primers annealing to DMPK exon 7 (SEQ ID NO:38) and the mScarlet ORF (SEQ ID NO:39) of cDNA of cells. FIG. 11B shows that precise amplification of target DNA yielded a band at ~1 kb as observed exclusively in Lane 4 of the gel.

[0017] FIG. 12 shows the 3 ’ DMPK Sanger sequencing results, which confirmed the trans-spliced product. Alignment of the cDNA obtained from wild HEK293 cells against the trans-spliced PCR product from the lane 4 of FIG. 11. Alignment of sanger sequencing traces of cis (top) and trans (bottom) spliced RNA. Notable in the trans-spliced PCR product a silent G > T mutation was observed and highlighted. Briefly, cis-spliced RNA sample corresponded to the same transfection and harvest conditions as Lane 1 of FIG. 12, and trans-spliced sample was gel extracted from the band observed in Lane 4 of FIG. 11. These samples were amplified via PCR with primers comprising the sequence of SEQ ID NO:40 and SEQ ID NO:41.

[0018] FIG. 13A - FIG. 13B show the validation of 3 ’ trans-splicing in the LMNA transcript via binary PCR based readout. Briefly, HEK293 cells were transfected with the following DNA constructs: Lane 1 (SEQ ID NO: 16), Lane 2 (SEQ ID NO:01 and SEQ ID NO: 15), Lane 3 (SEQ ID NO:01 and SEQ ID NO: 16). 72 hours post-transfection, RNA was harvested with TriZOL reagent following manufacturer’s directions. Purified RNA was converted to cDNA with Applied Biosciences’ High-Capacity RNA-to-cDNA kit. Trans-splicing was then detected via PCR amplification using primers annealing to LMNA exon 6 (SEQ ID NO:44) and the mScarlet ORF (SEQ ID NO:45) of cDNA of cells. Precise amplification of target DNA yielded a band at ~1 kb as observed exclusively in Lane 3 of the gel (FIG. 13B).

[0019] FIG. 14 shows 3’ LMNA Sanger sequencing confirmation of the trans-spliced product. Alignment of the cDNA obtained from wild HEK293 cells (top) against the trans-spliced PCR product (bottom) from the lane 3 of FIG. 13. Notable in the trans-spliced PCR product a silent G > A mutation was observed and is highlighted.

[0020] FIG. 15 shows 3’ LMNA codon optimized replacement, which demonstrated the complete rewriting of replaced DNA sequence. Briefly, HEK293 cells were transfected with SEQ ID NO:01 and SEQ ID NO:22. Then, 72 hours post-transfection, RNA was harvested with TriZOL reagent following the manufacturer’s directions. Purified RNA was converted to cDNA with Applied Biosciences’ High-Capacity RNA-to-cDNA kit. Trans-splicing was then detected via PCR amplification using primers annealing to LMNA exon 10 (SEQ ID NO:46) and the 3’ UTR (SEQ ID NO:47) of cDNA of cells. Sanger sequencing was completed with a primer corresponding to SEQ ID NO:46. At the top is a schematic showing the trans-spliced RNA molecule generated comprising the endogenous exons 1-10 of human LMNA, followed by codon optimized exons 11-12 of human LMNA. Below is an alignment of the codon optimized sequence to the hg38 reference transcript, and the exon 10-11 exon junction is denoted. Notably the alignment to exon 10 is perfect, whereas the alignment to exon 11 displays a difference at the codon optimized sequence. A representative Sanger sequencing trace is shown.

[0021] FIG. 16A - FIG. 16B show 5’ LMNA editing gel, validating the 5’ trans-splicing in the LMNA transcript via binary PCR based readout. Briefly, HEK293 cells were transfected with the following DNA constructs: Lane 1 (SEQ ID NO:03), Lane 2 (SEQ ID NO:20), Lane 3 (SEQ ID NO:03 and SEQ ID NO: 19), and Lane 4 (SEQ ID N0:03 and SEQ ID NO:20). Then, 72 hours pos-transfection, RNA was harvested with TriZOL reagent following manufacturer’s directions. Purified RNA was converted to cDNA with Applied Biosciences’ High-Capacity RNA-to-cDNA kit. Trans-splicing was then detected via PCR amplification using primers annealing to LMNA exon 4 (SEQ ID NO:50) and the mScarlet ORF (SEQ ID NO:51) of cDNA of cells. Precise amplification of target DNA yielded a band at ~1 kb as observed exclusively in Lane 4 of the gel. [0022] FIG. 17 shows 5’ LMNA Sanger sequencing, which confirmed the trans-spliced product. Alignment of the cDNA obtained from wild HEK293 cells (top) against the trans-spliced PCR product from the lane 4 of FIG. 16B. Notable in the trans-spliced PCR product, a silent G > C mutation was observed and is highlighted. Briefly, cis-spliced RNA sample corresponded to the same transfection and harvest conditions as Lane 1 of FIG. 16A, and trans-spliced sample was gel extracted from the band observed in Lane 4 of FIG. 16B. The primer corresponding to SEQ ID NO. 51 was used to sequence these samples.

[0023] FIG. 18A - FIG. 18B provide the RNA editing strategy and HTS data for DMPK editing. The editing efficiency was based on amplicon sequencing of total cDNA from cells amplified with sequencing primer set (SEQ ID NO:40 and SEQ ID NO:41). Efficiency was quantified as the percent of transcripts containing the silent T > A (P593) mutation. Briefly, in FIG. 18B, HEK293 cells were transfected with the following DNA constructs: Lane 1 (SEQ ID NO:01), Lane 2 (SEQ ID NO: 14), Lane 3 (SEQ ID NO:01 and SEQ ID NO: 13), and Lane 4 (SEQ ID NO:01 and SEQ ID NO: 14). Then, 72 hours post-transfection, RNA was harvested with TriZOL reagent following the manufacturer’s directions. Purified RNA was converted to cDNA with Applied Biosciences’ High-Capacity RNA-to-cDNA kit and amplified via PCR amplification using primers SEQ ID NO:42 and SEQ ID NO:43 of cDNA of cells. Amplicons were then processed on an Illumina Hi- Seq and was analyzed using CRISPRESSO2 software.

[0024] FIG. 19A - FIG. 19B provide the RNA editing strategy and HTS data for LMNA editing. The editing efficiency was based on amplicon sequencing of total cDNA from cells amplified with sequencing primer set (SEQ ID NO:48 and SEQ ID NO:49). Efficiency was quantified as the percent of transcripts containing the silent T > C (A577) mutation. Briefly, in FIG. 19B, HEK293 cells were transfected with the following DNA constructs: Lane 1 (SEQ ID NO:01), Lane 2 (SEQ ID NO: 18), Lane 3 (SEQ ID NO:01 and SEQ ID NO: 17), and Lane 4 (SEQ ID NO:01 and SEQ ID NO: 18). Then, 72 hours post-transfection, RNA was harvested with TriZOL reagent following manufacturer’s directions. Purified RNA was converted to cDNA with Applied Biosciences’ High-Capacity RNA-to-cDNA kit and was amplified via PCR amplification using primers SEQ ID NO:48 and SEQ ID NO:49 of cDNA of cells. Amplicons were then processed on an Illumina Hi-Seq and was analyzed using CRISPRESSO2 software.

[0025] FIG. 20 (left side) shows a mechanism of 5’ trans-splicing while FIG. 20 (right side) shows the mechanism of 3 ’ trans-splicing. The schematic on the left side shows first the constructs that were transfected to demonstrate trans-splicing for 5’ replacement. The stop codon in the first half of the open reading frame blocks translation if the RNA species splices in cis. If the trans- splicing RNA successfully edits the RNA, then the open reading frame of EGFP is restored and fluorescent expression is restored. The schematic on the right side shows first the constructs that were transfected to demonstrate trans-splicing for 3’ replacement. The stop codon in the second half of the open reading frame blocks translation if the RNA species splices in cis. If the trans- splicing RNA successfully edits the RNA, the open reading frame of EGFP is restored and fluorescent expression is restored. GRAFT is Guide RNA Assisted Fragment Trans-splicing.

[0026] FIG. 21A - FIG. 21B show a panel of 5 ’-splicing motifs. These are flow cytometry data from co-transfection experiment of 5’ trans-splicing candidate molecules. In FIG. 21 A, the percent of cells that express green fluorescence when trans-splicing RNA candidates were delivered to cells was measured. RNA structures 1-11 are plotted against the x axis, and percent GFP positive cells is plotted on the y axis. In FIG. 21B, the mean fluorescent intensity of cells when trans-splicing RNA candidates were delivered was measured. RNA structures 1-11 were plotted against the x axis, and mean fluorescent intensity is plotted on the y axis.

[0027] FIG. 22A - FIG. 22D show a panel of 3 ’ trans-splicing motifs. Flow cytometry data from co-transfection experiment of 3’ trans-splicing candidate molecules. In FIG. 22A, the percent of cells that express green fluorescence when trans-splicing RNA candidates were delivered to cells was measured. RNA structures 1-11 are plotted against the x axis, and percent GFP positive cells is plotted on the y axis. In FIG. 22B, the mean fluorescent intensity of cells when trans-splicing RNA candidates were delivered was measured. RNA structures 1-11 are plotted against the x axis, and mean fluorescent intensity is plotted on the y axis. The data in FIG. 22C and FIG. 22D were measured in the same way for a different target intron.

[0028] FIG. 23A - FIG. 23B show repeat validation of top 3’ Trans-splicing RNA candidates. Flow cytometry data from co-transfection experiment of 3’ trans-splicing candidate molecules. In FIG. 23A, the percent of cells that express green fluorescence when trans-splicing RNA candidates were delivered to cells was measured. RNA structures are plotted against the x axis, and percent GFP positive cells is plotted on the y axis. In FIG. 23B, the mean fluorescent intensity of cells when trans-splicing RNA candidates were delivered was measured. RNA structures are plotted against the x axis, and mean fluorescent intensity is plotted on the y axis.

[0029] FIG. 24A - FIG. 24B show repeat validation of top 3’ trans-splicing RNA candidates against new target. Flow cytometry data from co-transfection experiment of 3’ trans-splicing candidate molecules was measured. In FIG. 24A, the percent of cells that express green fluorescence when trans-splicing RNA candidates we are delivered to cells was measured. RNA structures are plotted against the x axis, and percent GFP positive cells is plotted on the y axis. In FIG. 24B, the mean fluorescent intensity of cells when trans-splicing RNA candidates were delivered was measured. RNA structures are plotted against the x axis, and mean fluorescent intensity is plotted on the y axis.

[0030] FIG. 25A - FIG. 25G provide plasmid maps for the constructs used in Example 1 and/or disclosed herein. FIG. 25A shows an exemplary 3’ Replacement Construct (Null). FIG. 25B the 3’ Replacement Construct for RYR2 (3-GRAFT-RYR2) while FIG. 25C shows the 5’ Replacement Construct for RYR2 (5-GRAFT-RYR2). FIG. 25D shows the 3’ Replacement Construct for LMNA (3-GRAFT-LMNA) while FIG. 25E shows the 3’ Replacement Construct for FXN (3-GRAFT-FXN). FIG. 25F shows the Split GFP Reporter Construct for LMNA while FIG. 25G shows Split GFP Reporter Construct for RYR2.

[0031] FIG. 26A shows the 5’ replacement construct for DMD in Example 2. FIG. 26B shows the 3’ replacement construct for DMD in Example 10.

[0032] FIG. 27A - FIG. 27D show high-throughput guide selection through guide coupled barcode, validation, and comparison to existing strategy. FIG. 27A shows a schematic of the disclosed barcode approach. FIG. 27B shows an enrichment plot for each guide targeting Imna intron 10/11 as a function of position. FIG. 27C shows the frequency of 3’-CRAFT editing in endogenous Imna transcripts. FIG. 27D shows the optimization of 3’ rcRNA guide position using the former SplitGFP reporter system.

[0033] FIG. 28A - FIG. 28C show the reproducibility of barcode strategy using DMPK. FIG. 28A is an enrichment plot for each guide targeting dmpk intron 13/14 as a function of position. FIG. 28B is a schematic of endogenous dmpk transcript (left) and edited dmpk transcript (right). FIG. 28C shows the frequency of 3 ’-CRAFT editing in endogenous dmpk transcripts. V. BRIEF SUMMARY

[0034] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence to be transspliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein.

[0035] Disclosed herein is a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive PspdCasl3b, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive PspdCasl3b; and a polyadenylation signal.

[0036] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence to be trans- spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron; a 5’ hemi intron; two or more guide RNA sequences; a promoter operably linked to the two or more guide RNA sequences; and a nucleic acid sequence encoding one or more RNA binding proteins.

[0037] Disclosed herein is a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d; a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal.

[0038] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence to be trans- spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein.

[0039] Disclosed herein is a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d; a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal.

[0040] Disclosed herein is a transcriptome engineering system comprising (i) a first isolated nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3 ’ hemi intron linked to the nucleic acid sequence to be trans- spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein; and (ii) a second isolated nucleic acid molecule comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal.

[0041] In an aspect, a disclosed first isolated nucleic acid molecule and a disclosed second isolated nucleic acid molecule can form a ternary complex with the endogenous pre-mRNA molecule. In an aspect, a disclosed resulting chimeric RNA molecule can comprise the trans-spliced nucleic acid sequence.

[0042] Disclosed herein is a transcriptome engineering system comprising (i) a first isolated nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the nucleic acid sequence to be trans- spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein; and (ii) a second isolated nucleic acid molecule comprising a nucleic acid sequence encoding a catalytically inactive PspdCasl3b, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive PspdCasl3b; and a polyadenylation signal.

[0043] Disclosed herein is a transcriptome engineering system, comprising (i) a first isolated nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron; a 5’ hemi intron; two or more guide RNA sequences; a promoter operably linked to the two or more guide RNA sequences; and a nucleic acid sequence encoding one or more RNA binding proteins; and (ii) a second isolated nucleic acid molecule comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal.

[0044] Disclosed herein is a vector comprising a disclosed isolated nucleic acid molecule. Disclosed herein is a vector comprising one or more disclosed isolated nucleic acid molecules.

[0045] Disclosed herein is a vector comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein.

[0046] Disclosed herein is a vector comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron; a 5’ hemi intron; two or more guide RNA sequences; a promoter operably linked to the two or more guide RNA sequences; and a nucleic acid sequence encoding one or more RNA binding proteins. [0047] Disclosed herein is a vector comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein.

[0048] Disclosed herein is a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d; a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal.

[0049] Disclosed herein is a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a catalytically inactive PspdCasl3b, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive PspdCasl3b; and a polyadenylation signal.

[0050] Disclosed herein is a pharmaceutical formulation comprising a vector comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre- mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein.

[0051] Disclosed herein is a pharmaceutical formulation comprising a vector comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron; a 5’ hemi intron; two or more guide RNA sequences; a promoter operably linked to the two or more guide RNA sequences; and a nucleic acid sequence encoding one or more RNA binding proteins.

[0052] Disclosed herein is a pharmaceutical formulation comprising a vector comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre- mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein. [0053] Disclosed herein is a pharmaceutical formulation comprising a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d; a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal.

[0054] Disclosed herein is a pharmaceutical formulation comprising a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a catalytically inactive PspdCasl3b, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive PspdCasl3b; and a polyadenylation signal.

[0055] Disclosed herein is a method of generating a chimeric RNA molecule in a cell, the method comprising contacting an endogenous pre-mRNA in a cell with (i) a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein, and (ii) a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive PspdCasl3b, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive PspdCasl3b; and a polyadenylation signal, wherein the isolated nucleic acid molecules form a ternary complex with the endogenous pre-mRNA molecule, and wherein the resulting chimeric RNA molecule comprises the trans-spliced nucleic acid sequence.

[0056] Disclosed herein is a method of generating a chimeric RNA molecule in a cell, the method comprising contacting an endogenous pre-mRNA in a cell with (i) a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron; a 5’ hemi intron; two or more guide RNA sequences; a promoter operably linked to the two or more guide RNA sequences; and a nucleic acid sequence encoding one or more RNA binding proteins; and (ii) a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d; a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal, wherein the isolated nucleic acid molecules form a ternary complex with the endogenous pre-mRNA molecule, and wherein the resulting chimeric RNA molecule comprises the trans-spliced nucleic acid sequence. [0057] Disclosed herein is a method of generating a chimeric RNA molecule in a cell, the method comprising contacting an endogenous pre-mRNA in a cell with (i) a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein; and (ii) a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d; a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal, wherein the isolated nucleic acid molecules form a ternary complex with the endogenous pre-mRNA molecule, and wherein the resulting chimeric RNA molecule comprises the trans- spliced nucleic acid sequence.

[0058] Disclosed herein is a nucleic acid molecule comprising an exogenous RNA to be trans- spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron; one or more RNA targeting motifs; and one or more RNA structures.

[0059] Disclosed herein is a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs).

[0060] Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron; one or more RNA targeting motifs; one or more RNA structures.

[0061] Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs).

[0062] Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or a viral vector comprising a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron; one or more RNA targeting motifs; one or more RNA structures.

[0063] Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or a viral vector comprising a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs).

VI. DETAILED DESCRIPTION

[0064] The present disclosure describes formulations, compounded compositions, kits, capsules, containers, and/or methods thereof. It is to be understood that the inventive aspects of which are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0065] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

A. Definitions

[0066] Before the present compounds, compositions, articles, systems, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0067] This disclosure describes inventive concepts with reference to specific examples. However, the intent is to cover all modifications, equivalents, and alternatives of the inventive concepts that are consistent with this disclosure.

[0068] As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0069] The phrase “consisting essentially of’ limits the scope of a claim to the recited components in a composition or the recited steps in a method as well as those that do not materially affect the basic and novel characteristic or characteristics of the claimed composition or claimed method. The phrase “consisting of’ excludes any component, step, or element that is not recited in the claim. The phrase “comprising” is synonymous with “including”, “containing”, or “characterized by”, and is inclusive or open-ended. “Comprising” does not exclude additional, unrecited components or steps.

[0070] As used herein, when referring to any numerical value, the term “about” means a value falling within a range that is ± 10% of the stated value.

[0071] Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0072] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0073] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. In an aspect, a disclosed method can optionally comprise one or more additional steps, such as, for example, repeating an administering step or altering an administering step.

[0074] As used herein, “isolated” refers to a nucleic acid molecule or a nucleic acid sequence that has been substantially separated, produced apart from, or purified away from other biological components in the cell or tissue of an organism in which the component occurs, such as other cells, chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins. [0075] As used herein, the term “subject” refers to the target of administration, e.g., a human being. The term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.). Thus, the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Alternatively, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not denote a particular age or sex, and thus, adult and child subjects, as well as fetuses, whether male or female, are intended to be covered. In an aspect, a subject can be a human patient. In an aspect, a subject can have a disease or disorder, be suspected of having a disease or disorder, or be at risk of developing a disease or disorder (e.g., a genetic disease or disorder). In an aspect, a subject can be treatment-naive.

[0076] As used herein, a “regulatory element” can refer to promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Regulatory elements can include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).

[0077] As used herein, the term “diagnosed” means having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods. For example, “diagnosed with a disease or disorder” means having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition (such as a genetic disease or disorder) that can be treated by one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods. For example, “suspected of having a disease or disorder” can mean having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition (such as a genetic disease or disorder) that can likely be treated by one or more of by one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods. In an aspect, an examination can be physical, can involve various tests (e.g., blood tests, genotyping, biopsies, etc.) and assays (e.g., enzymatic assay), or a combination thereof.

[0078] A “patient” refers to a subject afflicted with a disease or disorder (e.g., a genetic disease or disorder). In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder. In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder and is seeking treatment or receiving treatment for a disease or disorder.

[0079] As used herein, the phrase “identified to be in need of treatment for a disease or disorder,” or the like, refers to selection of a subject based upon need for treatment of the disease or disorder. For example, a subject can be identified as having a need for treatment of a disease or disorder (e.g., a genetic disease or disorder) based upon an earlier diagnosis by a person of skill and thereafter subj ected to treatment for the genetic disease or disorder. In an aspect, the identification can be performed by a person different from the person making the diagnosis. In an aspect, the administration can be performed by one who performed the diagnosis.

[0080] As used herein, “inhibit,” “inhibiting”, and “inhibition” mean to diminish or decrease an activity, level, response, condition, severity, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, level, response, condition, severity, disease, or other biological parameter. This can also include, for example, a 10% inhibition or reduction in the activity, level, response, condition, severity, disease, or other biological parameter as compared to the native or control level (e.g., a subject not having a disease or disorder such as a genetic disease or disorder). Thus, in an aspect, the inhibition or reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of reduction in between as compared to native or control levels. In an aspect, the inhibition or reduction can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% as compared to native or control levels. In an aspect, the inhibition or reduction can be 0-25%, 25- 50%, 50-75%, or 75-100% as compared to native or control levels. In an aspect, a native or control level can be a pre-disease or pre-disorder level.

[0081] The words “treat” or “treating” or “treatment” include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In an aspect, the terms cover any treatment of a subject, including a mammal e.g., a human), and includes: (i) preventing the undesired physiological change, disease, pathological condition, or disorder from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the physiological change, disease, pathological condition, or disorder, i.e., arresting its development; or (iii) relieving the physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease. For example, in an aspect, treating a disease or disorder can reduce the severity of an established a disease or disorder in a subject by 1%- 100% as compared to a control (such as, for example, an individual not having a genetic disease or disorder). In an aspect, treating can refer to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of a disease or disorder (such as a genetic disease or disorder). For example, treating a disease or disorder can reduce one or more symptoms of a disease or disorder in a subject by l%-100% as compared to a control (such as, for example, an individual not having a genetic disease or disorder). In an aspect, treating can refer to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% reduction of one or more symptoms of an established a disease or disorder. It is understood that treatment does not necessarily refer to a cure or complete ablation or eradication of a disease or disorder. However, in an aspect, treatment can refer to a cure or complete ablation or eradication of a disease or disorder.

[0082] As used herein, the term “prevent” or “preventing” or “prevention” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. In an aspect, preventing a disease or disorder having chromatin deregulation and/or chromatin dysregulation is intended. The words “prevent”, “preventing”, and “prevention” also refer to prophylactic or preventative measures for protecting or precluding a subject (e.g., an individual) not having a given a disease or disorder (such as a genetic disease or disorder) or related complication from progressing to that complication.

[0083] As used herein, the terms “administering” and “administration” refer to any method of providing one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, the following: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can also include hepatic intra-arterial administration or administration through the hepatic portal vein (HPV). Administration of a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical composition, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed small molecule, a disclosed endonuclease, a disclosed oligonucleotide, and/or a disclosed RNA therapeutic can comprise administration directly into the CNS or the PNS. Administration can be continuous or intermittent. Administration can comprise a combination of one or more route.

[0084] In an aspect, the skilled person can determine an efficacious dose, an efficacious schedule, and an efficacious route of administration for one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof to treat or prevent a disease or disorder (such as genetic disease or disorder). In an aspect, the skilled person can also alter, change, or modify an aspect of an administering step to improve efficacy of one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof.

[0085] By “determining the amount” is meant both an absolute quantification of a particular analyte (e.g., an mRNA sequence containing a particular tag) or a determination of the relative abundance of a particular analyte (e.g., an amount as compared to a mRNA sequence including a different tag). The phrase includes both direct or indirect measurements of abundance (e.g., individual mRNA transcripts may be quantified or the amount of amplification of an mRNA sequence under certain conditions for a certain period may be used a surrogate for individual transcript quantification) or both.

[0086] As used herein, “modifying the method” can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof administered to a subject, or by changing the frequency of administration of one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof to a subject, by changing the duration of time one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination are administered to a subject, or by substituting for one or more of the disclosed components and/or reagents with a similar or equivalent component and/or reagent. The same applies to all disclosed therapeutic agents, immune modulators, immunosuppressive agents, proteosome inhibitors, etc.

[0087] As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. In an aspect, a pharmaceutical carrier employed can be a solid, liquid, or gas. In an aspect, examples of solid carriers can include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. In an aspect, examples of liquid carriers can include sugar syrup, peanut oil, olive oil, and water. In an aspect, examples of gaseous carriers can include carbon dioxide and nitrogen. In preparing a disclosed composition for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers. [0088] As used herein, the term “excipient” refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.). See, also, for reference, Remington’s Pharmaceutical Sciences, (1990) Mack Publishing Co., Easton, Pa., which is hereby incorporated by reference in its entirety.

[0089] As used herein, “concurrently” means (1) simultaneously in time, or (2) at different times during the course of a common treatment schedule.

[0090] The term “contacting” as used herein refers to bringing one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof together with a target area or intended target area in such a manner that the one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof exert an effect on the intended target or targeted area either directly or indirectly. A target area can comprise one or more cells, and in an aspect, one or more cells can be in a subject. A target area or intended target area can be one or more of a subject’s organs (e.g., lungs, heart, liver, kidney, brain, etc.). In an aspect, a target area or intended target area can be any cell or any organ infected by a disease or disorder (such as a genetic disease or disorder). In an aspect, a target area or intended target area can be any organ, tissue, or cells that are affected by a disease or disorder (such as a genetic disease or disorder).

[0091] As used herein, “determining” can refer to measuring or ascertaining the presence and severity of a disease or disorder, such as, for example, a genetic disease or disorder. Methods and techniques used to determine the presence and/or severity of a disease or disorder are typically known to the medical arts. For example, the art is familiar with the ways to identify and/or diagnose the presence, severity, or both of a disease or disorder (such as, for example, a genetic disease or disorder).

[0092] As used herein, “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired result such as, for example, the treatment and/or prevention of a disease or disorder (e.g., a genetic disease or disorder) or a suspected disease or disorder. As used herein, the terms “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired an effect on an undesired condition e.g., a disease or disorder). For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. In an aspect, “therapeutically effective amount” means an amount of a disclosed nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation; that (i) treats the particular disease, condition, or disorder (e.g., a genetic disease or disorder), (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder e.g., a genetic disease or disorder), or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein (e.g., a genetic disease or disorder). The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations employed; the disclosed methods employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations employed; the duration of the treatment; drugs used in combination or coincidental with the disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations employed, and other like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, then the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, a single dose of the disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition, such as, for example, a disease or disorder due to a missing, deficient, and/or mutant protein or enzyme.

[0093] As used herein, “RNA therapeutics” can refer to the use of oligonucleotides to target RNA. RNA therapeutics can offer the promise of uniquely targeting the precise nucleic acids involved in a particular disease with greater specificity, improved potency, and decreased toxicity. This could be particularly powerful for genetic diseases where it is most advantageous to aim for the RNA as opposed to the protein. In an aspect, a therapeutic RNA can comprise one or more expression sequences. As known to the art, expression sequences can comprise an RNAi, shRNA, mRNA, non-coding RNA (ncRNA), an antisense such as an antisense RNA, miRNA, morpholino oligonucleotide, peptide-nucleic acid (PNA) or ssDNA (with natural, and modified nucleotides, including but not limited to, LNA, BNA, 2’-0-Me-RNA, 2’-ME0-RNA, 2’-F-RNA), or analog or conjugate thereof. In an aspect, a disclosed therapeutic RNA can comprise one or more long non-coding RNA (IncRNA), such as, for example, a long intergenic non-coding RNA (lincRNA), pre-transcript, pre-miRNA, pre-mRNA, competing endogenous RNA (ceRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), pseudo-gene, rRNA, or tRNA. In an aspect, ncRNA can be pi wi -interacting RNA (piRNA), primary miRNA (pri-miRNA), or premature miRNA (pre-miRNA). In an aspect, a disclosed therapeutic RNA or an RNA therapeutic can comprise antisense oligonucleotides (ASOs) that inhibit mRNA translation, oligonucleotides that function via RNA interference (RNAi) pathway, RNA molecules that behave like enzymes (ribozymes), RNA oligonucleotides that bind to proteins and other cellular molecules, and ASOs that bind to mRNA and form a structure that is recognized by RNase H resulting in cleavage of the mRNA target. In an aspect, RNA therapeutics can comprise RNAi and ASOs that inhibit mRNA translation. Generally speaking, as known to the art, RNAi operates sequence specifically and post-transcriptionally by activating ribonucleases which, along with other enzymes and complexes, coordinately degrade the RNA after the original RNA target has been cut into smaller pieces while antisense oligonucleotides bind to their target nucleic acid via Watson-Crick base pairing, and inhibit or alter gene expression via steric hindrance, splicing alterations, initiation of target degradation, or other events.

[0094] As used herein, “small molecule” can refer to any organic or inorganic material that is not a polymer. Small molecules exclude large macromolecules, such as large proteins (e.g., proteins with molecular weights over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000), large nucleic acids (e.g., nucleic acids with molecular weights of over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000), or large polysaccharides (e.g., polysaccharides with a molecular weight of over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000). In an aspect, a “small molecule”, for example, can be a drug that can enter cells easily because it has a low molecular weight. In an aspect, a small molecule can be used in conjunction with a disclosed composition in a disclosed method.

[0095] In an aspect, the term “ex vivo” can refer generally to activities that take place outside an organism or subject such as experimentation, modification, differentiation, manipulation, and/or measurement done in or on living tissue in an artificial environment outside the organism. In an aspect, ex vivo experimentation, ex vivo modification, ex vivo differentiation, ex vivo manipulation, and/or ex vivo measurement can occur with a minimum alteration of the natural conditions. In an aspect, “ex vivo” can comprise living cells, tissues, or organs (e.g., cells in need of trans-splicing for one or more protein coding genes) taken from a subject in need thereof or a donor subject and cultured and/or maintained and/or perfused in a laboratory apparatus, usually under sterile conditions, and typically for a limited duration of time (e.g., a few hours or up to about 24 hours, up to about 48 hours, up to about 72 hours, up to about 96 hours, up to about 120 hours, up to about 144 hours, up to about 168 hours, or more depending on the circumstances and/or the desired characteristics. In an aspect, tissues, cells, or organs can be collected, frozen, and later thawed for ex vivo treatment.

[0096] As used herein, “operably linked” means that expression of a gene or a transgene is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5’ (upstream) or 3’ (downstream) of a gene under its control. The distance between the promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance can be accommodated without loss of promoter function.

[0097] As used herein, “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein must contain at least two amino acids and there is no limitation on the maximum number of amino acids that can comprise a protein’s sequence. The term “peptide” can refer to a short chain of amino acids including, for example, natural peptides, recombinant peptides, synthetic peptides, or any combination thereof. Proteins and peptides can include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others.

[0098] “Nucleic acid” or “oligonucleotide” or “polynucleotide” as used herein means at least two nucleotides covalently linked together. The depiction of a single strand can also define the sequence of the complementary strand. Thus, a nucleic acid can encompass the complementary strand of a depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid can encompass substantially identical nucleic acids and complements thereof. A single strand can provide a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid can encompass a probe that hybridizes under stringent hybridization conditions. A nucleic acid can be single-stranded, or double-stranded, or can contain portions of both double-stranded and single-stranded sequence. The nucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods. Also as used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid construct,” “nucleotide sequence”, and “polynucleotide” can refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term can encompass RNA/DNA hybrids. When dsRNA is produced synthetically, less common bases, such as inosine, 5- methylcytosine, 6-methyladenine, hypoxanthine and others can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modification to the phosphodiester backbone, or the 2’ -hydroxy in the ribose sugar group of the RNA can also be made. A “synthetic” nucleic acid or polynucleotide, as used herein, refers to a nucleic acid or polynucleotide that is not found in nature but is constructed by the hand of man and therefore is not a product of nature.

[0099] A “polynucleotide” is a sequence of nucleotide bases, and may be RNA, DNA, or DNA- RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotides).

[0100] A “fragment” or “portion” of a nucleotide sequence can be understood to mean a nucleotide sequence of reduced length relative (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,

13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides) to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of, or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence. Such a nucleic acid fragment or portion according to the disclosure can be, where appropriate, included in a larger polynucleotide of which it is a constituent. In an aspect, a fragment or portion of a nucleotide sequence or nucleic acid sequence can comprise the sequence encoding an exon having one or more mutations.

[0101] A “fragment” or “portion” of an amino acid sequence can be understood to mean an amino acid sequence of reduced length relative (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or more amino acids) to a reference amino acid sequence and comprising, consisting essentially of, or consisting of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the reference amino acid sequence. Such an amino acid fragment or portion according to the disclosure can be, where appropriate, included in a larger amino acid sequence of which it is a constituent.

[0102] A “heterologous” or a “recombinant” nucleotide or amino acid sequence as used interchangeably herein can refer to a nucleotide or an amino acid sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleotide or amino acid sequence.

[0103] As used herein, the term “endogenous” can refer to a gene, protein, compound, or activity that is normally present in a host cell (e.g., a pre-mRNA). As used herein, an “exogenous” nucleic acid molecule, construct, or sequence (e.g., an RNA sequence to be trans-spliced) can refer to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to a host cell, but may be homologous to a nucleic acid molecule or portion of a nucleic acid molecule from the host cell.

[0104] Different nucleic acids or proteins having homology can be referred to as “homologues”. The term homologue includes homologous sequences from the same and other species and orthologous sequences from the same and other species. “Homology” refers to the level of similarity between two or more nucleic acid and/or amino acid sequences in terms of percent of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties among different nucleic acids or proteins. Thus, the disclosed compositions and disclosed methods can comprise homologues to the disclosed nucleotide sequences and/or disclosed polypeptide sequences.

[0105] “Orthologous,” as used herein, can refer to homologous nucleotide sequences and/or amino acid sequences in different species that arose from a common ancestral gene during speciation. A homologue of a disclosed nucleotide sequence or a disclosed polypeptide can have substantial sequence identity (e.g., at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100%) to a disclosed nucleotide sequence or a disclosed polypeptide.

[0106] “Complement” or “complementary” as used herein means a nucleic acid can mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. “Complementarity” refers to a property shared between two nucleic acid sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.

[0107] As used herein, “promoter” or “promoters” are known to the art. Depending on the level and tissue-specific expression desired, a variety of promoter elements can be used. A promoter can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the gene expression desired. A promoter can be native (endogenous) or foreign (exogenous) and can be a natural or a synthetic sequence. By foreign or exogenous, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

[0108] “Tissue-specific promoters” are known to the art and include, but are not limited to, neuron-specific promoters, muscle-specific promoters, liver-specific promoters, skeletal musclespecific promoters, and heart-specific promoters.

[0109] “Liver-specific promoters” are known to the art and include, but are not limited to, the thyroxin binding globulin (TBG) promoter, the al-microglobulin/bikunin enhancer/thyroid hormone-binding globulin promoter, the human albumin (hALB) promoter, the thyroid hormone- binding globulin promoter, the a- 1 -anti -trypsin promoter, the bovine albumin (bAlb) promoter, the murine albumin (mAlb) promoter, the human al -antitrypsin (hAAT) promoter, the ApoEhAAT promoter comprising the ApoE enhancer and the hAAT promoter, the transthyretin (TTR) promoter, the liver fatty acid binding protein promoter, the hepatitis B virus (HBV) promoter, the DC 172 promoter comprising the hAAT promoter and the al -microglobulin enhancer, the DC 190 promoter comprising the human albumin promoter and the prothrombin enhancer, or any other natural or synthetic liver-specific promoter. In an aspect, a liver specific promoter can comprise about 845-bp and comprise the thyroid hormone-binding globulin promoter sequences (2382 to 13), two copies of al-microglobulin/bikunin enhancer sequences (22,804 through 22,704), and a 71-bp leader sequence as described by Ill CR, et al. (1997).

[0110] Ubiquitous/constitutive promoters” are known to the art and include, but are not limited to, a CMV major immediate-early enhancer/chicken beta-actin promoter, a cytomegalovirus (CMV) major immediate-early promoter, an Elongation Factor 1-a (EFl -a) promoter, a simian vacuolating virus 40 (SV40) promoter, an AmpR promoter, a PyK promoter, a human ubiquitin C gene (Ubc) promoter, a MFG promoter, a human beta actin promoter, a CAG promoter, a EGR1 promoter, a FerH promoter, a FerL promoter, a GRP78 promoter, a GRP94 promoter, a HSP70 promoter, a [3-kin promoter, a murine phosphoglycerate kinase (mPGK) or human PGK (hPGK) promoter, a ROSA promoter, human Ubiquitin B promoter, a Rous sarcoma virus promoter, or any other natural or synthetic ubiquitous/constitutive promoters.

[0111] As used herein, an “inducible promoter” refers to a promoter that can be regulated by positive or negative control. Factors that can regulate an inducible promoter include, but are not limited to, chemical agents (e.g., the metallothionein promoter or a hormone inducible promoter), temperature, and light. [0112] As used herein, the term “serotype” is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serologic distinctiveness can be determined by the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences/antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequence differences of AAV serotypes).

[0113] As used herein, “tropism” refers to the specificity of an AAV capsid protein present in an AAV viral particle, for infecting a particular type of cell or tissue. The tropism of an AAV capsid for a particular type of cell or tissue may be determined by measuring the ability of AAV vector particles comprising the hybrid AAV capsid protein to infect or to transduce a particular type of cell or tissue, using standard assays that are well-known in the art such as those disclosed in the examples of the present application. As used herein, the term “liver tropism” or “hepatic tropism” refers to the tropism for liver or hepatic tissue and cells, including hepatocytes.

[0114] “Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as “substantially identical” or “essentially similar” when they are optimally aligned. For example, sequence similarity or identity can be determined by searching against databases such as FASTA, BLAST, etc., but hits should be retrieved and aligned pairwise to compare sequence identity. Two proteins or two protein domains, or two nucleic acid sequences can have “substantial sequence identity” if the percentage sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, preferably 90%, 95%, 98%, 99% or more. Such sequences are also referred to as “variants” herein, e.g., other variants of a missing, deficient, and/or mutant protein or enzyme. It should be understood that sequence with substantial sequence identity do not necessarily have the same length and may differ in length. For example, sequences that have the same nucleotide sequence but of which one has additional nucleotides on the 3’- and/or 5’-side are 100% identical.

[0115] As used herein, “codon optimization” can refer to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing one or more codons or more of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. As contemplated herein, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database.” Many methods and software tools for codon optimization have been reported previously. (See, for example, genomes.urv.es/OPTIMIZER/).

[0116] As used herein, “CRISPR or clustered regularly interspaced short palindromic repeat” is an ideal tool for correction of genetic abnormalities as the system can be designed to target genomic DNA directly. Cas9 is well-known to the art. The CRISPR/Cas methods disclosed herein, such as those that use an Cast 3d, can be used to edit the sequence of one or more target RNAs, such as one associated with a disease or disorder disclosed herein (e.g., a genetic disease or disorder).

[0117] The diverse Casl3 family contains at least four known subtypes, including Casl3a (formerly C2c2), Cast 3b, Cast 3 c, and Cast 3d. All known Cast 3 family members contain two HEPN domains, which confer RNase activity. Casl3 can be reprogrammed to cleave a targeted ssRNA molecule through a short guide RNA with complementarity to the target sequence. Cas 13s function similarly to Cas9, using a ~64-nucleotide guide RNA to encode target specificity. The Cas 13 protein complexes with the guide RNA via recognition of a short hairpin in the crRNA, and target specificity is encoded by a 28-nucleotide to a 30-nucleotide spacer that is complementary to the target region. In addition to programmable RNase activity, all Casl3s exhibit collateral activity after recognition and cleavage of a target transcript, leading to nonspecific degradation of any nearby transcripts regardless of complementarity to the spacer. While Cas 13a showed some activity for RNA knockdown, certain orthologs of Cas 13b proved more stable and robust in mammalian cells for RNA knockdown and editing. More recently, additional orthologs of Casl3 have been discovered, including Casl3d, which has been leveraged for efficient and robust knockdown across many endogenous transcripts. Casl3d can be used to modulate splicing of endogenous transcripts and that the coding sequence for Casl3d is small enough to fit within the packaging limits of AAV for in vivo delivery.

[0118] In an aspect, Cas 13 can be considered an outlier in the CRISPR world because it targets RNA, not DNA. Once it is activated by a ssRNA sequence bearing complementarity to its crRNA spacer, it unleashes a nonspecific RNase activity and destroys all nearby RNA regardless of their sequence. As disclosed herein, this property can be harnessed in vitro for precision diagnostics. Generally, Casl3 can be found in Leptotrichia buccalis, Leptotrichia shahii, Ruminococcus flavefaciens, Bergeyella zoohelcum, Prevotella buccae, and Listeria seeligeri and can have a size of about 900 to about 1300 amino acids. In an aspect, the guide spacer length can be about 22 to about 30 nucleotides while the total guide length can be about 52 to about 66 nucleotides. In an aspect, a PAM can be 3-H for LshCasl3a, 5-D and 3-NAN or NNA for BzCasl3b, and none for RfCasl3d. In an aspect, a disclosed Casl3 can cut ssRNA. [0119] As known to the skilled person in the cart, a Casl3d ortholog can be from a prokaryotic genome or metagenome, gut metagenome, an activated sludge metagenome, an anaerobic digester metagenome, a chicken gut metagenome, a human gut metagenome, a pig gut metagenome, a bovine gut metagenome, a sheep gut metagenome, a goat gut metagenome, a capybara gut metagenome, a primate gut metagenome, a termite gut metagenome, a fecal metagenome, a genome from the Order Clostridiales, or the Family Ruminococcaceae. In an aspect, a disclosed Cast 3d ortholog can include an Cast 3d ortholog from Ruminococcus albus, Eubacterium siraeum, a Ruminococcus flavefaciens strain XPD3002, Ruminococcus flavefaciens FD-1, uncultured Eubacterium sp TS28-c4095, uncultured Ruminococcus sp., Ruminococcus bicirculans, or Ruminococcus sp CAG57.

[0120] In an aspect, a disclosed Casl3 can comprise RfxCasl3d (see, for example, US Patent Publication No. 2020/0244609, which is incorporated by reference for its teachings of RfxCasl3d and relevant sequences). In an aspect, a disclosed Casl3 can comprise PspCasl3b (see, for example, US Patent Publication No. 2020/0231975, which is incorporated by reference for its teachings of PspCasl3b and relevant sequences).

As known to the art, RNA binding proteins consist of multiple repetitive sequences that contain only a few specific basic domains. Structurally, common RNA-binding domains mainly include RNA-recognition motif (RRM), K homology (KH) domain, double-stranded RBD (dsRBD), coldshock domain (CSD), arginine-glycine-glycine (RGG) motif, tyrosine-rich domain, and zinc fingers (ZnF) of the CCHC, CCCH, ZZ type etc. According to the different functions of RBPs in cells, RBPs can be divided into epithelial splicing regulatory proteins (ESRP1), cytoplasmic polyadenylation element binding protein family (CPEB1/2), Hu-antigen R (HuR), heterogeneous nuclear ribonucleoprotein family members (hnRNP A/D/H/K/MZE/L), insulin-like growth factor 2 mRNA family members (IMP1/2/3), zfh family of transcription factors (ZEB 1/2), KH-type splicing regulatory protein (KHSRP), La ribonucleoprotein domain family members (LARP 1/6/7), Lin-28 homolog proteins (Lin28), Musashi protein family (MSI1/2), Pumilio protein family (PUM1/2), Quaking (QK), RNA-binding motif protein family (4/10/38/47), Src- associated substrate during mitosis of 68 kDa (SAM68), serine and arginine rich splicing factor (SRSF1/3), T cell intracellular antigens (TIA1/TIAR), and Upstream of N-Ras (UNR).

[0121] In an aspect, “RNA editing” can be a post-transcriptional modification where a precursor mRNA (pre-mRNA) nucleotide sequence is changed by base insertion, deletion, or modification. The extent of RNA editing varies from a few hundred bases, in mitochondrial DNA of trypanosomes, to a just single base, in nuclear genes of mammals. Even a single base change in the pre-mRNA can convert a codon for one amino acid into the codon for another amino acid or a stop codon. This type of re-coding can significantly affect the structure and function of a protein and may lead to the production of multiple variants of a protein from a single gene.

[0122] In an aspect, insertional and deletional RNA editing can involve the addition and deletion of specific nucleotides or sequences of nucleotides from pre-mRNA. In an aspect, substitutional RNA editing by base modifications is observed in higher eukaryotes, where the base is modified without changing the length of the pre-mRNA.

[0123] In an aspect, also disclosed herein are partial self-complementary parvovirus (e.g., a disclosed AAV) genomes, plasmid vectors encoding the parvovirus genomes, and parvovirus (e.g., a disclosed AAV) particles including such genomes. In an aspect, provided herein is a plasmid vector comprising a nucleotide sequence encoding a disclosed parvovirus genome such as for example, a disclosed AAV. In an aspect, provided herein is a partial self-complementary parvovirus genome including a payload construct, parvovirus ITRs flanking the payload construct, and a self-complementary region flanking one of the ITRs. A self-complementary region can comprise a nucleotide sequence that is complementary to the payload construct. A disclosed self- complementary region can have a length that is less the entire length of the payload construct.

[0124] In an aspect, a disclosed self-complementary region of a disclosed parvovirus genome can comprise a minimum length, while still having a length that is less the entire length of the payload construct. In an aspect, a disclosed self-complementary region can comprise at least 50 bases in length, at least 100 bases in length, at least 200 in length, at least 300 bases in length, at least 400 bases in length, at least 500 bases in length, at least 600 bases in length, at least 700 bases in length, at least 800 bases in length, at least 900 bases in length, or at least 1,000 bases in length.

[0125] In an aspect, a “self-complementary parvovirus genome” can be a single stranded polynucleotide having, in the 5’ to 3’ direction, a first parvovirus ITR sequence, a heterologous sequence (e.g., payload construct comprising, for example, a desired gene), a second parvovirus ITR sequence, a second heterologous sequence, wherein the second heterologous sequence is complementary to the first heterologous sequence, and a third parvovirus ITR sequence. In contrast to a self-complementary genome, a “partial self-complementary genome” does not include three parvovirus ITRs and the second heterologous sequence that is complementary to the first heterologous sequence has a length that is less than the entire length of the first heterologous sequence (e.g., payload construct). Accordingly, a partial self-complementary genome is a single stranded polynucleotide having, in the 5’ to 3’ direction or the 3’ to 5’ direction, a first parvovirus ITR sequence, a heterologous sequence (e.g., payload construct), a second parvovirus ITR sequence, and a self-complementary region that is complementary to a portion of the heterologous sequence and has a length that is less than the entire length the heterologous sequence. [0126] As used herein, “immune-modulating” refers to the ability of a disclosed nucleic acid molecules, a disclosed vector, a disclosed pharmaceutical formulation, or a disclosed agent to alter (modulate) one or more aspects of the immune system. The immune system functions to protect the organism from infection and from foreign antigens by cellular and humoral mechanisms involving lymphocytes, macrophages, and other antigen-presenting cells that regulate each other by means of multiple cell-cell interactions and by elaborating soluble factors, including lymphokines and antibodies, that have autocrine, paracrine, and endocrine effects on immune cells.

[0127] As used herein, the term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.

[0128] As used herein, the term “in combination” in the context of the administration of other therapies (e.g., other agents) includes the use of more than one therapy (e.g., drug therapy). Administration “in combination with” one or more further therapeutic agents includes simultaneous (e.g., concurrent) and consecutive administration in any order. The use of the term “in combination” does not restrict the order in which therapies are administered to a subject. By way of non-limiting example, a first therapy (e.g., a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof) may be administered prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks), concurrently, or after (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or longer) the administration of a second therapy (e.g., agent) to a subject having or diagnosed with a disease or disorder (such as a genetic disease or disorder).

[0129] Disclosed are the components to be used to prepare the disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations as well as the disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C- D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention.

B. Compositions for Transcriptome Engineering (Barcoded CRAFT)

[0130] In eukaryotic organisms, chromosomal DNA is transcribed into precursor RNA messages (pre-mRNA) which contain protein coding regions (exons) and intervening non-protein coding regions (introns). Prior to processing, these pre-mRNA molecules do not possess a sequence primed for translation by the ribosome, due to the retention of non-coding intronic sequences. Thus, prior to nuclear export, the exons of pre-mRNA transcripts are joined through a cellular mechanism known as splicing. This mechanism features dual transesterifications mediated by a large multi ribonucleoprotein structure, called the spliceosome. In the first transesterification, the branch point sequence of the intervening intron attacks the 5’ splice site, forming a lariat structure. This reaction frees the 5’ splice site to attack the 3’ splice site removing the intervening intron, joining the adjacent exons. Upon removal of all intronic sequences, the precursor message matures into a translation competent mature RNA transcript, which is trafficked to the ribosome where it is decoded to manufacture cellular proteins.

[0131] In mammalian cells, mutations in transcriptionally active regions of chromosomal DNA give rise to pre-mRNA bearing identical mutations. If the mutation is located in a non-coding region, then processing of the pre-mRNA may be altered or abolished. If the mutation is located in an exonic region of the pre-mRNA, then that mutation will be passed to the mature mRNA sequence. These mutations can contribute to inhibition of complete protein translation of the encoded protein (non-sense mutation) or modify the primary structure of the encoded protein in a counter-productive manner (missense mutation). Collectively, these genetically encoded mutations may function to contribute to pathogenesis in eukaryotes. 1. Nucleic Acid Molecules

5’ Replacement Constructs

[0132] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence to be transspliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein.

[0133] In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 20 SNPs. In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 10 SNPs. In an aspect, a disclosed series of SNPs can comprise 5 SNPs.

[0134] In an aspect, a disclosed isolated nucleic acid molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, a disclosed isolated nucleic acid molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0135] In an aspect, a disclosed isolated nucleic acid molecule can further comprise a polyadenylation signal.

[0136] In an aspect, a disclosed isolated nucleic acid molecule can further comprise a spacer region. In an aspect, a disclosed spacer region can separate the 5’ splice region from the one or more guide RNA sequences. In an aspect, a disclosed spacer region can comprise any known spacer. In an aspect, a disclosed spacer region can comprise a consensus splicing motif (e.g., such as U1 or U2). In an aspect, a disclosed spacer region can comprise a limited number of consensus splicing motifs (e.g., such as U1 or U2).

[0137] In an aspect, a disclosed isolated nucleic acid molecule can further comprise one or more stem loops. In an aspect, a disclosed stem loop can be a cognate aptamer for a disclosed RNA binding protein. For example, in an aspect, a disclosed stem loop can be a direct repeat of the guide RNA scaffold for a disclosed Casl3d. In an aspect, a disclosed stem loop can facilitate interaction between a disclosed RNA molecule and a disclosed Cas protein.

[0138] In an aspect, a disclosed isolated nucleic acid molecule can further comprise a nuclear localization signal (NLS). In an aspect, a disclosed NLS can be comprise the sequence set froth in SEQ ID NO:60. In an aspect, a disclosed NLS can comprise any NLS known to the art. As known to the art (see, e.g., Lu J, et al. (2021) Cell Commun Signal. 19:60, which is incorporated herein by reference for its teachings of NLS), nuclear localization signals (NLS) are generally short peptides that act as a signal fragment that mediates the transport of proteins from the cytoplasm into the nucleus.

[0139] In an aspect, the one or more disclosed guide RNA sequences can be directed the intron immediately 5’ to the first exon of the target endogenous pre-mRNA.

[0140] In an aspect of a disclosed isolated nucleic acid molecule, a disclosed 5’ hemi intron can comprise a consensus 5’ splice site. In an aspect, a disclosed 5’ splice site can comprise the sequence set forth in SEQ ID NO:59 (GT). In an aspect, a disclosed consensus 5’ splice site can comprise the sequence set forth in SEQ ID NO:61. In an aspect, a disclosed consequence 5’ splice site can comprise MAG|GURAGU (SEQ ID NO:61), wherein | denotes the exon intron junction, wherein M = A or C, and wherein R = A or G.

[0141] In an aspect, a disclosed 5’ hemi intron can be recognized by nuclear splicing components within a host cell. In an aspect, a disclosed nucleic acid sequence encoding the RNA binding protein can interact with the one or more stem loops and/or can stabilize the one or more guide RNA sequences.

[0142] As known to the skilled person, RNA binding proteins (RBPs) can be important effectors of gene expression. RBPs can recognize hundreds of transcripts and form extensive regulatory networks that help to maintain cell homeostasis. Accordingly, the malfunction of RBPs underlies the origin of many diseases. In an aspect, a disclosed RNA binding protein can be any RNA binding protein having bi specific affinity for the trans-splicing RNA and the target pre-mRNA of interest. In an aspect, this affinity can be mediated by riboncleoprotein interactions by, for example, Type VI CRISPR enzymes, or through direct RNA protein interactions by, for example, Pumillo and FBF (PUF) proteins. In an aspect, these interactions can be mediated by protein/ aptamer interactions. RNA binding proteins are discussed in depth supra.

[0143] In an aspect, a disclosed RNA binding protein can comprise bispecific affinity for a disclosed target pre-mRNA as well as a disclosed Casl3 or a disclosed catalytically inactive Casl3. In an aspect, a disclosed Cast 3 can comprise any catalytically inactive Casl3. For example, in an aspect, a disclosed Casl3 can comprise a catalytically inactive RfxCasl3d or a catalytically inactive PspdCasl3b. For example, in an aspect, a Casl3 alternative can comprise alternatives known to the art including, but not limited to CRISPR-Inspired RNA Targeting System (CIRTS), Pumillo and FBF (PUF), rCas9, and/or any bifunctional RNA binding protein that can both bind to the trans-splicing RNA molecule and the target RNA to facilitate interaction between the two RNA species. RNA binding proteins are discussed in depth supra.

[0144] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode a translatable protein or a portion thereof. In an aspect, a disclosed portion can comprise one or more exons comprising a mutation. In an aspect, a disclosed portion can comprise some part of the gene sequence but not the complete sequence. For example, in an aspect, a disclosed portion can comprise the nucleic acid sequence having one or more mutations.

[0145] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode LMNA/C or a portion thereof. In an aspect, the one or more disclosed guide RNA sequences can comprise the sequence set forth in SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:32 or a fragment thereof.

[0146] LMNA/C is known to the art (e.g., Gene ID 4000) and this nucleotide sequence can comprise nucleotides 4974 - 62517 in Accession No. NG008692.2. The nuclear lamina consists of a two-dimensional matrix of proteins located next to the inner nuclear membrane. The lamin family of proteins make up the matrix and are highly conserved in evolution. During mitosis, the lamina matrix is reversibly disassembled as the lamin proteins are phosphorylated. Lamin proteins are involved in nuclear stability, chromatin structure and gene expression. Vertebrate lamins consist of two types, A and B. Alternative splicing results in multiple transcript variants. Mutations in this gene lead to several diseases: Emery-Dreifuss muscular dystrophy, familial partial lipodystrophy, limb girdle muscular dystrophy, dilated cardiomyopathy, Charcot-Marie- Tooth disease, and Hutchinson-Gilford progeria syndrome.

[0147] In an aspect, a disclosed encoded Lamin A/C (LMNA/C) can comprise the sequence set forth in SEQ ID NO:55 or a fragment thereof. In an aspect, a disclosed encoded Lamin A/C can comprise a sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the sequence set forth in SEQ ID NO:55.

[0148] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode DP71 or a portion thereof. DP71 is known to the art (e.g., Gene ID 13405).

[0149] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode CFTR or a portion thereof. CFTR is known to the art (e.g., Gene ID 1080) and this nucleotide sequence can comprise nucleotides 19180 - 207882 in Accession No. NG016465.4. This gene encodes a member of the ATP -binding cassette (ABC) transporter superfamily. The encoded protein functions as a chloride channel, making it unique among members of this protein family, and controls ion and water secretion and absorption in epithelial tissues. Channel activation is mediated by cycles of regulatory domain phosphorylation, ATP -binding by the nucleotide-binding domains, and ATP hydrolysis. Mutations in this gene cause cystic fibrosis, the most common lethal genetic disorder in populations of Northern European descent. The most frequently occurring mutation in cystic fibrosis, DeltaF508, results in impaired folding and trafficking of the encoded protein. Multiple pseudogenes have been identified in the human genome. [0150] In an aspect, a disclosed encoded CFTR can comprise the sequence set forth in SEQ ID NO:54 or a fragment thereof. In an aspect, a disclosed encoded CFTR can comprise a sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the sequence set forth in SEQ ID NO:54.

[0151] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode DMPK or a portion thereof. In an aspect, the one or more disclosed guide RNA sequences can comprise the sequence set forth in SEQ ID NO:29 or SEQ ID NO:30 or a fragment thereof. DMPK is known to the art (e.g., Gene ID 1760) and this nucleotide sequence can comprise nucleotides 5068 - 17841 in Accession No. NG009784.1. DMPK is a serine-threonine kinase that is closely related to other kinases that interact with members of the Rho family of small GTPases. Substrates for this enzyme include myogenin, the beta-subunit of the L-type calcium channels, and phospholemman. The 3’ untranslated region of this gene contains 5-38 copies of a CTG trinucleotide repeat. Expansion of this unstable motif to 50-5,000 copies causes myotonic dystrophy type I, which increases in severity with increasing repeat element copy number. Repeat expansion is associated with condensation of local chromatin structure that disrupts the expression of genes in this region. Several alternatively spliced transcript variants of this gene have been described, but the full- length nature of some of these variants has not been determined.

[0152] In an aspect, a disclosed encoded DMPK can comprise the sequence set forthin SEQ ID NO:56 or a fragment thereof. In an aspect, a disclosed encoded DMPK can comprise a sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the sequence set forth in SEQ ID NO:56.

[0153] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode DMD or a portion thereof. In an aspect, the one or more disclosed guide RNA sequences can comprise the sequence set forth in SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28 or a fragment thereof. [0154] In an aspect, a disclosed gene can be DMD (dystrophin). DMD is known to the art (e.g., Gene ID 1756) and this nucleotide sequence can comprise nucleotides 5001 - 2225382 in Accession No. NG012232.1. DMD spans a genomic range of greater than 2 Mb and encodes a large protein containing an N-terminal actin-binding domain and multiple spectrin repeats. The encoded protein forms a component of the dystrophin-glycoprotein complex (DGC), which bridges the inner cytoskeleton and the extracellular matrix. Deletions, duplications, and point mutations at this gene locus may cause Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or cardiomyopathy. Alternative promoter usage and alternative splicing result in numerous distinct transcript variants and protein isoforms for this gene. [0155] In an aspect, a disclosed encoded DMD can comprise the sequence set forth in SEQ ID NO:52 or a fragment thereof. In an aspect, a disclosed encoded DMD can comprise a sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the sequence set forth in SEQ ID NO:52.

[0156] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode LRRK2 or a portion thereof. LRRK2 is known to the art (e.g., Gene ID 120892) and this nucleotide sequence can comprise nucleotides 5001 - 149275 in Accession No. NG011709.1. LRRK2 is a member of the leucine-rich repeat kinase family and encodes a protein with an ankryin repeat region, a leucine-rich repeat (LRR) domain, a kinase domain, a DFG-like motif, a RAS domain, a GTPase domain, a MLK-like domain, and a WD40 domain. The protein is present largely in the cytoplasm but also associates with the mitochondrial outer membrane. Mutations in this gene have been associated with Parkinson’s disease.

[0157] In an aspect, a disclosed encoded LRRK2 can comprise the sequence set forth in SEQ ID NO:53 or a fragment thereof. In an aspect, a disclosed encoded LRRK2 can comprise a sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the sequence set forth in SEQ ID NO:53.

[0158] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode the protein or a portion thereof (such as, for example, Exon 1 or Exon 4, etc.) associated with the following genes: ABCA1, ABCA12, ABCA13, ABCA2, ABCA3, ABCA4, ABCA5, ABCC1, ABCC2, ABCC6, ABCC8, ABCC9, ACAN, ADAMTS13, ADCY10, ADGRV1, AGL, AGRN, AHDC1, ALK, ALMS1, ALPK3, ALS2, ANAPC1, ANK1, ANK2, ANK3, ANKRD11, ANKRD26, APC, APC2, APOB, ARFGEF2, ARHGAP31, ARHGEF10, ARHGEF18, ARID ! A, ARID I B, ARID2, ASH1L, ASPM, ASXL1, ASXL2, ASXL3, ATM, ATP7A, ATP7B, ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, BLM, BPTF, BRCA1, BRCA2, BRD4, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD, CAMTAI, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44, CFAP65, CFTR/ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1, CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1, CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1, DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11, DNAH17, DNAH2, DNAH5, DNAH7, DNAH8, DNAH9, DNMBP, DNMT1, DOCK2, DOCK3, DOCK6, D0CK7, D0CK8, DSCAM, DSP, DST, DU0X2, DYNC1H1, DYNC2H1, DYSF, EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, EYS, F5, F8, FANCA, FANCD2, FANCM, FAT1, FAT4, FBN1, FBN2, FLG, FLG2, FLNA, FLNB, FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2, FSIP2, FYC01, GLI2, GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1, HERC2, HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R, IGF2R, IGSF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A, KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220, KIF14, KIF1A, KIF1B, KIF21A, KIF26B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1, LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMC3, LCT, L0XHD1, LPA, LRBA, LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD, MAGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5, MCM3AP, MED12, MED12L, MED13, MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MYH11, MYH14, MYH2, MYH3, MYH6, MYH7, MYH7B, MYH8, MYH9, MYLK, MYO 15 A, MYO18B, MYO3A, MYO5A, MYO5B, MYO7A, MYO9A, NALCN, NBAS, NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF, NF1, NFASC, NHS, NIN, NIPBL, NLRP1, NOTCH1, NOTCH2, NOTCH3, NPHP4, NRXN1, NRXN3, NSD1, NSD2, NUP155, NUP188, NUP205, OBSCN, OBSL1, OTOF, OTOG, OTOGL, PARD3, PBRM1, PCDH15, PCLO, PCNT, PHIP, PI4KA, PIEZO1, PIEZO2, PIK3C2A, PIKFYVE, PKD1, PKD1L1, PKHD1, PLCE1, PLEC, PLEKHG2, PNPLA6, POGZ, POLA1, POLE, POLR1A, POLR2A, POLR3A, PRG4, PRKDC, PRPF8, PRR12, PRX, PTCHI, PTPN23, PTPRF, PTPRJ, PTPRQ, PXDN, QRICH2, RAB3GAP2, RAH, RALGAPA1, RANBP2, RB1CC1, RELN, RERE, REV3L, RIC1, RIMS1, RIMS2, RNF213, ROBO1, ROBO2, ROBO3, ROS1, RP1, RP1L1, RTTN, RUSC2, RYR1, RYR2, SACS, SAMD9, SAMD9L, SBF2, SCAPER, SCN10A, SCN11A, SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SETBP1, SETD1A, SETD1B, SETD2, SETD5, SETX, SHANK2, SHANK3, SHROOM4, SI, SIPA1L3, SLIT2, SLX4, SMARCA2, SMARCA4, SMCHD1, SNRNP200, SON, SPEF2, SPEG, SPG11, SPTA1, SPTAN1, SPTB, SPTBN2, SPTBN4, SRCAP, STRC, SVIL, SYNE1, SYNGAP1, SYNJ1, SZT2, TAF1, TANC2, TCF20, TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B, TNXB, TOGARAMI, TONSL, TRIO, TRIOBP, TRIP11, TRIP12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, TTN, TUBGCP6, UBR1, UNC80, USH2A, USP9X, VCAN, VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62, WDR81, WNK1, WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292, ZNF335, ZNF407, ZNF462, ZNF469, or a portion thereof.

[0159] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode the protein or a portion thereof (such as, for example, Exon 1 or Exon 4, etc.) associated with the following genes: DMD, TTN, TCAP, SGCA, SGCB, SGCG, SGCD, Al-AT, MYH6, MYH7, MYH11, ML2, ML3, MYLK2, MYBPC3, DES, DNM2, LAMA2, LMNA, LMNB, LBR, DYSF, EMD, EPO, LPL, SERCA2A, S100A1, MTM, DM1 DMPK, PYGL„ PYGM, GYSI, GYS2, NAGA, GAA, SMPD1, LIPA, COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, PLOD1, LIPA, FXN, MSTN, HEXA, HEXB, GBA, AMPD1, HBB, IDUA, IDS, TNNI3, TNNT2, TNNC1, TPM1, TPM3, NAGLU, SGSH, HGSNAT, IGTA7, IGTA9, N-acetyl, GNS, N-acetyl, GALNS, GLB1, GUSB, HYAL1, ASAHI, GALC, CTSA, CTSA, CTSK, GM2A, ARSA, ARSB, SUMFI, NEU1 GNPTA, GNPTB, GNPTG, MCOLN1, NPC1, NPC2, CLN5, CLN6, CLN8, PPT1, TPP1, CLN3, DNAJC5, MFSD8, MAN2B1, MANBA, AGA, FUCA1, CTNS, SLC2A10, SLC17A5, SLC6A19, SLC22A5, SLC37A4, LAMP2, SCN4A, SCN4B, SCN5A, SCN4A, CACNA1C, CACNA1S, PGK1, PGAM2, AGL, KCNE1, KCNE2, KCNJ2, KCNJ5, KCNH2, KCNQ1, HCN4, CLCN1, CPT1A, RYR1, RYR2, BINI, LARGE1, DOK7, FKTN, FKRP, SELENON, POMT1, POMT2, POMGNT1, POMGNT2, POMK, ISPD, PLEC, CHRNE, CHAT, CHKB, COLQ, RAPSN, FHL1, B4GAT1, B3GALNT2, DAGI, TMEM5, TMEM43, SECISBP2, UDP-N-acetyl, GNE, ANO5, SMCHD1, LDHA, LHDB, CAPN3, CAV3, TRIM32, CNBP, NEB, ACTA1, ACTC1, ACTN2, PABPN1, LEMD3, ZMPSTE24, MTTP, CHRNA1, CHRNA2, CHRNA3, CHRNA4, CHRNA5, CHRNA6, CHRNA7, CHRNA8, CHRNA9, CHRNA10, CHRNB1, CHRNB2, CHRNB3, CHRNB4, CHRNG1, CHRND, CHRNE1, ABCA1, ABCC6, ABCC9, ABCD1, ATP2A1, ATM, TTP A, KIF21A, PHOX2A, HSPG2, STIM1, NOTCHI, NOTCH3, DTNA, PRKAG2, CSRP3, VCL, MyoZ2, MYPN, JPH2, PLN, CALR3, NEXN, LDB3, EYA4, HTT, AR, PTPN11, JUP, DSP, PKP2, DSG2, DSC2, CTNNA3, NKX2-5, AKAP9, AKAP10, GNAI2, ANK2, SNTAT, CALM1, CALM2, HTRA1, FBN1, FBN2, XYLT1, XYLT2, TAZ, HGD, G6PC, GBE1, PFKM, PHKA1, PHKA2, PHKB, PHKG2, PGAM2, CBS, MTHFR, MTR, MTRR, MMADHC, MT-ND1, MT-ND5, MT-TE, MT- TH, MT-TL1, MT-TK, MT-TS1, MT-TV, MAP2K1, BRAF, RAFI, IGF-1, TGF[33, TGFpRl, TGFPR2, FGF2, FGF4, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, or VEGFR2.

[0160] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can be CpG depleted and codon-optimized for expression in a human cell. In an aspect, “CpG-free” can mean completely free of CpGs or partially free of CpGs. In an aspect, “CpG-free” can mean “CpG- depleted”. In an aspect, “CpG-depleted” can mean “CpG-free”. In an aspect, “CpG-depleted” can mean completely depleted of CpGs or partially depleted of CpGs. In an aspect, “CpG-free” can mean “CpG-optimized” for a desired and/or ideal expression level. CpG depletion and/or optimization is known to the skilled person in the art.

[0161] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode an RNA. In an aspect, a disclosed encoded RNA can comprise ribosomal RNA (rRNA), transfer RNA (tRNA), heterogeneous nuclear RNA (hnRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), micro RNA (miRNA), Piwi-interacting RNA (piRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), singe guide RNA (sgRNA), non-coding RNA (ncRNA), long non-coding RNA (IncRNA), 7SL, Xist, short enhancer RNA (eRNA), circular RNA, intergenic RNA, or any combination thereof. In an aspect, a disclosed encoded RNA can comprise IncRNA, siRNA, shRNA, sgRNA, circular RNA, snoRNA, miRNA, or any combination thereof. In an aspect, a disclosed encoded RNA can comprise a functional non-coding RNA element.

[0162] In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the gene expression desired. A promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. In an aspect, a disclosed promoter can be a promoter/enhancer. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter/enhancer. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can generally be obtained from a non-coding region upstream of a transcription initiation site of a gene of interest. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can be used for constitutive and efficient expression of a disclosed gene. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be a CMV promoter or a CMV promoter/enhancer. CMV promoters and CMV promoters/enhancers are well known to the art. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be any eukaryotic RNA polymerase II promoter.

[0163] Disclosed herein is an expression cassette, comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein. [0164] Disclosed herein is a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive PspdCasl3b, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive PspdCasl3b; and a polyadenylation signal.

[0165] In an aspect, a disclosed Casl3d can further comprise one or more other agents or domains (e.g., is a fusion protein), such as one or more subcellular localization signals, one or more effector domains, or any combinations thereof.

[0166] In an aspect, a disclosed promoter for a catalytically inactive PspdCasl3b can be tissuespecific or ubiquitous and can be constitutive or inducible, depending on the pattern of the gene expression desired. A promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. In an aspect, a disclosed promoter for a catalytically inactive PspdCasl3b can be a promoter/enhancer. In an aspect, a disclosed promoter can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter/enhancer. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can generally be obtained from a non-coding region upstream of a transcription initiation site of a gene of interest. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can be used for constitutive and efficient expression of a disclosed gene. In an aspect, a disclosed promoter for a catalytically inactive PspdCasl3b can be a CMV promoter or a CMV promoter/enhancer. CMV promoters and CMV promoters/enhancers are well known to the art. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be any eukaryotic RNA polymerase II promoter.

[0167] In an aspect, a disclosed isolated nucleic acid molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, a disclosed isolated nucleic acid molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0168] Disclosed herein is an expression cassette, a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive PspdCasl3b, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive PspdCasl3b; and a polyadenylation signal.

[0169] Disclosed herein is a nucleic acid molecule, comprising a nucleic acid sequence encoding a Cast 3 alternative, a promoter operably linked to the nucleic acid sequence encoding the Cast 3 alternative; and a polyadenylation signal. Disclosed herein is an expression cassette, a nucleic acid molecule, comprising a nucleic acid sequence encoding a Cast 3 alternative, a promoter operably linked to the nucleic acid sequence encoding the Cast 3 alternative; and a polyadenylation signal. In an aspect, a Casl3 alternative can comprise alternatives known to the art including, but not limited to CRISPR-Inspired RNA Targeting System (CIRTS), Pumillo and FBF (PUF), rCas9, and/or any bifunctional RNA binding protein that can both bind to the transsplicing RNA molecule and the target RNA to facilitate interaction between the two RNA species. RNA binding proteins are discussed in depth supra.

Internal Replacement Constructs

[0170] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence to be transspliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron; a 5’ hemi intron; two or more guide RNA sequences; a promoter operably linked to the two or more guide RNA sequences; and a nucleic acid sequence encoding one or more RNA binding proteins.

[0171] In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 20 SNPs. In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 10 SNPs. In an aspect, a disclosed series of SNPs can comprise 5 SNPs.

[0172] In an aspect, a disclosed isolated nucleic acid molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, a disclosed isolated nucleic acid molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0173] In an aspect, a disclosed isolated nucleic acid molecule can further comprise a polyadenylation signal.

[0174] In an aspect, a disclosed isolated nucleic acid molecule can further comprise a spacer region. In an aspect, a disclosed spacer region can separate the 3’ splice region from the one or more guide RNA sequences. In an aspect, a disclosed spacer region can separate the 5’ splice region from the one or more guide RNA sequences. In an aspect, a disclosed spacer region can comprise any known spacer. In an aspect, a disclosed spacer region can comprise a consensus splicing motif (e.g., such as U1 or U2). In an aspect, a disclosed spacer region can comprise a limited number of consensus splicing motifs (e.g., such as U1 or U2).

[0175] In an aspect, a disclosed isolated nucleic acid molecule can further comprise two or more stem loops. In an aspect, a disclosed stem loop can be a cognate aptamer for a disclosed RNA binding protein. For example, in an aspect, a disclosed stem loop can be a direct repeat of the guide RNA scaffold for a disclosed Casl3d. In an aspect, a disclosed stem loop can facilitate interaction between a disclosed RNA molecule and a disclosed Cas protein. [0176] In an aspect, a disclosed isolated nucleic acid molecule can further comprise a nuclear localization signal. In an aspect, a disclosed NLS can be comprise the sequence set forth in SEQ ID NO:60. In an aspect, a disclosed NLS can comprise any NLS known to the art. As known to the art (see, e.g., Lu J, et al. (2021) Cell Commun Signal. 19:60, which is incorporated herein by reference for its teachings of NLS), nuclear localization signals (NLS) are generally short peptides that act as a signal fragment that mediates the transport of proteins from the cytoplasm into the nucleus.

[0177] In an aspect, the two or more disclosed guide RNA sequences can directed the intron immediately 3’ to the target exon of the target endogenous pre-mRNA and the intron immediately 5’ to the target exon of the target endogenous pre-mRNA.

[0178] In an aspect, a disclosed 3’ hemi intron can comprise a branch point sequence, a polypyrimidine tract, and a 3’ splice acceptor site. In an aspect, a disclosed branch point sequence can comprise the sequence set forth in SEQ ID NO:57 (YNYYRAY, wherein Y is a pyrimidine and R is a purine). In an aspect, a disclosed branch point sequence can be any eukaryotic branch point sequence known to the art. In an aspect, a disclosed 3’ splice acceptor site can comprise the sequence set forth in SEQ ID NO:58 (YAG, wherein Y is a pyrimidine).

[0179] In an aspect of a disclosed isolated nucleic acid molecule, a disclosed 5’ hemi intron can comprise a consensus 5’ splice site. In an aspect, a disclosed 5’ splice site can comprise the sequence set forth in SEQ ID NO:59 (GT). In an aspect, a disclosed consensus 5’ splice site can comprise the sequence set forth in SEQ ID NO:61. In an aspect, a disclosed consequence 5’ splice site can comprise MAG|GURAGU (SEQ ID NO:61), wherein | denotes the exon intron junction, wherein M = A or C, and wherein R = A or G.

[0180] In an aspect, a disclosed 3’ hemi intron and/or a disclosed 5’ hemi intron can be recognized by nuclear splicing components within a host cell. In an aspect, a disclosed nucleic acid sequence encoding the RNA binding protein can interact with the two or more stem loops and/or can stabilize the two or more guide RNA sequences.

[0181] As known to the skilled person, RNA binding proteins (RBPs) can be important effectors of gene expression. RBPs can recognize hundreds of transcripts and form extensive regulatory networks that help to maintain cell homeostasis. Accordingly, the malfunction of RBPs underlies the origin of many diseases. In an aspect, a disclosed RNA binding protein can be any RNA binding protein having bi specific affinity for the trans-splicing RNA and the target pre-mRNA of interest. In an aspect, this affinity can be mediated by ribonucleoprotein interactions by, for example, Type VI CRISPR enzymes, or through direct RNA protein interactions by, for example, Pumillo and FBF (PUF) proteins. In an aspect, these interactions can be mediated by protein/ aptamer interactions. RNA binding proteins are discussed in depth supra.

[0182] In an aspect, a disclosed RNA binding protein can comprise bispecific affinity for a disclosed target pre-mRNA as well as a disclosed Casl3 or a disclosed catalytically inactive Casl3. In an aspect, a disclosed Cast 3 can comprise any catalytically inactive Casl3. For example, in an aspect, a disclosed Casl3 can comprise a catalytically inactive RfxCasl3d or a catalytically inactive PspdCasl3b. For example, in an aspect, a Casl3 alternative can comprise alternatives known to the art including, but not limited to CRISPR-Inspired RNA Targeting System (CIRTS), Pumillo and FBF (PUF), rCas9, and/or any bifunctional RNA binding protein that can both bind to the trans-splicing RNA molecule and the target RNA to facilitate interaction between the two RNA species. RNA binding proteins are discussed in depth supra.

[0183] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode a translatable protein or a portion thereof. In an aspect, a disclosed portion can comprise one or more exons comprising a mutation. In an aspect, a disclosed portion can comprise some part of the gene sequence but not the complete sequence. For example, in an aspect, a disclosed portion can comprise the nucleic acid sequence having one or more mutations.

[0184] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode DP71 or a portion thereof. DP71 is known to the art and discussed supra.

[0185] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode DMPK or a portion thereof. DMPK is known to the art and discussed supra. In an aspect, the one or more disclosed guide RNA sequences can comprise the sequence set forth in SEQ ID NO:29 or SEQ ID NO:30 or a fragment thereof.

[0186] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode DMD or a portion thereof. DMD is known to the art and discussed supra. In an aspect, the one or more disclosed guide RNA sequences can comprise the sequence set forth in SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28 or a fragment thereof.

[0187] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode LMNA/C or a portion thereof. LMNA/C is known to the art and discussed supra. In an aspect, the one or more disclosed guide RNA sequences can comprise the sequence set forth in SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:32 or a fragment thereof.

[0188] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode CFTR or a portion thereof. CFTR is known to the art and discussed supra. In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode LRRK2 or a portion thereof. LRRK2 is known to the art and discussed supra. [0189] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode the protein or a portion thereof (such as, for example, Exon 1 or Exon 4, etc.) associated with the following genes: ABCA1, ABCA12, ABCA13, ABCA2, ABCA3, ABCA4, ABCA5, ABCC1, ABCC2, ABCC6, ABCC8, ABCC9, ACAN, ADAMTS13, ADCY10, ADGRV1, AGL, AGRN, AHDC1, ALK, ALMS1, ALPK3, ALS2, ANAPC1, ANK1, ANK2, ANK3, ANKRD11, ANKRD26, APC, APC2, APOB, ARFGEF2, ARHGAP31, ARHGEF10, ARHGEF18, ARID ! A, ARID I B, ARID2, ASH1L, ASPM, ASXL1, ASXL2, ASXL3, ATM, ATP7A, ATP7B, ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, BLM, BPTF, BRCA1, BRCA2, BRIM, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD, CAMTAI, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44, CFAP65, CFTR/ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1, CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1, CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1, DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11, DNAH17, DNAH2, DNAH5, DNAH7, DNAH8, DNAH9, DNMBP, DNMT1, DOCK2, DOCK3, DOCK6, DOCK7, DOCK8, DSCAM, DSP, DST, DUOX2, DYNC1H1, DYNC2H1, DYSF, EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, EYS, F5, F8, FANCA, FANCD2, FANCM, FAT1, FAT4, FBN1, FBN2, FLG, FLG2, FLNA, FLNB, FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2, FSIP2, FYCO1, GLI2, GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1, HERC2, HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R, IGF2R, IGSF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A, KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220, KIF14, KIF1A, KIF1B, KIF21A, KIF26B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1, LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMC3, LCT, LOXHD1, LPA, LRBA, LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD, MAGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5, MCM3AP, MED12, MED12L, MED13, MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MYH11, MYH14, MYH2, MYH3, MYH6, MYH7, MYH7B, MYH8, MYH9, MYLK, MYO 15 A, MYO18B, MYO3A, MYO5A, MYO5B, MYO7A, MYO9A, NALCN, NBAS, NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF, NF1, NFASC, NHS, NIN, NIPBL, NLRP1, N0TCH1, NOTCH2, NOTCH3, NPHP4, NRXN1, NRXN3, NSD1, NSD2, NUP155, NUP188, NUP205, OBSCN, OBSL1, OTOF, OTOG, OTOGL, PARD3, PBRM1, PCDH15, PCLO, PCNT, PHIP, PI4KA, PIEZO1, PIEZO2, PIK3C2A, PIKFYVE, PKD1, PKD1L1, PKHD1, PLCE1, PLEC, PLEKHG2, PNPLA6, POGZ, POLA1, POLE, POLR1A, POLR2A, POLR3A, PRG4, PRKDC, PRPF8, PRR12, PRX, PTCHI, PTPN23, PTPRF, PTPRJ, PTPRQ, PXDN, QRICH2, RAB3GAP2, RAH, RALGAPA1, RANBP2, RB1CC1, RELN, RERE, REV3L, RIC1, RIMS1, RIMS2, RNF213, ROBO1, ROBO2, ROBO3, ROS1, RP1, RP1L1, RTTN, RUSC2, RYR1, RYR2, SACS, SAMD9, SAMD9L, SBF2, SCAPER, SCN10A, SCN11A, SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SETBP1, SETD1A, SETD1B, SETD2, SETD5, SETX, SHANK2, SHANK3, SHR00M4, SI, SIPA1L3, SLIT2, SLX4, SMARCA2, SMARCA4, SMCHD1, SNRNP200, SON, SPEF2, SPEG, SPG11, SPTA1, SPTAN1, SPTB, SPTBN2, SPTBN4, SRCAP, STRC, SVIL, SYNE1, SYNGAP1, SYNJ1, SZT2, TAF1, TANC2, TCF20, TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B, TNXB, TOGARAMI, TONSL, TRIO, TRIOBP, TRIP11, TRIP12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, TTN, TUBGCP6, UBR1, UNC80, USH2A, USP9X, VCAN, VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62, WDR81, WNK1, WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292, ZNF335, ZNF407, ZNF462, ZNF469, or a portion thereof.

[0190] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode the protein or a portion thereof (such as, for example, Exon 1 or Exon 4, etc.) associated with the following genes: DMD, TTN, TCAP, SGCA, SGCB, SGCG, SGCD, Al-AT, MYH6, MYH7, MYH11, ML2, ML3, MYLK2, MYBPC3, DES, DNM2, LAMA2, LMNA, LMNB, LBR, DYSF, EMD, EPO, LPL, SERCA2A, S100A1, MTM, DM1 DMPK, PYGL„ PYGM, GYSI, GYS2, NAGA, GAA, SMPD1, LIPA, COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, PLOD1, LIPA, FXN, MSTN, HEXA, HEXB, GBA, AMPD1, HBB, IDUA, IDS, TNNI3, TNNT2, TNNC1, TPM1, TPM3, NAGLU, SGSH, HGSNAT, IGTA7, IGTA9, N-acetyl, GNS, N-acetyl, GALNS, GLB1, GUSB, HYAL1, ASAHI, GALC, CTSA, CTSA, CTSK, GM2A, ARSA, ARSB, SUMFI, NEU1 GNPTA, GNPTB, GNPTG, MCOLN1, NPC1, NPC2, CLN5, CLN6, CLN8, PPT1, TPP1, CLN3, DNAJC5, MFSD8, MAN2B1, MANBA, AGA, FUCA1, CTNS, SLC2A10, SLC17A5, SLC6A19, SLC22A5, SLC37A4, LAMP2, SCN4A, SCN4B, SCN5A, SCN4A, CACNA1C, CACNA1S, PGK1, PGAM2, AGL, KCNE1, KCNE2, KCNJ2, KCNJ5, KCNH2, KCNQ1, HCN4, CLCN1, CPT1A, RYR1, RYR2, BINI, LARGE1, DOK7, FKTN, FKRP, SELENON, POMT1, POMT2, POMGNT1, POMGNT2, POMK, ISPD, PLEC, CHRNE, CHAT, CHKB, COLQ, RAPSN, FHL1, B4GAT1, B3GALNT2, DAGI, TMEM5, TMEM43, SECISBP2, UDP-N-acetyl, GNE, AN05, SMCHD1, LDHA, LHDB, CAPN3, CAV3, TRIM32, CNBP, NEB, ACTA1, ACTC1, ACTN2, PABPN1, LEMD3, ZMPSTE24, MTTP, CHRNA1, CHRNA2, CHRNA3, CHRNA4, CHRNA5, CHRNA6, CHRNA7, CHRNA8, CHRNA9, CHRNA10, CHRNB1, CHRNB2, CHRNB3, CHRNB4, CHRNG1, CHRND, CHRNE1, ABCA1, ABCC6, ABCC9, ABCD1, ATP2A1, ATM, TTP A, KIF21A, PHOX2A, HSPG2, STIM1, NOTCHI, NOTCH3, DTNA, PRKAG2, CSRP3, VCL, MyoZ2, MYPN, JPH2, PLN, CALR3, NEXN, LDB3, EYA4, HTT, AR, PTPN11, JUP, DSP, PKP2, DSG2, DSC2, CTNNA3, NKX2-5, AKAP9, AKAP10, GNAI2, ANK2, SNTAT, CALM1, CALM2, HTRA1, FBN1, FBN2, XYLT1, XYLT2, TAZ, HGD, G6PC, GBE1, PFKM, PHKA1, PHKA2, PHKB, PHKG2, PGAM2, CBS, MTHFR, MTR, MTRR, MMADHC, MT-ND1, MT-ND5, MT-TE, MT- TH, MT-TL1, MT-TK, MT-TS1, MT-TV, MAP2K1, BRAF, RAFI, IGF-1, TGFP3, TGFpRl, TGFPR2, FGF2, FGF4, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, or VEGFR2.

[0191] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can be CpG depleted and codon-optimized for expression in a human cell. In an aspect, “CpG-free” can mean completely free of CpGs or partially free of CpGs. In an aspect, “CpG-free” can mean “CpG- depleted”. In an aspect, “CpG-depleted” can mean “CpG-free”. In an aspect, “CpG-depleted” can mean completely depleted of CpGs or partially depleted of CpGs. In an aspect, “CpG-free” can mean “CpG-optimized” for a desired and/or ideal expression level. CpG depletion and/or optimization is known to the skilled person in the art.

[0192] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode an RNA. In an aspect, a disclosed encoded RNA can comprise ribosomal RNA (rRNA), transfer RNA (tRNA), heterogeneous nuclear RNA (hnRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), micro RNA (miRNA), Piwi-interacting RNA (piRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), singe guide RNA (sgRNA), non-coding RNA (ncRNA), long non-coding RNA (IncRNA), 7SL, Xist, short enhancer RNA (eRNA), circular RNA, intergenic RNA, or any combination thereof. In an aspect, a disclosed encoded RNA can comprise IncRNA, siRNA, shRNA, sgRNA, circular RNA, snoRNA, miRNA, or any combination thereof. In an aspect, a disclosed encoded RNA can comprise a functional non-coding RNA element.

[0193] In an aspect, a disclosed promoter for the two or more disclosed guide RNA sequences can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the gene expression desired. A promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. In an aspect, a disclosed promoter can be a promoter/enhancer. In an aspect, a disclosed promoter for the two or more disclosed guide RNA sequences can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter/enhancer. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can generally be obtained from a non-coding region upstream of a transcription initiation site of a gene of interest. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can be used for constitutive and efficient expression of a disclosed gene. In an aspect, a disclosed promoter for the two or more disclosed guide RNA sequences can be a CMV promoter or a CMV promoter/enhancer. CMV promoters and CMV promoters/enhancers are well known to the art. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be any eukaryotic RNA polymerase II promoter.

[0194] Disclosed herein is an expression cassette comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron; a 5’ hemi intron; two or more guide RNA sequences; a promoter operably linked to the two or more guide RNA sequences; and a nucleic acid sequence encoding one or more RNA binding proteins.

[0195] Disclosed herein is a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d; a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal.

[0196] In an aspect, a disclosed isolated nucleic acid molecule comprising a catalytically inactive RfxCasl3d can further comprise a nuclear localization signal. In an aspect, a disclosed catalytically inactive RfxCasl3d can comprise one or more inactivation mutations. In an aspect, a disclosed inactivation mutation can comprise R295A, H300A, R849A, H854A, or any combination thereof.

[0197] In an aspect, a disclosed Casl3d can further comprise one or more other agents or domains (e.g., is a fusion protein), such as one or more subcellular localization signals, one or more effector domains, or any combinations thereof.

[0198] In an aspect, a disclosed promoter for a catalytically inactive RfxCasl3d can be tissuespecific or ubiquitous and can be constitutive or inducible, depending on the pattern of the gene expression desired. A promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. In an aspect, a disclosed promoter for a catalytically inactive RfxCasl3d can be a promoter/enhancer. In an aspect, a disclosed promoter can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter/enhancer. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can generally be obtained from a non-coding region upstream of a transcription initiation site of a gene of interest. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can be used for constitutive and efficient expression of a disclosed gene. In an aspect, a disclosed promoter for a catalytically inactive RfxCasl3d can be a CMV promoter or a CMV promoter/enhancer. CMV promoters and CMV promoters/enhancers are well known to the art. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be any eukaryotic RNA polymerase II promoter.

[0199] In an aspect, a disclosed isolated nucleic acid molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, a disclosed isolated nucleic acid molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0200] Disclosed herein is an expression cassette comprising a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d; a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal.

[0201] Disclosed herein is a nucleic acid molecule, comprising a nucleic acid sequence encoding a Cast 3 alternative; a promoter operably linked to the nucleic acid sequence encoding the a Cast 3 alternative; and a polyadenylation signal. Disclosed herein is an expression cassette comprising a nucleic acid molecule, comprising a nucleic acid sequence encoding a Casl3 alternative; a promoter operably linked to the nucleic acid sequence encoding the Cast 3 alternative; and a polyadenylation signal. In an aspect, a Casl3 alternative can comprise alternatives known to the art including, but not limited to CRISPR-Inspired RNA Targeting System (CIRTS), Pumillo and FBF (PUF), rCas9, and/or any bifunctional RNA binding protein that can both bind to the transsplicing RNA molecule and the target RNA to facilitate interaction between the two RNA species. RNA binding proteins are discussed in depth supra.

3’ Replacement Constructs

[0202] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence to be transspliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein.

[0203] In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 20 SNPs. In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 10 SNPs. In an aspect, a disclosed series of SNPs can comprise 5 SNPs.

[0204] In an aspect, a disclosed isolated nucleic acid molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, a disclosed isolated nucleic acid molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0205] In an aspect, a disclosed isolated nucleic acid molecule can further comprise a polyadenylation signal.

[0206] In an aspect, a disclosed isolated nucleic acid molecule can further comprise a spacer region. In an aspect, a disclosed spacer region can separate the 3’ splice region from the one or more guide RNA sequences. In an aspect, a disclosed spacer region can comprise any known spacer. In an aspect, a disclosed spacer region can comprise a consensus splicing motif (e.g., such as U1 or U2). In an aspect, a disclosed spacer region can comprise a limited number of consensus splicing motifs (e.g., such as U1 or U2).

[0207] In an aspect, a disclosed isolated nucleic acid molecule can further comprise one or more stem loops. In an aspect, a disclosed stem loop can be a cognate aptamer for a disclosed RNA binding protein. For example, in an aspect, a disclosed stem loop can be a direct repeat of the guide RNA scaffold for a disclosed Casl3d. In an aspect, a disclosed stem loop can facilitate interaction between a disclosed RNA molecule and a disclosed Cas protein.

[0208] In an aspect, a disclosed isolated nucleic acid molecule can further comprise a nuclear localization signal. In an aspect, a disclosed NLS can be comprise the sequence set forth in SEQ ID NO:60. In an aspect, a disclosed NLS can comprise any NLS known to the art. As known to the art (see, e.g., Lu J, et al. (2021) Cell Commun Signal. 19:60, which is incorporated herein by reference for its teachings of NLS), nuclear localization signals (NLS) are generally short peptides that act as a signal fragment that mediates the transport of proteins from the cytoplasm into the nucleus.

[0209] In an aspect, the one or more disclosed guide RNA sequences can direct the intron immediately 3’ to the last exon of the target endogenous pre-mRNA.

[0210] In an aspect, a disclosed 3’ hemi intron can comprise a branch point sequence, a polypyrimidine tract, and a 3’ splice acceptor site. In an aspect, a disclosed branch point sequence can comprise the sequence set forth in SEQ ID NO:57 (YNYYRAY, wherein Y is a pyrimidine and R is a purine). In an aspect, a disclosed branch point sequence can be any eukaryotic branch point sequence known to the art. In an aspect, a disclosed 3’ splice acceptor site can comprise the sequence set forth in SEQ ID NO:58 (YAG, wherein Y is a pyrimidine).

[0211] In an aspect, a disclosed 3’ hemi intron can be recognized by nuclear splicing components within a host cell. In an aspect, a disclosed nucleic acid sequence encoding the RNA binding protein can interact with the one or more stem loops and/or can stabilize the one or more guide RNA sequences.

[0212] As known to the skilled person, RNA binding proteins (RBPs) can be important effectors of gene expression. RBPs can recognize hundreds of transcripts and form extensive regulatory networks that help to maintain cell homeostasis. Accordingly, the malfunction of RBPs underlies the origin of many diseases. In an aspect, a disclosed RNA binding protein can be any RNA binding protein having bi specific affinity for the trans-splicing RNA and the target pre-mRNA of interest. In an aspect, this affinity can be mediated by riboncleoprotein interactions by, for example, Type VI CRISPR enzymes, or through direct RNA protein interactions by, for example, Pumillo and FBF (PUF) proteins. In an aspect, these interactions can be mediated by protein/ aptamer interactions. RNA binding proteins are discussed in depth supra.

[0213] In an aspect, a disclosed RNA binding protein can comprise bispecific affinity for a disclosed target pre-mRNA as well as a disclosed Casl3 or a disclosed catalytically inactive Casl3. In an aspect, a disclosed Cast 3 can comprise any catalytically inactive Casl3. For example, in an aspect, a disclosed Casl3 can comprise a catalytically inactive RfxCasl3d or a catalytically inactive PspdCasl3b. For example, in an aspect, a Casl3 alternative can comprise alternatives known to the art including, but not limited to CRISPR-Inspired RNA Targeting System (CIRTS), Pumillo and FBF (PUF), rCas9, and/or any bifunctional RNA binding protein that can both bind to the trans-splicing RNA molecule and the target RNA to facilitate interaction between the two RNA species. RNA binding proteins are discussed in depth supra.

[0214] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode a translatable protein or a portion thereof. In an aspect, a disclosed portion can comprise one or more exons comprising a mutation. In an aspect, a disclosed portion can comprise some part of the gene sequence but not the complete sequence. For example, in an aspect, a disclosed portion can comprise the nucleic acid sequence having one or more mutations.

[0215] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode DP71 or a portion thereof. [0216] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode DMPK or a portion thereof. In an aspect, the one or more disclosed guide RNA sequences can comprise the sequence set forth in SEQ ID NO:29 or SEQ ID NO:30 or a fragment thereof.

[0217] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode DMD or a portion thereof. In an aspect, the one or more disclosed guide RNA sequences can comprise the sequence set forth in SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28 or a fragment thereof.

[0218] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode LMNA/C or a portion thereof. In an aspect, the one or more disclosed guide RNA sequences can comprise the sequence set forth in SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:32 or a fragment thereof.

[0219] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode CFTR or a portion thereof. In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode LRRK2 or a portion thereof.

[0220] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode the protein or a portion thereof (such as, for example, Exon 1 or Exon 4, etc.) associated with the following genes: ABCA1, ABCA12, ABCA13, ABCA2, ABCA3, ABCA4, ABCA5, ABCC1, ABCC2, ABCC6, ABCC8, ABCC9, ACAN, ADAMTS13, ADCY10, ADGRV1, AGL, AGRN, AHDC1, ALK, ALMS1, ALPK3, ALS2, ANAPC1, ANK1, ANK2, ANK3, ANKRD11, ANKRD26, APC, APC2, APOB, ARFGEF2, ARHGAP31, ARHGEF10, ARHGEF18, ARID ! A, ARID I B, ARID2, ASH1L, ASPM, ASXL1, ASXL2, ASXL3, ATM, ATP7A, ATP7B, ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, BLM, BPTF, BRCA1, BRCA2, BRD4, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD, CAMTAI, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44, CFAP65, CFTR/ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1, CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1, CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1, DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11, DNAH17, DNAH2, DNAH5, DNAH7, DNAH8, DNAH9, DNMBP, DNMT1, DOCK2, DOCK3, DOCK6, DOCK7, DOCK8, DSCAM, DSP, DST, DU0X2, DYNC1H1, DYNC2H1, DYSF, EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, EYS, F5, F8, FANCA, FANCD2, FANCM, FAT1, FAT4, FBN1, FBN2, FLG, FLG2, FLNA, FLNB, FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2, FSIP2, FYCO1, GLI2, GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1, HERC2, HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R, IGF2R, IGSF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A, KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220, KIF14, KIF1A, KIF1B, KIF21A, KIF26B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1, LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMC3, LCT, L0XHD1, LPA, LRBA, LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD, MAGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5, MCM3AP, MED12, MED12L, MED13, MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MYH11, MYH14, MYH2, MYH3, MYH6, MYH7, MYH7B, MYH8, MYH9, MYLK, MYO 15 A, MYO18B, MYO3A, MYO5A, MYO5B, MYO7A, MYO9A, NALCN, NBAS, NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF, NF1, NFASC, NHS, NIN, NIPBL, NLRP1, NOTCH1, NOTCH2, NOTCH3, NPHP4, NRXN1, NRXN3, NSD1, NSD2, NUP155, NUP188, NUP205, OBSCN, OBSL1, OTOF, OTOG, OTOGL, PARD3, PBRM1, PCDH15, PCLO, PCNT, PHIP, PI4KA, PIEZO1, PIEZO2, PIK3C2A, PIKFYVE, PKD1, PKD1L1, PKHD1, PLCE1, PLEC, PLEKHG2, PNPLA6, POGZ, POLA1, POLE, POLR1A, POLR2A, POLR3A, PRG4, PRKDC, PRPF8, PRR12, PRX, PTCHI, PTPN23, PTPRF, PTPRJ, PTPRQ, PXDN, QRICH2, RAB3GAP2, RAH, RALGAPA1, RANBP2, RB1CC1, RELN, RERE, REV3L, RIC1, RIMS1, RIMS2, RNF213, ROBO1, ROBO2, ROBO3, ROS1, RP1, RP1L1, RTTN, RUSC2, RYR1, RYR2, SACS, SAMD9, SAMD9L, SBF2, SCAPER, SCN10A, SCN11A, SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SETBP1, SETD1A, SETD1B, SETD2, SETD5, SETX, SHANK2, SHANK3, SHROOM4, SI, SIPA1L3, SLIT2, SLX4, SMARCA2, SMARCA4, SMCHD1, SNRNP200, SON, SPEF2, SPEG, SPG11, SPTA1, SPTAN1, SPTB, SPTBN2, SPTBN4, SRCAP, STRC, SVIL, SYNE1, SYNGAP1, SYNJ1, SZT2, TAF1, TANC2, TCF20, TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B, TNXB, TOGARAMI, TONSL, TRIO, TRIOBP, TRIP11, TRIP12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, TTN, TUBGCP6, UBR1, UNC80, USH2A, USP9X, VCAN, VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62, WDR81, WNK1, WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292, ZNF335, ZNF407, ZNF462, ZNF469, or a portion thereof.

[0221] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode the protein or a portion thereof (such as, for example, Exon 1 or Exon 4, etc.) associated with the following genes: DMD, TTN, TCAP, SGCA, SGCB, SGCG, SGCD, Al-AT, MYH6, MYH7, MYH11, ML2, ML3, MYLK2, MYBPC3, DES, DNM2, LAMA2, LMNA, LMNB, LBR, DYSF, EMD, EPO, LPL, SERCA2A, S100A1, MTM, DM1 DMPK, PYGL„ PYGM, GYSI, GYS2, NAGA, GAA, SMPD1, LIPA, COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, PLOD1, LIPA, FXN, MSTN, HEXA, HEXB, GBA, AMPD1, HBB, IDUA, IDS, TNNI3, TNNT2, TNNC1, TPM1, TPM3, NAGLU, SGSH, HGSNAT, IGTA7, IGTA9, N-acetyl, GNS, N-acetyl, GALNS, GLB1, GUSB, HYAL1, ASAHI, GALC, CTSA, CTSA, CTSK, GM2A, ARSA, ARSB, SUMFI, NEU1 GNPTA, GNPTB, GNPTG, MCOLN1, NPC1, NPC2, CLN5, CLN6, CLN8, PPT1, TPP1, CLN3, DNAJC5, MFSD8, MAN2B1, MANBA, AGA, FUCA1, CTNS, SLC2A10, SLC17A5, SLC6A19, SLC22A5, SLC37A4, LAMP2, SCN4A, SCN4B, SCN5A, SCN4A, CACNA1C, CACNA1S, PGK1, PGAM2, AGL, KCNE1, KCNE2, KCNJ2, KCNJ5, KCNH2, KCNQ1, HCN4, CLCN1, CPT1A, RYR1, RYR2, BINI, LARGE1, DOK7, FKTN, FKRP, SELENON, POMT1, POMT2, POMGNT1, POMGNT2, POMK, ISPD, PLEC, CHRNE, CHAT, CHKB, COLQ, RAPSN, FHL1, B4GAT1, B3GALNT2, DAGI, TMEM5, TMEM43, SECISBP2, UDP-N-acetyl, GNE, AN05, SMCHD1, LDHA, LHDB, CAPN3, CAV3, TRIM32, CNBP, NEB, ACTA1, ACTC1, ACTN2, PABPN1, LEMD3, ZMPSTE24, MTTP, CHRNA1, CHRNA2, CHRNA3, CHRNA4, CHRNA5, CHRNA6, CHRNA7, CHRNA8, CHRNA9, CHRNA10, CHRNB1, CHRNB2, CHRNB3, CHRNB4, CHRNG1, CHRND, CHRNE1, ABC Al, ABCC6, ABCC9, ABCD1, ATP2A1, ATM, TTP A, KIF21A, PHOX2A, HSPG2, STIM1, NOTCHI, NOTCH3, DTNA, PRKAG2, CSRP3, VCL, MyoZ2, MYPN, JPH2, PLN, CALR3, NEXN, LDB3, EYA4, HTT, AR, PTPN11, JUP, DSP, PKP2, DSG2, DSC2, CTNNA3, NKX2-5, AKAP9, AKAP10, GNAI2, ANK2, SNTAT, CALM1, CALM2, HTRA1, FBN1, FBN2, XYLT1, XYLT2, TAZ, HGD, G6PC, GBE1, PFKM, PHKA1, PHKA2, PHKB, PHKG2, PGAM2, CBS, MTHFR, MTR, MTRR, MMADHC, MT-ND1, MT-ND5, MT-TE, MT- TH, MT-TL1, MT-TK, MT-TS1, MT-TV, MAP2K1, BRAF, RAFI, IGF-1, TGFP3, TGFpRl, TGFPR2, FGF2, FGF4, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, or VEGFR2.

[0222] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode a gene or a portion thereof (e.g., a specific exon such as an exon having a mutation) with a gene product that is directly or indirectly linked to one or more genetic diseases.

[0223] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can be CpG depleted and codon-optimized for expression in a human cell. In an aspect, “CpG-free” can mean completely free of CpGs or partially free of CpGs. In an aspect, “CpG-free” can mean “CpG- depleted”. In an aspect, “CpG-depleted” can mean “CpG-free”. In an aspect, “CpG-depleted” can mean completely depleted of CpGs or partially depleted of CpGs. In an aspect, “CpG-free” can mean “CpG-optimized” for a desired and/or ideal expression level. CpG depletion and/or optimization is known to the skilled person in the art.

[0224] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode an RNA. In an aspect, a disclosed encoded RNA can comprise ribosomal RNA (rRNA), transfer RNA (tRNA), heterogeneous nuclear RNA (hnRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), micro RNA (miRNA), Piwi-interacting RNA (piRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), singe guide RNA (sgRNA), non-coding RNA (ncRNA), long non-coding RNA (IncRNA), 7SL, Xist, short enhancer RNA (eRNA), circular RNA, intergenic RNA, or any combination thereof. In an aspect, a disclosed encoded RNA can comprise IncRNA, siRNA, shRNA, sgRNA, circular RNA, snoRNA, miRNA, or any combination thereof. In an aspect, a disclosed encoded RNA can comprise a functional non-coding RNA element.

[0225] In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the gene expression desired. A promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. In an aspect, a disclosed promoter can be a promoter/enhancer. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter/enhancer. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can generally be obtained from a non-coding region upstream of a transcription initiation site of a gene of interest. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can be used for constitutive and efficient expression of a disclosed gene. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be a CMV promoter or a CMV promoter/enhancer. CMV promoters and CMV promoters/enhancers are well known to the art. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be any eukaryotic RNA polymerase II promoter.

[0226] Disclosed herein is an expression cassette comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein. [0227] Disclosed herein is a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d; a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal.

[0228] In an aspect, a disclosed isolated nucleic acid molecule comprising a catalytically inactive RfxCasl3d can further comprise a nuclear localization signal. In an aspect, a disclosed catalytically inactive RfxCasl3d can comprise one or more inactivation mutations. In an aspect, a disclosed inactivation mutation can comprise R295A, H300A, R849A, H854A, or any combination thereof.

[0229] In an aspect, a disclosed Casl3d can further comprise one or more other agents or domains (e.g., is a fusion protein), such as one or more subcellular localization signals, one or more effector domains, or any combinations thereof.

[0230] In an aspect, a disclosed promoter for a catalytically inactive RfxCasl3d can be tissuespecific or ubiquitous and can be constitutive or inducible, depending on the pattern of the gene expression desired. A promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. In an aspect, a disclosed promoter can be a promoter/enhancer. In an aspect, a disclosed promoter for a catalytically inactive RfxCasl3d can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter/enhancer. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can generally be obtained from a non-coding region upstream of a transcription initiation site of a gene of interest. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can be used for constitutive and efficient expression of a disclosed gene. In an aspect, a disclosed promoter for a catalytically inactive RfxCasl3d can be a CMV promoter or a CMV promoter/enhancer. CMV promoters and CMV promoters/enhancers are well known to the art. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be any eukaryotic RNA polymerase II promoter.

[0231] In an aspect, a disclosed isolated nucleic acid molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, a disclosed isolated nucleic acid molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0232] Disclosed herein is an expression cassette comprising a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d; a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal.

[0233] Disclosed herein is a nucleic acid molecule, comprising a nucleic acid sequence encoding a Cast 3 alternative; a promoter operably linked to the nucleic acid sequence encoding the Cast 3 alternative; and a polyadenylation signal. Disclosed herein is an expression cassette comprising a nucleic acid molecule, comprising a nucleic acid sequence encoding a Casl3 alternative; a promoter operably linked to the nucleic acid sequence encoding the Cast 3 alternative; and a polyadenylation signal. In an aspect, a Casl3 alternative can comprise alternatives known to the art including, but not limited to CRISPR-Inspired RNA Targeting System (CIRTS), Pumillo and FBF (PUF), rCas9, and/or any bifunctional RNA binding protein that can both bind to the transsplicing RNA molecule and the target RNA to facilitate interaction between the two RNA species. RNA binding proteins are discussed in depth supra.

[0234] In an aspect, a disclosed resulting chimeric molecule is non-functional due to the series of SNPs. In an aspect, a disclosed resulting chimeric molecule is non-functional due to the series of SNPs. In an aspect, a disclosed nucleic acid can be used in one or more methods of trans-splicing, including, for example, SMaRT, CRAFT, and GRAFT (disclosed herein).

[0235] In an aspect, a disclosed targeted endogenous pre-mRNA can be encoded by one or more relevant genes (such as, for example, those listed below in Table 1).

Table 1 - Larges Gene with Identification of Affected Chromosomes and # of Mutations

2. Transcriptome Engineering Systems

[0236] Disclosed herein is a transcriptome engineering system comprising (i) a first isolated nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3 ’ hemi intron linked to the nucleic acid sequence to be trans- spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein; and (ii) a second isolated nucleic acid molecule comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal. [0237] In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 20 SNPs. In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 10 SNPs. In an aspect, a disclosed series of SNPs can comprise 5 SNPs.

[0238] In an aspect, a disclosed first isolated nucleic acid molecule and a disclosed second isolated nucleic acid molecule can form a ternary complex with the endogenous pre-mRNA molecule. In an aspect, a disclosed resulting chimeric RNA molecule can comprise the trans-spliced nucleic acid sequence.

[0239] Disclosed herein is a transcriptome engineering system comprising (i) a first isolated nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the nucleic acid sequence to be trans- spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein; and (ii) a second isolated nucleic acid molecule comprising a nucleic acid sequence encoding a catalytically inactive PspdCasl3b, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive PspdCasl3b; and a polyadenylation signal.

[0240] In an aspect, a disclosed first isolated nucleic acid molecule and a disclosed second isolated nucleic acid molecule can form a ternary complex with the endogenous pre-mRNA molecule. In an aspect, a disclosed resulting chimeric RNA molecule can comprise the trans-spliced nucleic acid sequence.

[0241] Disclosed herein is a transcriptome engineering system, comprising (i) a first isolated nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron; a 5’ hemi intron; two or more guide RNA sequences; a promoter operably linked to the two or more guide RNA sequences; and a nucleic acid sequence encoding one or more RNA binding proteins; and (ii) a second isolated nucleic acid molecule comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal. In an aspect, a disclosed first isolated nucleic acid molecule and a disclosed second isolated nucleic acid molecule can form a ternary complex with the endogenous pre-mRNA molecule. In an aspect, a disclosed resulting chimeric RNA molecule can comprise the trans-spliced nucleic acid sequence.

[0242] Disclosed herein is a transcriptome engineering system comprising (i) a first isolated nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3 ’ hemi intron linked to the nucleic acid sequence to be transspliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein; and (ii) a second isolated nucleic acid molecule comprising a nucleic acid sequence encoding a Casl3 alternative, a promoter operably linked to the nucleic acid sequence encoding the Cast 3 alternative; and a polyadenylation signal. In an aspect, a disclosed first isolated nucleic acid molecule and a disclosed second isolated nucleic acid molecule can form a ternary complex with the endogenous pre-mRNA molecule. In an aspect, a disclosed resulting chimeric RNA molecule can comprise the trans-spliced nucleic acid sequence.

[0243] Disclosed herein is a transcriptome engineering system comprising (i) a first isolated nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the nucleic acid sequence to be trans- spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein; and (ii) a second isolated nucleic acid molecule comprising a nucleic acid sequence encoding a Casl3 alternative, a promoter operably linked to the nucleic acid sequence encoding the Cast 3 alternative; and a polyadenylation signal. In an aspect, a disclosed first isolated nucleic acid molecule and a disclosed second isolated nucleic acid molecule can form a ternary complex with the endogenous pre-mRNA molecule. In an aspect, a disclosed resulting chimeric RNA molecule can comprise the trans-spliced nucleic acid sequence.

[0244] Disclosed herein is a transcriptome engineering system, comprising (i) a first isolated nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron; a 5’ hemi intron; two or more guide RNA sequences; a promoter operably linked to the two or more guide RNA sequences; and a nucleic acid sequence encoding one or more RNA binding proteins; and (ii) a second isolated nucleic acid molecule comprising a nucleic acid sequence encoding a Cast 3 alternative, a promoter operably linked to the nucleic acid sequence encoding the Cast 3 alternative; and a polyadenylation signal. In an aspect, a disclosed first isolated nucleic acid molecule and a disclosed second isolated nucleic acid molecule can form a ternary complex with the endogenous pre-mRNA molecule. In an aspect, a disclosed resulting chimeric RNA molecule can comprise the trans-spliced nucleic acid sequence. [0245] In an aspect, a Casl3 alternative can comprise alternatives known to the art including, but not limited to CRISPR-Inspired RNA Targeting System (CIRTS), Pumillo and FBF (PUF), rCas9, and/or any bifunctional RNA binding protein that can both bind to the trans-splicing RNA molecule and the target RNA to facilitate interaction between the two RNA species. RNA binding proteins are discussed in depth supra.

[0246] In an aspect, a disclosed transcription engineering system can be used in one or more methods of trans-splicing, including, for example, SMaRT, CRAFT, and GRAFT (disclosed herein).

3. Vectors

[0247] Disclosed herein is a vector comprising a disclosed isolated nucleic acid molecule. Disclosed herein is a vector comprising one or more disclosed isolated nucleic acid molecules. Disclosed herein is a vector comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein.

[0248] Disclosed herein is a vector comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron; a 5’ hemi intron; two or more guide RNA sequences; a promoter operably linked to the two or more guide RNA sequences; and a nucleic acid sequence encoding one or more RNA binding proteins. [0249] Disclosed herein is a vector comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein.

[0250] Disclosed herein is a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d; a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal.

[0251] Disclosed herein is a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a catalytically inactive PspdCasl3b, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive PspdCasl3b; and a polyadenylation signal. [0252] Disclosed herein is a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a Casl3 alternative; a promoter operably linked to the nucleic acid sequence encoding the Cast 3 alternative; and a polyadenylation signal. In an aspect, a Cast 3 alternative can comprise alternatives known to the art including, but not limited to CRISPR-Inspired RNA Targeting System (CIRTS), Pumillo and FBF (PUF), rCas9, and/or any bifunctional RNA binding protein that can both bind to the trans-splicing RNA molecule and the target RNA to facilitate interaction between the two RNA species. RNA binding proteins are discussed in depth supra.

[0253] Disclosed herein is a vector comprising an expression cassette comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre- mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein.

[0254] Disclosed herein is a vector comprising an expression cassette comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre- mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein.

[0255] Disclosed herein is a vector comprising an expression cassette comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre- mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron; a 5’ hemi intron; two or more guide RNA sequences; a promoter operably linked to the two or more guide RNA sequences; and a nucleic acid sequence encoding one or more RNA binding proteins.

[0256] Disclosed herein is a vector comprising an expression cassette comprising a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d; a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal.

[0257] Disclosed herein is a vector comprising an expression cassette a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive PspdCasl3b, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive PspdCasl3b; and a polyadenylation signal. [0258] Disclosed herein is a vector comprising an expression cassette a nucleic acid molecule, comprising a nucleic acid sequence encoding a Cast 3 alternative, a promoter operably linked to the nucleic acid sequence encoding the Cast 3 alternative; and a polyadenylation signal. In an aspect, a Cast 3 alternative can comprise alternatives known to the art including, but not limited to CRISPR-Inspired RNA Targeting System (CIRTS), Pumillo and FBF (PUF), rCas9, and/or any bifunctional RNA binding protein that can both bind to the trans-splicing RNA molecule and the target RNA to facilitate interaction between the two RNA species. RNA binding proteins are discussed in depth supra.

[0259] In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 20 SNPs. In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 10 SNPs. In an aspect, a disclosed series of SNPs can comprise 5 SNPs.

[0260] In an aspect, a disclosed vector can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, a disclosed vector can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0261] In an aspect, a disclosed nucleic acid sequence can have a coding sequence that is less than about 4.5 kilobases. In an aspect, a disclosed vector can be a viral vector or a non-viral vector. In an aspect, a disclosed non-viral vector can be a polymer-based vector, a peptide-based vector, a lipid nanoparticle, a solid lipid nanoparticle, or a cationic lipid-based vector. In an aspect, a disclosed vector can comprise exosomes, extracellular vesicles, and virus like particles. In an aspect, a disclosed viral vector can be an adenovirus vector, an AAV vector, a herpes simplex virus vector, a retrovirus vector, a lentivirus vector, and alphavirus vector, a Flavivirus vector, a rhabdovirus vector, a measles virus vector, a Newcastle disease viral vector, a poxvirus vector, or a picornavirus vector.

[0262] In an aspect, a disclosed viral vector can be an adeno-associated virus (AAV) vector In an aspect, a disclosed AAV vector can include naturally isolated serotypes including, but not limited to, AAV1, AAV2, AAV3 (including 3a and 3b), AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAVrh39, AAVrh43, AAVcy.7 as well as bovine AAV, caprine AAV, canine AAV, equine AAV, ovine AAV, avian AAV, primate AAV, non-primate AAV, and any other virus classified by the International Committee on Taxonomy of Viruses (ICTV) as an AAV. In an aspect, an AAV capsid can be a chimera either created by capsid evolution or by rational capsid engineering from a naturally isolated AAV variants to capture desirable serotype features such as enhanced or specific tissue tropism and/or a host immune response escape. Naturally isolated AAV variants include, but not limited to, AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV-1829, AAV2 Y/F, AAV2 T/V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A-String (e.g., AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep, and AAV9.47-AS, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, and AAVcc.81. In an aspect, a disclosed AAV vector can be AAV-Rh74 or a related variant (e.g., capsid variants like RHM4-1). In an aspect, a disclosed AAV vector can be AAV8. In an aspect, a disclosed AAV vector can be AAVhum.8. In an aspect, a disclosed AAV vector can be a self-complementary AAV as disclosed herein.

[0263] In an aspect, a disclosed vector can comprise one or more ITRs (such as, for example, ITRs from AAV2).

[0264] In an aspect, a disclosed vector can further comprise one or more nuclear localization signals (NLS). NLS are known to the skilled person in the art. In an aspect, a disclosed NLS can comprise any NLS known to the art. As known to the art (see, e.g., Lu J, et al. (2021) Cell Commun Signal. 19:60, which is incorporated herein by reference for its teachings of NLS), nuclear localization signals (NLS) are generally short peptides that act as a signal fragment that mediates the transport of proteins from the cytoplasm into the nucleus.

[0265] In an aspect, a disclosed vector can further comprise one or more nuclear retention elements (NRE). NRE are known to the skilled person in the art. In an aspect, a disclosed NRE can comprise SIRLOIN (SEQ ID NO:96) or BORG (SEQ ID NO:97).

[0266] In an aspect, a disclosed vector can further comprise one or more Flavivirus genetic elements. In an aspect, Flavivirus genetic elements can comprise one or more Flavivirus 3’ untranslated region (3’ UTR), one or more subgenomic Flavivirus RNA (sfRNA) elements, one or more Flavivirus XRN1 -resistant RNA (xrRNA) elements, one or more Flavivirus dumbbell (DB) RNA elements, one or more Flavivirus 3’ stem loop (3’ SL) elements, or any combination thereof. (See WO 2022/182835 for a description of Flavivirus gene elements).

[0267] In an aspect, a disclosed vector can comprise one or more promoters operably linked to a disclosed transgene, a disclosed sequence to be trans-spliced, a disclosed isolated nucleic acid molecule, a disclosed catalytically inactive Casl3 (e.g., RfxdCasl3 or PspdCasl3b), and/or a disclosed nucleic acid sequence. In an aspect, a disclosed promoter can be positioned 5’ (upstream) or 3’ (downstream) of a disclosed transgene, a disclosed sequence to be trans-spliced, a disclosed isolated nucleic acid molecule, a disclosed catalytically inactive Cast 3 (e.g., RfxdCasl3 or PspdCasl3b), and/or a disclosed nucleic acid sequence under its control. The distance between a disclosed promoter and a disclosed transgene, a disclosed sequence to be trans- spliced, a disclosed isolated nucleic acid molecule, a disclosed catalytically inactive Casl3 (e.g., RfxdCasl3 or PspdCasl3b), and/or a disclosed nucleic acid sequence can be approximately the same as the distance between that promoter and to the disclosed transgene, the disclosed sequence to be trans-spliced, the disclosed isolated nucleic acid molecule, the disclosed catalytically inactive Casl3 (e.g., RfxdCasl3 orPspdCasl3b or Casl3 alternative), and/or the disclosed nucleic acid sequence under its control. As is known in the art, variation in this distance can be accommodated without loss of promoter function.

[0268] In an aspect, a disclosed promoter for the one or more disclosed isolated nucleic acid molecules or the one or more disclosed guide RNA sequences can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the gene expression desired. A promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. In an aspect, a disclosed promoter can be a promoter/enhancer. In an aspect, a disclosed promoter for the one or more disclosed isolated nucleic acid molecules or the one or more disclosed guide RNA sequences can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter/enhancer. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can generally be obtained from a non-coding region upstream of a transcription initiation site of a gene of interest. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can be used for constitutive and efficient expression of a disclosed gene. In an aspect, a disclosed promoter for the one or more disclosed isolated nucleic acid molecules or the one or more disclosed guide RNA sequences can be a CMV promoter or a CMV promoter/enhancer. CMV promoters and CMV promoters/enhancers are well known to the art. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be any eukaryotic RNA polymerase II promoter.

[0269] In an aspect, a disclosed AAV vector can be used to generate AAV particles. In an aspect, a disclosed AAV vector can be used to generate AAV particles comprising a disclosed nucleic acid molecule (e.g., a 5’ replacement construct and/or 3’ replacement construct), a disclosed transgene, a disclosed sequence to be trans-spliced, and/or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and/or a disclosed therapeutic RNA) under its control.

[0270] Disclosed herein is an AAV particle comprising a disclosed nucleic acid molecule (e.g., a 5’ replacement construct and/or 3’ replacement construct), a disclosed transgene, a disclosed sequence to be trans-spliced, and/or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and/or a disclosed therapeutic RNA) under its control.

[0271] In an aspect, a disclosed vector can be used in one or more methods of trans-splicing, including, for example, SMaRT, CRAFT, and GRAFT (disclosed herein). 4. Pharmaceutical Formulations

[0272] Disclosed herein is a pharmaceutical formulation comprising a disclosed isolated nucleic acid molecule. Disclosed herein is a pharmaceutical formulation comprising a disclosed isolated nucleic acid molecule and a pharmaceutically acceptable carrier. Disclosed herein is a pharmaceutical formulation comprising a disclosed vector. Disclosed herein is a pharmaceutical formulation comprising a disclosed vector and a pharmaceutically acceptable carrier.

[0273] Disclosed herein is a pharmaceutical formulation comprising a vector comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre- mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein.

[0274] Disclosed herein is a pharmaceutical formulation comprising a vector comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron; a 5’ hemi intron; two or more guide RNA sequences; a promoter operably linked to the two or more guide RNA sequences; and a nucleic acid sequence encoding one or more RNA binding proteins.

[0275] Disclosed herein is a pharmaceutical formulation comprising a vector comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre- mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein.

[0276] Disclosed herein is a pharmaceutical formulation comprising a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d; a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal.

[0277] Disclosed herein is a pharmaceutical formulation comprising a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a catalytically inactive PspdCasl3b, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive PspdCasl3b; and a polyadenylation signal.

[0278] Disclosed herein is a pharmaceutical formulation comprising a vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a Cast 3 alternative, a promoter operably linked to the nucleic acid sequence encoding the Cast 3 alternative; and a polyadenylation signal. In an aspect, a Casl3 alternative can comprise alternatives known to the art including, but not limited to CRISPR-Inspired RNA Targeting System (CIRTS), Pumillo and FBF (PUF), rCas9, and/or any bifunctional RNA binding protein that can both bind to the transsplicing RNA molecule and the target RNA to facilitate interaction between the two RNA species. RNA binding proteins are discussed in depth supra.

[0279] Disclosed herein is a pharmaceutical formulation comprising a vector comprising an expression cassette comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein.

[0280] Disclosed herein is a pharmaceutical formulation comprising a vector comprising an expression cassette comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein.

[0281] Disclosed herein is a pharmaceutical formulation comprising a vector comprising an expression cassette comprising a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron; a 5’ hemi intron; two or more guide RNA sequences; a promoter operably linked to the two or more guide RNA sequences; and a nucleic acid sequence encoding one or more RNA binding proteins.

[0282] Disclosed herein is a pharmaceutical formulation comprising a vector comprising an expression cassette comprising a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d; a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal.

[0283] Disclosed herein is a pharmaceutical formulation comprising a vector comprising an expression cassette a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive PspdCasl3b, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive PspdCasl3b; and a polyadenylation signal. [0284] Disclosed herein is a pharmaceutical formulation comprising a vector comprising an expression cassette a nucleic acid molecule, comprising a nucleic acid sequence encoding a Cast 3 alternative, a promoter operably linked to the nucleic acid sequence encoding the Cast 3 alternative; and a polyadenylation signal. In an aspect, a Casl3 alternative can comprise alternatives known to the art including, but not limited to CRISPR-Inspired RNA Targeting System (CIRTS), Pumillo and FBF (PUF), rCas9, and/or any bifunctional RNA binding protein that can both bind to the trans-splicing RNA molecule and the target RNA to facilitate interaction between the two RNA species. RNA binding proteins are discussed in depth supra.

[0285] In an aspect, a disclosed pharmaceutical formulation can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, a disclosed pharmaceutical formulation can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0286] In an aspect, a disclosed formulation can comprise (i) one or more active agents, (ii) biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In an aspect, a disclosed composition can comprise one or more immune modulators. In an aspect, a disclosed composition can comprise one or more proteasome inhibitors. In an aspect, a disclosed composition can comprise one or more immunosuppressives or immunosuppressive agents. In an aspect, an immunosuppressive agent can be anti -thymocyte globulin (ATG), cyclosporine (CSP), my cophenolate mofetil (MMF), or a combination thereof. In an aspect, a disclosed formulation can comprise an anaplerotic agent (such as, for example, C7 compounds like triheptanoin or MCT).

[0287] In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 20 SNPs. In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 10 SNPs. In an aspect, a disclosed series of SNPs can comprise 5 SNPs.

[0288] In an aspect, a disclosed formulation can comprise an RNA therapeutic. An RNA therapeutic can comprise RNA-mediated interference (RNAi) and/or antisense oligonucleotides (ASO). In an aspect, a disclosed RNA therapeutic can be directed at any protein or enzyme that is overexpressed or is overactive due to a missing, deficient, and/or mutant protein or enzyme. In an aspect, a disclosed RNA therapeutic can comprise therapy delivered via LNPs. In an aspect, a disclosed formulation can comprise an enzyme or enzyme precursor for enzyme replacement therapy (ERT). [0289] In an aspect, a disclosed formulation can comprise a disclosed small molecule. In an aspect, a disclosed small molecule can assist in restoring the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0290] In an aspect, any disclosed pharmaceutical formulation can comprise one or more excipients and/or pharmaceutically acceptable carriers. Excipients and/or pharmaceutically acceptable carriers are known to the art and are discussed supra.

[0291] In an aspect, a disclosed pharmaceutical formuation can be used in one or more methods of trans-splicing, including, for example, SMaRT, CRAFT, and GRAFT (disclosed herein).

5. Plasmids

[0292] Disclosed herein is a plasmid comprising one or more disclosed isolated nucleic acid molecules. Disclosed herein is a plasmid comprising one or more disclosed vectors. Disclosed here are plasmids used in methods of making a disclosed composition such as, for example, a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. Plasmids and using plasmids are known to the art.

[0293] Disclosed herein is a plasmid comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:22 or a fragment thereof. Disclosed herein is a plasmid comprising a sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:22 or a fragment thereof. Disclosed herein is a plasmid comprising a sequence having at least 40%-60%, at least 60%-80%, at least 80%-90%, or at least 90%-100% identity to the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:22 or a fragment thereof.

[0294] In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 20 SNPs. In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 10 SNPs. In an aspect, a disclosed series of SNPs can comprise 5 SNPs. In an aspect, a disclosed plasmid can be used in one or more methods of trans-splicing, including, for example, SMaRT, CRAFT, and GRAFT (disclosed herein).

6. Cells

[0295] Disclosed herein are cells comprising a disclosed isolated nucleic acid molecule, a disclosed vector, and/or a disclosed plasmid. Disclosed herein are cells transduced by one or more disclosed viral vectors. Disclosed herein are cells transfected with one or more disclosed isolated nucleic acid molecules. In an aspect, a disclosed cell has been transfected with one or more nucleic acid sequences having the sequence set forth in any of SEQ ID NO:01 - SEQ ID NO:22. Techniques to achieve transfection and transduction are known to the art and using transfected or transduced cells are known to the art. In an aspect, disclosed herein are human immortalized cells lines transduced by one or more disclosed viral vectors or transfected with one or more disclosed isolated nucleic acids or disclosed plasmids. In an aspect, disclosed herein are human immortalized cells lines contacted with one or more disclosed pharmaceutical formulations. Disclosed herein are cells obtained for a subject treated with one or more disclosed isolated nucleic acid molecule, one or more disclosed vectors, one or more disclosed plasmids, or one or more disclosed pharmaceutical formulations. Disclosed herein are cells used to identify the most effective or most efficacious guide RNA sequence or guide RNA sequences.

7. Animals

[0296] Disclosed herein are animals treated with one or more disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, and/or one or more disclosed plasmids (e.g., SEQ ID NO:01 - SEQ ID NO:22). Transgenic animals are known to the art as are the techniques to generate transgenic animals.

8. Libraries

[0297] Disclosed herein is a library of one or more disclosed barcoded nucleic acid molecules for use in CRAFT. Disclosed herein is a library of one or more disclosed barcoded oligonucleotides for use in CRAFT. Disclosed herein is a library of one or more disclosed barcoded 5’ replacement constructs for use in CRAFT. Disclosed herein is a library of one or more disclosed barcoded 3’ replacement constructs for use in CRAFT. Disclosed herein is a library of one or more disclosed barcoded 5’ replacement constructs and/or disclosed barcoded 3’ replacement constructs for use in CRAFT. Disclosed herein is a library of one or more barcoded disclosed vectors for use in CRAFT. Disclosed herein is a library of one or more disclosed vectors comprising one or more disclosed barcoded 5’ replacement constructs, one or more disclosed barcoded 3’ constructs, or any combination thereof for use in CRAFT. Disclosed herein is a library of one or more disclosed AAV particles comprising one or more disclosed barcoded 5’ replacement constructs, one or more disclosed barcoded 3 ’ constructs, or any combination thereof for use in CRAFT. Disclosed herein is a library of one or more disclosed barcoded plasmids for use in CRAFT.

9. Kits

[0298] Disclosed herein is a kit comprising one or more disclosed barcoded nucleic acid molecules, disclosed barcoded vectors or disclosed barcoded AAV particles, disclosed barcoded pharmaceutical formulations, or any combination thereof. Disclosed herein is a kit comprising one or more disclosed barcoded nucleic acid molecules, one or more disclosed barcoded vectors, one or more disclosed barcoded pharmaceutical formulations, or any combination thereof. In an aspect, a kit can comprise a disclosed barcoded nucleic acid molecule, a disclosed barcoded vector or disclosed barcoded AAV particle, a disclosed barcoded pharmaceutical formulation, a disclosed barcoded therapeutic agent, or a combination thereof, and one or more agents. “Agents” and “Therapeutic Agents” are known to the art and are described supra.

[0299] In an aspect, the one or more agents can treat, prevent, inhibit, and/or ameliorate one or more comorbidities in a subject. In an aspect, one or more active agents can treat, inhibit, prevent, and/or ameliorate cellular and/or metabolic complications related to a missing, deficient, and/or mutant protein or enzyme.

[0300] In an aspect, a disclosed kit can comprise at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose (such as, for example, treating a subject diagnosed with or suspected of having a genetic disease or genetic disorder). Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. In an aspect, a kit for use in a disclosed method can comprise one or more containers holding a disclosed barcoded nucleic acid molecule, a disclosed vector, a disclosed barcoded pharmaceutical formulation, a disclosed RNA therapeutic, or a combination thereof, and a label or package insert with instructions for use. In an aspect, suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers can be formed from a variety of materials such as glass or plastic. The container can hold a disclosed barcoded nucleic acid molecule, a disclosed barcoded vector, a disclosed barcoded pharmaceutical formulation, or a combination thereof, and can have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert can indicate that a disclosed barcoded nucleic acid molecule, a barcoded disclosed vector, a disclosed barcoded AAV particle, a disclosed barcoded pharmaceutical formulation, a disclosed RNA therapeutic agent, or a combination thereof can be used for treating, preventing, inhibiting, and/or ameliorating a disease or disorder or complications and/or symptoms associated with a disease or disorder. A disclosed kit can comprise additional components necessary for administration such as, for example, other buffers, diluents, filters, needles, and syringes. In an aspect, a disclosed kit can be used in any disclosed method. In an aspect, a disclosed kit can be used to generate one or more chimeric RNA molecules. In an aspect, a disclosed kit can be used to treat a genetic disease or genetic disorder. In an aspect, a disclosed kit can be used to inhibit and/or minimize disease progression. C. Methods of Generating a Chimeric RNA Molecule

[0301] Disclosed herein is a method of generating a chimeric RNA molecule in a cell, the method comprising contacting an endogenous pre-mRNA in a cell with (i) a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein, and (ii) a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive PspdCasl3b, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive PspdCasl3b; and a polyadenylation signal, wherein the isolated nucleic acid molecules form a ternary complex with the endogenous pre-mRNA molecule, and wherein the resulting chimeric RNA molecule comprises the trans-spliced nucleic acid sequence.

[0302] Disclosed herein is a method of generating a chimeric RNA molecule in a cell, the method comprising contacting an endogenous pre-mRNA in a cell with (i) a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron; a 5’ hemi intron; two or more guide RNA sequences; a promoter operably linked to the two or more guide RNA sequences; and a nucleic acid sequence encoding one or more RNA binding proteins; and (ii) a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d; a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal, wherein the isolated nucleic acid molecules form a ternary complex with the endogenous pre-mRNA molecule, and wherein the resulting chimeric RNA molecule comprises the trans-spliced nucleic acid sequence. [0303] Disclosed herein is a method of generating a chimeric RNA molecule in a cell, the method comprising contacting an endogenous pre-mRNA in a cell with (i) a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein; and (ii) a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d; a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal, wherein the isolated nucleic acid molecules form a ternary complex with the endogenous pre-mRNA molecule, and wherein the resulting chimeric RNA molecule comprises the transspliced nucleic acid sequence.

[0304] Disclosed herein is a method of generating a chimeric RNA molecule in a cell, the method comprising contacting an endogenous pre-mRNA in a cell with (i) a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein, and (ii) a nucleic acid molecule, comprising a nucleic acid sequence encoding a Cast 3 alternative, a promoter operably linked to the nucleic acid sequence encoding the Cast 3 alternative; and a polyadenylation signal, wherein the isolated nucleic acid molecules form a ternary complex with the endogenous pre-mRNA molecule, and wherein the resulting chimeric RNA molecule comprises the trans-spliced nucleic acid sequence. [0305] Disclosed herein is a method of generating a chimeric RNA molecule in a cell, the method comprising contacting an endogenous pre-mRNA in a cell with (i) a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron; a 5’ hemi intron; two or more guide RNA sequences; a promoter operably linked to the two or more guide RNA sequences; and a nucleic acid sequence encoding one or more RNA binding proteins; and (ii) a nucleic acid molecule, comprising a nucleic acid sequence encoding a Cast 3 alternative; a promoter operably linked to the nucleic acid sequence encoding the Cast 3 alternative; and a polyadenylation signal, wherein the isolated nucleic acid molecules form a ternary complex with the endogenous pre-mRNA molecule, and wherein the resulting chimeric RNA molecule comprises the trans-spliced nucleic acid sequence.

[0306] Disclosed herein is a method of generating a chimeric RNA molecule in a cell, the method comprising contacting an endogenous pre-mRNA in a cell with (i) a nucleic acid molecule comprising a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein; and (ii) a nucleic acid molecule, comprising a nucleic acid sequence encoding a Cast 3 alternative; a promoter operably linked to the nucleic acid sequence encoding the Cast 3 alternative; and a polyadenylation signal, wherein the isolated nucleic acid molecules form a ternary complex with the endogenous pre-mRNA molecule, and wherein the resulting chimeric RNA molecule comprises the trans-spliced nucleic acid sequence. [0307] In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 20 SNPs. In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 10 SNPs. In an aspect, a disclosed series of SNPs can comprise 5 SNPs.

[0308] In an aspect, a Casl3 alternative can comprise alternatives known to the art including, but not limited to CRISPR-Inspired RNA Targeting System (CIRTS), Pumillo and FBF (PUF), rCas9, and/or any bifunctional RNA binding protein that can both bind to the trans-splicing RNA molecule and the target RNA to facilitate interaction between the two RNA species. RNA binding proteins are discussed in depth supra.

[0309] In an aspect, a disclosed method of generating a chimeric RNA molecule in a cell can comprise validating the trans-splicing and/or the generation of the chimeric RNA molecule. Validation of the trans-splicing event and/or generation of the chimeric RNA molecule can be accomplished using methods and techniques known to the art (e.g., sequencing, northern blots, FISH, PCR, RNA-Seq, 3’ RACE, 5’ RACE, etc ).

[0310] In an aspect, a disclosed method of generating a chimeric RNA molecule can comprise preparing a disclosed 5’ replacement construct, a disclosed 3’ replacement construct, a disclosed non-viral vector or disclosed viral vector, a disclosed nucleic acid molecule, a disclosed pharmaceutical formulation, or any combination thereof.

[0311] In an aspect, a disclosed method of generating a chimeric RNA molecule in cells can comprise identifying the most effective or most efficacious guide RNA sequence or guide RNA sequences. In an aspect, the most effective or most efficacious guide RNA sequence or guide RNA sequences can achieve the highest level of trans-splicing.

[0312] In an aspect, a disclosed method of generating a chimeric RNA molecule in cells can comprise identifying guide RNA sequence or guide RNA sequences that are most effective at generating a chimeric molecule through trans-splicing. In an aspect, the one or more RNA targeting motifs identified as effective at generating a chimeric molecule through trans-splicing can then be prepared and packaged as part of a transcriptome engineering system. In an aspect, a disclosed transcriptome engineering system can then be packaged in a pharmaceutical formulation that can be administered to a subject in need thereof.

[0313] In an aspect, a disclosed chimeric molecule is non-functional due to the series of SNPs.

[0314] In an aspect, a disclosed method can be reported in a library of replacement constructs to identify the top performing guide RNAs (i.e., those that have the highest trans-splicing efficacy). [0315] In an aspect, once a disclosed method identifies the effective or most efficacious guide RNA sequence or guide RNA sequences, that guide RNA sequence or guide RNA sequences can be used to generate a chimeric RNA molecule in cells. In an aspect, the cells can be in a subject. In an aspect, the cells can be cells affected by a disease or disorder. In an aspect, the effective or most efficacious guide RNA sequence or guide RNA sequences can be used in a disclosed method that can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, a disclosed method can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra). In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi-systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity.

D. Compositions for Transcriptome Engineering (Barcoded GRAFT)

1. Nucleic Acid Molecules

5’ Replacement Constructs

[0316] Disclosed herein is a nucleic acid molecule, comprising an exogenous RNA to be transspliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures.

[0317] Disclosed herein is a nucleic acid molecule, comprising an exogenous RNA to be trans- spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95. [0318] Disclosed herein is a nucleic acid molecule, comprising an exogenous RNA to be transspliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA.

[0319] Disclosed herein is a nucleic acid molecule, comprising an exogenous RNA to be trans- spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA.

[0320] In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 20 SNPs. In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 10 SNPs. In an aspect, a disclosed series of SNPs can comprise 5 SNPs.

[0321] In an aspect, a disclosed targeted endogenous pre-mRNA can comprise one or more mutations. In an aspect of a disclosed targeted endogenous pre-mRNA, one or more disclosed mutations can be in the 5 ’ portion of the pre-mRNA. In an aspect, one or more disclosed mutations in one or more exons can contribute to pathogenesis of one or more cells.

[0322] In an aspect, the disclosed cells can be in a subject. In an aspect, a subject can be a human patient and can be male or female. In an aspect, a subject can have a genetic disease or disorder. In an aspect, a subject can be treatment-naive.

[0323] In an aspect, one or more disclosed mutations can inhibit translation of the encoded protein. In an aspect, one or more disclosed mutations can modify translation of the encoded protein. In an aspect, one or more disclosed mutations can generate an encoded protein having a non-sense mutation or a missense mutation.

[0324] In an aspect, a disclosed targeted endogenous pre-mRNA can comprise one or more mutations in one or more exons. In an aspect, a disclosed targeted endogenous pre-mRNA can comprise one or more mutations in one or more introns. In an aspect, one or more disclosed exonic mutations can contribute to pathogenesis in one or more cells. In an aspect, one or more disclosed intronic mutations can contribute to pathogenesis in one or more cells.

[0325] In an aspect, a disclosed targeted endogenous pre-mRNA can be a primary transcript of a protein coding gene. In an aspect of a disclosed targeted endogenous pre-mRNA, a disclosed protein coding gene can comprise one or more coding regions of [0326] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode the protein or a portion thereof (such as, for example, Exon 1 or Exon 4, etc.) associated with the following genes: ABCA1, ABCA12, ABCA13, ABCA2, ABCA3, ABCA4, ABCA5, ABCC1, ABCC2, ABCC6, ABCC8, ABCC9, ACAN, ADAMTS13, ADCY10, ADGRV1, AGL, AGRN, AHDC1, ALK, ALMS1, ALPK3, ALS2, ANAPC1, ANK1, ANK2, ANK3, ANKRD11, ANKRD26, APC, APC2, APOB, ARFGEF2, ARHGAP31, ARHGEF10, ARHGEF18, ARID ! A, ARID I B, ARID2, ASH1L, ASPM, ASXL1, ASXL2, ASXL3, ATM, ATP7A, ATP7B, ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, BLM, BPTF, BRCA1, BRCA2, BRIM, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD, CAMTAI, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44, CFAP65, CFTR/ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1, CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1, CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1, DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11, DNAH17, DNAH2, DNAH5, DNAH7, DNAH8, DNAH9, DNMBP, DNMT1, DOCK2, DOCK3, DOCK6, DOCK7, DOCK8, DSCAM, DSP, DST, DUOX2, DYNC1H1, DYNC2H1, DYSF, EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, EYS, F5, F8, FANCA, FANCD2, FANCM, FAT1, FAT4, FBN1, FBN2, FLG, FLG2, FLNA, FLNB, FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2, FSIP2, FYCO1, GLI2, GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1, HERC2, HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R, IGF2R, IGSF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A, KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220, KIF14, KIF1A, KIF1B, KIF21A, KIF26B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1, LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMC3, LCT, LOXHD1, LPA, LRBA, LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD, MAGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5, MCM3AP, MED12, MED12L, MED13, MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MYH11, MYH14, MYH2, MYH3, MYH6, MYH7, MYH7B, MYH8, MYH9, MYLK, MYO 15 A, MYO18B, MYO3A, MYO5A, MYO5B, MYO7A, MYO9A, NALCN, NBAS, NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF, NF1, NFASC, NHS, NIN, NIPBL, NLRP1, N0TCH1, NOTCH2, NOTCH3, NPHP4, NRXN1, NRXN3, NSD1, NSD2, NUP155, NUP188, NUP205, OBSCN, OBSL1, OTOF, OTOG, OTOGL, PARD3, PBRM1, PCDH15, PCLO, PCNT, PHIP, PI4KA, PIEZO1, PIEZO2, PIK3C2A, PIKFYVE, PKD1, PKD1L1, PKHD1, PLCE1, PLEC, PLEKHG2, PNPLA6, POGZ, POLA1, POLE, POLR1A, POLR2A, POLR3A, PRG4, PRKDC, PRPF8, PRR12, PRX, PTCHI, PTPN23, PTPRF, PTPRJ, PTPRQ, PXDN, QRICH2, RAB3GAP2, RAH, RALGAPA1, RANBP2, RB1CC1, RELN, RERE, REV3L, RIC1, RIMS1, RIMS2, RNF213, ROBO1, ROBO2, ROBO3, ROS1, RP1, RP1L1, RTTN, RUSC2, RYR1, RYR2, SACS, SAMD9, SAMD9L, SBF2, SCAPER, SCN10A, SCN11A, SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SETBP1, SETD1A, SETD1B, SETD2, SETD5, SETX, SHANK2, SHANK3, SHR00M4, SI, SIPA1L3, SLIT2, SLX4, SMARCA2, SMARCA4, SMCHD1, SNRNP200, SON, SPEF2, SPEG, SPG11, SPTA1, SPTAN1, SPTB, SPTBN2, SPTBN4, SRCAP, STRC, SVIL, SYNE1, SYNGAP1, SYNJ1, SZT2, TAF1, TANC2, TCF20, TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B, TNXB, TOGARAMI, TONSL, TRIO, TRIOBP, TRIP11, TRIP12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, TTN, TUBGCP6, UBR1, UNC80, USH2A, USP9X, VCAN, VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62, WDR81, WNK1, WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292, ZNF335, ZNF407, ZNF462, ZNF469, or a portion thereof.

[0327] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode the protein or a portion thereof (such as, for example, Exon 1 or Exon 4, etc.) associated with the following genes: DMD, TTN, TCAP, SGCA, SGCB, SGCG, SGCD, Al-AT, MYH6, MYH7, MYH11, ML2, ML3, MYLK2, MYBPC3, DES, DNM2, LAMA2, LMNA, LMNB, LBR, DYSF, EMD, EPO, LPL, SERCA2A, S100A1, MTM, DM1 DMPK, PYGL„ PYGM, GYSI, GYS2, NAGA, GAA, SMPD1, LIPA, COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, PLOD1, LIPA, FXN, MSTN, HEXA, HEXB, GBA, AMPD1, HBB, IDUA, IDS, TNNI3, TNNT2, TNNC1, TPM1, TPM3, NAGLU, SGSH, HGSNAT, IGTA7, IGTA9, N-acetyl, GNS, N-acetyl, GALNS, GLB1, GUSB, HYAL1, ASAHI, GALC, CTSA, CTSA, CTSK, GM2A, ARSA, ARSB, SUMFI, NEU1 GNPTA, GNPTB, GNPTG, MCOLN1, NPC1, NPC2, CLN5, CLN6, CLN8, PPT1, TPP1, CLN3, DNAJC5, MFSD8, MAN2B1, MANBA, AGA, FUCA1, CTNS, SLC2A10, SLC17A5, SLC6A19, SLC22A5, SLC37A4, LAMP2, SCN4A, SCN4B, SCN5A, SCN4A, CACNA1C, CACNA1S, PGK1, PGAM2, AGL, KCNE1, KCNE2, KCNJ2, KCNJ5, KCNH2, KCNQ1, HCN4, CLCN1, CPT1A, RYR1, RYR2, BINI, LARGE1, DOK7, FKTN, FKRP, SELENON, POMT1, POMT2, POMGNT1, POMGNT2, POMK, ISPD, PLEC, CHRNE, CHAT, CHKB, COLQ, RAPSN, FHL1, B4GAT1, B3GALNT2, DAGI, TMEM5, TMEM43, SECISBP2, UDP-N-acetyl, GNE, AN05, SMCHD1, LDHA, LHDB, CAPN3, CAV3, TRIM32, CNBP, NEB, ACTA1, ACTC1, ACTN2, PABPN1, LEMD3, ZMPSTE24, MTTP, CHRNA1, CHRNA2, CHRNA3, CHRNA4, CHRNA5, CHRNA6, CHRNA7, CHRNA8, CHRNA9, CHRNA10, CHRNB1, CHRNB2, CHRNB3, CHRNB4, CHRNG1, CHRND, CHRNE1, ABCA1, ABCC6, ABCC9, ABCD1, ATP2A1, ATM, TTP A, KIF21A, PHOX2A, HSPG2, STIM1, NOTCHI, NOTCH3, DTNA, PRKAG2, CSRP3, VCL, MyoZ2, MYPN, JPH2, PLN, CALR3, NEXN, LDB3, EYA4, HTT, AR, PTPN11, JUP, DSP, PKP2, DSG2, DSC2, CTNNA3, NKX2-5, AKAP9, AKAP10, GNAI2, ANK2, SNTAT, CALM1, CALM2, HTRA1, FBN1, FBN2, XYLT1, XYLT2, TAZ, HGD, G6PC, GBE1, PFKM, PHKA1, PHKA2, PHKB, PHKG2, PGAM2, CBS, MTHFR, MTR, MTRR, MMADHC, MT-ND1, MT-ND5, MT-TE, MT- TH, MT-TL1, MT-TK, MT-TS1, MT-TV, MAP2K1, BRAF, RAFI, IGF-1, TGFP3, TGFpRl, TGFPR2, FGF2, FGF4, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, or VEGFR2.

[0328] In an aspect, a disclosed protein coding gene can comprise one or more coding regions of FXN, LMNA, or RYR2. In an aspect, a disclosed protein coding gene can comprise a portion of a disclosed protein coding gene (such as, for example, Exon 1 or Exon 4, etc.)

[0329] In an aspect, a disclosed 3’ portion of the targeted endogenous pre-mRNA can be transspliced with the exogenous RNA. In an aspect, a disclosed RNA targeting motif can bind to the targeted endogenous pre-mRNA. In an aspect, a disclosed RNA targeting motif can bind to the 5’ end of the targeted endogenous pre-mRNA. In an aspect, a disclosed RNA targeting motif can be specific for an endogenous pre-mRNA having one or more mutations. In an aspect, a disclosed RNA targeting motif can be specific for an endogenous pre-mRNA having one or more exonic mutations. In an aspect, a disclosed RNA targeting motif can be specific for an endogenous pre- mRNA having one or more intronic mutations.

[0330] In an aspect, a disclosed RNA targeting motif can comprise an antisense oligonucleotide. In an aspect, a disclosed antisense oligonucleotide can comprise about 15 nucleotides to about 50 nucleotides. In an aspect, a disclosed antisense oligonucleotide can comprise about 30 nucleotides. In an aspect, a disclosed RNA targeting motif can be directed to the intron immediately 5’ to the exon of the targeted endogenous pre-mRNA with which it is to be spliced. [0331] In an aspect, a disclosed 5’ hemi intron can comprise a 5’ splice site. In an aspect, a disclosed 5’ splice site can comprise a consensus 5’ splice site. In an aspect, a disclosed consequence 5’ splice site can comprise MAG | GURAGU (SEQ ID NO:61), wherein | denotes the exon intron junction, wherein M = A or C, and wherein R = A or G. In an aspect, a disclosed 5’ hemi intron can be recognized by nuclear splicing components in a host cell. In an aspect, a disclosed 5’ hemi intron can be recognized by the spliceosome in a host cell. In an aspect, a disclosed 5’ hemi intron can facilitate the trans-splicing of the exogenous RNA to the exon immediately 3 ’ to the targeted intron in the endogenous pre-mRNA.

[0332] In an aspect, a disclosed exogenous RNA to be trans-spliced to the targeted endogenous pre-mRNA can comprise one or more exons of the protein coding gene. In an aspect, a disclosed exogenous RNA to be trans-spliced to the targeted endogenous pre-mRNA can comprise the primary sequence of the coding sequence of one or more exons having the one or more mutations. In an aspect, a disclosed exogenous RNA can be trans-spliced to a 3’ end of the targeted endogenous pre-mRNA.

[0333] In an aspect of a disclosed exogenous RNA, a disclosed protein coding gene can comprise one or more coding regions of ABCA1, ABCA12, ABCA13, ABCA2, ABCA3, ABCA4, ABCA5, ABCC1, ABCC2, ABCC6, ABCC8, ABCC9, ACAN, ADAMTS13, ADCY10, ADGRV1, AGL, AGRN, AHDC1, ALK, ALMS1, ALPK3, ALS2, ANAPC1, ANK1, ANK2, ANK3, ANKRD11, ANKRD26, APC, APC2, APOB, ARFGEF2, ARHGAP31, ARHGEF10, ARHGEF18, ARID! A, ARID1B, ARID2, ASH1L, ASPM, ASXL1, ASXL2, ASXL3, ATM, ATP7A, ATP7B, ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, BLM, BPTF, BRCA1, BRCA2, BRIM, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD, CAMTAI, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP 152, CEP 164, CEP250, CEP290, CFAP43, CFAP44, CFAP65, CFTR/ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1, CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1, CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1, DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11, DNAH17, DNAH2, DNAH5, DNAH7, DNAH8, DNAH9, DNMBP, DNMT1, DOCK2, DOCK3, DOCK6, DOCK7, DOCK8, DSCAM, DSP, DST, DUOX2, DYNC1H1, DYNC2H1, DYSF, EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, EYS, F5, F8, FANCA, FANCD2, FANCM, FAT1, FAT4, FBN1, FBN2, FLG, FLG2, FLNA, FLNB, FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2, FSIP2, FXN, FYCO1, GLI2, GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1, HERC2, HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R, IGF2R, IGSF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A, KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220, KIF14, KIF1A, KIF1B, KIF21A, KIF26B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1, LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMC3, LCT, LMNA, L0XHD1, LPA, LRBA, LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD, MAGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5, MCM3AP, MED12, MED12L, MED13, MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MYH11, MYH14, MYH2, MYH3, MYH6, MYH7, MYH7B, MYH8, MYH9, MYLK, MYO15A, MYO18B, MYO3 A, MYO5A, MYO5B, MYO7A, MYO9A, NALCN, NBAS, NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF, NF1, NFASC, NHS, NIN, NIPBL, NLRP1, NOTCH1, NOTCH2, NOTCH3, NPHP4, NRXN1, NRXN3, NSD1, NSD2, NUP155, NUP188, NUP205, OBSCN, OBSL1, OTOF, OTOG, OTOGL, PARD3, PBRM1, PCDH15, PCLO, PCNT, PHIP, PI4KA, PIEZO1, PIEZO2, PIK3C2A, PIKFYVE, PKD1, PKD1L1, PKHD1, PLCE1, PLEC, PLEKHG2, PNPLA6, POGZ, POLA1, POLE, POLR1A, POLR2A, POLR3A, PRG4, PRKDC, PRPF8, PRR12, PRX, PTCHI, PTPN23, PTPRF, PTPRJ, PTPRQ, PXDN, QRICH2, RAB3GAP2, RAH, RALGAPA1, RANBP2, RB1CC1, RELN, RERE, REV3L, RIC1, RIMS1, RIMS2, RNF213, ROBO1, ROBO2, ROBO3, ROS1, RP1, RP1L1, RTTN, RUSC2, RYR1, RYR2, SACS, SAMD9, SAMD9L, SBF2, SCAPER, SCN10A, SCN11A, SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SETBP1, SETD1A, SETD1B, SETD2, SETD5, SETX, SHANK2, SHANK3, SHROOM4, SI, SIPA1L3, SLIT2, SLX4, SMARCA2, SMARCA4, SMCHD1, SNRNP200, SON, SPEF2, SPEG, SPG11, SPTA1, SPTAN1, SPTB, SPTBN2, SPTBN4, SRCAP, STRC, SVIL, SYNE1, SYNGAP1, SYNJ1, SZT2, TAF1, TANC2, TCF20, TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B, TNXB, TOGARAMI, TONSL, TRIO, TRIOBP, TRIP11, TRIP12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, TTN, TUBGCP6, UBR1, UNC80, USH2A, USP9X, VCAN, VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62, WDR81, WNK1, WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292, ZNF335, ZNF407, ZNF462, or ZNF469. In an aspect, a disclosed protein coding gene can comprise one or more coding regions of CFTR, MDX, DYSF/TTN, DMPK, COL7A1, K14, MAPT, FVIII, HTT, RHO, DNA-PKcs, SMN2, or CD40L. In an aspect, a disclosed protein coding gene can comprise one or more coding regions of FXN_x LMNA, or RYR2.

[0334] In an aspect of a disclosed exogenous RNA, a disclosed protein coding gene can comprise one or more coding regions of DMD, TTN, TCAP, SGCA, SGCB, SGCG, SGCD, Al -AT, MYH6, MYH7, MYH11, ML2, ML3, MYLK2, MYBPC3, DES, DNM2, LAMA2, LMNA, LMNB, LBR, DYSF, EMD, EPO, LPL, SERCA2A, S100A1, MTM, DM1 DMPK, PYGL„ PYGM, GYSI, GYS2, NAGA, GAA, SMPD1, LIPA, C0L1A1, COL1A2, COL3A1, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, PLOD1, LIPA, FXN, MSTN, HEXA, HEXB, GBA, AMPD1, HBB, IDUA, IDS, TNNI3, TNNT2, TNNC1, TPM1, TPM3, NAGLU, SGSH, HGSNAT, IGTA7, IGTA9, N-acetyl, GNS, N-acetyl, GALNS, GLB1, GUSB, HYAL1, ASAHI, GALC, CTSA, CTSA, CTSK, GM2A, ARSA, ARSB, SUMFI, NEU1 GNPTA, GNPTB, GNPTG, MC0LN1, NPC1, NPC2, CLN5, CLN6, CLN8, PPT1, TPP1, CLN3, DNAJC5, MFSD8, MAN2B1, MANBA, AGA, FUCA1, CTNS, SLC2A10, SLC17A5, SLC6A19, SLC22A5, SLC37A4, LAMP2, SCN4A, SCN4B, SCN5A, SCN4A, CACNA1C, CACNA1S, PGK1, PGAM2, AGL, KCNE1, KCNE2, KCNJ2, KCNJ5, KCNH2, KCNQ1, HCN4, CLCN1, CPT1A, RYR1, RYR2, BINI, LARGE1, D0K7, FKTN, FKRP, SELENON, POMT1, POMT2, POMGNT1, POMGNT2, POMK, ISPD, PLEC, CHRNE, CHAT, CHKB, COLQ, RAPSN, FHL1, B4GAT1, B3GALNT2, DAGI, TMEM5, TMEM43, SECISBP2, UDP-N-acetyl, GNE, AN05, SMCHD1, LDHA, LHDB, CAPN3, CAV3, TRIM32, CNBP, NEB, ACTA1, ACTC1, ACTN2, PABPN1, LEMD3, ZMPSTE24, MTTP, CHRNA1, CHRNA2, CHRNA3, CHRNA4, CHRNA5, CHRNA6, CHRNA7, CHRNA8, CHRNA9, CHRNA10, CHRNB1, CHRNB2, CHRNB3, CHRNB4, CHRNG1, CHRND, CHRNE1, ABC Al, ABCC6, ABCC9, ABCD1, ATP2A1, ATM, TTP A, KIF21A, PHOX2A, HSPG2, STIM1, NOTCHI, NOTCH3, DTNA, PRKAG2, CSRP3, VCL, MyoZ2, MYPN, JPH2, PLN, CALR3, NEXN, LDB3, EYA4, HTT, AR, PTPN11, JUP, DSP, PKP2, DSG2, DSC2, CTNNA3, NKX2-5, AKAP9, AKAP10, GNAI2, ANK2, SNTAT, CALM1, CALM2, HTRA1, FBN1, FBN2, XYLT1, XYLT2, TAZ, HGD, G6PC, GBE1, PFKM, PHKA1, PHKA2, PHKB, PHKG2, PGAM2, CBS, MTHFR, MTR, MTRR, MMADHC, MT-ND1, MT- ND5, MT-TE, MT-TH, MT-TL1, MT-TK, MT-TS1, MT-TV, MAP2K1, BRAF, RAFI, IGF-1, TGFP3, TGFPR1, TGFPR2, FGF2, FGF4, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, or VEGFR2.

[0335] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode LMNA/C (SEQ ID NO:55) or a portion thereof. LMNA/C is known to the art (e.g., Gene ID 4000) and this nucleotide sequence can comprise nucleotides 4974 - 62517 in Accession No. NG008692.2. The nuclear lamina consists of a two-dimensional matrix of proteins located next to the inner nuclear membrane. The lamin family of proteins make up the matrix and are highly conserved in evolution. During mitosis, the lamina matrix is reversibly disassembled as the lamin proteins are phosphorylated. Lamin proteins are involved in nuclear stability, chromatin structure and gene expression. Vertebrate lamins consist of two types, A and B. Alternative splicing results in multiple transcript variants. Mutations in this gene lead to several diseases: Emery-Dreifuss muscular dystrophy, familial partial lipodystrophy, limb girdle muscular dystrophy, dilated cardiomyopathy, Charcot-Marie-Tooth disease, and Hutchinson-Gilford progeria syndrome.

[0336] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode DP71 or a portion thereof. DP71 is known to the art (e.g., Gene ID 13405).

[0337] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode CFTR (SEQ ID NO:54) or a portion thereof. CFTR is known to the art (e.g., Gene ID 1080) and this nucleotide sequence can comprise nucleotides 19180 - 207882 in Accession No. NG016465.4. This gene encodes a member of the ATP -binding cassette (ABC) transporter superfamily. The encoded protein functions as a chloride channel, making it unique among members of this protein family, and controls ion and water secretion and absorption in epithelial tissues. Channel activation is mediated by cycles of regulatory domain phosphorylation, ATP -binding by the nucleotide-binding domains, and ATP hydrolysis. Mutations in this gene cause cystic fibrosis, the most common lethal genetic disorder in populations of Northern European descent. The most frequently occurring mutation in cystic fibrosis, DeltaF508, results in impaired folding and trafficking of the encoded protein. Multiple pseudogenes have been identified in the human genome.

[0338] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode DMPK (SEQ ID NO:56) or a portion thereof. DMPK is known to the art (e.g., Gene ID 1760) and this nucleotide sequence can comprise nucleotides 5068 - 17841 in Accession No. NG009784.1. DMPK is a serine-threonine kinase that is closely related to other kinases that interact with members of the Rho family of small GTPases. Substrates for this enzyme include myogenin, the beta-subunit of the L-type calcium channels, and phosphol emman. The 3’ untranslated region of this gene contains 5-38 copies of a CTG trinucleotide repeat. Expansion of this unstable motif to 50-5,000 copies causes myotonic dystrophy type I, which increases in severity with increasing repeat element copy number. Repeat expansion is associated with condensation of local chromatin structure that disrupts the expression of genes in this region. Several alternatively spliced transcript variants of this gene have been described, but the full-length nature of some of these variants has not been determined.

[0339] In an aspect, a disclosed gene can be DMD (dystrophin) (SEQ ID NO: 52). DMD is known to the art (e.g., Gene ID 1756) and this nucleotide sequence can comprise nucleotides 5001 - 2225382 in Accession No. NG012232.1. DMD spans a genomic range of greater than 2 Mb and encodes a large protein containing an N-terminal actin-binding domain and multiple spectrin repeats. The encoded protein forms a component of the dystrophin-glycoprotein complex (DGC), which bridges the inner cytoskeleton and the extracellular matrix. Deletions, duplications, and point mutations at this gene locus may cause Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or cardiomyopathy. Alternative promoter usage and alternative splicing result in numerous distinct transcript variants and protein isoforms for this gene.

[0340] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode LRRK2 (SEQ ID NO:53) or a portion thereof. LRRK2 is known to the art (e.g., Gene ID 120892) and this nucleotide sequence can comprise nucleotides 5001 - 149275 in Accession No. NG011709.1. LRRK2 is a member of the leucine-rich repeat kinase family and encodes a protein with an repeat region, a leucine-rich repeat (LRR) domain, a kinase domain, a DFG-like motif, a RAS domain, a GTPase domain, a MLK-like domain, and a WD40 domain. The protein is present largely in the cytoplasm but also associates with the mitochondrial outer membrane. Mutations in this gene have been associated with Parkinson’s disease.

[0341] In an aspect, a disclosed exogenous RNA to be trans-spliced can further comprise a UTR. [0342] In an aspect, one or more disclosed RNA structures can bind to one or more RNA binding proteins. In an aspect, one or more disclosed RNA structures can bind to one or more doublestranded RNA binding proteins (dsRBP). In an aspect, dsRBPs are known to the skilled person in the art and include, but are not limited to, AD ARI, ADAR2, DICER, NEAR, PACT, PKR, RHA RNaselll, Stauffen, TRBP, TSEN, or any combination thereof.

[0343] In an aspect, one or more disclosed RNA structures can comprise the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95. In an aspect, one or more disclosed RNA structures can improve and/or can enhance trans-splicing efficiency. In an aspect, one or more disclosed RNA structures can stabilize the pre-mRNA. In an aspect, one or more disclosed RNA structures can localize the RNA to the nucleus. In an aspect, one or more disclosed RNA structures can stabilize the interaction between the targeted endogenous pre-mRNA molecule and the exogenous RNA to be trans-spliced. In an aspect, a disclosed nucleic acid molecule can lack a CRISPR-associated protein.

[0344] In an aspect, a disclosed resulting chimeric RNA transcript can comprise the 5’ portion of the targeted endogenous pre-mRNA and the 3’ portion of the exogenous RNA. In an aspect, a disclosed resulting chimeric RNA transcript can comprise the 3’ portion of the targeted endogenous pre-mRNA and the 5’ portion of the exogenous RNA.

[0345] In an aspect, a disclosed targeted endogenous pre-mRNA and a disclosed exogenous RNA can encode the same protein coding gene. In an aspect, a disclosed targeted endogenous pre- mRNA and a disclosed exogenous RNA can comprise one or more exons of the same protein coding gene.

[0346] In an aspect, a disclosed nucleic acid molecule can be packaged into a viral vector. In an aspect, a disclosed viral vector can comprise an AAV vector. In an aspect, a disclosed nucleic acid molecule can be packaged into a non-viral carrier. In an aspect, a disclosed nucleic acid molecule can be incorporated into a plasmid. In an aspect, a disclosed nucleic acid molecule can be incorporated into lipid nanoparticles.

[0347] In an aspect, a disclosed nucleic acid molecule can further comprise a polyadenylation sequence. In an aspect, a disclosed nucleic acid molecule can further comprise a sequence for a promoter. In an aspect, a disclosed nucleic acid molecule can further comprise a spacer region. In an aspect, a disclosed spacer region can separate the 5’ splice region from the one or more RNA structures. In an aspect, a disclosed spacer region can comprise any known spacer. In an aspect, a disclosed spacer region can comprise a consensus splicing motif (e.g., such as U1 or U2). In an aspect, a disclosed spacer region can comprise a limited number of consensus splicing motifs (e.g., such as U1 or U2).

[0348] In an aspect, a disclosed nucleic acid molecule can further comprise one or more nuclear localization signals (NLS). NLS are known to the skilled person in the art. In an aspect, a disclosed NLS can comprise any NLS known to the art. As known to the art (see, e.g., Lu J, et al. (2021) Cell Commun Signal. 19:60, which is incorporated herein by reference for its teachings of NLS), nuclear localization signals (NLS) are generally short peptides that act as a signal fragment that mediates the transport of proteins from the cytoplasm into the nucleus.

[0349] In an aspect, a disclosed nucleic acid molecule can further comprise one or more nuclear retention elements (NRE). NRE are known to the skilled person in the art. In an aspect, a disclosed NRE can comprise SIRLOIN (SEQ ID NO:96) or BORG (SEQ ID NO:97).

[0350] In an aspect, a disclosed nucleic acid molecule can further comprise one or more Flavivirus genetic elements. In an aspect, Flavivirus genetic elements can comprise one or more Flavivirus 3’ untranslated region (3’ UTR), one or more subgenomic Flavivirus RNA (sfRNA) elements, one or more Flavivirus XRN1 -resistant RNA (xrRNA) elements, one or more Flavivirus dumbbell (DB) RNA elements, one or more Flavivirus 3’ stem loop (3’ SL) elements, or any combination thereof. (See WO 2022/182835 for a description of Flavivirus gene elements).

[0351] In an aspect, a disclosed exogenous RNA can induce a splice event. In an aspect, a disclosed 5’ hemi intron can be recognized by nuclear splicing components within a host cell.

[0352] In an aspect, a disclosed promoter for the 5’ replacement construct can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the expression desired. A promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. In an aspect, a disclosed promoter can be a promoter/enhancer. In an aspect, a disclosed promoter for the disclosed nucleic acid molecule can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter/enhancer. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can generally be obtained from a non-coding region upstream of a transcription initiation site of a gene of interest. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can be used for constitutive and efficient expression of a disclosed protein coding gene. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be a CMV promoter or a CMV promoter/enhancer. CMV promoters and CMV promoters/enhancers are well known to the art. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be any eukaryotic RNA polymerase II promoter.

[0353] Disclosed herein is an expression cassette comprising an exogenous RNA to be transspliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures. Disclosed herein is an expression cassette comprising an exogenous RNA to be trans- spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95. Disclosed herein is an expression cassette comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA. Disclosed herein is an expression cassette comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA.

[0354] In an aspect, expression of a disclosed protein coding gene can be restored and/or returned to a wild-type, normal, or control expression level. In an aspect, a disclosed nucleic acid molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, a disclosed nucleic acid molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra). In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi- systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity.

[0355] In an aspect, restoring the activity and/or functionality of a missing, deficient, and/or mutant protein or enzyme can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and/or mutant protein or enzyme). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is similar to that of a wild-type or control level.

3’ Replacement Constructs

[0356] Disclosed herein is a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3 ’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs). Disclosed herein is a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs). Disclosed herein is a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA. Disclosed herein is a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO: 87 - SEQ ID NO: 95; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans- spliced comprises a series of single nucleotide polymorphisms (SNPs), wherein the nucleic acid molecule enables the 3 ’ replacement of the targeted endogenous pre-mRNA.

[0357] In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 20 SNPs. In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 10 SNPs. In an aspect, a disclosed series of SNPs can comprise 5 SNPs.

[0358] In an aspect, a disclosed targeted endogenous pre-mRNA can comprise one or more mutations in one or more exons. In an aspect, one or more disclosed mutations can be in the 3’ portion of the one or more exons of the pre-mRNA. In an aspect, a disclosed targeted endogenous pre-mRNA can comprise one or more mutations in one or more introns. In an aspect, one or more disclosed mutations in one or more exons can contribute to pathogenesis of one or more cells. In an aspect, disclosed cells can be in a subject. In an aspect, a subject can be a human patient and can be male or female. In an aspect, a subject can have a genetic disease or disorder. In an aspect, a subject can be treatment-naive. In an aspect, the one or more disclosed mutations can inhibit translation of the encoded protein. In an aspect, the one or more disclosed mutations can modify translation of the encoded protein. In an aspect, during and/or following translation the one or more disclosed mutations can generate a protein having a non-sense mutation or a missense mutation. In an aspect, the one or more disclosed exonic mutations can contribute to pathogenesis in one or more cells. In an aspect, the one or more disclosed intronic mutations can contribute to pathogenesis in one or more cells. In an aspect, a disclosed targeted endogenous pre-mRNA can encode a protein coding gene.

[0359] In an aspect of a disclosed targeted endogenous pre-mRNA, a disclosed protein coding gene can comprise one or more coding regions of ABCA1, ABCA12, ABCA13, ABCA2, ABCA3, ABCA4, ABCA5, ABCC1, ABCC2, ABCC6, ABCC8, ABCC9, ACAN, ADAMTS13, ADCY10, ADGRV1, AGL, AGRN, AHDC1, ALK, ALMS1, ALPK3, ALS2, ANAPC1, ANK1, ANK2, ANK3, ANKRD11, ANKRD26, APC, APC2, APOB, ARFGEF2, ARHGAP31, ARHGEF10, ARHGEF18, ARID! A, ARID I B, ARID2, ASH1L, ASPM, ASXL1, ASXL2, ASXL3, ATM, ATP7A, ATP7B, ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, BLM, BPTF, BRCA1, BRCA2, BRD4, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD, CAMTAI, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP 152, CEP 164, CEP250, CEP290, CFAP43, CFAP44, CFAP65, CFTR/ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1, CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1, CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1, DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11, DNAH17, DNAH2, DNAH5, DNAH7, DNAH8, DNAH9, DNMBP, DNMT1, DOCK2, DOCK3, DOCK6, DOCK7, DOCK8, DSCAM, DSP, DST, DUOX2, DYNC1H1, DYNC2H1, DYSF, EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, EYS, F5, F8, FANCA, FANCD2, FANCM, FAT1, FAT4, FBN1, FBN2, FLG, FLG2, FLNA, FLNB, FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2, FSIP2, FXN, FYCO1, GLI2, GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1, HERC2, HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R, IGF2R, IGSF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A, KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220, KIF14, KIF1A, KIF1B, KIF21A, KIF26B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1, LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMC3, LCT, LMNA, LOXHD1, LPA, LRBA, LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD, MAGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5, MCM3AP, MED12, MED12L, MED13, MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MYH11, MYH14, MYH2, MYH3, MYH6, MYH7, MYH7B, MYH8, MYH9, MYLK, MYO 15 A, MYO18B, MY03A, MY05A, MYO5B, MY07A, MY09A, NALCN, NBAS, NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF, NF1, NFASC, NHS, NIN, NIPBL, NLRP1, NOTCH1, NOTCH2, NOTCH3, NPHP4, NRXN1, NRXN3, NSD1, NSD2, NUP155, NUP188, NUP205, OBSCN, OBSL1, OTOF, OTOG, OTOGL, PARD3, PBRM1, PCDH15, PCLO, PCNT, PHIP, PI4KA, PIEZO1, PIEZO2, PIK3C2A, PIKFYVE, PKD1, PKD1L1, PKHD1, PLCE1, PLEC, PLEKHG2, PNPLA6, POGZ, POLA1, POLE, POLR1A, POLR2A, POLR3A, PRG4, PRKDC, PRPF8, PRR12, PRX, PTCHI, PTPN23, PTPRF, PTPRJ, PTPRQ, PXDN, QRICH2, RAB3GAP2, RAH, RALGAPA1, RANBP2, RB1CC1, RELN, RERE, REV3L, RIC1, RIMS1, RIMS2, RNF213, ROBO1, ROBO2, ROBO3, ROS1, RP1, RP1L1, RTTN, RUSC2, RYR1, RYR2, SACS, SAMD9, SAMD9L, SBF2, SCAPER, SCN10A, SCN11A, SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SETBP1, SETD1A, SETD1B, SETD2, SETD5, SETX, SHANK2, SHANK3, SHROOM4, SI, SIPA1L3, SLIT2, SLX4, SMARCA2, SMARCA4, SMCHD1, SNRNP200, SON, SPEF2, SPEG, SPG11, SPTA1, SPTAN1, SPTB, SPTBN2, SPTBN4, SRCAP, STRC, SVIL, SYNE1, SYNGAP1, SYNJ1, SZT2, TAF1, TANC2, TCF20, TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B, TNXB, TOGARAMI, TONSL, TRIO, TRIOBP, TRIP11, TRIP12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, TTN, TUBGCP6, UBR1, UNC80, USH2A, USP9X, VCAN, VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62, WDR81, WNK1, WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292, ZNF335, ZNF407, ZNF462, or ZNF469. In an aspect, a disclosed protein coding gene can comprise one or more coding regions of CFTR, MDX, DYSF/TTN, DMPK, COL7A1, K14, MAPT, FVIII, HTT, RHO, DNA-PKcs, SMN2, or CD40L. In an aspect, a disclosed protein coding gene can comprise one or more coding regions of FXN, LMNA, or RYR2. In an aspect, a disclosed protein coding gene can comprise a portion of a disclosed protein coding gene (such as, for example, Exon 1 or Exon 4, etc.).

[0360] In an aspect of a disclosed targeted endogenous pre-mRNA, a disclosed protein coding gene can comprise one or more coding regions of DMD, TTN, TCAP, SGCA, SGCB, SGCG, SGCD, Al-AT, MYH6, MYH7, MYH11, ML2, ML3, MYLK2, MYBPC3, DES, DNM2, LAMA2, LMNA, LMNB, LBR, DYSF, EMD, EPO, LPL, SERCA2A, S100A1, MTM, DM1 DMPK, PYGL„ PYGM, GYSI, GYS2, NAGA, GAA, SMPD1, LIPA, COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, PLOD1, LIPA, FXN, MSTN, HEXA, HEXB, GBA, AMPD1, HBB, IDUA, IDS, TNNI3, TNNT2, TNNC1, TPM1, TPM3, NAGLU, SGSH, HGSNAT, IGTA7, IGTA9, N-acetyl, GNS, N-acetyl, GALNS, GLB1, GUSB, HYAL1, ASAHI, GALC, CTSA, CTSA, CTSK, GM2A, ARSA, ARSB, SUMFI, NEU1 GNPTA, GNPTB, GNPTG, MCOLN1, NPC1, NPC2, CLN5, CLN6, CLN8, PPT1, TPP1, CLN3, DNAJC5, MFSD8, MAN2B1, MANBA, AGA, FUCA1, CTNS, SLC2A10, SLC17A5, SLC6A19, SLC22A5, SLC37A4, LAMP2, SCN4A, SCN4B, SCN5A, SCN4A, CACNA1C, CACNA1S, PGK1, PGAM2, AGL, KCNE1, KCNE2, KCNJ2, KCNJ5, KCNH2, KCNQ1, HCN4, CLCN1, CPT1A, RYR1, RYR2, BINI, LARGE1, DOK7, FKTN, FKRP, SELENON, POMT1, POMT2, POMGNT1, POMGNT2, POMK, ISPD, PLEC, CHRNE, CHAT, CHKB, COLQ, RAPSN, FHL1, B4GAT1, B3GALNT2, DAGI, TMEM5, TMEM43, SECISBP2, UDP-N-acetyl, GNE, AN05, SMCHD1, LDHA, LHDB, CAPN3, CAV3, TRIM32, CNBP, NEB, ACTA1, ACTC1, ACTN2, PABPN1, LEMD3, ZMPSTE24, MTTP, CHRNA1, CHRNA2, CHRNA3, CHRNA4, CHRNA5, CHRNA6, CHRNA7, CHRNA8, CHRNA9, CHRNA10, CHRNB1, CHRNB2, CHRNB3, CHRNB4, CHRNG1, CHRND, CHRNE1, ABCA1, ABCC6, ABCC9, ABCD1, ATP2A1, ATM, TTP A, KIF21A, PHOX2A, HSPG2, STIM1, NOTCHI, NOTCH3, DTNA, PRKAG2, CSRP3, VCL, MyoZ2, MYPN, JPH2, PLN, CALR3, NEXN, LDB3, EYA4, HTT, AR, PTPN11, JUP, DSP, PKP2, DSG2, DSC2, CTNNA3, NKX2-5, AKAP9, AKAP10, GNAI2, ANK2, SNTAT, CALM1, CALM2, HTRA1, FBN1, FBN2, XYLT1, XYLT2, TAZ, HGD, G6PC, GBE1, PFKM, PHKA1, PHKA2, PHKB, PHKG2, PGAM2, CBS, MTHFR, MTR, MTRR, MMADHC, MT-ND1, MT-ND5, MT-TE, MT-TH, MT-TL1, MT-TK, MT-TS1, MT- TV, MAP2K1, BRAF, RAFI, IGF-1, TGFP3, TGFpRl, TGFPR2, FGF2, FGF4, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2

[0361] In an aspect, a disclosed 5’ portion of the targeted endogenous pre-mRNA can be transspliced with the exogenous RNA.

[0362] In an aspect, a disclosed RNA targeting motif can bind to the targeted endogenous pre- mRNA. In an aspect, a disclosed RNA targeting motif can bind to the 3’ end of the targeted endogenous pre-mRNA. In an aspect, a disclosed RNA targeting motif can be specific for an endogenous pre-mRNA having one or more mutations. In an aspect, a disclosed RNA targeting motif can be specific for an endogenous pre-mRNA having one or more exonic mutations. In an aspect, a disclosed RNA targeting motif can be specific for an endogenous pre-mRNA having one or more intronic mutations. In an aspect, a disclosed RNA targeting motif can comprise an antisense oligonucleotide.

[0363] In an aspect, a disclosed antisense oligonucleotide can comprise about 15 nucleotides to about 50 nucleotides. In an aspect, a disclosed antisense oligonucleotide can comprise about 30 nucleotides. In an aspect, a disclosed RNA targeting motif can be directed to the intron immediately 3’ to the exon of the targeted endogenous pre-mRNA with which it is to be spliced. [0364] In an aspect, a disclosed 3’ hemi intron can comprise (i) a 3’ splice region comprising a branch point, (ii) a polypyrimidine tract, and (iii) a 3’ splice acceptor site. In an aspect, a disclosed branch point can comprise the sequence of SEQ ID NO: 57 (YNYYRAY, wherein Y is a pyrimidine and R is a purine). In an aspect, a disclosed 3’ splice acceptor site can comprise the sequence YAG, where Y is a pyrimidine (SEQ ID NO:58). In an aspect, a disclosed 3’ hemi intron can be recognized by nuclear splicing components in a host cell. In an aspect, a disclosed 3’ hemi intron can be recognized by the spliceosome in a host cell. In an aspect, a disclosed 3’ hemi intron can facilitate the trans-splicing of the exogenous RNA to the exon immediately 5’ to the targeted intron in the endogenous pre-mRNA.

[0365] In an aspect, a disclosed exogenous RNA to be trans-spliced to the targeted endogenous pre-mRNA can comprise one or more exons of the protein coding gene. In an aspect, a disclosed exogenous RNA to be trans-spliced to the targeted endogenous pre-mRNA can comprise the primary sequence of the coding sequence of one or more exons having the one or more mutations. In an aspect, a disclosed exogenous RNA can be trans-spliced to a 5’ end of the targeted endogenous pre-mRNA.

[0366] In an aspect of a disclosed exogenous RNA, a disclosed protein coding gene can comprise one or more coding regions of ABCA1, ABCA12, ABCA13, ABCA2, ABCA3, ABCA4, ABCA5, ABCC1, ABCC2, ABCC6, ABCC8, ABCC9, ACAN, ADAMTS13, ADCY10, ADGRV1, AGL, AGRN, AHDC1, ALK, ALMS1, ALPK3, ALS2, ANAPC1, ANK1, ANK2, ANK3, ANKRD11, ANKRD26, APC, APC2, APOB, ARFGEF2, ARHGAP31, ARHGEF10, ARHGEF18, ARID! A, ARID1B, ARID2, ASH1L, ASPM, ASXL1, ASXL2, ASXL3, ATM, ATP7A, ATP7B, ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, BLM, BPTF, BRCA1, BRCA2, BRIM, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD, CAMTAI, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP 152, CEP 164, CEP250, CEP290, CFAP43, CFAP44, CFAP65, CFTR/ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1, CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1, CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1, DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11, DNAH17, DNAH2, DNAH5, DNAH7, DNAH8, DNAH9, DNMBP, DNMT1, DOCK2, DOCK3, DOCK6, DOCK7, DOCK8, DSCAM, DSP, DST, DUOX2, DYNC1H1, DYNC2H1, DYSF, EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, EYS, F5, F8, FANCA, FANCD2, FANCM, FAT1, FAT4, FBN1, FBN2, FLG, FLG2, FLNA, FLNB, FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2, FSIP2, FXN, FYCO1, GLI2, GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1, HERC2, HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R, IGF2R, IGSF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A, KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220, KIF14, KIF1A, KIF1B, KIF21A, KIF26B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1, LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMC3, LCT, LMNA, L0XHD1, LPA, LRBA, LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD, MAGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5, MCM3AP, MED12, MED12L, MED13, MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MYH11, MYH14, MYH2, MYH3, MYH6, MYH7, MYH7B, MYH8, MYH9, MYLK, MYO15A, MYO18B, MYO3 A, MYO5A, MYO5B, MYO7A, MYO9A, NALCN, NBAS, NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF, NF1, NFASC, NHS, NIN, NIPBL, NLRP1, NOTCH1, NOTCH2, NOTCH3, NPHP4, NRXN1, NRXN3, NSD1, NSD2, NUP155, NUP188, NUP205, OBSCN, OBSL1, OTOF, OTOG, OTOGL, PARD3, PBRM1, PCDH15, PCLO, PCNT, PHIP, PI4KA, PIEZO1, PIEZO2, PIK3C2A, PIKFYVE, PKD1, PKD1L1, PKHD1, PLCE1, PLEC, PLEKHG2, PNPLA6, POGZ, POLA1, POLE, POLR1A, POLR2A, POLR3A, PRG4, PRKDC, PRPF8, PRR12, PRX, PTCHI, PTPN23, PTPRF, PTPRJ, PTPRQ, PXDN, QRICH2, RAB3GAP2, RAH, RALGAPA1, RANBP2, RB1CC1, RELN, RERE, REV3L, RIC1, RIMS1, RIMS2, RNF213, ROBO1, ROBO2, ROBO3, ROS1, RP1, RP1L1, RTTN, RUSC2, RYR1, RYR2, SACS, SAMD9, SAMD9L, SBF2, SCAPER, SCN10A, SCN11A, SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SETBP1, SETD1A, SETD1B, SETD2, SETD5, SETX, SHANK2, SHANK3, SHROOM4, SI, SIPA1L3, SLIT2, SLX4, SMARCA2, SMARCA4, SMCHD1, SNRNP200, SON, SPEF2, SPEG, SPG11, SPTA1, SPTAN1, SPTB, SPTBN2, SPTBN4, SRCAP, STRC, SVIL, SYNE1, SYNGAP1, SYNJ1, SZT2, TAF1, TANC2, TCF20, TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B, TNXB, TOGARAMI, TONSL, TRIO, TRIOBP, TRIP11, TRIP12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, TTN, TUBGCP6, UBR1, UNC80, USH2A, USP9X, VCAN, VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62, WDR81, WNK1, WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292, ZNF335, ZNF407, ZNF462, or ZNF469. In an aspect, a disclosed protein coding gene can comprise one or more coding regions of CFTR, MDX, DYSF/TTN, DMPK, COL7A1, K14, MAPT, FVIII, HTT, RHO, DNA-PKcs, SMN2, or CD40L. In an aspect, a disclosed protein coding gene can comprise one or more coding regions of FXN, LMNA, or RYR2.

[0367] In an aspect of a disclosed exogenous RNA, a disclosed protein coding gene can comprise one or more coding regions of DMD, TTN, TCAP, SGCA, SGCB, SGCG, SGCD, Al -AT, MYH6, MYH7, MYH11, ML2, ML3, MYLK2, MYBPC3, DES, DNM2, LAMA2, LMNA, LMNB, LBR, DYSF, EMD, EPO, LPL, SERCA2A, S100A1, MTM, DM1 DMPK, PYGL„ PYGM, GYSI, GYS2, NAGA, GAA, SMPD1, LIPA, COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, PLOD1, LIPA, FXN, MSTN, HEXA, HEXB, GBA, AMPD1, HBB, IDUA, IDS, TNNI3, TNNT2, TNNC1, TPM1, TPM3, NAGLU, SGSH, HGSNAT, IGTA7, IGTA9, N-acetyl, GNS, N-acetyl, GALNS, GLB1, GUSB, HYAL1, ASAHI, GALC, CTSA, CTSA, CTSK, GM2A, ARSA, ARSB, SUMFI, NEU1 GNPTA, GNPTB, GNPTG, MCOLN1, NPC1, NPC2, CLN5, CLN6, CLN8, PPT1, TPP1, CLN3, DNAJC5, MFSD8, MAN2B1, MANBA, AGA, FUCA1, CTNS, SLC2A10, SLC17A5, SLC6A19, SLC22A5, SLC37A4, LAMP2, SCN4A, SCN4B, SCN5A, SCN4A, CACNA1C, CACNA1S, PGK1, PGAM2, AGL, KCNE1, KCNE2, KCNJ2, KCNJ5, KCNH2, KCNQ1, HCN4, CLCN1, CPT1A, RYR1, RYR2, BINI, LARGE1, DOK7, FKTN, FKRP, SELENON, POMT1, POMT2, POMGNT1, POMGNT2, POMK, ISPD, PLEC, CHRNE, CHAT, CHKB, COLQ, RAPSN, FHL1, B4GAT1, B3GALNT2, DAGI, TMEM5, TMEM43, SECISBP2, UDP-N-acetyl, GNE, ANO5, SMCHD1, LDHA, LHDB, CAPN3, CAV3, TRIM32, CNBP, NEB, ACTA1, ACTC1, ACTN2, PABPN1, LEMD3, ZMPSTE24, MTTP, CHRNA1, CHRNA2, CHRNA3, CHRNA4, CHRNA5, CHRNA6, CHRNA7, CHRNA8, CHRNA9, CHRNA10, CHRNB1, CHRNB2, CHRNB3, CHRNB4, CHRNG1, CHRND, CHRNE1, ABC Al, ABCC6, ABCC9, ABCD1, ATP2A1, ATM, TTP A, KIF21A, PHOX2A, HSPG2, STIM1, NOTCHI, NOTCH3, DTNA, PRKAG2, CSRP3, VCL, MyoZ2, MYPN, JPH2, PLN, CALR3, NEXN, LDB3, EYA4, HTT, AR, PTPN11, JUP, DSP, PKP2, DSG2, DSC2, CTNNA3, NKX2-5, AKAP9, AKAP10, GNAI2, ANK2, SNTAT, CALM1, CALM2, HTRA1, FBN1, FBN2, XYLT1, XYLT2, TAZ, HGD, G6PC, GBE1, PFKM, PHKA1, PHKA2, PHKB, PHKG2, PGAM2, CBS, MTHFR, MTR, MTRR, MMADHC, MT-ND1, MT- ND5, MT-TE, MT-TH, MT-TL1, MT-TK, MT-TS1, MT-TV, MAP2K1, BRAF, RAFI, IGF-1, TGFP3, TGFPR1, TGFPR2, FGF2, FGF4, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, or VEGFR2.

[0368] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode LMNA/C (SEQ ID NO:55) or a portion thereof. LMNA/C is discussed supra. In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode DP71 or a portion thereof. DP71 is discussed supra. In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode CFTR (SEQ ID NO:54) or a portion thereof. CFTR is discussed supra. In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode DMPK (SEQ ID NO:56) or a portion thereof. DMPK is discussed supra. In an aspect, a disclosed gene can be DMD (dystrophin) (SEQ ID NO:52). DMD is discussed supra.

[0369] In an aspect, a disclosed exogenous RNA to be trans-spliced can further comprise a UTR. [0370] In an aspect, one or more disclosed RNA structures can bind to one or more RNA binding proteins. In an aspect, one or more disclosed RNA structures can bind to one or more double- stranded RNA binding proteins (dsRBP). In an aspect, dsRBPs are known to the skilled person in the art and include, but are not limited to, AD ARI, ADAR2, DICER, NEAR, PACT, PKR, RHA RNaselll, Stauffen, TRBP, TSEN, or any combination thereof.

[0371] In an aspect, one or more disclosed RNA structures can bind to one or more RNA binding proteins. In an aspect, one or more disclosed RNA structures can bind to one or more doublestranded RNA binding proteins. In an aspect, one or more disclosed RNA structures can comprise the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95. In an aspect, one or more disclosed RNA structures can improve and/or can enhance trans-splicing efficiency. In an aspect, one or more disclosed RNA structures can stabilize the pre-mRNA. In an aspect, one or more disclosed RNA structures can localize the RNA to the nucleus. In an aspect, one or more disclosed RNA structures can stabilize the interaction between the targeted endogenous pre-mRNA molecule and the exogenous RNA to be trans-spliced.

[0372] In an aspect, a disclosed nucleic acid molecule can lack a CRISPR-associated protein.

[0373] In an aspect, a disclosed resulting chimeric RNA transcript can comprise the 5’ portion of the targeted endogenous pre-mRNA and the 3’ portion of the exogenous RNA. In an aspect, a disclosed resulting chimeric RNA transcript can comprise the 3’ portion of the targeted endogenous pre-mRNA and the 5’ portion of the exogenous RNA. In an aspect, a disclosed targeted endogenous pre-mRNA and a disclosed exogenous RNA can encode the same protein coding gene. In an aspect, a disclosed targeted endogenous pre-mRNA and a disclosed exogenous RNA can comprise one or more exons of the same protein coding gene.

[0374] In an aspect, a disclosed nucleic acid molecule can be packaged into a viral vector. In an aspect, a disclosed viral vector can comprise an AAV vector. In an aspect, a disclosed nucleic acid molecule can be packaged into a non-viral carrier. In an aspect, a disclosed nucleic acid molecule can be incorporated into a plasmid. In an aspect, a disclosed nucleic acid molecule can be incorporated into lipid nanoparticles.

[0375] In an aspect, a disclosed nucleic acid molecule can further comprise a polyadenylation sequence. In an aspect, a disclosed nucleic acid molecule can further comprise a sequence for a promoter. In an aspect, a disclosed 3’ hemi intron can be recognized by nuclear splicing components within a host cell. In an aspect, a disclosed exogenous RNA can induce a splice event. In an aspect, a disclosed nucleic acid molecule can further comprise a spacer region. In an aspect, a disclosed spacer region can separate the 5’ splice region from the one or more RNA structures. In an aspect, a disclosed spacer region can comprise any known spacer. In an aspect, a disclosed spacer region can comprise a consensus splicing motif (e.g., such as U1 or U2). In an aspect, a disclosed spacer region can comprise a limited number of consensus splicing motifs (e.g., such as U1 or U2). In an aspect, a disclosed nucleic acid molecule can further comprise a nuclear localization signal (NLS). In an aspect, a disclosed nucleic acid molecule can further comprise one or more nuclear retention elements (NRE). NRE are known to the skilled person in the art. In an aspect, a disclosed NRE can comprise SIRLOIN (SEQ ID NO:96) or BORG (SEQ ID NO:97).

[0376] In an aspect, a disclosed nucleic acid molecule can further comprise one or more Flavivirus genetic elements. In an aspect, Flavivirus genetic elements can comprise one or more Flavivirus 3’ untranslated region (3’ UTR), one or more subgenomic Flavivirus RNA (sfRNA) elements, one or more Flavivirus XRN1 -resistant RNA (xrRNA) elements, one or more Flavivirus dumbbell (DB) RNA elements, one or more Flavivirus 3’ stem loop (3’ SL) elements, or any combination thereof. (See WO 2022/182835 for a description of Flavivirus gene elements).

[0377] In an aspect, a disclosed nucleic acid molecule can comprise the sequence for one or more regulatory elements (e.g., Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulator Element (WPRE), triplex from MALAT1, the PRE of Hepatitis B virus (HPRE), and an iron response element). For example, a disclosed regulatory element can comprise a promoter operably linked to a disclosed nucleic acid molecule, wherein the promoter drives the expression of a disclosed variant capsid protein, a disclosed encoded polypeptide, a disclosed encoded therapeutic agent, or both.

[0378] In an aspect, a disclosed promoter for the 3’ replacement construct can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the expression desired. A promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. In an aspect, a disclosed promoter can be a promoter/enhancer. In an aspect, a disclosed promoter for the disclosed nucleic acid molecule can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter/enhancer. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can generally be obtained from a non-coding region upstream of a transcription initiation site of a gene of interest. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can be used for constitutive and efficient expression of a disclosed gene. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be a CMV promoter or a CMV promoter/enhancer. CMV promoters and CMV promoters/enhancers are well known to the art. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be any eukaryotic RNA polymerase II promoter. [0379] Disclosed herein is an expression cassette comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs). Disclosed herein is an expression cassette comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs). Disclosed herein is an expression cassette comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA. Disclosed herein is an expression cassette comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO: 87 - SEQ ID NO: 95; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans- spliced comprises a series of single nucleotide polymorphisms (SNPs), wherein the nucleic acid molecule enables the 3 ’ replacement of the targeted endogenous pre-mRNA.

[0380] In an aspect, expression of a disclosed protein coding gene can be restored and/or returned to a wild-type, normal, or control expression level. In an aspect, a disclosed nucleic acid molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, a disclosed nucleic acid molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra). In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi- systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity. In an aspect, restoring the activity and/or functionality of a missing, deficient, and/or mutant protein or enzyme can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration can be 10- 20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and/or mutant protein or enzyme). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is similar to that of a wild-type or control level.

[0381] In an aspect, a disclosed nucleic acid molecule can be used in one or more methods of trans-splicing, including, for example, SMaRT, CRAFT, and GRAFT (disclosed herein).

[0382] In an aspect, a disclosed targeted endogenous pre-mRNA can be encoded by one or more relevant genes (such as, for example, those listed above in Table 1).

2. Transcriptome Engineering Systems

[0383] Disclosed herein is a transcriptome engineering system comprising one or more disclosed 3’ replacement construct, one or more disclosed 5’ replacement constructs, or any combination thereof. Disclosed herein is a transcriptome engineering system comprising one or more of (i) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans- spliced; one or more RNA targeting motifs; one or more RNA structures, (ii) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95, (iii) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans- spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA, and (iv) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be transspliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA.

[0384] Disclosed herein is a transcriptome engineering system comprising one or more (i) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans- spliced comprises a series of single nucleotide polymorphisms (SNPs);, (ii) a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO: 87 - SEQ ID NO: 95; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), (iii) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre- mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA, and (iv) a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), wherein the nucleic acid molecule enables the 3 ’ replacement of the targeted endogenous pre-mRNA.

[0385] Disclosed herein is a transcriptome engineering system comprising one or more of (i) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans- spliced; one or more RNA targeting motifs; one or more RNA structures, (ii) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95, (iii) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be transspliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA, (iv) a nucleic acid molecule comprising an exogenous RNA to be trans- spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), (v) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), (vi) a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), (vii) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA, and (viii) a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA. [0386] In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 20 SNPs. In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 10 SNPs. In an aspect, a disclosed series of SNPs can comprise 5 SNPs. In an aspect, a disclosed transcriptome engineering system can be used in one or more methods of trans-splicing, including, for example, SMaRT, CRAFT, and GRAFT (disclosed herein).

3. Vectors

[0387] Disclosed herein is a vector comprising a disclosed nucleic acid molecule. In an aspect, a disclosed vector can be a non-viral vector or a viral vector. Disclosed herein is a non-viral vector comprising a disclosed nucleic acid molecule. Disclosed herein is a non-viral vector comprising one or more disclosed nucleic acid molecules. Disclosed herein is a viral vector comprising a disclosed nucleic acid molecule. Disclosed herein is a viral vector comprising one or more disclosed nucleic acid molecules. Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans- spliced; one or more RNA targeting motifs; one or more RNA structures. Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans- spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95.

[0388] Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA. Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA. Disclosed herein is a non- viral or viral vector comprising a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be transspliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs). Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre- mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs). Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), wherein the nucleic acid molecule enables the 3 ’ replacement of the targeted endogenous pre-mRNA. Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO: 87 - SEQ ID NO: 95; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans- spliced comprises a series of single nucleotide polymorphisms (SNPs);, wherein the nucleic acid molecule enables the 3 ’ replacement of the targeted endogenous pre-mRNA.

[0389] Disclosed herein is a non-viral or viral vector comprising one or more 5’ replacement constructs. Disclosed herein is a non-viral or viral vector comprising one or more 3’ replacement constructs. Disclosed herein is a non-viral or viral vector comprising one or more 5’ replacement constructs and/or one or more 3’ replacement constructs. In an aspect, a disclosed 5’ replacement construct to be used in combination with one or more other replacement constructs can comprise any disclosed 5’ replacement construct. In an aspect, a disclosed 5’ replacement construct can comprise (i) nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, (ii) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95, (iii) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be transspliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA, or (iv) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans- spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA, or (v) any combination thereof.

[0390] In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 20 SNPs. In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 10 SNPs. In an aspect, a disclosed series of SNPs can comprise 5 SNPs.

[0391] In an aspect, a disclosed 3’ replacement construct to be used in combination with one or more other replacement constructs can comprise any disclosed 3’ replacement construct. In an aspect, a disclosed 3’ replacement construct can comprise (i) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), (ii) a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), (iiii) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA, (iv) a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA, or (v) any combination thereof.

[0392] In an aspect, a disclosed nucleic acid sequence can have a coding sequence that is less than about 4.5 kilobases.

[0393] In an aspect, a disclosed vector can be a viral vector or a non-viral vector. In an aspect, a disclosed non-viral vector can be a polymer-based vector, a peptide-based vector, a lipid nanoparticle, a solid lipid nanoparticle, or a cationic lipid-based vector. In an aspect, a disclosed vector can comprise exosomes, extracellular vesicles, and virus like particles. In an aspect, a disclosed viral vector can be an adenovirus vector, an AAV vector, a herpes simplex virus vector, a retrovirus vector, a lentivirus vector, and alphavirus vector, a Flavivirus vector, a rhabdovirus vector, a measles virus vector, a Newcastle disease viral vector, a poxvirus vector, or a picomavirus vector.

[0394] In an aspect, a disclosed viral vector can be an adeno-associated virus (AAV) vector In an aspect, a disclosed AAV vector can include naturally isolated serotypes including, but not limited to, AAV1, AAV2, AAV3 (including 3a and 3b), AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAVrh39, AAVrh43, AAVcy.7 as well as bovine AAV, caprine AAV, canine AAV, equine AAV, ovine AAV, avian AAV, primate AAV, non-primate AAV, and any other virus classified by the International Committee on Taxonomy of Viruses (ICTV) as an AAV. In an aspect, an AAV capsid can be a chimera either created by capsid evolution or by rational capsid engineering from a naturally isolated AAV variants to capture desirable serotype features such as enhanced or specific tissue tropism and/or a host immune response escape. Naturally isolated AAV variants include, but not limited to, AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV-1829, AAV2 Y/F, AAV2 T/V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A-String (e.g., AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep, and AAV9.47-AS, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, and AAVcc.81. In an aspect, a disclosed AAV vector can be AAV-Rh74 or a related variant (e.g., capsid variants like RHM4-1). In an aspect, a disclosed AAV vector can be AAV8. In an aspect, a disclosed AAV vector can be AAVhum.8. In an aspect, a disclosed AAV vector can be a self-complementary AAV as disclosed herein.

[0395] In an aspect, a disclosed vector can comprise one or more ITRs (such as, for example, ITRs from AAV2).

[0396] In an aspect, a disclosed vector can further comprise one or more nuclear localization signals (NLS). NLS are known to the skilled person in the art. In an aspect, a disclosed NLS can comprise any NLS known to the art. As known to the art (see, e.g., Lu J, et al. (2021) Cell Commun Signal. 19:60, which is incorporated herein by reference for its teachings of NLS), nuclear localization signals (NLS) are generally short peptides that act as a signal fragment that mediates the transport of proteins from the cytoplasm into the nucleus.

[0397] In an aspect, a disclosed vector can further comprise one or more nuclear retention elements (NRE). NRE are known to the skilled person in the art. In an aspect, a disclosed NRE can comprise SIRLOIN (SEQ ID NO:96) or BORG (SEQ ID NO:97).

[0398] In an aspect, a disclosed vector can further comprise one or more Flavivirus genetic elements. In an aspect, Flavivirus genetic elements can comprise one or more Flavivirus 3’ untranslated region (3’ UTR), one or more subgenomic Flavivirus RNA (sfRNA) elements, one or more Flavivirus XRN1 -resistant RNA (xrRNA) elements, one or more Flavivirus dumbbell (DB) RNA elements, one or more Flavivirus 3’ stem loop (3’ SL) elements, or any combination thereof. (See WO 2022/182835 for a description of Flavivirus gene elements).

[0399] In an aspect, a disclosed vector can comprise one or more promoters operably linked to a disclosed nucleic acid molecule (e.g., a 5’ replacement construct and/or 3’ replacement construct), a disclosed transgene, a disclosed sequence to be trans-spliced, and/or a disclosed nucleic acid sequence. In an aspect of a disclosed vector, a disclosed nucleic acid molecule can be operably linked to one or more transcription regulatory elements. In an aspect, the one or more transcription regulatory elements (e.g., Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulator Element (WPRE), triplex from MALAT1, the PRE of Hepatitis B virus (HPRE), and an iron response element) can increase the transcription and/or expression of a disclosed nucleic acid molecule (e.g., a 5’ replacement construct and/or 3 ’ replacement construct), a disclosed transgene, a disclosed sequence to be trans-spliced, and/or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and/or a disclosed therapeutic RNA). In an aspect, a disclosed promoter can be positioned 5’ (upstream) or 3’ (downstream) of a disclosed nucleic acid molecule (e.g., a 5’ replacement construct and/or 3’ replacement construct), a disclosed transgene, a disclosed sequence to be trans-spliced, and/or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and/or a disclosed therapeutic RNA) under its control. The distance between a disclosed promoter and a disclosed nucleic acid molecule (e.g., a 5’ replacement construct and/or 3’ replacement construct), a disclosed transgene, a disclosed sequence to be trans- spliced, and/or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and/or a disclosed therapeutic RNA) can be approximately the same as the distance between that promoter and to the disclosed nucleic acid molecule (e.g., a 5’ replacement construct and/or 3’ replacement construct), the disclosed transgene, the disclosed sequence to be trans-spliced, and/or

I l l the disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and/or a disclosed therapeutic RNA) under its control. As is known in the art, variation in this distance can be accommodated without loss of promoter function.

[0400] In an aspect of a disclosed vector, a disclosed promoter can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the expression desired. A disclosed promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. In an aspect, a disclosed promoter can be a promoter/enhancer. In an aspect, a disclosed promoter for a disclosed nucleic acid molecule (e.g., a 5’ replacement construct and/or 3 ’ replacement construct), a disclosed transgene, a disclosed sequence to be trans-spliced, and/or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and/or a disclosed therapeutic RNA) can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter/enhancer. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can generally be obtained from a non-coding region upstream of a transcription initiation site of a gene of interest. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can be used for constitutive and efficient expression of a disclosed protein coding gene. In an aspect, a disclosed promoter for a disclosed nucleic acid molecule (e.g., a 5’ replacement construct and/or 3’ replacement construct), a disclosed transgene, a disclosed sequence to be trans-spliced, and/or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and/or a disclosed therapeutic RNA) can be a CMV promoter or a CMV promoter/enhancer. CMV promoters and CMV promoters/enhancers are well known to the art. In an aspect, a disclosed promoter for a disclosed nucleic acid molecule (e.g., a 5’ replacement construct and/or 3’ replacement construct), a disclosed transgene, a disclosed sequence to be trans- spliced, and/or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and/or a disclosed therapeutic RNA) can be any eukaryotic RNA polymerase II promoter.

[0401] In an aspect, a disclosed AAV vector can be used to generate AAV particles. In an aspect, a disclosed AAV vector can be used to generate AAV particles comprising a disclosed nucleic acid molecule (e.g., a 5’ replacement construct and/or 3’ replacement construct), a disclosed transgene, a disclosed sequence to be trans-spliced, and/or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and/or a disclosed therapeutic RNA) under its control. [0402] Disclosed herein is an AAV particle comprising a disclosed nucleic acid molecule (e.g., a 5’ replacement construct and/or 3’ replacement construct), a disclosed transgene, a disclosed sequence to be trans-spliced, and/or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and/or a disclosed therapeutic RNA) under its control.

[0403] In an aspect, a disclosed vector can be used in one or more methods of trans-splicing, including, for example, SMaRT, CRAFT, and GRAFT (disclosed herein).

4. Pharmaceutical Formulations

[0404] Disclosed herein is a pharmaceutical formulation comprising a disclosed nucleic acid molecule. Disclosed herein is a pharmaceutical formulation comprising a disclosed nucleic acid molecule and a pharmaceutically acceptable carrier. Disclosed herein is a pharmaceutical formulation comprising a disclosed vector. Disclosed herein is a pharmaceutical formulation comprising a disclosed vector and a pharmaceutically acceptable carrier. Disclosed herein is a pharmaceutical formulation comprising a disclosed AAV particle. Disclosed herein is a pharmaceutical formulation comprising a disclosed AAV particle and a pharmaceutically acceptable carrier.

[0405] Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or a viral vector comprising a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures. Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or a viral vector comprising a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95. Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or a viral vector comprising a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA. Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or a viral vector comprising a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO: 87 - SEQ ID NO: 95, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA. Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or a viral vector comprising a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs). Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or viral vector comprising a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs). Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or a viral vector comprising a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA. Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or a viral vector comprising a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO: 87 - SEQ ID NO: 95; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans- spliced comprises a series of single nucleotide polymorphisms (SNPs), wherein the nucleic acid molecule enables the 3 ’ replacement of the targeted endogenous pre-mRNA.

[0406] In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 20 SNPs. In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 10 SNPs. In an aspect, a disclosed series of SNPs can comprise 5 SNPs.

[0407] In an aspect, a disclosed pharmaceutical formulation can comprise (i) one or more active agents, (ii) biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In an aspect, a disclosed composition can comprise one or more immune modulators. In an aspect, a disclosed composition can comprise one or more proteasome inhibitors. In an aspect, a disclosed composition can comprise one or more immunosuppressives or immunosuppressive agents. In an aspect, an immunosuppressive agent can be anti-thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), or a combination thereof. In an aspect, a disclosed formulation can comprise an anaplerotic agent (such as, for example, C7 compounds like triheptanoin or MCT).

[0408] In an aspect, a disclosed formulation can comprise an RNA therapeutic. An RNA therapeutic can comprise RNA-mediated interference (RNAi) and/or antisense oligonucleotides (ASO). In an aspect, a disclosed RNA therapeutic can be directed at any protein or enzyme that is overexpressed or is overactive due to a missing, deficient, and/or mutant protein or enzyme. In an aspect, a disclosed RNA therapeutic can comprise therapy delivered via LNPs. In an aspect, a disclosed formulation can comprise an enzyme or enzyme precursor for enzyme replacement therapy (ERT).

[0409] In an aspect, a disclosed formulation can comprise a disclosed small molecule. In an aspect, a disclosed small molecule can assist in restoring the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0410] In an aspect, any disclosed pharmaceutical formulation can comprise one or more excipients and/or pharmaceutically acceptable carriers. Excipients and/or pharmaceutically acceptable carriers are known to the art and are discussed supra.

[0411] In an aspect, a therapeutically effective amount of a disclosed pharmaceutical formulation can comprise a range of about 1 x 10¹⁰ vg/kg to about 2 x 10¹⁴vg/kg of a disclosed vector and/or a disclosed AAV particle. In an aspect, for example, a dose of a disclosed pharmaceutical formulation can comprise about 1 x 10¹¹ to about 8 x 10¹³ vg/kg or about 1 x 10¹² to about 8 x 10¹³ vg/kg. In an aspect, a dose of a disclosed pharmaceutical formulation can comprise about 1 x 10¹³ to about 6 x 10¹³ vg/kg. In an aspect, a dose of a disclosed pharmaceutical formulation can comprise at least about 1 x 10¹⁰, at least about 5 x 10¹⁰, at least about 1 x 10¹¹, at least about 5 x 10¹¹, at least about 1 x 10¹², at least about 5 x 10¹², at least about 1 x 10¹³, at least about 5 x 10¹³, or at least about 1 x 10¹⁴ vg/kg. In an aspect, a dose of a disclosed pharmaceutical formulation can comprise no more than about 1 x 10¹⁰, no more than about 5 x 10¹⁰, no more than about 1 x 10¹¹, no more than about 5 x 10¹¹, no more than about 1 x 10¹², no more than about 5 x 10¹², no more than about 1 x 10¹³, no more than about 5 x 10¹³, or no more than about 1 x 10¹⁴ vg/kg. In an aspect, a dose of a disclosed pharmaceutical formulation can comprise about 1 x 10¹² vg/kg. In an aspect, a dose of a disclosed pharmaceutical formulation can comprise about 1 x 10¹¹ vg/kg. In an aspect, a dose of a disclosed pharmaceutical formulation can comprise a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results.

[0412] In an aspect, a therapeutically effective amount of a disclosed pharmaceutical formulation can comprise a range of about 1 x 10¹² vg per subject total to about 1 x 10¹⁷ vg per subject total. In an aspect, a therapeutically effective amount of a disclosed pharmaceutical formulation can comprise a range of about 1 x 10¹² vg per subject total, about 1 x 10¹³ vg per subject total, about 1 x 10¹⁴ vg per subject total, about 1 x 10¹⁵ vg per subject total, about 1 x 10¹⁶ vg per subject total, or about 1 x 10¹⁷ vg per subject total. In an aspect, a therapeutically effective amount of a disclosed pharmaceutical formulation can comprise about 1 x 10⁶ DRP/mL to about 1 x 10¹⁴ DRP/mL. In an aspect, a therapeutically effective amount of a disclosed pharmaceutical formulation can comprise about 1 x 10⁶ DRP/mL, 1 x 10⁷ DRP/mL, 1 x 10⁸ DRP/mL, 1 x 10⁹ DRP/mL, 1 x IO¹⁰ DRP/mL, 1 x 10¹¹ DRP/mL, 1 x 10¹² DRP/mL, 1 x 10¹³ DRP/mL, or 1 x 10¹⁴ DRP/mL.

[0413] In an aspect, a therapeutically effective amount of a disclosed pharmaceutical formulation can comprise a range determined by a skilled person.

[0414] In an aspect, a disclosed pharmaceutical formulation can be used to restore and/or return expression of a disclosed protein coding gene to a wild-type, normal, or control expression level. In an aspect, a disclosed pharmaceutical formulation can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, a disclosed nucleic acid molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra). In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi -systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity. In an aspect, restoring the activity and/or functionality of a missing, deficient, and/or mutant protein or enzyme can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and/or mutant protein or enzyme). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is similar to that of a wild-type or control level.

5. Plasmids

[0415] Disclosed herein is a plasmid comprising one or more disclosed nucleic acid molecules. Disclosed herein is a plasmid comprising one or more disclosed vectors. Disclosed here are plasmids used in methods of making a disclosed composition such as, for example, a disclosed nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. Plasmids and using plasmids are known to the art. Disclosed herein is a plasmid comprising the sequence set forth in any one of SEQ ID NO: 100 - SEQ ID NO: 108 or a fragment thereof. Disclosed herein is a plasmid comprising a sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the sequence set forth in any one of SEQ ID NO: 100 - SEQ ID NO: 108 or a fragment thereof. Disclosed herein is a plasmid comprising a sequence having at least 40%-60%, at least 60%-80%, at least 80%-90%, or at least 90%-100% identity to the sequence set forth in any one of SEQ ID NO: 100 - SEQ ID NO: 108 or a fragment thereof.

[0416] In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 20 SNPs. In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 10 SNPs. In an aspect, a disclosed series of SNPs can comprise 5 SNPs. In an aspect, a disclosed pharmaceutical formulation can be used in one or more methods of trans-splicing, including, for example, SMaRT, CRAFT, and GRAFT (disclosed herein).

6. Cells

[0417] Disclosed herein are cells comprising a disclosed nucleic acid molecule, a disclosed vector, and/or a disclosed plasmid. Disclosed herein are cells transduced by one or more disclosed viral vectors. Disclosed herein are cells transfected with one or more disclosed nucleic acid molecules. Techniques to achieve transfection and transduction are known to the art and using transfected or transduced cells are known to the art. In an aspect, disclosed herein are human cells lines transduced by one or more disclosed viral vectors or transfected with one or more disclosed nucleic acids, one or more disclosed non-viral vectors, or one or more disclosed plasmids. In an aspect, disclosed herein are human cells lines having one or more genetic diseases or genetic disorders contacted with one or more nucleic acid molecules, one or more disclosed vectors, and/or one or more disclosed pharmaceutical formulations. Disclosed herein are cells obtained for a subject treated with one or more disclosed nucleic acid molecules, one or more disclosed vectors, one or more disclosed plasmids, and/or one or more disclosed pharmaceutical formulations.

In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 20 SNPs. In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 10 SNPs. In an aspect, a disclosed series of SNPs can comprise 5 SNPs. Disclosed herein are cells used to identify the most effective or most efficacious RNA targeting motif or RNA targeting motifs

7. Animals

[0418] Disclosed herein are animals treated with one or more disclosed nucleic acid molecules, one or more disclosed replacement constructs, one or more disclosed vectors, one or more disclosed AAV particles, one or more disclosed pharmaceutical formulations, and/or one or more disclosed plasmids. Transgenic animals are known to the art as are the techniques to generate transgenic animals.

8. Libraries

[0419] Disclosed herein is a library of one or more disclosed barcoded nucleic acid molecules for use in GRAFT. Disclosed herein is a library of one or more disclosed barcoded oligonucleotides for use in GRAFT. Disclosed herein is a library of one or more disclosed barcoded 5’ replacement constructs for use in GRAFT. Disclosed herein is a library of one or more disclosed barcoded 3’ replacement constructs for use in GRAFT. Disclosed herein is a library of one or more disclosed barcoded 5’ replacement constructs and/or disclosed barcoded 3’ replacement constructs for use in GRAFT. Disclosed herein is a library of one or more barcoded disclosed vectors. Disclosed herein is a library of one or more disclosed vectors comprising one or more disclosed barcoded 5’ replacement constructs, one or more disclosed barcoded 3’ constructs, or any combination thereof for use in GRAFT. Disclosed herein is a library of one or more disclosed AAV particles comprising one or more disclosed barcoded 5’ replacement constructs, one or more disclosed barcoded 3’ constructs, or any combination thereof for use in GRAFT. Disclosed herein is a library of one or more disclosed barcoded plasmids for use in GRAFT.

9. Kits

[0420] Disclosed herein is a kit comprising one or more disclosed barcoded nucleic acid molecules, disclosed barcoded vectors or disclosed barcoded AAV particles, disclosed barcoded pharmaceutical formulations, or any combination thereof. Disclosed herein is a kit comprising one or more disclosed barcoded nucleic acid molecules, one or more disclosed barcoded vectors, one or more disclosed barcoded pharmaceutical formulations, or any combination thereof. In an aspect, a kit can comprise a disclosed barcoded nucleic acid molecule, a disclosed barcoded vector or disclosed barcoded AAV particle, a disclosed barcoded pharmaceutical formulation, a disclosed barcoded therapeutic agent, or a combination thereof, and one or more agents. “Agents” and “Therapeutic Agents” are known to the art and are described supra.

[0421] In an aspect, the one or more agents can treat, prevent, inhibit, and/or ameliorate one or more comorbidities in a subject. In an aspect, one or more active agents can treat, inhibit, prevent, and/or ameliorate cellular and/or metabolic complications related to a missing, deficient, and/or mutant protein or enzyme.

[0422] In an aspect, a disclosed kit can comprise at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose (such as, for example, treating a subject diagnosed with or suspected of having a genetic disease or genetic disorder). Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. In an aspect, a kit for use in a disclosed method can comprise one or more containers holding a disclosed barcoded nucleic acid molecule, a disclosed vector, a disclosed barcoded pharmaceutical formulation, a disclosed RNA therapeutic, or a combination thereof, and a label or package insert with instructions for use. In an aspect, suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers can be formed from a variety of materials such as glass or plastic. The container can hold a disclosed barcoded nucleic acid molecule, a disclosed barcoded vector, a disclosed barcoded pharmaceutical formulation, or a combination thereof, and can have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert can indicate that a disclosed barcoded nucleic acid molecule, a barcoded disclosed vector, a disclosed barcoded AAV particle, a disclosed barcoded pharmaceutical formulation, a disclosed RNA therapeutic agent, or a combination thereof can be used for treating, preventing, inhibiting, and/or ameliorating a disease or disorder or complications and/or symptoms associated with a disease or disorder. A disclosed kit can comprise additional components necessary for administration such as, for example, other buffers, diluents, filters, needles, and syringes. In an aspect, a disclosed kit can be used in any disclosed method. In an aspect, a disclosed kit can be used to generate one or more chimeric RNA molecules. In an aspect, a disclosed kit can be used to treat a genetic disease or genetic disorder. In an aspect, a disclosed kit can be used to inhibit and/or minimize disease progression.

E. Methods of Generating a Chimeric RNA Molecule

[0423] Disclosed herein is a method of generating a chimeric RNA molecule in a cell, the method comprising contacting an endogenous pre-mRNA in a cell with a disclosed 5’ replacement construct, wherein the resulting chimeric RNA transcript comprises the 3’ portion of the targeted endogenous pre-mRNA and the 5’ portion of the exogenous RNA.

[0424] Disclosed herein is a method of generating a chimeric RNA molecule, the method comprising contacting one or more cells with a disclosed 5’ replacement construct, wherein the resulting chimeric RNA transcript comprise the 3’ portion of the targeted endogenous pre-mRNA and the 5’ portion of the exogenous RNA.

[0425] Disclosed herein is a method of generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the resulting chimeric RNA transcript comprise the 3’ portion of the targeted endogenous pre-mRNA and the 5’ portion of the exogenous RNA.

[0426] Disclosed herein is a method of generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95, wherein the resulting chimeric RNA transcript comprise the 3’ portion of the targeted endogenous pre- mRNA and the 5’ portion of the exogenous RNA.

[0427] Disclosed herein is a method of generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA transcript comprise the 3’ portion of the targeted endogenous pre-mRNA and the 5’ portion of the exogenous RNA.

[0428] Disclosed herein is a method of generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA transcript comprise the 3’ portion of the targeted endogenous pre-mRNA and the 5’ portion of the exogenous RNA comprising a series of single nucleotide polymorphisms (SNPs).

[0429] Disclosed herein is a method of generating a chimeric RNA molecule in a cell, the method comprising contacting an endogenous pre-mRNA in a cell with a disclosed 3’ replacement construct, wherein the resulting chimeric RNA transcript comprises the 5’ portion of the targeted endogenous pre-mRNA and the 3’ portion of the exogenous RNA comprising a series of single nucleotide polymorphisms (SNPs).

[0430] Disclosed herein is a method of generating a chimeric RNA molecule, the method comprising contacting one or more cells with a disclosed 3’ replacement construct, wherein the resulting chimeric RNA transcript can comprise the 5’ portion of the targeted endogenous pre- mRNA and the 3’ portion of the exogenous RNA comprising a series of single nucleotide polymorphisms (SNPs).

[0431] Disclosed herein is a method of generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre- mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), wherein the resulting chimeric RNA transcript can comprise the 5’ portion of the targeted endogenous pre-mRNA and the 3’ portion of the exogenous RNA comprising a series of single nucleotide polymorphisms (SNPs).

[0432] Disclosed herein is a method of generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), wherein the resulting chimeric RNA transcript can comprise the 5’ portion of the targeted endogenous pre-mRNA and the 3’ portion of the exogenous RNA comprising a series of single nucleotide polymorphisms (SNPs).

[0433] Disclosed herein is a method of generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre- mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA transcript can comprise the 5’ portion of the targeted endogenous pre-mRNA and the 3’ portion of the exogenous RNA comprising a series of single nucleotide polymorphisms (SNPs).

[0434] Disclosed herein is a method of generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:87 - SEQ ID NO:95; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA transcript can comprise the 5’ portion of the targeted endogenous pre-mRNA and the 3’ portion of the exogenous RNA comprising a series of single nucleotide polymorphisms (SNPs).

[0435] In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 20 SNPs. In an aspect, a disclosed series of SNPs can comprise 2 SNPs to 10 SNPs. In an aspect, a disclosed series of SNPs can comprise 5 SNPs. In an aspect, a disclosed method of generating a chimeric RNA molecule in cells can comprise validating the trans-splicing event and/or the generation of the chimeric RNA molecule. Validation of the trans-splicing event and/or generation of the chimeric RNA molecule can be accomplished using methods and techniques known to the art (e.g., sequencing, northern blots, FISH, PCR, RNA-Seq, 3’ RACE, 5’ RACE, etc.).

[0436] In an aspect, a disclosed method of generating a chimeric RNA molecule can comprise preparing a disclosed 5’ replacement construct, a disclosed 3’ replacement construct, a disclosed non-viral vector or disclosed viral vector, a disclosed nucleic acid molecule, a disclosed pharmaceutical formulation, or any combination thereof.

[0437] In an aspect, a disclosed method of generating a chimeric RNA molecule in cells can comprise identifying the most effective or most efficacious RNA targeting motif or RNA targeting motifs. In an aspect, the most effective or most efficacious RNA targeting motif or RNA targeting motifs can achieve the highest level of trans-splicing. In an aspect, a disclosed method of generating a chimeric RNA molecule in cells can comprise identifying the RNA targeting motif or RNA targeting motifs that are most effective at generating a chimeric molecule through trans- splicing. In an aspect, the one or more RNA targeting motifs identified as effective at generating a chimeric molecule through trans-splicing can then be prepared and packaged as part of a transcriptome engineering system. In an aspect, a disclosed transcriptome engineering system can then be packaged in a pharmaceutical formulation that can be administered to a subject in need thereof. In an aspect, a disclosed chimeric molecule is non-functional due to the series of SNPs. [0438] In an aspect, a disclosed method can be reported in a library of replacement constructs to identify the top performing RNA targeting motifis (i.e., those that have the highest trans-splicing efficacy).

[0439] In an aspect, once a disclosed method identifies the effective or most efficacious RNA targeting motif or RNA targeting motifs, that RNA targeting motif or RNA targeting motifs can be used to generate a chimeric RNA molecule in cells. In an aspect, the cells can be in a subject. In an aspect, the cells can be cells affected by a disease or disorder. In an aspect, the effective or most efficacious RNA targeting motif or RNA targeting motifs can be used in a disclosed method that can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, a disclosed method can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra). In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi-systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity.

VII. EXAMPLES

[0440] As there are pathogenic mutations in more than 500 genes exceeding the packaging capacity of AAV, several efforts have been aimed at circumventing this barrier to expression of large genes. While these techniques differ, the general approach remains broadly similar between strategies. Briefly, a dual AAV vector approach is taken; wherein the DNA sequence encoding the protein of interest is split and packaged into separate vectors. Upon co-infection of target cells by the two vectors, the genomes of the two vectors recombine with each other via inverted repeat sequences or overlapping complementary sequences forming a single genome bearing the reconstituted DNA sequence expressing the full protein of interest. While this strategy is feasible the efficiency of recombination between genomes has limited the viability of its widespread adoption.

[0441] Separately, other efforts to effect phenotypic correction of genes ineligible for classical AAV mediated gene therapy have inspired an approach involving the manipulation of endogenous messenger RNA, the conduit between DNA and protein. The RNA editing strategy known as spliceosome mediated RNA trans-splicing (SMART) has been developed as a strategy to introduce large precise modifications to the primary structure of RNA transcripts independent of target transcript length. The aim of this approach is to hijack the cellular RNA processing machinery for incorporation of a desired sequence into an endogenous transcript. In this strategy, a recombinant RNA molecule is introduced to the cell comprising 3 essential components: an RNA targeting motif, a hemi intron sequence, and the primary sequence of the desired RNA to be joined to an endogenous RNA transcript. The RNA targeting motif is comprised of a stretch of oligonucleotides anti-sense to an intron of an endogenous target pre-mRNA. Upon Watson-Crick base pairing of the RNA targeting motif with the endogenous intron, the hemi intron is then recognized by the spliceosome. Depending on the desired splicing reaction either a 5’ hemi intron facilitates the splicing of the trans-splicing molecule to the exon immediately 3’ to the targeted intron, or a 3’ hemi intron linked to the exogenous RNA to be trans-spliced facilitates the splicing of the trans-splicing molecule to the exon immediately 5’ to the targeted intron. This produces a mature chimeric RNA transcript comprised of the recombinant 5’ start of a transcript joined to an endogenous 3’ sequence or endogenous 5’ start of a transcript joined to a recombinant 3 ’ sequence, respectively. The utility of this strategy is that a single AAV vector needs only to package a genome capable of producing a trans-splicing RNA molecule containing the sequence for part of a gene, obviating the need to deliver the full-length protein coding sequence to a cell. As the entire recombinant RNA region of a chimeric RNA product may be specified by the user, the trans-splicing RNA may contain the wild type sequence of a target RNA, an inactivating mutation in the target RNA, or a modified RNA sequence encoding a novel protein. However, similar to the split- AAV vector approach, the low specificity and efficiency of RNA targeting by anti-sense RNA sequences has precluded the widespread use of this technology in research and clinical settings. Although promising, expressing a CRISPR protein indefinitely (e.g., long term expression) as a therapeutic presents challenges arising from immunogenicity and off-target effects. Moreover, CRISPR-based approaches often require two constructs to be delivered (namely the CRISPR effector protein and the RNA trans-splicing construct), thereby leading to high dose requirements and low efficiency of correction. Disclosed herein is a system for transcriptome engineering that does not need the CRISPR system. There are many endogenous RNA binding ribonucleoproteins in human cells, and the system disclosed herein exploits several RNA structures that interact with known human ribonucleoproteins for enabling trans-splicing. As detailed herein, by incorporating these RNA structures into trans-splicing RNA, effective trans-splicing of many targeted endogenous pre-mRNAs was achieved. This allowed for the rewriting of large stretches of mRNA.

EXAMPLE 1 - CRAFT Validation of 3’ Trans-Splicing in the DP71 Transcript

[0442] A PCR based validation of 3’ editing at the DMD locus in HEK293 cells was developed. FIG. 7 (top) depicts what a trans-spliced DP71 transcript would look like comprised of the endogenous 5’ exons of the transcript (black) the trans-spliced remaining exons (gray) and an additional mScarlett tag on the trans-spliced exons. RT-PCR across the splice junction was used as a primary endpoint of trans-splicing validation. Cells transfected with either the dCasRx (lane 1) or trans-splicing RNA (lane 2) expression plasmid did not yield detectable trans-splicing. Further, when the dCasRx expression plasmid is transfected with a trans-splicing RNA expression plasmid lacking a targeting sequence to the transcript of interest (lane 3), trans-splicing was not detected. But, when dCasRx expression plasmid was transfected with the trans-splicing expression plasmid containing an on-target guide sequence, trans-splicing was detected by RT- PCR (lane 4). This band was confirmed to be the trans-spliced RNA by sanger sequencing of the band to detect a single nucleotide polymorphism (A > G [E3580]) encoded uniquely in the trans- spliced RNA product (FIG. 8).

[0443] The efficiency of this editing strategy was measured by unbiased amplicon sequencing across the exon 74/75 splice junction. The percent of reads containing the encoded silent mutation correspond to the percent of transcripts that are trans-spliced (FIG. 9). Based on this editing strategy, the percent of trans-spliced reads was 41.33% when a dCasRx expression plasmid was transfected with the trans-splicing expression plasmid containing an on-target guide sequence (n = 3) and was significantly greater than any other condition tested (p < 0.0002).

EXAMPLE 2 - CRAFT CRISPR RNP Machine Increased Trans-Splicing Efficiency

[0444] To demonstrate the benefit of guiding the trans-splicing machinery with a CRISPR RNP, the trans-splicing efficiencies of the technology disclosed was compared to a previous trans- splicing technology (i.e., SMART (Spliceosome Mediated RNA Trans-Splicing)). RNA editing efficiency was evaluated in the same manner as described in Example 1 (supra) and compared at three guide sequences along intron 74 or DP71. The 3 guide sequences were A, B, and C (FIG. 10). Across all three guide sequences an increase in trans-splicing efficiency is noted in with the present technology of a 5.2, 2.3 and 7.7-fold at guides A, B, and C respectively (n = 3).

EXAMPLE 3 - CRAFT Validation of 3’ Trans-Splicing in the DMPK Transcript

[0445] A PCR based validation of 3 ’ editing at the DMPK locus in HEK293 cells was developed. FIG. 11A depicts what a trans-spliced DMPK transcript would look like comprised of the endogenous 5’ exons of the transcript (black) the trans-spliced remaining exons (gray) and an additional mScarlett tag on the trans-spliced exons. RT-PCR across the splice junction was used as a primary endpoint of trans-splicing validation. Cells transfected with either the dCasRx (lane 1) or trans-splicing RNA (lane 2) expression plasmid did not yield detectable trans-splicing. Further, when the dCasRx expression plasmid was transfected with a trans-splicing RNA expression plasmid lacking a targeting sequence to the transcript of interest (lane 3), trans-splicing was not detected. FIG. 11B shows that only when dCasRx expression plasmid was transfected with the trans-splicing expression plasmid containing an on-target guide sequence, was trans- splicing detected by RT-PCR (lane 4). This band was confirmed to be the trans-spliced RNA by Sanger sequencing of the band to detect a silent G > T encoded uniquely in the trans-spliced RNA product (FIG. 12).

EXAMPLE 4 - CRAFT RT-PCR Confirmed dCasRX Expression Generated Trans-Splicing

[0446] FIG. 13 depicts a PCR based validation of 3 ’ editing at the LMNA locus in HEK293 cells. FIG. 13 (top) depicts what a trans-spliced LMNA transcript would look like comprised of the endogenous 5’ exons of the transcript (black) the trans-spliced remaining exons (gray) and an additional mScarlett tag on the trans-spliced exons. RT-PCR across the splice junction was used as a primary endpoint of trans-splicing validation. Cells transfected with only the trans-splicing RNA (lane 1) expression plasmid did not yield detectable trans-splicing. Further, when the dCasRx expression plasmid was transfected with a trans-splicing RNA expression plasmid lacking a targeting sequence to the transcript of interest (lane 2), trans-splicing was not detected. Only when dCasRx expression plasmid was transfected with the trans-splicing expression plasmid containing an on-target guide sequence, was trans-splicing detected by RT-PCR (lane 3). This band is confirmed to be the trans-spliced RNA by Sanger sequencing of the band to detect a silent G > A encoded uniquely in the trans-spliced RNA product (FIG. 14).

EXAMPLE 5 - CRAFT Editing Strategy Generated Full Replacement of 3’ Exons

[0447] The final two exons of the human LMNA transcript were replaced with codon optimized sequenced using the proposed RNA editing machinery (FIG. 15).

EXAMPLE 6 - CRAFT Validation of 5’ Trans-Splicing in the LMNA Transcript

[0448] FIG. 16A - FIG. 16B depict a PCR based validation of 5’ editing at the LMNA locus in HEK293 cells. FIG. 16A depicts what a trans-spliced LMNA transcript would look like comprised of mScarlett tag (white) linked to the trans-spliced exons (gray) followed by the endogenous 3’ exons of the transcript (black). RT-PCR across the splice junction was used as a primary endpoint of trans-splicing validation. In FIG. 16B, cells transfected with either the dCasl3b (lane 1) or trans-splicing RNA (lane 2) expression plasmid did not yield detectable trans- splicing. Further, when the dCasl3b expression plasmid was transfected with a trans-splicing RNA expression plasmid lacking a targeting sequence to the transcript of interest (lane 3), trans- splicing was not detected. Only when dCasl3b expression plasmid was transfected with the trans- splicing expression plasmid containing an on-target guide sequence, was trans-splicing detected by RT-PCR (lane 4). This band was confirmed to be the trans-spliced RNA by Sanger sequencing of the band to detect a silent G > C encoded uniquely in the trans-spliced RNA product (FIG. 17).

EXAMPLE 7 - CRAFT

Quantification of 3’ Trans-Splicing Efficiency at the DMPK Locus [0449] RNA editing efficiency of the proposed system at the DMPK locus in accordance with one embodiment of the present disclosure. A trans-splicing strategy was designed to replace exon 14 of the DMPK transcript such a recombinant exon 14 is joined to the endogenous exons 1-13. The efficiency of this editing strategy was measured by unbiased amplicon sequencing across the exon 13/14 splice junction. The percent of reads containing the encoded silent T > A (P593) mutation correspond to the percent of transcripts that are trans-spliced (FIG. 18A - FIG. 18B). Based on this editing strategy the percent of trans-spliced reads is 24.65% when a dCasRx expression plasmid was transfected with the trans-splicing expression plasmid containing an on-target guide sequence (n = 3) and was significantly greater than any other condition tested (p < 0.0001).

EXAMPLE 8 - CRAFT Quantification of 3’ Trans-Splicing Efficiency at the LMNA Locus

[0450] RNA editing efficiency at the LMNA locus using the methodology disclosed herein was explored. A trans-splicing strategy was designed to replace exons 11-12 of the LMNA transcript such recombinant exons 11-12 are joined to the endogenous exons 1-10. The efficiency of this editing strategy was measured by unbiased amplicon sequencing across the exon 10/11 splice junction. The percent of reads containing the encoded silent T > C (A577) mutation correspond to the percent of transcripts that were trans-spliced (FIG. 19A - FIG. 19B). Based on this editing strategy, the percent of trans-spliced reads was 23.07% when a dCasRx expression plasmid was transfected with the trans-splicing expression plasmid containing an on-target guide sequence (n = 3) and was significantly greater than any other condition tested (p < 0.0013).

SUMMARY OF CRAFT EXAMPLES

[0451] As demonstrated by the Examples, the compositions and methods disclosed herein are superior to previously disclosed compositions and methods. For at least three reasons, the data provided herein show that CRISPR Assisted Fragment Trans-Splicing (CRAFT) provides surprisingly exceptional results when compared to known technologies.

[0452] First, the nuclear localization signal on Cast 3 promoted retention of the RNA editing machinery in the nucleus, where the target endogenous pre-mRNA existed. This represents an engineered improvement over other technologies such as Spliceosome Mediated RNA TransSplicing (SMART), which lacks a NLS, and therefore has a lower concentration of trans-splicing RNA within the nucleus where splicing occurs.

[0453] Second, the Cas enzyme stabilized the interaction of the guide RNA with the target endogenous pre-RNA molecule, both through optimal presentation of the guide sequence and a conformation change in the enzyme upon target recognition to stabilized RNA binding. The enhanced stability of this interaction promoted association of the trans-splicing RNA and target endogenous pre-mRNA and enhanced the efficiency of the tool due to the proximity of the splicing signals. Third, the CAS enzyme also inhibited cis splicing upon binding to a target endogenous RNA molecule. As the editing strategy is predicated on tipping the balance of splicing from cis to trans by reducing cis splicing, trans splicing rates can increase using this methodology.

EXAMPLE 9 - GRAFT Validation of Trans-Splicing Efficiency

[0454] To assay trans-splicing efficiency, a green fluorescence-based screening system was designed and built. The first step was to construct a reporter in which the green fluorescent protein (EGFP) open reading frame was split into two halves. An intron was inserted between the two halves. Accordingly, if this construct spliced in cis, then it would make a mature RNA that encoded EGFP and, upon translation, would express the green fluorophore.

[0455] Next, we abolished fluorescent expression by introducing a premature stop codon into the first “exon” of the EGFP open reading frame (FIG. 20 (left side)). If this construct spliced in cis, then there would be no green fluorescence expression because the premature stop codon in the first half of the EGFP open reading frame stopped translation of the full-length protein.

[0456] By delivering a trans-splicing RNA that encoded the correct open reading frame for the first half of EGFP followed by a hemi-intron and guide RNA sequence, green fluorescence expression could be restored. Thus, green fluorescence was used as an indirect readout of trans- splicing efficiency (FIG. 20). This reporter system was the workhorse of our downstream assays. [0457] The first trans-splicing RNA delivered to these cells (hereinafter - SMaRT or Spliceosome mediated RNA trans-splicing) had only the first half of EGFP, a hemi-intron and 30 bp antisense targeting domain. Sequences were then cloned into a trans-splicing RNA vector to ascertain whether trans-splicing efficiency could be enhanced. These new constructs were transfected into HEK293 cells with the split GFP reporter and 48 hours later green fluorescence intensity (percent GFP% and mean fluorescence intensity) was assayed by flow cytometry as a proxy for trans- splicing efficiency (FIG. 21A and FIG. 21B). Through this process, three (3) RNA structures that yielded significantly enhanced trans-splicing efficiency (RNA Structure 4, 5 and 7). In FIG. 21A - FIG. 21B, the RNA targeting motif for RYR2 was set forth in SEQ ID NO:98.

[0458] Whether any member of this panel of RNA sequences enhanced the 3’ trans-splicing approach was next examined. To do this, the same split GFP reporter was used, but for a slight modification in that the stop codon was moved from the first half of the open reading frame to the second half (FIG. 20 (right side)). This construct, on its own, does not express green fluorescence when delivered to HEK293 cells because cis splicing retains the premature stop codon. To restore EGFP expression, a trans-splicing RNA that completed the open reading frame of EGFP upon proper splicing was used. Like the 5’ editing approach, a trans-splicing RNA was used and contained a 30 bp anti-sense targeting motif, followed by a hemi-intron, and the second half of the EGFP open reading frame (this construct was SMaRT). The same panel of RNA structures was then introduced into this RNA molecule with the intent of improving trans-splicing efficiency. [0459] The new constructs were co-transfected into HEK293 cells with the split GFP reporter. Then, 48 hrs. later, green fluorescence intensity (percent GFP% and mean fluorescence intensity) was assayed by flow cytometry (as a proxy for trans-splicing efficiency). (FIG. 22A - FIG. 22B). Through this process, 4 RNA structures that yielded significantly enhanced trans-splicing efficiency (RNA Structure 1, 2, 10, and 11) were identified. This panel was repeated on a second intron target intron (FIG. 22C - FIG. 22D) and a similar trend in fold change of editing efficiency was observed. In FIG. 22A - FIG. 22B, the RNA targeting motif for RYR2 is in SEQ ID NO:98 while the RNA targeting motif for LMNA in FIG. 22C - FIG. 22D was is in SEQ ID NO:32.

[0460] Following these results, the trans-splicing assay for the top hits (RNA Structures 1 and 2) were repeated. A second control RNA structure (i.e., the direct repeat from Ruminococcus flavefaciens XPD3002 (RfxCasl3d)) was also used. The rationale for this control was to ensure that the boost in RNA editing was unique to these RNA structures and not an artifact of including additional sequence length/complexity in the trans-splicing RNA. Indeed, only RNA structures 1 and 2 appeared to boost editing efficiency, while the Cast 3 direct repeat had a slightly detrimental effect of trans-splicing efficiency (FIG. 23A - FIG. 23B).

[0461] Finally, a new target intron was cloned into the split GFP reporter and assayed for editing efficiency with RNA structure 2 and several guide RNA candidates. At this new target, editing was observed in excess of 50% GFP positive cells (FIG. 24 A - FIG. 24B). In FIG. 24 A - FIG. 24B, the RNA targeting motif for FXN was set forth in SEQ ID NO:99. In Table 2, Structures 4 and 5 contain a cloning site within them for where guide RNA is inserted this is the bold sequence.

Table 2 - List of RNA Structures

Table 3 - List of Plasmids Used in Examples

EXAMPLE 10 - GRAFT Trans-Splicing of a Mutations in DMD

[0462] DMD (dystrophin) is known to the art (e.g., Gene ID 1756) and this nucleotide sequence can comprise nucleotides 5001 - 2225382 in Accession No. NG012232.1. DMD spans a genomic range of greater than 2 Mb and encodes a large protein containing an N-terminal actin-binding domain and multiple spectrin repeats. The encoded protein (SEQ ID NO: 52) forms a component of the dystrophin-glycoprotein complex (DGC), which bridges the inner cytoskeleton and the extracellular matrix. Deletions, duplications, and point mutations at this gene locus may cause Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or cardiomyopathy. Currently, about 1750 pathogenic mutations of DMD have been reported. Detailed below are constructs to address over 1000 of those pathogenic mutations. Here, 5’ replacement constructs for DMD are generated using a plasmid based on the schematic presented in FIG. 26A. The 5’ replacement construct is directed at Exons 1-23 of DMD. (SEQ ID NO: 108). The 5’ replacement construct redresses one or more of the 618 mutations identified below in Table 4 of International Publication No. WO 2024/206891, which is incorporated by reference in its entirety, and provides a list of mutations in Exons 1-23 of DMD.

[0463] An AAV vector based on the plasmid construct show in in FIG. 7A is delivered to a subject having one or more 5’ mutations in DMD (e.g., Table 4 in WO 2024/206891)). Following administration of a therapeutically effective amount of the AAV vector (e.g., about 1 x 10¹⁰ vg/kg to about 2 x 10¹⁴. vg/kg), one or more subject’s cells, tissues, and/or organs generate a chimeric RNA molecule encoding a corrected and/or restored DMD. Confirmation of the generation of the chimeric RNA molecule is performed by obtaining a sample from the subject and confirming the expression level of the corrected and/or restored DMD (by comparing the post-treatment level of operative/functional DMD to the subject’s pre-treatm ent level of operative/functional DMD). The subject experiences an inhibition and/or minimization of DMD disease progression. The subject’s quality of life improves. Here, 3’ replacement constructs for DMD are generated using a plasmid based on the schematic presented in FIG. 26B. The 3’ replacement construct is directed at Exons 53-79 of DMD. (SEQ ID NO: 107). The 3’ replacement construct redresses one or more of the 446 mutations identified below in Table 5 of International Publication No. WO 2024/206891, which is incorporated by reference in its entirety, and provides a list of mutations in Exons 53-79 of DMD.

[0464] An AAV vector based on the plasmid construct show in in FIG. 26B is delivered to a subject having one or more 3’ mutations (e.g., Table 5 in WO 2024/206891). Following administration of a therapeutically effective amount of the AAV vector (e.g., about 1 x 10¹⁰ vg/kg to about 2 x 10¹⁴. vg/kg), one or more subject’s cells, tissues, and/or organs generate a chimeric RNA molecule encoding a corrected and/or restored DMD. Confirmation of the generation of the chimeric RNA molecule is performed by obtaining a sample from the subject and confirming the expression level of the corrected and/or restored DMD (by comparing the post-treatment level of operative/functional DMD to the subject’s pre-treatm ent level of operative/functional DMD). The subject experiences an inhibition and/or minimization of DMD disease progression. The subject’s quality of life improves.

SUMMARY OF GRAFT EXAMPLES

[0465] The compositions and methods disclosed herein are superior to previously disclosed compositions and methods such as SMART. The advantages conferred by the disclosed system are numerous. These surprising and unexpected advantages include (i) the lack of need for an additional effector protein (e.g., CRISPR-based to enable trans-splicing); (ii) the ability to modify the effector RNA structure to bind endogenous splicing machinery and enable either 3’ or 5’ end replacement of pre-spliced mRNA; (iii) the ability to deliver the construct in a single AAV vector or as a trans-splicing RNA fragment using a non-viral delivery system (e.g., LNP); (iv) the ability to edit large stretches of mRNA constituting multiple exons; (v) the ability to correct mRNA in a single “knockdown and replace” approach, of particular utility in autosomal dominant inheritance disorders using a single trans-splicing construct without having to provide two distinct constructs for knockdown and replacement; and (vi) the ability to prevent over-expression of any gene, correction is based on the trans-splicing fragment provided, which is dependent on endogenous mutant transcript levels (hence the corrected mRNA will never be expressed at levels higher than the endogenous disease transcript). While comprehensive, this list of advantages is not exhaustive.

[0466] Overall, described herein is a platform for efficient RNA editing that does not rely on CRISPR-based targeting and outperforms simple antisense base pairing. This enables efficient rewrite of large stretches of RNA, which has implications for human health and basic biology. As a therapeutic, this would allow for rewriting of genes that exceed the packaging capacity of AAV, correct dominant negative mutations, all while maintaining expression of the target transcript at endogenous levels.

EXAMPLE 11 - BARCODED CRAFT

[0467] As demonstrated herein, the exons of a trans-splicing RNA can be modified to contain single nucleotide polymorphisms (SNPs) that can differentiate edited transcripts from non-edited transcripts. As a step towards expanding the adaptability of CRAFT as an RNA editing tool, this observation was exploited to design a high-throughput screen comprised of a library of guide sequences tiling the intron 10/11 of Imna (Gene ID 16905). Specifically, a pool of oligos that contained a spacer (directed repeats) sequence, a hemi-intron, and the beginning of exon 11 of Imna. Each oligo in the pool contained a unique spacer (directed repeat that was operably linked to a “barcode” of SNPs in exon 11 of Imna. This oligo pool was then cloned into the rcRNA expression plasmid between the direct repeat and remaining exon 11 sequence. The overall approach, CRISPR Assisted RNA Fragment Trans-splicing (CRAFT) involves co-expression of a recombinant trans-splicing CRAFT RNA fragment (rcRNA) and a modified, cognate type VI CRISPR nuclease.

[0468] Guide RNA spacers were cloned into rcRNA vectors by digesting plasmids with Esp3i restriction enzyme (New England Biolabs) for 1 hour at 37 °C according to manufacturer’s directions. This plasmid pool was transfected along with Rfx-dCasl3d to HEK293 cells. Three days later, the same targeted amplicon sequencing was performed on the RNA from these cells as was done for measuring the efficiency of CRAFT at endogenous targets (FIG. 27A). Thus, by knowing the barcode of a guide and sequencing across the exon 10/11 splice junction in the mature transcript, the relative efficiency of each guide was determined by calculating the abundance of its associated barcode in the mRNA and comparing that to its abundance in the plasmid pool delivered to the cells. (FIG. 27B) The trend of relative efficiencies corroborated the low- throughput screen using the splitGFP reporter (FIG. 27D). This library technique was then applied to rewrite the last exon of dystrophia myotonica protein kinase (dmpk) (Gene ID 13400) in HEK 293 cells using CRAFT.

[0469] An optimal guide that achieved efficient trans-splicing exceeding 24% was identified. Interestingly, without the expression of Cas, significant RNA trans-splicing in the dmpk RNA was not detected, which corroborated the markedly improved trans-splicing efficiency enabled by an effector protein.

[0470] FIG. 27A - FIG. 27D show high-throughput guide selection through guide coupled barcode, validation, and comparison to existing strategy. FIG. 27A shows a schematic of the disclosed barcode approach. Briefly, a library of unique barcodes corresponding a specific spacer sequence was delivered to HEK293 cells with Rfx-dCasl3d. Functional RNPs target to intron 10/11 of Imna and undergo trans-splicing. RNA is harvested and the abundance of each barcode at the start of exon 11 was measured by targeted amplicon sequencing. This barcode corresponded to the guide associated with its trans-splicing. FIG. 27B shows an enrichment plot for each guide targeting Imna intron 10/11 as a function of position. Y-axis is barcode enrichment calculated by the (%barcode in trans-spliced RNA / %barcode in input plasmid DNA), and x-axis is the position along Imna intron 10/11 of each guide. Each unique barcode (3) for a single guide is plotted as a triangle and the average of these enrichment scores is plotted as a black circle. The best guide is plotted in purple. FIG. 27C shows the frequency of 3 ’-CRAFT editing in endogenous Imna transcripts. The y-axis is the percent of reads from targeted amplicon sequencing containing the C > G mutation. The x-axis refers to specific treatment: dCasl3 alone, rcRNA alone, dCasl3 with an rcRNA that does not target the Imna intron, and dCasl3 with an rcRNA that targets the Imna intron (n = 3 individual samples, **** is p < 0.0001, one-way Anova).

Table 4 - Endogenous 3’ Imna Editing Percentages

[0471] FIG. 27D shows the optimization of 3’ rcRNA guide position using the former SplitGFP reporter system. Schematic guide tiling for 5’CRAFT rcRNAs along the target intron (top). Target position and color of guide target sequence correlates with the quantification by flow cytometry presented as percent GFP positive cells and MFI. (n = 3 individual samples). Data is presented in the table below.

Table 5 - 3’ Guide Position Imna GFP

EXAMPLE 12 - BARCODED GRAFT

[0472] FIG. 28A - FIG. 28C show the reproducibility of barcode strategy using DMPK. FIG. 28A is an enrichment plot for each guide targeting dmpk intron 13/14 as a function of position. FIG. 28B is a schematic of endogenous dmpk transcript (left) and edited dmpk transcript (right). Notably, there is a single G>T transition mutation installed between the edited transcript and the endogenous transcript. FIG. 28C shows the frequency of 3 ’ -CRAFT editing in endogenous dmpk transcripts. The y-axis is the percent of reads from targeted amplicon sequencing containing the snp mutation. The x-axis refers to specific treatment: dCasl3 alone, rcRNA alone, dCasl3 with an rcRNA that does not target the dmpk intron, and dCasl3 with an rcRNA that targets the given intron (n =3 individual samples, **** means p < 0.0001, one-way Anova).

[0473] This method of optimizing the guide RNA sequence of trans-splicing molecules is used for other RNA trans-splicing technologies that utilize on anti-sense RNA binding to engage their target RNA. For example, this technology is used with GRAFT, in which an RNA molecule comprised of a non-coding RNA structure followed by an anti-sense “guide” sequence, a hemiintron, and the cargo exon or exons that will splice into the target RNA (described above). This barcode screening approach is used to optimize the anti-sense “guide” sequence for GRAFT. For example, to identify an optimal anti-sense “guide” sequence for Imna intron 10/11, the following strategy is used.

[0474] Briefly, a pool of oligos would be generated that contained an anti-sense “guide” sequence, hemi-intron, and beginning of exon 11. Each oligo in the pool contains an anti-sense “guide” that is operably linked to a “barcode” of SNPs in exon 11. This oligo pool is then cloned into a GRAFT RNA expression plasmid between the RNA structure and remaining exon 11 sequence. This plasmid pool is then transfected into an appropriate eukaryotic cell that expresses Imna (eg. HEK293). Three days later, RNA from these cells is harvested, converted to cDNA, and targeted PCR amplification of Imna with primers that span of the exon 10/11 junction and the “barcode” region. This amplicon is then sequenced with any next generation sequencing machine (e.g., Illumina, PacBio, Nanopore). As the barcode and anti-sense sequence are unique and operably linked, the abundance of each barcode in the sequencing data is proportional to the relative efficiency of each unique anti-sense “guide” sequence. Thus, the most abundant “barcode” in the sequencing data would be the optimal anti-sense “guide” from that pool trans-splicing GRAFT RNAs. This approach is extended to optimize other aspects of the trans-splicing RNA such as the hemi-intron composition or RNA structure. So long as the oligo pool has at least one unique barcode for each unique structure or hemi-intron. Thus, in these cases the anti-sense sequence is constant and the most abundant barcode from targeted amplicon sequencing would reveal the hemi-intron or RNA structure that facilitated the most efficient trans-splicing.

EXAMPLE 13 - BARCODED SMART

[0475] This method of optimizing the guide RNA sequence of trans-splicing molecules can be used for other RNA trans-splicing technologies that utilize on anti-sense RNA binding to engage their target RNA. Spliceosome mediated RNA Trans-Splicing (SMaRT - see US Publication No. 2002/0193580) is another such previously describe an RNA targeting technology. This technology contains an RNA molecule comprised of an anti-sense “guide” sequence, a hemi- intron, and the cargo exon or exons that will splice into the target RNA. This barcode screening approach can also optimize the anti-sense “guide” sequence for SMaRT. For example, to identify an optimal anti-sense “guide” sequence for Imna intron 10/11, one would employ the following strategy.

[0476] Briefly, a pool of oligos is generated to contain an anti-sense “guide” sequence, hemi- intron, and beginning of exon 11. Each oligo in the pool contains an anti-sense “guide” that is operably linked to a “barcode” of SNPs in exon 11. This oligo pool is then cloned into a SMaRT RNA expression plasmid between the promoter and remaining exon 11 sequence. This plasmid pool is then transfected into an appropriate eukaryotic cell that expresses Imna (e.g., HEK293). Three days later, RNA from these cells is harvested, converted to cDNA, and targeted PCR amplification of Imna with primers that span of the exon 10/11 junction and the “barcode” region. This amplicon is then sequenced with any next generation sequencing machine (e.g., Illumina, PacBio, Nanopore). As the barcode and anti-sense sequence are unique and operably linked, the abundance of each barcode in the sequencing data is proportional to the relative efficiency of each unique anti-sense “guide” sequence. Thus, the most abundant “barcode” in the sequencing data is the optimal anti-sense “guide” from that pool trans-splicing SMaRT RNAs.

Table 6 - Listing of Sequences

Claims

VIII. CLAIMS What is claimed is:

1. A nucleic acid molecule, comprising: a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 3’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein.

2. The isolated nucleic acid molecule of Claim 1, wherein the series of SNPs are in the 5’ end of the nucleic acid sequence to be trans-spliced.

3. The isolated nucleic acid molecule of Claim 1, further comprising one or more stem loops.

4. The isolated nucleic acid molecule of Claim 1, wherein the RNA binding protein has a bispecific affinity for the target endogenous pre-mRNA and a catalytically inactive Casl3.

5. The isolated nucleic acid molecule of Claim 1, wherein the one or more guide RNA sequences are directed to the intron immediately 3’ to the last exon of the target endogenous pre- mRNA.

6. The isolated nucleic acid molecule of Claim 1, wherein the 3’ hemi intron is recognized by nuclear splicing components within a host cell.

7. A nucleic acid molecule, comprising: a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); a 5’ hemi intron linked to the nucleic acid sequence to be trans-spliced; one or more guide RNA sequences; a promoter operably linked to the one or more guide RNA sequences; and a nucleic acid sequence encoding an RNA binding protein.

8. The isolated nucleic acid molecule of Claim 7, wherein the series of SNPs are in the 3’ end of the nucleic acid sequence to be trans-spliced.

9. The isolated nucleic acid molecule of Claim 7, further comprising one or more stem loops.

10. The isolated nucleic acid molecule of Claim 7, wherein the RNA binding protein has a bispecific affinity for the target endogenous pre-mRNA and a catalytically inactive Casl3.

11. The isolated nucleic acid molecule of Claim 7, wherein the one or more guide RNA sequences are directed to the intron immediately 5’ to the first exon of the target endogenous pre- mRNA.

12. The isolated nucleic acid molecule of Claim 7, wherein the 5’ hemi intron is recognized by nuclear splicing components within a host cell.

13. A nucleic acid molecule, comprising: a nucleic acid sequence to be trans-spliced to a target endogenous pre-mRNA, wherein the nucleic acid to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs), a 3 ’ hemi intron; a 5’ hemi intron; two or more guide RNA sequences; a promoter operably linked to the two or more guide RNA sequences; and a nucleic acid sequence encoding one or more RNA binding proteins.

14. The isolated nucleic acid molecule of Claim 13, wherein the series of SNPs are in the 5’ end of the nucleic acid sequence to be trans-spliced.

15. The isolated nucleic acid molecule of Claim 13, wherein the series of SNPs are in the 3’ end of the nucleic acid sequence to be trans-spliced.

16. The isolated nucleic acid molecule of Claim 13, further comprising two or more stem loops.

17. The isolated nucleic acid molecule of Claim 13, wherein the two or more RNA binding proteins have a bispecific affinity for the target endogenous pre-mRNA and a catalytically inactive Cast 3.

18. The isolated nucleic acid molecule of Claim 13, wherein the two or more guide RNA sequences are directed to the intron immediately 3’ to the target exon of the target endogenous pre-mRNA and to the intron immediately 5’ to the target exon of the target endogenous pre-mRNA.

19. The isolated nucleic acid molecule of Claim 13, wherein the 3’ hemi intron and the 5’ hemi intron are recognized by nuclear splicing components within a host cell.

20. A transcriptome engineering system, comprising:

(i) the isolated nucleic acid molecule of any one of Claims 1 - 19; and

(ii-a) a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive PspdCasl3b, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive PspdCasl3b; and a polyadenylation signal; or (ii-b) a nucleic acid molecule, comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal.

21. The transcriptome engineering system of Claim 20, wherein the isolated nucleic acid molecules form a ternary complex with the target endogenous pre-mRNA molecule, and wherein the resulting chimeric RNA molecule comprises the trans-spliced nucleic acid sequence.

22. A method of generating a chimeric RNA molecule in a cell, the method comprising: contacting a target endogenous pre-mRNA in a cell with the isolated nucleic acid molecule of any one of Claims 1 - 19; and contacting the target endogenous pre-mRNA in the cell with (i) a nucleic acid molecule comprising a nucleic acid sequence encoding a catalytically inactive PspdCasl3b, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive PspdCasl3b; and a polyadenylation signal; or (ii) a nucleic acid molecule comprising a nucleic acid sequence encoding a catalytically inactive RfxCasl3d, a promoter operably linked to the nucleic acid sequence encoding the catalytically inactive RfxCasl3d; and a polyadenylation signal. wherein the isolated nucleic acid molecules form a ternary complex with the target endogenous pre-mRNA molecule, and wherein the resulting chimeric RNA molecule comprises the trans-spliced nucleic acid sequence.

23. The method of Claim 22, further comprising characterizing the trans-splicing efficacy of one or more guide RNA sequences.

24. A nucleic acid molecule, comprising: one or more RNA structures comprising the sequence of any one of SEQ ID NO: 87 - SEQ ID NO:95; one or more RNA targeting motifs; a 3 ’ hemi intron; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs).

25. A nucleic acid molecule, comprising: a 5’ hemi intron; an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the exogenous RNA to be trans-spliced comprises a series of single nucleotide polymorphisms (SNPs); one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO: 87 - SEQ ID NO:95.

26. The nucleic acid molecule of Claim 24, wherein the series of SNPs are in the 5’ end of the exogenous RNA to be trans-spliced.

27. The nucleic acid molecule of Claim 25, wherein the series of SNPs are in the 3’ end of the exogenous RNA to be trans-spliced.

28. The nucleic acid molecule of Claim 24 or Claim 25, wherein the targeted endogenous pre- mRNA comprises one or more mutations in one or more exons.

29. The nucleic acid molecule of Claim 26, wherein the one or more mutations are in the 3’ portion of the targeted endogenous pre-mRNA.

30. The nucleic acid molecule of Claim 26, wherein the one or more mutations are in the 5’ portion of the targeted endogenous pre-mRNA.

31. The nucleic acid molecule of Claim 24, wherein the targeted endogenous pre-mRNA encodes a protein coding gene.

32. The nucleic acid molecule of any one of Claims 26 - 31, wherein the one or more mutations in one or more exons contribute to pathogenesis in one or more cells.

33. The nucleic acid molecule of Claim 24, wherein a 5’ portion of the targeted endogenous pre- mRNA is trans-spliced with the exogenous RNA.

34. The nucleic acid molecule of Claim 25, wherein a 3’ portion of the targeted endogenous pre- mRNA is trans-spliced with the exogenous RNA.

35. The nucleic acid molecule of Claim 24 or Claim 25, wherein the RNA targeting motif binds to the targeted endogenous pre-mRNA.

36. The nucleic acid molecule of Claim 35, wherein the RNA targeting motif comprises an antisense oligonucleotide.

37. The nucleic acid molecule of Claim 24, wherein the RNA targeting motif binds to the 3’ end of the targeted endogenous pre-mRNA,

38. The nucleic acid molecule of Claim 25, wherein the RNA targeting motifs binds to the 5’ end of the targeted endogenous pre-mRNA.

39. The nucleic acid molecule of Claim 26, wherein the RNA targeting motif is specific for an endogenous pre-mRNA having one or more mutations.

40. The nucleic acid molecule of Claim 24, wherein the RNA targeting motif is directed to the intron immediately 3’ to the exon of the targeted endogenous pre-mRNA with which it is to be spliced.

41. The nucleic acid molecule of Claim 25, wherein the RNA targeting motif is directed to the intron immediately 5’ to the exon of the targeted endogenous pre-mRNA with which it is to be spliced.

42. The nucleic acid molecule of Claim 24, wherein the 3’ hemi intron is linked to the exogenous

RNA to be trans-spliced with the endogenous mRNA.

43. The nucleic acid molecule of Claim 24, wherein the 3’ hemi intron is recognized by nuclear splicing components in a host cell.

44. The nucleic acid molecule of Claim 24, wherein the 3’ hemi intron comprises (i) a 3’ splice region comprising a branch point, (ii) a polypyrimidine tract, and (iii) a 3’ splice acceptor site.

45. The nucleic acid molecule of Claim 25, wherein the 5’ hemi intron is linked to the exogenous

RNA to be trans-spliced with the endogenous mRNA.

46. The nucleic acid molecule of Claim 25, wherein the 5’ hemi intron is recognized by nuclear splicing components in a host cell.

47. The nucleic acid molecule of Claim 25, wherein the 5’ hemi intron comprises a 5’ splice site.

48. The nucleic acid molecule of any preceding claim, wherein the one or more RNA structures bind to one or more RNA binding proteins.

49. The nucleic acid molecule of any preceding claim, wherein the one or more RNA structures

(i) improve and/or enhance trans-splicing efficiency, (ii) stabilize the resulting chimeric RNA transcript, (iii) localize the RNA to the nucleus, (iv) stabilize the interaction between the targeted endogenous pre-mRNA molecule and the exogenous RNA to be trans-spliced, or (v) any combination thereof.

50. A method of generating a chimeric RNA molecule, the method comprising: contacting a targeted endogenous pre-mRNA in one or more cells with the nucleic acid molecule of any one of Claim 24 - 49, wherein the resulting chimeric RNA molecule comprises a trans-spliced nucleic acid sequence; and characterizing the trans-splicing efficacy of one or more RNA targeting motifs.