WO2025085691A1

WO2025085691A1 - Trans-splicing nucleic acid molecules and methods of use

Info

Publication number: WO2025085691A1
Application number: PCT/US2024/051860
Authority: WO
Inventors: David Allen NELLES
Original assignee: Tacit Therapeutics Inc
Current assignee: Tacit Therapeutics Inc
Priority date: 2023-10-18
Filing date: 2024-10-17
Publication date: 2025-04-24
Anticipated expiration: 2026-04-18

Abstract

The present disclosure provides compositions, systems, and methods for promoting trans-splicing. In some examples, provided herein is a composition comprising or encoding a trans-splicing nucleic acid molecule comprising a 3' domain disclosed herein. In some embodiments, the 3' domain comprises one or more of the following domains: a nuclear retention domain, a stabilization domain, and a cleavage domain. In some embodiments, the 3' domain comprises a sequence or structure that functions as any one, any two, or all three of a nuclear retention domain, a stabilization domain, and a cleavage domain. In some embodiments, the 3' domain comprises: (i) a sequence or structure derived or isolated from a ribozyme (e.g., Twister or Twister Sister), and (ii) optionally a structured sequence or domain (e.g., a G-quadruplex, a pseudoknot, and/or a triplex), in the 3' to 5' direction.

Description

TRANS-SPLICING NUCLEIC ACID MOLECULES AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/567,850, filed March 20, 2024, entitled “NUCLEIC ACID MOLECULES TO PROMOTE TRANS-SPLICING,” and U.S. Provisional Patent Application No. 63/591,356, filed October 18, 2023, entitled “RNA STRUCTURAL MOTIFS TO PROMOTE TRANS-SPLICING,” each of which is herein incorporated by reference in its entirety for all purposes.

SUBMISSION OF SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (309222000840SEQLIST.xml; Size: 182,576 bytes; and Date of Creation: October 17, 2024) is herein incorporated by reference in its entirety.

FIELD

[0003] Effective treatment of human genetic diseases may require efficient repair of defective genetic sequences in human cells. Examples of human gene therapies include RNA trans-splicing.

BACKGROUND

[0004] Effective treatment of human genetic diseases may require efficient repair of defective genetic sequences in human cells. Examples of human gene therapies include RNA trans-splicing.

SUMMARY

[0005] In some aspects, herein is provided a trans-splicing nucleic acid molecule, comprising: (a) an exonic domain (e.g., one that encodes a therapeutic sequence); (b) an intronic domain configured to promote ribonucleic acid (RNA) trans-splicing; (c) an antisense domain configured to bind to a target RNA molecule; and (d) a sequence or structure derived or isolated from a ribozyme. In some embodiments, the ribozyme is selected from the group consisting of: a VS ribozyme, a twister sister ribozyme, a lantern ribozyme, a pistol ribozyme, a hairpin ribozyme, a leadzyme, a hatchet ribozyme, a GIRI branching ribozyme, a glmS ribozyme, a Class I intron, a Class II intron, a RNAse P, a CoTC ribozyme, a Hoylinc ribozyme, a Varkud satellite ribozyme, a CPEB3 ribozyme, and a riboswitch. In some embodiments, the trans- splicing molecule further comprises a G-quadruplex and/or a pseudoknot, and optionally a poly(A). In some embodiments, the trans- splicing molecule comprises, from 5’ to 3’: the G- quadruplex and/or the pseudoknot, and the sequence or structure derived or isolated from a ribozyme, and optionally the poly(A) at the 3’ end. In some embodiments, the trans-splicing molecule comprises, from 5’ to 3’: the sequence or structure derived or isolated from a ribozyme, the G-quadruplex and/or the pseudoknot, and optionally the poly(A) at the 3’ end. In any of the embodiments herein, the trans-splicing molecule may but does not need to comprise a hammerhead ribozyme, an HDV ribozyme, or a twister ribozyme.

[0006] In some aspects, herein is provided a trans-splicing nucleic acid molecule, comprising: (a) an exonic domain (e.g., one that encodes a therapeutic sequence); (b) an intronic domain configured to promote ribonucleic acid (RNA) trans-splicing; (c) an antisense domain configured to bind to a target RNA molecule; and (d) a sequence or structure derived or isolated from a ribozyme, wherein the trans-splicing nucleic acid molecule does not comprise a 3’ poly (A).

[0007] In some embodiments, the sequence or structure derived or isolated from the ribozyme is in a 3’ domain of the trans-splicing nucleic acid molecule. In some embodiments, the sequence or structure derived or isolated from the ribozyme is in a 5’ domain of the trans- splicing nucleic acid molecule.

[0008] In any of the embodiments herein, the ribozyme can be selected from the group consisting of a hammerhead ribozyme, an HDV ribozyme, a twister ribozyme, a twister sister ribozyme, a lantern ribozyme, a pistol ribozyme, a hairpin ribozyme, a VS ribozyme, a leadzyme, a hatchet ribozyme, a GIRI branching ribozyme, a glmS ribozyme, a Class I intron, a Class II intron, a RNAse P, a CoTC ribozyme, a Hoylinc ribozyme, a Varkud satellite ribozyme, a CPEB3 ribozyme, and a riboswitch.

[0009] In any of the embodiments herein, the trans-splicing nucleic acid molecule can comprise a sequence or structure derived or isolated from a twister sister ribozyme. In any of the embodiments herein, the trans-splicing nucleic acid molecule comprises a sequence or structure derived or isolated from a lantern ribozyme. In any of the embodiments herein, the trans-splicing nucleic acid molecule can comprise a sequence or structure derived or isolated from a CPEB3 ribozyme. In some embodiments the CPEB3 ribozyme is a mammalian CPEB3 ribozyme. In some embodiments the CPEB ribozyme is a human CPEB3 ribozyme or a Pan troglodytes CPEB3 ribozyme. In any of the embodiments herein, the trans-splicing nucleic acid molecule can comprise a sequence or structure derived or isolated from a VS ribozyme. In any of the embodiments herein, the trans- splicing nucleic acid molecule can comprise a sequence or structure derived or isolated from a hatchet ribozyme.

[0010] In any of the embodiments herein, the ribozyme can be a ribozyme derived or isolated from an eukaryote. In some embodiments, the eukaryote is a mammal. In some embodiments, the mammal is a human or Pan troglodytes. In any of the embodiments herein, the ribozyme can be a ribozyme derived or isolated from a virus. In any of the embodiments herein, the ribozyme can be a ribozyme derived or isolated from a prokaryote. In some embodiments, the prokaryote is a bacterium.

[0011] In any of the embodiments herein, the ribozyme can be a mutant ribozyme. In any of the embodiments herein, the ribozyme can be an engineered ribozyme. In any of the embodiments herein, the ribozyme can be derived from or encoded by a IncRNA. In any of the embodiments herein, the ribozyme can be a self-cleaving ribozyme. In any of the embodiments herein, the ribozyme can be a self-alkylating ribozyme.

[0012] In any of the embodiments herein, the ribozyme can comprise one or more pseudoknots. In any of the embodiments herein, the ribozyme can be regulated by or require one or more metal ion cofactors. In some embodiments, the one or more metal ion cofactors comprise a divalent cation. In some embodiments, the one or more metal ion cofactors comprise Mg²⁺.

[0013] In any of the embodiments herein, the trans- splicing nucleic acid molecule can comprise a 3’ domain which comprises a cleavage domain, wherein the sequence or structure derived or isolated from the ribozyme is a first ribozyme sequence or structure. In some embodiments, the cleavage domain comprises: a second ribozyme sequence or structure that is the same or different from the first ribozyme sequence or structure; a microprocessor substrate; an RNAse P/Z substrate; or any combination thereof. In some embodiments, the cleavage domain comprises the sequence or structure derived or isolated from the ribozyme. In some embodiments, the cleavage domain does not comprise the sequence or structure derived or isolated from the ribozyme.

[0014] In any of the embodiments herein, the trans- splicing nucleic acid molecule can comprise a 3’ domain which comprises a stabilization domain. In some embodiments, the stabilization domain comprises the sequence or structure derived or isolated from the ribozyme. In some embodiments, the stabilization domain does not comprise the sequence or structure derived or isolated from the ribozyme. In some embodiments, the stabilization domain comprises a G-quadruplex and/or a pseudoknot. In some embodiments, the stabilization domain comprises a G-quadruplex. In some embodiments, the stabilization domain comprises a pseudoknot.

[0015] In any of the embodiments herein, the trans- splicing nucleic acid molecule can comprise a 3’ domain which comprises a nuclear retention domain. Optionally, in some embodiments, the nuclear retention domain comprises a triple helix, a pseudoknot, a riboswitch, a G-quadruplex, an RNAse P RNA, a stem-loop structure, a snoRNA, or any combination thereof. In some embodiments, the triple helix is a viral helix. In some embodiments, the triple helix is a human triple helix. In some embodiments, the G-quadruplex is a telomerase G- quadruplex. In some embodiments, the nuclear retention domain comprises the sequence or structure derived or isolated from the ribozyme. In some embodiments, the nuclear retention domain does not comprise the sequence or structure derived or isolated from the ribozyme.

[0016] In any of the embodiments herein, the trans- splicing nucleic acid molecule can comprise a 3’ domain which comprises, from 5’ to 3’: the G-quadruplex and/or the pseudoknot and the sequence or structure derived or isolated from the ribozyme. In any of the embodiments herein, the trans- splicing nucleic acid molecule can comprise a 3’ domain which comprises, from 5’ to 3’: the sequence or structure derived or isolated from the ribozyme and the G- quadruplex and/or the pseudoknot.

[0017] In any of the embodiments herein, the sequence or structure derived or isolated from the ribozyme can increase trans-splicing efficiency of the trans-splicing nucleic acid molecule. In some embodiments, the increase in trans-splicing efficiency is by at least about 10%, by at least about 20%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 100%, by at least about 200%, by at least about 300%, by at least about 400%, by at least about 500%, by at least about 600%, by at least about 700%, by at least about 800%, by at least about 900%, or by 1000%, compared to a reference trans-splicing nucleic acid molecule without the sequence or structure derived or isolated from the ribozyme.

[0018] In any of the embodiments herein, the trans-splicing nucleic acid molecule can further comprise a G-quadruplex and/or the pseudoknot that increases trans-splicing efficiency of the trans-splicing nucleic acid molecule. In some embodiments, the increase in trans-splicing efficiency is by at least about 10%, by at least about 20%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 100%, by at least about 200%, by at least about 300%, by at least about 400%, by at least about 500%, by at least about 600%, by at least about 700%, by at least about 800%, by at least about 900%, or by 1000%, compared to a reference trans-splicing nucleic acid molecule without the sequence or structure derived or isolated from the ribozyme and the G-quadruplex and/or the pseudoknot.

[0019] In any of the embodiments herein, the target RNA molecule can be in a cell. In any of the embodiments herein, the target RNA molecule can be a messenger RNA (mRNA) or a pre-mRNA. In any of the embodiments herein, the target RNA molecule can comprise a mutation. In some embodiments, the mutation is selected from the group consisting of a missense mutation, a nonsense mutation, a frameshift mutation, an insertion, a duplication, an inversion, a deletion, a splice site mutation, and a truncating mutation. In some embodiments, the mutation is a disease-causing mutation.

[0020] In any of the embodiments herein, the trans-splicing nucleic acid molecule can be packaged in a viral vector for delivery to a subject in need thereof. In some embodiments, the viral vector is a herpes simplex virus (HSV) vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector.

[0021] In some aspects, herein is presented a composition comprising the viral vector of any of the above and a pharmaceutically acceptable carrier or excipient. In any of the embodiments herein, the trans-splicing nucleic acid molecule can be packaged in a lipid nanoparticle for delivery to a subject in need thereof.

[0022] In some aspects, herein is presented a composition comprising the lipid nanoparticle of the above and a pharmaceutically acceptable carrier or excipient. In any of the embodiments herein, the engineered nucleic acid can be packaged in a vesicle for delivery to a subject in need thereof. In some embodiments, herein is presented a composition comprising the vesicle of the above and a pharmaceutically acceptable carrier or excipient.

INCORPORATION BY REFERENCE

[0023] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “FIG.” and “FIG.” herein), of which:

[0025] FIGS. 1A-1C illustrate the problem of human genetic disease and provide a schematic of gene repair using a trans-splicing nucleic acid molecule comprising a 3’ domain. FIG. 1A illustrates the concept of human genetic disease where a mutated (“defective”) DNA sequence is transcribed into RNA which directly contributes to disease or is translated into a disease-causing protein. FIG. IB illustrates an exemplary gene repair approach utilizing trans- splicing as described herein. In this example, a mutated gene is repaired by a trans-splicing RNA that comprises a 3’ domain. FIG. 1C illustrates the subdomains present in the 3’ domain. Specifically, the 3’ domain comprises a nuclear retention domain, a stabilization domain and a cleavage domain.

[0026] FIGS. 2A-2C illustrate challenges associated with trans-splicing technology and solutions described herein. FIG. 2A describes the mechanism of truncated protein production by trans-splicing therapeutics. Specifically, the truncated mRNA generated by a trans-splicing RNA can cause toxicity in human cells. FIGS. 2B-2C describe two exemplary mechanisms by which a truncated protein can be eliminated through the use of a 3’ domain in a trans-splicing RNA. FIG. 2B describes the activity of the cleavage domain which can result in elimination of the polyadenylation tail present on the trans-splicing RNA, thereby preventing translation of the trans-splicing RNA in the absence of a trans-splicing reaction. FIG. 2C describes an activity of the 3’ domain, whereby the 3’ domain promotes nuclear retention of the trans-splicing RNA, thereby increasing trans-splicing efficiency.

[0027] FIG. 3 illustrates empirical data describing the relative ability of different 3’ domains to increase the editing efficiency of a target RNA (CEP290).

[0028] FIGS. 4A-4C illustrate a schematic of gene repair using a trans-splicing nucleic acid molecule (e.g., a trans-splicing RNA) comprising a 3’ domain. FIG. 4A illustrates the concept of human genetic disease where a mutated (“defective”) DNA sequence is transcribed into RNA which directly contributes to disease or is translated into a disease-causing protein. FIG. 4B illustrates an exemplary gene repair approach utilizing trans-splicing as described herein. In this example, a mutated gene is repaired by a trans-splicing RNA that comprises a 3’ domain. FIG. 4C illustrates the 3’ domain of the trans-splicing nucleic acid molecule. The 3’ domain can comprise a sequence or structure derived or isolated from a ribozyme and optionally a G- quadruplex and/or a pseudoknot.

[0029] FIG. 5 shows relative repair efficiencies of various 3’ domains.

[0030] FIGS. 6A-6B show trans-splicing efficiencies of trans-splicing systems targeting CEP290. FIG. 6A illustrates the distribution of trans-splicing efficiencies of trans-splicing systems comprising certain ribozymes. FIG. 6B shows trans-splicing efficiencies of specific trans-splicing nucleic acid molecules.

[0031] FIG. 7 shows relative editing efficiency of trans-splicing systems targeting intron 8, intron 11, or intron 17 of SCN1A.

DETAILED DESCRIPTION

[0032] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0033] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein comprises (and describes) embodiments that are directed to that value or parameter per se.

[0034] As used herein, the singular forms “a,” “an,” and “the” comprise plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.”

[0035] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Where values are described in terms of ranges, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be comprised in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range comprises one or both of the limits, ranges excluding either or both of those comprised limits are also comprised in the claimed subject matter. This applies regardless of the breadth of the range.

[0036] The term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise.

[0037] Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, use of a), b), etc., or i), ii), etc. does not by itself connote any priority, precedence, or order of steps in the claims. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

[0038] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Overview

[0039] The disclosure provides a nucleic acid molecule (e.g., an RNA molecule) that selectively binds to and promotes a trans- splicing reaction with a target RNA molecule and carries a 3’ domain that promotes the trans- splicing reaction. The disclosure provides vectors, compositions and cells comprising or encoding the trans- splicing RNA molecule. The disclosure provides methods of using the trans-splicing nucleic acid molecule, vectors, compositions and cells of the disclosure to treat a disease or disorder.

[0040] In one aspect, the invention is a trans-splicing nucleic acid molecule (e.g., a trans- splicing RNA molecule), wherein the trans-splicing nucleic acid molecule comprises a sequence or structure derived or isolated from a ribozyme and different types of domains. One of the domain types is the Exonic Domain which is inserted into a target RNA molecule via a trans- splicing reaction. A second domain type is the Antisense Domain which is complementary to a target RNA. A third domain type is the Intronic Domain which promotes the trans-splicing reaction between the trans-splicing RNA molecule and the target RNA. A fourth domain is a 3’ domain that carries sequences that increase trans-splicing efficiency.

[0041] In one aspect, provided herein is a trans-splicing nucleic acid molecule, comprising: (a) an exonic domain (e.g., one that encodes a therapeutic sequence); (b) an intronic domain configured to promote ribonucleic acid (RNA) trans-splicing; (c) an antisense domain configured to bind to a target RNA molecule; (d) a sequence or structure derived or isolated from a ribozyme, wherein the ribozyme is selected from the group consisting of wherein the ribozyme is selected from the group consisting of: a VS ribozyme, a twister sister ribozyme, a lantern ribozyme, a pistol ribozyme, a hairpin ribozyme, a leadzyme, a hatchet ribozyme, a GIRI branching ribozyme, a glmS ribozyme, a Class I intron, a Class II intron, a RNAse P, a CoTC ribozyme, a Hoylinc ribozyme, a Varkud satellite ribozyme, a mammalian CPEB3 ribozyme, and a riboswitch.

[0042] In one aspect, provided herein is a trans-splicing nucleic acid molecule, comprising: (a) an exonic domain (e.g., one that encodes a therapeutic sequence); (b) an intronic domain configured to promote ribonucleic acid (RNA) trans-splicing; (c) an antisense domain configured to bind to a target RNA molecule; (d) a sequence or structure derived or isolated from a ribozyme; e) a 3’ domain, wherein the 3’ domain does not comprise a poly(A). In some embodiments, the 3’ domain comprises the sequence or structure derived or isolated from a ribozyme. In some embodiments, the 3’ domain does not comprise the sequence or structure derived or isolated from a ribozyme. This novel domain increases trans-splicing efficiency by promoting RNA processing phenomena that are favorable for efficiency trans-splicing. The 3’ domain comprises up to three subdomains: the nuclear retention domain, the stabilization domain, and the cleavage domain. Without wishing to be bound by any theory, the nuclear retention domain promotes localization of the trans-splicing RNA to the nucleus which is the site of trans-splicing activity. The stabilization domain reduces the turnover of the trans-splicing RNA. And the cleavage domain removes the tail of the RNA which reduces nuclear export and concomitant RNA processing activities that interfere with trans-splicing. This novel combination of typical trans-splicing domains (Exonic, Intronic, and Antisense Domains) with the 3’ domain promotes RNA trans-splicing in a manner that is sufficient to replace diseasecausing RNA sequences in human cells to address disease. Indeed, low efficiency has been a major barrier to many nucleic acid editing approaches including RNA trans-splicing. The disclosure provides compositions and methods for specifically targeting disease-causing RNA molecules and replacing disease-causing RNA sequences within these RNA molecules with high efficiency. The trans-splicing RNA molecule implementations show utility in a variety of contexts including replacement of disease-causing sequences or insertion of engineered sequences into target RNAs. The engineered sequences can alter the translation or stability of target RNAs to increase or decrease protein production or target RNA levels. This disclosure provides vectors, compositions and cells comprising or encoding the trans-splicing nucleic acid molecule (e.g., trans-splicing RNA molecule) and methods of using the trans-splicing nucleic acid molecule (e.g., trans-splicing RNA molecule) compositions.

[0043] In one aspect, the invention is an RNA technology that enables replacement of arbitrary sequences within specific RNA molecules in living cells. The technology, based on RNA trans-splicing, utilizes the naturally-existing spliceosome in human cells to provide the catalytic activity for this trans-splicing process. Typically, RNA splicing occurs within RNA molecules where exons are concatenated and introns removed from immature messenger RNA molecules (pre-mRNAs) to form mature messenger RNA molecules (mRNAs). This process is referred to as cis- splicing and requires the set of enzymes and noncoding RNAs collectively known as the spliceosome. RNA trans-splicing is a process by which the spliceosome concatenates exons derived from distinct and separate RNA molecules. This process rarely occurs in human cells and state-of-the-art systems that promote RNA trans-splicing are active at low levels. The present invention comprises compositions that increase the efficiency of RNA trans-splicing. These improved RNA trans-splicing compositions could be used to replace mutated sequences within a target RNA molecule to address a human disease. Replacement of arbitrary RNA sequences is a general ability with innumerable specific applications a few of which have been explored as relevant demonstrations. RNA trans-splicing can insert engineered sequences into a target RNA to impart new activities to the target RNA such as altered RNA stability or altered RNA translation. This feature can be used to increase production of protein by a target RNA. In the broadest sense, this RNA trans-splicing technology can impart arbitrary changes to both coding and non-coding regions of target RNAs.

[0044] References describe the use of sequences derived from mRNA, long noncoding RNAs, and synthetic sequences to alter that localization of varied transcript types within the cellular nucleus (Espinoza et al., 2007; Guo et al., 2020; Long et al., 2017; Lubelsky and Ulitsky, 2018; Miyagawa et al., 2012; Shukla et al., 2018; Wilusz et al., 2012) Indeed, a variety of RNA sequences placed in a heterologous context are known to promote the accumulation of RNAs in the nucleus. Typically, these sequences are derived from long noncoding RNAs such as MALAT1 and cleave off the poly adenylated tail of an RNA and contain tertiary structures that stabilize the RNA and/or localization sequences that promote nuclear retention (Wilusz et al., 2012). But little is known of whether heterologous sequences derived from other sources can combine to generate a superior nuclear localization phenomenon. As the activity of many known RNA sequences is context-dependent, the present inventor conceived of a distinct group of 3’ domain sequences that would function in the context of trans-splicing. This was confirmed by experiments that indicate that activity of a 3’ domain in other contexts is not necessarily predictive of activity in trans-splicing.

[0045] An important safety aspect of associated with a trans-splicing systems is the ability to prevent protein production from the trans-splicing RNA before the trans-splicing reaction occurs. In some embodiments, the 3’ domain is configured to prevent the production of protein from the trans-splicing molecule in the absence of a trans-splicing reaction. In some embodiments, the presence of a 3’ domain comprises three subdomains: a stabilization domain, a nuclear retention domain, and a cleavage domain. In some embodiments, an activity of the 3’ domain is configured to eliminate an expression of a protein product encoded by the trans- splicing RNA molecule or a portion thereof. In some embodiments, an activity of the 3’ domain is configured to eliminate an expression of a protein product encoded by the trans-splicing RNA molecule or a portion thereof in an absence of the association of the two or more exonic domains. In some embodiments, an activity of the 3’ domain is configured to eliminate an expression of a protein product encoded by the trans-splicing RNA molecule or a portion thereof in an absence of a trans-splicing reaction of the trans-splicing RNA molecule with a target RNA. In some embodiments, the 3’ domain is configured to eliminate the exonic domain of the trans- splicing RNA molecule in an absence of a trans-splicing reaction of the trans-splicing RNA molecule with a target RNA.

[0046] In some embodiments, the stabilization domain forms a tertiary RNA structure. In some embodiments, the stabilization domain is isolated or derived from a sequence selected from the group consisting of: an RNA pseudoknot, an RNA triplex, a riboswitch, an aptamer. In some embodiments, the cleavage domain promotes cutting of the RNA molecule. In some embodiments, the stabilization domain comprises the sequence or structure derived or isolated from a ribozyme. In some embodiments, the ribozyme is selected from the group consisting of: a hammerhead ribozyme, an HDV ribozyme, a twister ribozyme, a twister sister ribozyme, a lantern ribozyme, a pistol ribozyme, a hairpin ribozyme, a VS ribozyme, a leadzyme, a hatchet ribozyme, a GIRI branching ribozyme, a glmS ribozyme, a Class I intron, a Class II intron, a RNAse P, a CoTC ribozyme, a Hoylinc ribozyme, a Varkud satellite ribozyme, a mammalian CPEB3 ribozyme, and a riboswitch.

[0047] In some embodiments, the cleavage domain comprises the sequence or structure derived or isolated from a ribozyme. In some embodiments, the ribozyme is selected from the group consisting of: a hammerhead ribozyme, an HDV ribozyme, a twister ribozyme, a twister sister ribozyme, a lantern ribozyme, a pistol ribozyme, a hairpin ribozyme, a VS ribozyme, a leadzyme, a hatchet ribozyme, a GIRI branching ribozyme, a glmS ribozyme, a Class I intron, a Class II intron, a RNAse P, a CoTC ribozyme, a Hoylinc ribozyme, a Varkud satellite ribozyme, a mammalian CPEB3 ribozyme, and a riboswitch.

[0048] In some embodiments, the nuclear retention domain promotes localization of the trans-splicing RNA to the cellular nucleus. In some embodiments, the nuclear retention domain is isolated or derived from a viral ENE (expression and nuclear retention element) sequence, a human ENE sequence, a NEAT1 ENE sequence, XIST, BORG, TUG1, MEG3, GAS5, a human IncRNA, a mouse IncRNA. In some embodiments, the nuclear retention domain comprises the sequence or structure derived or isolated from a ribozyme. In some embodiments, the ribozyme is selected from the group consisting of a hammerhead ribozyme, an HDV ribozyme, a twister ribozyme, a twister sister ribozyme, a lantern ribozyme, a pistol ribozyme, a hairpin ribozyme, a VS ribozyme, a leadzyme, a hatchet ribozyme, a GIRI branching ribozyme, a glmS ribozyme, a Class I intron, a Class II intron, a RNAse P, a CoTC ribozyme, a Hoylinc ribozyme, a Varkud satellite ribozyme, a mammalian CPEB3 ribozyme, and a riboswitch.

[0049] The trans-splicing nucleic acid molecule can comprise RNA, DNA, an DNA/RNA hybrid, a nucleic acid analog, a chemically modified nucleic acid, a chimera composed of two or more nucleic acids or nucleic acid analogs, or any combination thereof. A trans-splicing nucleic acid molecule comprising DNA may be transcribed from DNA into RNA. A trans-splicing nucleic acid molecule comprising DNA may be transcribed from DNA into RNA upon administration into a subject. The trans-splicing nucleic acid molecule (e.g., trans-splicing RNA molecule) may associate with a target RNA via trans-splicing. The target RNA sequence or portion thereof may have a mutated or missing sequence. The binding and/or replacing of the target RNA sequence or portion thereof with the one or more exonic domains may treat or restore a function of the target RNA sequence or a portion thereof.

[0050] The nucleic acid molecules of the present disclosure may be provided in a cell. The nucleic acid molecules provided herein may be administered to a cell. The nucleic acid molecules provided herein may be delivered into a cell. The cell may comprise a human cell. For example, a DNA molecule or an RNA molecule may be provided in a human cell or delivered into a human cell. The target RNA may be in a cell. For example, the target RNA may be in a human cell. The target RNA may be a messenger RNA (mRNA) or a pre-mRNA. The nucleic acid molecules provided herein can be for trans-splicing and can be referred to as transsplicing nucleic acid molecules.

[0051] The inclusion of localization sequences in trans-splicing nucleic acid molecules to form the present RNA trans-splicing technology is a general capability that further allows the alteration of non-coding sequences within target RNAs. By replacing the 5’ or 3’ untranslated regions of target RNAs with high efficiency, this invention allows the alteration of RNA behaviors such as translation or turnover. The net result of these effects is increased production of protein from target RNAs or other downstream effects associated with altered RNA levels.

[0052] The nucleic acid molecules may comprise or encode nucleic acid sequences that promote trans-splicing. For example, the trans-splicing nucleic acid molecule may comprise one or more intronic domains. The one or more intronic domains may carry binding sites that are preferentially targeted by RNA-binding proteins with disease-causing mutations. In some embodiments, a disease-causing mutation may be an insertion, a duplication, an inversion, a deletion, a splice site mutation, a missense mutation, a nonsense mutation, a frameshift mutation, or a truncating mutation. In some embodiments, the intronic domains comprise one or more trans-splicing enhancing sequences. In some embodiments, the trans-splicing enhancing sequences are configured to bind an engineered U1 snRNA (ESM). In some embodiments, the ESM comprises an engineered small nuclear RNA (esnRNA). The antisense domain is configured to bind to the target mRNA or pre-mRNA. The trans-splicing nucleic acid molecule may comprise one or more domains that bind RNA-binding proteins. The nucleic acid sequences provided herein may comprise or encode one or more engineered U1 snRNAs (ESM). The ESM may comprise one or more engineered non-coding RNA molecules that may enhance trans- splicing. The engineered non-coding RNA molecules can comprise engineered snRNA (e.g., esnRNA), which can recruit RNPs of the spliceosome. The trans-splicing nucleic acid molecule may comprise one or more trans-splicing enhancers.

[0053] In some embodiments, the nucleic acid sequences of the present disclosure are provided by one nucleic acid molecule. In some embodiments, the nucleic acid sequences are provided by two or more nucleic acid molecules. In some embodiments, the two or more nucleic acid molecules are provided to a cell in one vector. In some embodiments, the two or more nucleic acid molecules are provided to a cell in two or more vectors. In some embodiments, the vectors are recombinant viruses.

Compositions

[0054] In one aspect, provided herein are compositions comprising engineered nucleic acid molecules for RNA trans-splicing (e.g., trans- splicing nucleic acid molecules).

[0055] In one aspect, provided herein is a trans-splicing nucleic acid molecule, comprising: (a) an exonic domain (e.g., one that encodes a therapeutic sequence); (b) an intronic domain configured to promote ribonucleic acid (RNA) trans-splicing; (c) an antisense domain configured to bind to a target RNA molecule; (d) a sequence or structure derived or isolated from a ribozyme, wherein the ribozyme is selected from the group consisting of wherein the ribozyme is selected from the group consisting of: a VS ribozyme, a twister ribozyme, a twister sister ribozyme, a lantern ribozyme, a pistol ribozyme, a hairpin ribozyme, a leadzyme, a hatchet ribozyme, a GIRI branching ribozyme, a glmS ribozyme, a Class I intron, a Class II intron, a RNAse P, a CoTC ribozyme, a Hoylinc ribozyme, a Varkud satellite ribozyme, a mammalian CPEB3 ribozyme, and a riboswitch.

[0056] In one aspect, provided herein is a trans-splicing nucleic acid molecule, comprising: (a) an exonic domain (e.g., one that encodes a therapeutic sequence); (b) an intronic domain configured to promote ribonucleic acid (RNA) trans-splicing; (c) an antisense domain configured to bind to a target RNA molecule; (d) a sequence or structure derived or isolated from a ribozyme; (e) a 3’ domain, wherein the 3’ domain does not comprise a poly(A). In some embodiments, the 3’ domain comprises the sequence or structure derived or isolated from a ribozyme. In some embodiments, the 3’ domain does not comprise the sequence or structure derived or isolated from a ribozyme.

[0057] In some embodiments, the trans-splicing nucleic acid molecule comprises a trans- splicing RNA molecule. In some embodiments, the engineered nucleic acid molecules comprise two or more nucleic acid sequences each encoding a portion of a trans-splicing nucleic acid molecule (e.g., trans-splicing RNA molecule). The nucleic acid sequences may encode or comprise an antisense domain, a 3’ domain, an exonic domain, an intronic domain, an antisense domain, a splicing enhancer, a domain that binds an RNA-binding protein, or any combination thereof. In some embodiments, the composition further comprises an engineered U1 snRNA (ESM) that binds to a trans-splicing RNA molecule and promotes trans-splicing. [0058] In some embodiments, the trans-splicing molecule promotes trans-splicing in the absence of a CRISPR/Cas. In some embodiments, the trans-splicing molecule promotes trans- splicing in the presence of a CRISPR/Cas.

A ntisense domain

[0059] The present disclosure provides nucleic acid molecules comprising nucleic acid sequences encoding or comprising one or more antisense domains. The nucleic acid may comprise RNA, DNA, a DNA/RNA hybrid, and/or comprises at least one of a nucleic acid analog, a chemically modified nucleic acid, or a chimera composed of two or more nucleic acids or nucleic acid analogs. The nucleic acid may comprise RNA. The nucleic acid comprising RNA may encode the one or more antisense domains. The nucleic acid comprising RNA may be a trans-splicing RNA molecule. The nucleic acid may comprise DNA. The nucleic acid comprising DNA may encode one or more antisense domains. The one or more antisense domains of the nucleic acid comprising DNA may be transcribed into a trans-splicing RNA molecule comprising the one or more antisense domains. The antisense domains may promote the association of two or more exonic domains.

[0060] The one or more antisense domains may be provided on a nucleic acid molecule. A antisense domain can bind to another antisense domain. In some embodiments, the antisense domain binds to another antisense domain via covalent bonds. In some embodiments, the antisense domain binds to another antisense domain via non-covalent bonds. In some embodiments, the antisense domain comprises one or more nucleic acid sequences complementary to another antisense domain. A antisense domain can bind to a target RNA. In some embodiments, the antisense domain binds to a target RNA via covalent bonds. In some embodiments, the antisense domain binds to another a target RNA via non-covalent bonds. In some embodiments, the antisense domain comprises one or more nucleic acid sequences complementary to a target RNA.

[0061] In some embodiments of the compositions of the disclosure, the target RNA molecule is a pathogenic RNA molecule. In some embodiments, the target RNA comprises a target sequence that is complementary to an antisense domain of the trans-splicing RNA of the disclosure.

[0062] In some embodiments of the compositions and methods of the disclosure, the target sequence comprises or consists of between 5 and 500 nucleotides. In some embodiments, the target sequence comprises or consists of between 50 and 250 nucleotides. In some embodiments, the target sequence comprises or consists of between 5 and 50 nucleotides.

[0063] In some embodiments of the compositions and methods of the disclosure, a target sequence is contained within a single contiguous stretch of the target RNA. In some embodiments, the target sequence may consist of comprise of one or more nucleotides that are not spread among a single contiguous stretch of the target RNA.

[0064] In some embodiments of the disclosure, an Antisense Domain of the disclosure binds to a target sequence. In some embodiments of the disclosure, an antisense domain of the disclosure binds to a target RNA.

[0065] In some embodiments of the disclosure, the Antisense Domain is chosen so that successful trans-splicing causes removal of micro open reading frames in the target RNA. In this manner, the trans-splicing system removes micro open reading frames and increases the production of protein from the target RNA.

[0066] In some embodiments of the compositions of the disclosure, the sequence comprising the Antisense Domain has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or any percentage in between of complementarity to the target RNA sequence. In some embodiments, the Antisense Domain has 100% complementarity to the Target RNA sequence. In some embodiments, the Antisense Domain comprises or consists of about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 70 nucleotides, about 80 nucleotides, about 90 nucleotides, about 100 nucleotides, about 110 nucleotides, about 120 nucleotides, about 130 nucleotides, about 140 nucleotides, about 150 nucleotides, about 160 nucleotides, about 170 nucleotides, about 180 nucleotides, about 190 nucleotides, about 200 nucleotides, about 210 nucleotides, about 220 nucleotides, about 230 nucleotides, 240 nucleotides, about 250 nucleotides, 260 nucleotides, about 270 nucleotides, about 270 nucleotides, or more complementary to the Target RNA sequence.

[0067] In some embodiments, the Antisense Domain is reverse complementary to an RNA transcribed from a gene that is selected from the group consisting of: TNFRSF13B [ENSG00000240505] (common variable immune deficiency); ADA, CECR1 [ENSG00000196839, ENSG00000093072] (Adenosine deaminase deficiency); IL2RG [ENSG00000147168] (X-linked severe combined immunodeficiency); HBB [ENSG00000244734] (Beta-thassalemia); HBA1, HBA2 [ENSG00000206172, ENSG00000188536] (alpha-thassalemia); U2AF1 [ENSG00000160201] (myelodysplastic syndrome); SOD1, TARDBP, FUS, MATR3, SOD1, C9ORF72 [ENSG00000142168, ENSG00000120948, ENSG00000089280, ENSG00000015479, ENSG00000142168, ENSG00000147894] (Amyotrophic lateral sclerosis); MAPT, PGRN [ENSG00000186868, ENSG00000030582] (Frontotemporal dementia with parkinsonism); CDH23, MY07A, USH2A [ENSG00000107736, ENSG00000137474, ENSG00000042781] (Usher’s syndrome); GALC [ENSG00000054983] (Krabbe disease); SMPD1, NPC1, NPC2 [ENSG00000166311, ENSG00000141458, ENSG00000119655] (Niemann Pick disease); PRNP [ENSG00000171867] (prion disease); SCN1A [ENSG00000144285] (Dravet syndrome);

PINK1, ATPGAP2 [ENSG00000158828] (early-onset Parkinson’s disease); ATXN1, ATXN2, ATXN3, PLEKHG4, SPTBN2, CACNA1A, ATXN7, TTBK2, PPP2R2B, KCNC3, PRKCG, ITPR1, TBP, KCND1, FGF14 [ENSG00000124788, ENSG00000204842, ENSG00000066427, ENSG00000196155, ENSG00000173898, ENSG00000141837, ENSG00000163635, ENSG00000128881, ENSG00000156475, ENSG00000131398, ENSG00000126583, ENSG00000150995, ENSG00000112592, ENSG00000102057, ENSG00000102466] (spinocerebellar ataxias); SCN1A, SCN2A, CACNA1A, GRIN2B, GRIN2A, MECP2, FOXG1, SLC6A1, PRRT2, PTEN, KCNQ2, KCNQ3, STARD7, CLRN1 [ENSG00000144285, ENSG00000136531, ENSG00000141837, ENSG00000273079, ENSG00000183454, ENSG00000169057, ENSG00000176165, ENSG00000157103, ENSG00000167371, ENSG00000171862, ENSG00000075043, ENSG00000184156, ENSG00000084090, ENSG00000163646] (genetic epilepsy disorders); ATM [ENSG00000149311] (Ataxiatelangiectasia); GLB1 [ENSG00000170266] (GM1 gangliosidosis); GBA [ENSG00000177628] (Gaucher disease); GM2A [ENSG00000196743] (GM2 gangliosidosis); UBE3A [ENS G00000114062] (Angelman syndrome); SLC2A1 [ENSG00000117394] (glucose transporter deficiency type 1); LAMP2 [ENSG00000005893] (Danon disease); GLA [ENSG00000102393] (Fabry disease); PKD1, PKD2 [ENSG00000008710, ENSG00000118762] (Autosomal dominant polycystic kidney disease); GAA [ENSG00000171298] (Pompe disease); PCSK9, LDLR, APOB, APOE [ENSG00000169174, ENSG00000130164, ENSG00000084674, ENSG00000130203] (Familial hypercholesterolemia); MYOC, OPTN, TBK1, WDR36, CYPIB1 [ENSG00000034971, ENSG00000123240, ENSG00000183735, ENSG00000134987, ENSG00000138061] (Open Angle Glaucoma); IDUA [ENSG00000127415] (Hurler syndrome or Mucopolysaccharidosis 1); IDS [ENS G00000010404] (Hunter syndrome or Mucopolysaccharidosis 2); CLN3 [ENSG00000188603] (Batten disease); DMD [ENSG00000198947] (Duchenne muscular dystrophy); LMNA [ENSG00000160789] (Limb-girdle muscular dystrophy type IB); DYSF [ENSG00000135636] (Limb-girdle muscular dystrophy type 2B); SGCA [ENSG00000108823] (Limb-girdle muscular dystrophy type 2D); SGCB [ENSG00000163069] (Limb-girdle muscular dystrophy type 2E); SGCG [ENSG00000102683] (Limb-girdle muscular dystrophy type 2C); SGCD [ENSG00000170624] (Limb-girdle muscular dystrophy type 2F); DUX4 [ENSG00000260596] (Facioscapulohumeral muscular dystrophy); F9 [ENSG00000101981] (Hemophilia B); F8 [ENSG00000185010] (Hemophilia A ); USH2A, RPGR, RP2, RHO, PRPF31, USH1F, PRPF3, PRPF6 [ENSG00000156313, ENSG00000102218, ENSG00000163914, ENSG00000105618, ENSG00000150275, ENSG00000117360, ENSG00000101161] (Retinitis pigmentosa); CFTR [ENSG00000001626] (cystic fibrosis); GJB2, GJB6, STRC, DFNA1, WFS1 [ENSG00000165474, ENSG00000121742, ENSG00000242866, ENSG00000131504, ENSG00000109501] (autosomal dominant hearing impairment); POU3F3 [ENSG00000198914] (nonsyndromic hearing loss).

[0068] In some embodiments, the antisense domain(s) can be adjacent to a 5’ end of a transsplicing molecule. In some embodiments, the antisense domain(s) are at least: 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 110 nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, 150 nucleotides, 160 nucleotides, 170 nucleotides, 180 nucleotides, 190 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 400 nucleotides, or at least 500 nucleotides or more distant from the 5’ end of the trans-splicing molecule.

[0069] In some embodiments, the antisense domain(s) can be adjacent to the 3’ end of the trans-splicing molecule. In some embodiments, the antisense domain(s) are at least: 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 110 nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, 150 nucleotides, 160 nucleotides, 170 nucleotides, 180 nucleotides, 190 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 400 nucleotides, or at least 500 nucleotides or more distant from the 3’ end of the trans-splicing molecule.

[0070] In some embodiments, the antisense domain(s) can be at least: 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 110 nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, 150 nucleotides, 160 nucleotides, 170 nucleotides, 180 nucleotides, 190 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 400 nucleotides, or at least 500 nucleotides or more distant from the first nucleotide of the exonic domain or antisense domain in the 5’ direction.

[0071] In some embodiments, antisense domain(s) can be at least: 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 110 nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, 150 nucleotides, 160 nucleotides, 170 nucleotides, 180 nucleotides, 190 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 400 nucleotides, or at least 500 nucleotides or more distant from the last nucleotide of the exonic domain or antisense domain in the 3’ direction.

[0072] In some embodiments, the trans-splicing molecule may comprise a antisense domain. In some embodiments, the trans-splicing molecule may comprise 2 or more antisense domains. In some embodiments, the trans-splicing molecule comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 75, 100, 200, 300 or more antisense domains.

3’ domain

[0073] In some embodiments, the trans-splicing nucleic acid molecules of the present disclosure encodes or comprises one or more 3’ domains. The nucleic acid may comprise RNA, DNA, a DNA/RNA hybrid, and/or comprises at least one of a nucleic acid analog, a chemically modified nucleic acid, or a chimera composed of two or more nucleic acids or nucleic acid analogs. The nucleic acid may comprise RNA. The nucleic acid comprising RNA may comprise the one or more 3’ domains. The nucleic acid comprising RNA may be a trans-splicing RNA molecule. The nucleic acid may comprise DNA. The nucleic acid comprising DNA may encode the one or more 3’ domains. The nucleic acid comprising DNA that encodes the one or more 3’ domains may be transcribed into RNA, e.g., a trans-splicing RNA molecule.

[0074] 3’ domains are configured to promote processing of the trans-splicing RNA in a manner that increases the safety and efficiency of the trans-splicing therapeutic system. The 3’ domain can prevent the production of translated protein products from the trans-splicing RNA. If the trans-splicing RNA does not successfully react with the target RNA, the 3’ domains may prevent translation of the trans-splicing RNA and avoid generation of a truncated protein product encoded by the exonic domain. The 3’ domain can also promote nuclear retention of the trans-splicing RNA in a manner that increases the efficiency of the trans-splicing reaction. In this way, the 3’ domain may reduce a frequency of adverse events from exposure to the nucleic acid sequences provided herein. In some embodiments, a 3’ domain may reduce a frequency of systemic toxicity.

[0075] In some embodiments, the 3’ domain comprises three subdomains: a stabilization domain, a nuclear retention domain, and a cleavage domain.

[0076] In some embodiments, the cleavage domain is configured to remove the polyadenylation tail of an mRNA. In some embodiments, the 3’ domain is configured to eliminate the trans-splicing RNA molecule through non-sense mediated or non-stop RNA decay (NMD or NSD). In some embodiments, the cleavage domain comprises or encodes one more substrates for cellular nucleases. In some embodiments, the 3’ domain comprises or encodes one or more ribozymes (e.g., ribonucleic acid enzymes). In some embodiments a ribozyme encoded by the trans-splicing RNA provided herein cleaves the trans-splicing RNA molecule or a portion thereof. In some embodiments, the cleavage domain comprises or encodes a nucleolytic ribozyme, such as RNase P. In some embodiments, the nucleolytic ribozyme comprises a hairpin ribozyme, a VS ribozyme, a twister ribozyme, an HDV ribozyme, a TS ribozyme, a hammerhead ribozyme, a pistol ribozyme, or a glmS ribozyme. In some embodiments, the cleavage domain is isolated or derived from a sequence that is a substrate for small non-coding RNA processing such as the integrator complex, the microprocessor complex, or other small non-coding RNA processing enzymes.

[0077] In some embodiments, the 3’ domain disclosed herein comprises GGTCTCcggagCAGTCTCGGTAAGACACGGTCGTCTCtGATAgtttgaaaaatgtgaaggactttcgtaa cggaagtaattcaagatcaagagtaattaccaacttaatgtttttgcattggactttgagttaagattattttttaaatcctgaggactagcattaatt gacagctgacccaggtgctacacagaagtggattcagtgaatctaggaagacagcagcagacaggattccaggaaccagtgtttgatgaa gctaggactgaggagcaagcgagcaagcagcagttcgtggtgaagataggaaaagagtccaggagccagtgcgatttggtgaaggaa gctaggaagaaggaaggagcgctaacgatttggtggtgaagctaggaaaaaggattccaggaaggagcgagtgcaatCCCAAttttt cttttGAATTCTCTAGAGAATTCttttgctttttCTTCaaaaagcaaaagacgctggtggctggcactcctggtttccagga cggggttcaagtccctgcggtgtctttgcttTACGtGAGACGGCAGAACTTACGAGCCAGTGccataGAGA CC (SEQ ID NO: 103).

[0078] In some embodiments, the 3’ domain disclosed herein comprises GGTCTCcggagCAGTCTCGGTAAGACACGGTCGTCTCtGATAatttttaggtaaaatgctttttgttcatttct ggtggtgggaggggactgaagcctttagtcttttccagatgcaaccttaaaatcagtgacaagaaacattccaaacaagcaacagtcttcaa gaaattaaactggcaagtggaaatgtttaaacagttcagtgatctttagtgcattgtttatgtgtgggtttctctctcccctcccttggtcttaattct tacatgcaggaacactcagcagacacacgtatgcgaagggccagagaagccagacccagtaagaaaaaatagcctatttactttaaataa accaaacattccattttaaatgtggggattgggaaccactagttctttcagatggtattcttcagactatagaaggaggattcgtcagtagggtt gtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaagacgct ggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgcttTACGtGAGACGGCAGAACTTAC GAGCCAGTGccataGAGACC (SEQ ID NO: 104).

[0079] In some embodiments, the 3’ domain disclosed herein comprises

GGTCTCcggagCAGTCTCGGTAAGACACGGTCGTCTCtGATAatttttaggtaaaatgctttttgttcatttct ggtggtgggaggggactgaagcctttagtcttttccagatgcaaccttaaaatcagtgacaagaaacattccaaacaagcaacagtcttcaa gaaattaaactggcaagtggaaatgtttaaacagttcagtgatctttagtgcattgtttatgtgtgggtttctctctcccctcccttggtcttaattct tacatgcaggaacactcagcagacacacgtatgcgaagggccagagaagccagacccagtaagaaaaaatagcctatttactttaaataa accaaacattccattttaaatgtggggattgggaaccactagttctttcagatggtattcttcagactatagaaggagaagcaaagacaccgc agggacttgaaccccgtcctggaaaccaggagtgccagccaccagcgtcttttgcttttttttttttaaagctagggaaaggccaaaaagcaa aacctgagaaaacaaaaggttgttttctcaggaaaagaaaaacctttacaaccctactgacgaatcTACGtGAGACGGCAGA ACTTACGAGCCAGTGccataGAGACC (SEQ ID NO: 105).

[0080] In some embodiments, the 3’ domain disclosed herein comprises

GGTCTCcggagCAGTCTCGGTAAGACACGGTCGTCTCtGATAatttttaggtaaaatgctttttgttcatttct ggtggtgggaggggactgaagcctttagtcttttccagatgcaaccttaaaatcagtgacaagaaacattccaaacaagcaacagtcttcaa gaaattaaactggcaagtggaaatgtttaaacagttcagtgatctttagtgcattgtttatgtgtgggtttctctctcccctcccttggtcttaattct tacatgcaggaacactcagcagacacacgtatgcgaagggccagagaagccagacccagtaagaaaaaatagcctatttactttaaataa accaaacattccattttaaatgtggggattgggaaccactagttctttcagatggtattcttcagactatagaaggagaaaggtttttcttttcctg agaaatttctcaggttttgctttttaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcgg tgtctttgcttTACGtGAGACGGCAGAACTTACGAGCCAGTGccataGAGACC (SEQ ID NO: 106).

[0081] In some embodiments, the 3’ domain disclosed herein comprises

GGTCTCcggagCAGTCTCGGTAAGACACGGTCGTCTCtGATAatttttaggtaaaatgctttttgttcatttct ggtggtgggaggggactgaagcctttagtcttttccagatgcaaccttaaaatcagtgacaagaaacattccaaacaagcaacagtcttcaa gaaattaaactggcaagtggaaatgtttaaacagttcagtgatctttagtgcattgtttatgtgtgggtttctctctcccctcccttggtcttaattct tacatgcaggaacactcagcagacacacgtatgcgaagggccagagaagccagacccagtaagaaaaaatagcctatttactttaaataa accaaacattccattttaaatgtggggattgggaaccactagttctttcagatggtattcttcagactatagaaggagCCCAAtttttcttttG AATTCTCTAGAGAATTCttttgctttttCTTCaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggg gttcaagtccctgcggtgtctttgcttTACGtGAGACGGCAGAACTTACGAGCCAGTGccataGAGACC (SEQ ID NO: 107).

[0082] In some embodiments, the 3’ domain disclosed herein comprises

GGTCTCcggagCAGTCTCGGTAAGACACGGTCGTCTCtGATAtctgcagtattgcatgttagggataagt gcttatttttaagagctgtggagttcttaaatatcaaccatggcactttctcctgaccccttccctaggggatttcaggattgagaaatttttccatc gagcctttttaaaattgtaggacttgttcctgtgggcttcagtgatgggatagtacacttcactcagaggcatttgcatctttaaataatttcttaaa agcctctaaagtgatcagtgccttgatgccaactaaggaaatttgtttagcattgaatctctgaaggctctatgaaaggaatagcatgatgtgct gttagaatcagatgttactgctaaaatttacatgttgtgatgtaaattgtgtagaaaaccattaaatcattcaaaataataaactatttttattagaga atgtatacttttagaaagctgtctccttatttaaataaaatagtgtttgtctgtagttcagtgaaaggtttttcttttcctgagaaatttctcaggttttg ctttttaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgcttTACGtG AGACGGCAGAACTTACGAGCCAGTGccataGAGACC (SEQ ID NO: 108).

[0083] In some embodiments, the 3’ domain disclosed herein comprises

GGTCTCcggagCAGTCTCGGTAAGACACGGTCGTCTCtGATAtctgcagtattgcatgttagggataagt gcttatttttaagagctgtggagttcttaaatatcaaccatggcactttctcctgaccccttccctaggggatttcaggattgagaaatttttccatc gagcctttttaaaattgtaggacttgttcctgtgggcttcagtgatgggatagtacacttcactcagaggcatttgcatctttaaataatttcttaaa agcctctaaagtgatcagtgccttgatgccaactaaggaaatttgtttagcattgaatctctgaaggctctatgaaaggaatagcatgatgtgct gttagaatcagatgttactgctaaaatttacatgttgtgatgtaaattgtgtagaaaaccattaaatcattcaaaataataaactatttttattagaga atgtatacttttagaaagctgtctccttatttaaataaaatagtgtttgtctgtagttcagtgCCCAAtttttcttttGAATTCTCTAGA GAATTCttttgctttttCTTCaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgt ctttgcttTACGtGAGACGGCAGAACTTACGAGCCAGTGccataGAGACC(SEQ ID NO: 109).

[0084] In some embodiments, the 3’ domain disclosed herein comprises

GGTCTCcggagCAGTCTCGGTAAGACACGGTCGTCTCtGATAgattcttctctaatctttcagaaactttgtc tgcgaacactctttaatggaccagatcaggatttgagcggaagaacgaatgtaactttaaggcaggaaagacaaattttattcttcataaagtg atgagcatataataattccaggcacatggcaatagaggccctctaaataaggaataaataacctcttagacaggtgggagattatgatcaga gtaaaaggtaattacacattttatttccagaaagtcaggggtctataaattgacagtgattagagtaatactttttcacatttccaaagtttgcatgt taactttaaatgcttacaatcttagagtggtaggcaatgttttacactattgaccttatatagggaagggagggggtgcctgtggggttttaaag aattttcctttgcagaggcataaaggtttttcttttcctgagaaatttctcaggttttgctttttaaaaaaaaagcaaaagacgctggtggctggca ctcctggtttccaggacggggttcaagtccctgcggtgtctttgcttTACGtGAGACGGCAGAACTTACGAGCCAG TGccataGAGACC (SEQ ID NO: 110).

[0085] In some embodiments, the 3’ domain disclosed herein comprises

GGTCTCcggagCAGTCTCGGTAAGACACGGTCGTCTCtGATAgattcttctctaatctttcagaaactttgtc tgcgaacactctttaatggaccagatcaggatttgagcggaagaacgaatgtaactttaaggcaggaaagacaaattttattcttcataaagtg atgagcatataataattccaggcacatggcaatagaggccctctaaataaggaataaataacctcttagacaggtgggagattatgatcaga gtaaaaggtaattacacattttatttccagaaagtcaggggtctataaattgacagtgattagagtaatactttttcacatttccaaagtttgcatgt taactttaaatgcttacaatcttagagtggtaggcaatgttttacactattgaccttatatagggaagggagggggtgcctgtggggttttaaag aattttcctttgcagaggcatCCCAAtttttcttttGAATTCTCTAGAGAATTCttttgctttttCTTCaaaaagcaaaaga cgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgcttTACGtGAGACGGCAGAACT TACGAGCCAGTGccataGAGACC (SEQ ID NO: 111). [0086] In some embodiments, the 3’ domain disclosed herein comprises gtttgaaaaatgtgaaggactttcgtaacggaagtaattcaagatcaagagtaattaccaacttaatgtttttgcattggactttgagttaagatta ttttttaaatcctgaggactagcattaattgacagctgacccaggtgctacacagaagtggattcagtgaatctaggaagacagcagcagac aggattccaggaaccagtgtttgatgaagctaggactgaggagcaagcgagcaagcagcagttcgtggtgaagataggaaaagagtcca ggagccagtgcgatttggtgaaggaagctaggaagaaggaaggagcgctaacgatttggtggtgaagctaggaaaaaggattccagga aggagcgagtgcaataaaggtttttcttttcctgagaaatttctcaggttttgctttttaaaaaaaaagcaaaagacgctggtggctggcactcc tggtttccaggacggggttcaagtccctgcggtgtctttgctt (SEQ ID NO: 112).

[0087] In some embodiments, the 3’ domain disclosed herein comprises gtttgaaaaatgtgaaggactttcgtaacggaagtaattcaagatcaagagtaattaccaacttaatgtttttgcattggactttgagttaagatta ttttttaaatcctgaggactagcattaattgacagctgacccaggtgctacacagaagtggattcagtgaatctaggaagacagcagcagac aggattccaggaaccagtgtttgatgaagctaggactgaggagcaagcgagcaagcagcagttcgtggtgaagataggaaaagagtcca ggagccagtgcgatttggtgaaggaagctaggaagaaggaaggagcgctaacgatttggtggtgaagctaggaaaaaggattccagga aggagcgagtgcaatgattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttcc ctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt (SEQ ID NO: 113).

Exonic domain

[0088] In some embodiments, described herein is a trans- splicing ribonucleic acid comprising an exonic domain. In some embodiments, the exonic domain is derived or isolated from the target RNA. In some embodiments, the exonic domain is a heterologous sequence. In some embodiments, the exonic domain encodes an engineered protein. In some embodiments, the exonic domain encodes a molecule (e.g., a protein) that has a function in immunotherapy. In some embodiments, the exonic domain is a chimeric antigen receptor.

[0089] In some embodiments, the exonic domain is comprised of a sequence derived or isolated from a human gene. In some embodiments of the compositions of the disclosure, the sequence comprising the exonic domain has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 87%, 90%, 95%, 97%, 99% or any percentage in between of identity with a human gene. In some embodiments, the exonic domain has about 100% identity with a sequence derived or isolated from a human gene. In some embodiments, the exonic domain comprises or consists of about 2 nucleotides, about 5 nucleotides, about 10 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 70 nucleotides, about 80 nucleotides, about 90 nucleotides, about 100 nucleotides, about 110 nucleotides, about 120 nucleotides, about 130 nucleotides, about 140 nucleotides, about 150 nucleotides, about 160 nucleotides, about 170 nucleotides, about 180 nucleotides, about 190 nucleotides, about 200 nucleotides, about 210 nucleotides, about 220 nucleotides, about 230 nucleotides, about 240 nucleotides, about 250 nucleotides, about 260 nucleotides, about 270 nucleotides, about 270 nucleotides, or more.

[0090] The exonic domains can include, without limitation, nucleic acid (e.g., RNA) sequences derived or isolated from the following genes (with gene accession IDs in brackets and associated diseases in parentheses) such as TNFRSF13B [ENSG00000240505] (common variable immune deficiency); ADA, CECR1 [ENSG00000196839, ENSG00000093072] (Adenosine deaminase deficiency); IL2RG [ENSG00000147168] (X-linked severe combined immunodeficiency); HBB [ENSG00000244734] (Beta-thassalemia); HBA1, HBA2 [ENSG00000206172, ENSG00000188536] (alpha-thassalemia); U2AF1 [ENSG00000160201] (myelodysplastic syndrome); SOD1, TARDBP, FUS, MATR3, SOD1, C9ORF72 [ENSG00000142168, ENSG00000120948, ENSG00000089280, ENSG00000015479, ENSG00000142168, ENSG00000147894] (Amyotrophic lateral sclerosis); MAPT, PGRN [ENSG00000186868, ENSG00000030582] (Frontotemporal dementia with parkinsonism); CDH23, MYO7A, USH2A, PCDH15 [ENSG00000107736, ENSG00000137474, ENSG00000042781, ENSG00000150275] (Usher’s syndrome); GALC [ENSG00000054983] (Krabbe disease); SMPD1, NPC1, NPC2 [ENSG00000166311, ENSG00000141458, ENSG00000119655] (Niemann Pick disease); PRNP [ENSG00000171867] (prion disease); SCN1A [ENSG00000144285] (Dravet syndrome); PINK1, ATPGAP2 [ENSG00000158828] (early-onset Parkinson’s disease); ATXN1, ATXN2, ATXN3, PLEKHG4, SPTBN2, CACNA1A, ATXN7, TTBK2, PPP2R2B, KCNC3, PRKCG, ITPR1, TBP, KCND1, FGF14 [ENSG00000124788, ENSG00000204842, ENSG00000066427, ENSG00000196155, ENSG00000173898, ENSG00000141837, ENSG00000163635, ENSG00000128881, ENSG00000156475, ENSG00000131398, ENSG00000126583, ENSG00000150995, ENSG00000112592, ENS G00000102057, ENSG00000102466] (spinocerebellar ataxias); SCN1A, SCN2A, CACNA1A, GRIN2B, GRIN2A, MECP2, FOXG1, SLC6A1, PRRT2, PTEN, KCNQ2, KCNQ3, STARD7, CLRN1 [ENSG00000144285, ENSG00000136531, ENSG00000141837, ENSG00000273079, ENSG00000183454, ENSG00000169057, ENSG00000176165, ENSG00000157103, ENSG00000167371, ENSG00000171862, ENSG00000075043, ENSG00000184156, ENSG00000084090, ENSG00000163646] (genetic epilepsy disorders); ATM [ENSG00000149311] (Ataxia-telangiectasia); GLB1 [ENSG00000170266] (GM1 gangliosidosis); GBA [ENSG00000177628] (Gaucher disease); GM2A [ENSGOOOOO 196743] (GM2 gangliosidosis); UBE3A [ENS G00000114062] (Angelman syndrome); SLC2A1 [ENSGOOOOO 117394] (glucose transporter deficiency type 1); LAMP2 [ENSG00000005893] (Danon disease); GLA [ENSG00000102393] (Fabry disease); PKD1, PKD2 [ENSG00000008710, ENSGOOOOO 118762] (Autosomal dominant polycystic kidney disease); GAA [ENSGOOOOO 171298] (Pompe disease); PCSK9, LDLR, APOB, APOE [ENSG00000169174, ENSGOOOOO 130164, ENSG00000084674, ENSGOOOOO 130203] (Familial hypercholesterolemia); MYOC, OPTN, TBK1, WDR36, CYPIB1 [ENSG00000034971, ENSG00000123240, ENSG00000183735, ENSG00000134987, ENSG00000138061] (Open Angle Glaucoma); IDUA [ENSGOOOOO 127415] (Hurler syndrome or Mucopolysaccharidosis 1); IDS [ENS G00000010404] (Hunter syndrome or Mucopolysaccharidosis 2); CLN3 [ENSG00000188603] (Batten disease); DMD [ENSGOOOOO 198947] (Duchenne muscular dystrophy); LMNA [ENSG00000160789] (Limb-girdle muscular dystrophy type IB); DYSF [ENSG00000135636] (Limb-girdle muscular dystrophy type 2B); SGCA [ENSG00000108823] (Limb-girdle muscular dystrophy type 2D); SGCB [ENSGOOOOO 163069] (Limb-girdle muscular dystrophy type 2E); SGCG [ENSGOOOOO 102683] (Limb-girdle muscular dystrophy type 2C); SGCD [ENSG00000170624] (Limb-girdle muscular dystrophy type 2F); DUX4 [ENSG00000260596] (Facioscapulohumeral muscular dystrophy); F9 [ENSG00000101981] (Hemophilia B); F8 [ENSG00000185010] (Hemophilia A ); USH2A, RPGR, RP2, RHO, PRPF31, USH1F, PRPF3, PRPF6 [ENSG00000156313, ENSGOOOOO 102218, ENSG00000163914, ENSGOOOOO 105618, ENSG00000150275, ENSG00000117360, ENSG00000101161] (Retinitis pigmentosa); CFTR [ENSG00000001626] (cystic fibrosis); GJB2, GJB6, STRC, DFNA1, WFS1 [ENSGOOOOO 165474, ENSGOOOOO 121742, ENSG00000242866, ENSG00000131504, ENSG00000109501] (autosomal dominant hearing impairment); POU3F3 [ENSGOOOOO 198914] (nonsyndromic hearing loss), CEP290 [ENSGOOOOO 198707] (LCA10), COL7A1 [ENSG00000114270] (Epidermolysis bullosa), ATP7B [ENSG00000123191] (Wilson’s disease), ABCA4 [ENSGOOOOO 198691] (Stargardt’s disease), OTOF [ENSG00000115155] (Otoferlin syndrome).

[0091] In some embodiments, the exonic domain can be codon optimized. In some embodiments, the exonic domain can be codon optimized that can increase the stability, translation, or other desirable features.

[0092] In addition to nucleic acid sequences derived or isolated from human genes, exonic domains can comprise nucleic acid sequences derived or isolated from other organisms in order to alter the stability, translation, processing, or localization of a target RNA. In some embodiments, exonic domain derived or isolated from non-human sources can include, without limitation, nucleic acid sequences that increase protein production such as those derived or isolated from Woodchuck Hepatitis Virus (WHV) Post-transcriptional Regulatory Element (WPRE), triplex from MALAT1, the PRE of Hepatitis B virus (HPRE), and an iron response element of the form CAGYCX (Y = U or A; X = U, C, or A).

[0093] Compositions as described herein may modulate the level of protein produced. In addition to replacing specific mutated sequences within a target RNA with non-mutated sequences, another useful operation of compositions as described herein can be increasing the production of a protein encoded by a target RNA. To that end, the one or more exonic domains can be configured to amplify a translation of a target RNA. Furthermore, exonic domains as described herein may have greater target specificity to effect therapy to the appropriate target RNA, and thereby may increase production of a protein encoded by a target RNA.

[0094] The exonic domain may comprise one or more untranslated regions that enhances a translation of the exonic domain. In some embodiments, the exonic domain further comprises a 3’ untranslated region and/or a 5’ untranslated region. In some embodiments, the untranslated region that enhances the translation of the exonic domain comprises a sequence derived or isolated from the group consisting of: Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE), triplex from MALAT1, the PRE of Hepatitis B virus (HPRE), and an iron response element.

[0095] The exonic domain may encode a regulatory element. In some embodiments, the regulatory element comprises a promoter capable of expressing the exonic domains or the transsplicing RNA molecules in a eukaryotic cell. In some embodiments, the promoter is a tissue specific promoter. In some embodiments, tissue specific expression of a trans- splicing RNA molecule can be achieved through a tissue specific promoter.

Intron ic domain

[0096] The composition provided herein can comprise nucleic acid sequences encoding one or more intronic domains. The nucleic acid may comprise RNA, DNA, a DNA/RNA hybrid, and/or comprises at least one of a nucleic acid analog, a chemically modified nucleic acid, or a chimera composed of two or more nucleic acids or nucleic acid analogs. The nucleic acid may comprise a DNA encoding the one or more intronic domains. The one or more intronic domains may be transcribed into RNA. The nucleic acid comprising DNA encoding the one or more intronic domains may be transcribed into a trans-splicing RNA. The nucleic acid may comprise an RNA encoding the one or more intronic domains. The intronic domain may be configured to promote RNA splicing of the one or more exonic domains of the trans-splicing RNA molecule or a portion thereof. In some embodiments, the intronic domains can carry binding sites that are preferentially targeted by RNA-binding proteins with disease-causing mutations. In some embodiments, the dissociation constant of these mutated RNA-binding proteins and the intronic domain can be lower than the dissociation constant of the non-mutated RNA-binding protein and the intronic domain.

[0097] In some embodiments, the intronic domains further comprise one or more nucleic acid sequences configured to enhance the trans-splicing of the one or more exonic domains. In some embodiments, the one or more sequences configured to enhance the trans-splicing of the exonic domains can be trans-splicing enhancer sequences (or trans-splicing enhancing sequences). In some embodiments, the one or more sequences may be configured to bind an engineered U1 snRNA (ESM). In some embodiments, the ESM may comprise an engineered small nuclear RNA (esnRNA). In some embodiments, the one or more sequences can comprise binding sites that are preferentially targeted by an engineered snRNA. In some embodiments, the engineered small nuclear RNA can be a modified version of U1 snRNA. In some embodiments, this modified U1 snRNA can increase the trans-splicing efficiency of the trans-splicing nucleic acid molecule. In some embodiments, an engineered snRNA (esnRNA) disclosed herein includes any of the esnRNAs described in US 2024/0011026 Al, which is incorporated herein by reference in its entirety for all purposes.

[0098] In some embodiments, the trans-splicing enhancer sequences comprise 5’- X1X2X3X4X5X6-3’ wherein Xi is uracil (U) or guanine (G); X2 is adenine (A), uracil (U) or guanine (G); X3 is adenine (A), uracil (U) and guanine (G); X4 is adenine (A), uracil (U), cytosine (C) or guanine (G); X5 is adenine (A), cytosine (C), uracil (U) or guanine (G); and Xf> is adenine (A), uracil (U) or guanine (G).

[0099] In some embodiments, the trans-splicing enhancer sequences comprise 5’- X1X2X3X4X5X6-3’ wherein; Xi is selected from the group including adenine (A), uracil (U) and guanine (G); X2 is selected from the group including adenine (A), uracil (U) and guanine (G); X3 is selected from the group including adenine (A), uracil (U) and guanine (G); X4 is selected from the group including adenine (A), uracil (U) and guanine (G); X5 is selected from the group including adenine (A), uracil (U) and guanine (G); and X<> is selected from the group including uracil (U) and guanine (G). [0100] In some embodiments, the trans-splicing enhancer sequences comprise 5’- X1X2X3X4X5X6-3’ wherein; Xi is selected from the group including adenine (A), uracil (U) and guanine (G); X2 is selected from the group including uracil (U) and guanine (G); X3 is selected from the group including adenine (A), uracil (U) and guanine (G); X4 is selected from the group including uracil (U) and guanine (G); X5 is selected from the group including uracil (U) and guanine (G); and Xe is selected from the group including uracil (U) and guanine (G).

[0101] In some embodiments, the trans-splicing enhancing sequences (trans-splicing enhancer sequences) described herein may include any sequences that promote trans-splicing in an efficient manner. In some embodiments, trans-splicing enhancer sequences can comprise any one or more of TTACGG (UUACGG in RNA sequence), TAACGG (UAACGG in RNA sequence), GGGTTT (GGGUUU in RNA sequence), GTTTTG in (GUUUUG RNA sequence), GGTTTT (GGUUUU in RNA sequence), GGTTTG (GGUUUG in RNA sequence), GGTTGG (GGUUGG in RNA sequence), GTTAGG (GUUAGG in RNA sequence), TGGTTG (UGGUUG in RNA sequence), GGGTAG (GGGUAG in RNA sequence), GGTAGG (GGUAGG in RNA sequence), GGTAGT (GGUAGU in RNA sequence), GTAGTT (GUAGUU in RNA sequence), GTTGGT (GUUGGU in RNA sequence), GTGGTT (GUGGUU in RNA sequence), GGTGGT (GGUGGU in RNA sequence), TGGTGG (UGGUGG in RNA sequence), TTGGTG (UUGGUG in RNA sequence), GTAAGG (GUAAGG in RNA sequence), TAAGGG (UAAGGG in RNA sequence), TTAGGG (UUAGGG in RNA sequence), TAGGGG (UAGGGG in RNA sequence), TTGGGG (UUGGGG in RNA sequence), GTTGGG (GUUGGG in RNA sequence), GTAGGG (GUAGGG in RNA sequence), TATTGG (UAUUGG in RNA sequence), TGTTGG (UGUUGG in RNA sequence), TATGGG (UAUGGG in RNA sequence), TTTGGG (UUUGGG in RNA sequence), TGTGGG (UGUGGG in RNA sequence), TTGTGG (UUGUGG in RNA sequence), GAGTGT (GAGUGU in RNA sequence), GAGGTA (GAGGUA in RNA sequence), GGAGGT (GGAGGU in RNA sequence), TGGGAG (UGGGAG in RNA sequence), GGGGTG (GGGGUG in RNA sequence), GGGGGA (GGGGGA in RNA sequence), GGGGGT (GGGGGU in RNA sequence), GGGGTA (GGGGUA in RNA sequence), GGGAGG (GGGAGG in RNA sequence), GGGTGG (GGGUGG in RNA sequence), GGAGGG (GGAGGG in RNA sequence), GGTGGG (GGUGGG in RNA sequence), GAGGGG (GAGGGG in RNA sequence), GTGGGG (GUGGGG in RNA sequence), GAGTGG (GAGUGG in RNA sequence), GTATGG (GUAUGG in RNA sequence), GGTATT (GGUAUU in RNA sequence), GTATTT (GUAUUU in RNA sequence), GTATTG (GUAUUG in RNA sequence), AGTTTA (AGUUUA in RNA sequence), AGGTTA (AGGUUA in RNA sequence), GTAACG (GUAACG in RNA sequence), AGGTAA (AGGUAA in RNA sequence), GGTAAG (GGUAAG in RNA sequence), TGGGGG (UGGGGG in RNA sequence), AGGGTT (AGGGUU in RNA sequence), AGGTTG (AGGUUG in RNA sequence), AGGTAG (AGGUAG in RNA sequence), ATTTGG (AUUUGG in RNA sequence), AGTTGG (AGUUGG in RNA sequence), TCTGGG (UCUGGG in RNA sequence), AGAGTG (AGAGUG in RNA sequence), AGAGGG (AGAGGG in RNA sequence), AGTGTG (AGUGUG in RNA sequence), AGAGGT (AGAGGU in RNA sequence), AGGGAG (AGGGAG in RNA sequence), AGGGTG (AGGGUG in RNA sequence), AGGGGG (AGGGGG in RNA sequence), AGGGGT (AGGGGU in RNA sequence), AGTGGG (AGUGGG in RNA sequence), AGTATG (AGUAUG in RNA sequence), AGGTAT (AGGUAU in RNA sequence), GTATTC (GUAUUC in RNA sequence), and GGTAAC (GGUAAC in RNA sequence).

[0102] In some embodiments, none, some, or all, of the thymidine bases of the trans-splicing enhancing sequences may be replaced with uracil.

Nuclear retention domain

[0103] The compositions provided herein can further comprise one or more nucleic acid sequences encoding or comprising a nuclear retention domain. The nucleic acid may comprise RNA, DNA, a DNA/RNA hybrid, and/or comprises at least one of a nucleic acid analog, a chemically modified nucleic acid, or a chimera composed of two or more nucleic acids or nucleic acid analogs. The nucleic acid sequence may comprise RNA. The nucleic acid sequence comprising RNA may comprise the nuclear retention domain. In some embodiments, the nucleic acid sequence may comprise DNA encoding a nuclear retention domain. In some embodiments, the DNA encoding the nuclear retention domain may be transcribed into a trans-splicing RNA molecule or a portion thereof. The nuclear retention domain can increase the accumulation of an exonic domain in the nucleus or within specific structures in the nucleus, such as nuclear speckles or paraspeckles. In some embodiments, the nuclear retention domains are configured to promote the activity of trans-splicing of the exonic domains into the target RNA. In some embodiments, the nuclear retention domain is configured to promote the occurrence of trans- splicing events. In some embodiments, the nuclear retention domain is configured to reduce the translation of the trans splicing RNA. [0104] In some embodiments, the trans-splicing molecule (e.g., trans-splicing RNA molecule) or a portion thereof comprises a nuclear retention domain. In some embodiments, the trans-splicing molecule provided herein does not comprise a nuclear retention domain. In some embodiments, nuclear retention domains that increase trans-splicing activity or trans-splicing occurrence can also increase the levels of trans-splicing molecules within the nuclei. In some embodiments, a nuclear retention domain is derived or isolated from an mRNA, long noncoding RNAs, or synthetic sequences that can alter the localization of varied transcript types within the cellular nucleus. In some embodiments, the nuclear retention domain function specifically within the context of RNA trans-splicing. In some embodiments, a localization sequence described herein can function universally (e.g., in any systems).

[0105] The nuclear retention domain is configured to promote transport of the trans-splicing nucleic acid molecule to the cellular nucleus or to specific locations within the cellular nucleus. The nuclear retention domain may comprise one or more localization sequences that bind to enzymes involved in transcription (such as polymerase II or transcription-associated enzymes), RNA splicing, or the formation of nuclear speckles. There exist various means to promote RNA trans-splicing and the present disclosure focuses on RNA trans-splicing that is mediated by the cellular spliceosome. As the components on the spliceosome may be located inside and within the cellular nucleus, the nuclear retention domain is configured to increase RNA trans-splicing activity by promoting accumulation of the RNA trans-splicing molecule to the location of the spliceosome. In other embodiments, the present disclosure provides a composition comprising a nucleic acid sequence encoding the trans-splicing nucleic acid molecule.

[0106] In some embodiments, the nuclear retention domain can carry sequences that promote nuclear localization of the trans-splicing molecule and is derived or isolated from a gene selected from the group consisting of: CDKN2B-AS1 [NR_003529]; BANCR [NR_047671]; CASC15 [NR_015410]; CRNDE [NR_034105]; EMX2OS [NR_002791]; EVF2 [NR_015448]; FENDRR [NR.036444]; FTX [NR_028379]; GAS5 [NR_002578]; HOTAIR [NR_003716]; HOTAIRM1 [NR.038366]; HOXA-AS3 [NR_038832]; HOXA11-AS [NR_002795]; JPX [NR.024582]; LHX5-AS1 [NR_126425]; LINC01578 [NR_037600];

LINC00261 [NR_001558]; MALAT1 [NR_002819.4]; MEG3 [NR_046473]; TUNAR [NR_038861]; MIAT [NR_033320]; NEAT1 [NR.028272]; NR2F1-AS1 [NR_021490]; LINC- PINT [NR_015431]; PSMA3-AS1 [NR.029434]; EMX2OS [ENSG00000229847]; PVT1 [NR_003367]; MEG8 [NR_024149]; RMST [NR_024037]; SENCR [NR_038908]; SIX3-AS1 [NR_103786]; SOX21-AS1 [NR_046514]; TERC [NR_001566]; TUG1 [NR_002323]; XIST [NR_001564], malatl [NR.002847.3], Nfxl [NM.023739.3], Ogt [NM_139144.4], Nlrp6 [NM_133946.2], Mlxipl [NM.021455.5], Leng8 [NM_001374609.1], Gcgr [NM_008101.2], Gck [NM_001287386.1], Acly [NM_001199296.1], Ccnll [NM_001355433.1], Ccnl2 [NM_207678.2], Chkb [NM_007692.6].

[0107] In some embodiments, the nuclear retention domain can bind to polymerase II and is derived or isolated from an aptamer or long noncoding RNA.

[0108] In some embodiments, the nuclear retention domain is derived or isolated from a short interspersed element (SINE). In some embodiments, the SINE is derived or isolated from a gene selected from the group consisting of: ENSMUST00000064097, ENSMUST00000066988, ENSMUST00000074862, ENSMUST00000093950, ENSMUST00000095448, ENSMUST00000099693, ENSMUST00000105109, ENSMUST00000108741, ENSMUST00000109431, ENSMUST00000123368, ENSMUST00000124068, ENSMUST00000124095, ENSMUST00000124363, ENSMUST00000124434, ENSMUST00000124813, ENSMUST00000124848, ENSMUST00000125374, ENSMUST00000126063, ENSMUST00000126467, ENSMUST00000127001, ENSMUST00000127328, ENSMUST00000128305, ENSMUST00000129082, ENSMUST00000129910, ENSMUST00000130092, ENSMUST00000130362, ENSMUST00000130582, ENSMUST00000130679, ENSMUST00000131042, ENSMUST00000132070, ENSMUST00000132337, ENSMUST00000132370, ENSMUST00000132414, ENSMUST00000133960, ENSMUST00000134264, ENSMUST00000134795, ENSMUST00000134921, ENSMUST00000135423, ENSMUST00000135564, ENSMUST00000135987, ENSMUST00000136555, ENSMUST00000136749, ENSMUST00000137629, ENSMUST00000137706, ENSMUST00000137776, ENSMUST00000138291, ENSMUST00000138295, ENSMUST00000138574, ENSMUST00000139190, ENSMUST00000139424, ENSMUST00000139529, ENSMUST00000139576, ENSMUST00000139973, ENSMUST00000140009, ENSMUST00000140203, ENSMUST00000140298, ENSMUST00000141088, ENSMUST00000141452, ENSMUST00000141869, ENSMUST00000142279, ENSMUST00000142569, ENSMUST00000142581, ENSMUST00000143133, ENSMUST00000143260, ENSMUST00000143346, ENSMUST00000143649, ENSMUST00000143964, ENSMUST00000144006, ENSMUST00000144043, ENSMUST00000144368, ENSMUST00000144607, ENSMUST00000145549, ENSMUST00000146043, ENSMUST00000146372, ENSMUST00000146404, ENSMUST00000146531, ENSMUST00000146587, ENSMUST00000146644, ENSMUST00000146690, ENSMUST00000146963, ENSMUSTOOOOO 147541 , ENSMUST00000147722, ENSMUST00000148405, ENSMUST00000148534, ENSMUST00000148548, ENSMUST00000149025, ENSMUST00000149382, ENSMUSTOOOOO 149481, ENSMUST00000149618, ENSMUST00000149815, ENSMUST00000150171, ENSMUSTOOOOO 150265, ENSMUSTOOOOO 150455, ENSMUSTOOOOO 150482, ENSMUSTOOOOO 150628, ENSMUST00000151038, ENSMUST00000151599, ENSMUST00000151979, ENSMUSTOOOOO 152025 , ENSMUSTOOOOO 152172, ENSMUSTOOOOO 152439, ENSMUST00000152815, ENSMUSTOOOOO 152825, ENSMUST00000152987, ENSMUST00000153589, ENSMUST00000153817, ENSMUSTOOOOO 154085, ENSMUSTOOOOO 155540, ENSMUST00000155758, ENSMUST00000156149, ENSMUSTOOOOO 156150, ENSMUSTOOOOO 156331, ENSMUSTOOOOO 156350, ENSMUST00000156633, ENSMUSTOOOOO 162565, ENSMUSTOOOOO 163052, ENSMUSTOOOOO 163302, ENSMUSTOOOOO 164379, ENSMUST00000165505, ENSMUST00000170933, ENSMUST00000172285, ENSMUST00000172817, ENSMUST00000172838, ENSMUST00000174057, ENSMUST00000174630, ENSMUST00000174768, ENSMUSTOOOOO 176201, ENSMUST00000176366, ENSMUST00000177104, ENSMUST00000177482, ENSMUST00000178424, ENSMUST00000178920, ENSMUST00000179324, ENSMUSTOOOOO 180379, ENSMUST00000180382, ENSMUST00000180383, ENSMUSTOOOOO 180404, ENSMUST00000180410, ENSMUSTOOOOO 180411, ENSMUSTOOOOO 180426, ENSMUSTOOOOO 180434, ENSMUSTOOOOO 180445 , ENSMUSTOOOOO 180452, ENSMUSTOOOOO 180466, ENSMUSTOOOOO 180467 , ENSMUST00000180468, ENSMUSTOOOOO 180477, ENSMUST00000180485, ENSMUSTOOOOO 180495, ENSMUSTOOOOO 180505, ENSMUSTOOOOO 180506, ENSMUSTOOOOO 180509, ENSMUST00000180512, ENSMUST00000180518, ENSMUST00000180527, ENSMUSTOOOOO 180529, ENSMUSTOOOOO 180534, ENSMUST00000180538, ENSMUST00000180558, ENSMUSTOOOOO 180562, ENSMUSTOOOOO 180576, ENSMUST00000180586, ENSMUSTOOOOO 180590, ENSMUST00000180595, ENSMUST00000180598, ENSMUSTOOOOO 180599, ENSMUSTOOOOO 180601, ENSMUSTOOOOO 180609, ENSMUST00000180613, ENSMUSTOOOOO 180623 , ENSMUSTOOOOO 180650, ENSMUST00000180653, ENSMUSTOOOOO 180670, ENSMUSTOOOOO 180671, ENSMUSTOOOOO 180679, ENSMUSTOOOOO 180682, ENSMUST00000180685, ENSMUST00000180691, ENSMUST00000180693, ENSMUSTOOOOO 180712, ENSMUSTOOOOO 180732, ENSMUST00000180733, ENSMUST00000180738, ENSMUSTOOOOO 180741, ENSMUSTOOOOO 180748, ENSMUSTOOOOO 180750, ENSMUSTOOOOO 180751, ENSMUSTOOOOO 180779, ENSMUST00000180783, ENSMUST00000180785, ENSMUST00000180797, ENSMUSTOOOOO 180800, ENSMUSTOOOOO 180807, ENSMUSTOOOOO 180809, ENSMUST00000180812, ENSMUST00000180815, ENSMUSTOOOOO 180832, ENSMUSTOOOOO 180834, ENSMUSTOOOOO 180841, ENSMUSTOOOOO 180842, ENSMUST00000180855, ENSMUSTOOOOO 180860, ENSMUSTOOOOO 180864, ENSMUST00000180865, ENSMUST00000180875, ENSMUSTOOOOO 180876, ENSMUST00000180882, ENSMUSTOOOOO 180892, ENSMUSTOOOOO 180896, ENSMUSTOOOOO 180908, ENSMUST00000180917, ENSMUSTOOOOO 180926, ENSMUSTOOOOO 180927 , ENSMUSTOOOOO 180936, ENSMUSTOOOOO 180942, ENSMUSTOOOOO 180969, ENSMUSTOOOOO 180970, ENSMUST00000180975, ENSMUSTOOOOO 180981, ENSMUST00000181000, ENSMUST00000181003, ENSMUST00000181005, ENSMUST00000181020, ENSMUSTOOOOO 181022, ENSMUST00000181029, ENSMUST00000181030, ENSMUSTOOOOO 181041, ENSMUST00000181052, ENSMUST00000181056, ENSMUST00000181066, ENSMUST00000181073, ENSMUST00000181083, ENSMUST00000181085, ENSMUST00000181090, ENSMUST00000181097, ENSMUST00000181106, ENSMUST00000181113, ENSMUST00000181119, ENSMUSTOOOOO 181124, ENSMUST00000181125, ENSMUST00000181133, ENSMUSTOOOOO 181140, ENSMUSTOOOOO 181144, ENSMUST00000181148, ENSMUST00000181149, ENSMUST00000181152, ENSMUST00000181153, ENSMUST00000181160, ENSMUST00000181167, ENSMUST00000181175, ENSMUSTOOOOO18118O, ENSMUST00000181191, ENSMUST00000181193, ENSMUSTOOOOO 181197, ENSMUST00000181200, ENSMUST00000181203, ENSMUST00000181206, ENSMUST00000181207, ENSMUSTOOOOO 181220, ENSMUST00000181230, ENSMUST00000181255, ENSMUSTOOOOO 181262, ENSMUST00000181265, ENSMUSTOOOOO 181270, ENSMUSTOOOOO 181274, ENSMUST00000181301, ENSMUST00000181302, ENSMUST00000181303, ENSMUST00000181304, ENSMUST00000181305, ENSMUSTOOOOO 181307, ENSMUST00000181311, ENSMUST00000181315, ENSMUST00000181317, ENSMUST00000181328, ENSMUST00000181371, ENSMUST00000181382, ENSMUST00000181395, ENSMUST00000181400, ENSMUST00000181405, ENSMUST00000181416, ENSMUST00000181418, ENSMUST00000181425, ENSMUST00000181426, ENSMUST00000181440, ENSMUST00000181453, ENSMUST00000181454, ENSMUST00000181457, ENSMUST00000181458, ENSMUST00000181460, ENSMUST00000181462, ENSMUST00000181481, ENSMUST00000181482, ENSMUST00000181486, ENSMUST00000181491, ENSMUST00000181498, ENSMUST00000181499, ENSMUST00000181500, ENSMUST00000181503, ENSMUST00000181506, ENSMUST00000181522, ENSMUST00000181526, ENSMUST00000181531, ENSMUST00000181534, ENSMUST00000181538, ENSMUST00000181539, ENSMUST00000181540, ENSMUST00000181546, ENSMUST00000181552, ENSMUST00000181555, ENSMUST00000181556, ENSMUST00000181561, ENSMUST00000181570, ENSMUST00000181574, ENSMUST00000181576, ENSMUST00000181578, ENSMUST00000181587, ENSMUST00000181612, ENSMUST00000181617, ENSMUST00000181631, ENSMUST00000181637, ENSMUST00000181664, ENSMUST00000181668, ENSMUST00000181680, ENSMUST00000181682, ENSMUST00000181687, ENSMUST00000181706, ENSMUST00000181713, ENSMUST00000181717, ENSMUST00000181719, ENSMUST00000181720, ENSMUST00000181723, ENSMUST00000181727, ENSMUST00000181729, ENSMUST00000181732, ENSMUST00000181746, ENSMUST00000181765, ENSMUST00000181769, ENSMUST00000181771, ENSMUSTOOOOO1818O1, ENSMUST00000181803, ENSMUST00000181805, ENSMUST00000181807, ENSMUSTOOOOO181811, ENSMUST00000181831, ENSMUST00000181842, ENSMUST00000181846, ENSMUST00000181858, ENSMUST00000181866, ENSMUST00000181872, ENSMUST00000181875, ENSMUST00000181885, ENSMUST00000181890, ENSMUST00000181891, ENSMUST00000181915, ENSMUST00000181918, ENSMUST00000181920, ENSMUST00000181925, ENSMUST00000181928, ENSMUSTOOOOO 181942, ENSMUST00000181973.

[0109] In some embodiments, the nuclear retention domain can bind to proteins involved in transcription. In some embodiments, the nuclear retention domain can bind to proteins involved in RNA splicing. [0110] In some embodiments, the nuclear retention domain can promote accumulation of the trans-splicing molecule in nuclear paraspeckles. In some embodiments, the nuclear retention domain is configured to promote accumulation of the trans-splicing molecule in nuclear paraspeckles can be derived or isolated from a gene selected from the group consisting of: Inc- LTBP3-10 [lnc-LTBP3-10], SLC29A2 [ENSG00000174669.12], SNHG1 [ENSG00000255717.7], MUS81 [ENSG00000172732.12], TCIRG1 [ENSG00000110719.10], INPPL1 [ENSG00000165458.14], lnc-ANAPCl l-7 [lnc-ANAPCl l-7], IL18BP [ENSG00000137496.18], POLA2 [ENSG00000014138.9], PCNX3 [ENSG00000197136.4], PC [ENSG00000173599.15], RBM4 [ENSG00000173933.20], lnc-KCNK7-6 [lnc-KCNK7-6], EML3 [ENSG00000149499.i l], PGGHG [ENSG00000142102.16], RBM14 [ENSG00000239306.4], LTBP3 [ENSG00000168056.16], ATG2A [ENSG00000110046.13], XLOC_026224 [XLOC_026224], HERC2P2 [ENSG00000276550.4], WDR90 [ENSG00000161996.19], lnc-LTBP3-2 [lnc-LTBP3-2], LENG8 [ENSG00000167615.16], TPCN2 [ENSG00000162341.18], lnc-TCIRGl-1 [lnc-TCIRGl-1], ATG16L2 [ENSG00000168010.i l], MROH1 [ENSG00000179832.17], CCDC57 [ENSG00000176155.19], lnc-LTBP3-l l [lnc-LTBP3-l l], PIDD1 [ENSG00000177595.18], Inc- VSTM5-1 [lnc-VSTM5-l], NEAT1 [ENSG00000245532.9], XLOC_079850 [XLOC_079850], XLOC_028656 [XLOC_028656], DNHD1 [ENSG00000179532.12], ABCA7 [ENSG00000064687.12], XLOC_000636 [XLOC_000636], MAN2C1 [ENSG00000140400.17], lnc-SSH3-5 [lnc-SSH3-5], MIRLET7BHG [ENSG00000197182.14], MAMDC4 [ENSG00000177943.14], NAA40 [ENSG00000110583.13], ANKRD13D [ENSG00000172932.14], lnc-NUMAl-3 [lnc-NUMAl-3], ADAMTS10 [ENSG00000142303.14], XLOC_083799 [XLOC_083799], ARHGEF17 [ENSG00000110237.5], CDC42BPG [ENSG00000171219.9], SNAPC4 [ENSG00000165684.4], lnc-CFLl-1 [lnc-CFLl-1], B4GALNT4 [ENSG00000182272.12], XLOC_027567 [XLOC_027567], XLOC_000644 [XLOC_000644], XLOC_024022 [XLOC_024022], LTO1 [ENSG00000149716.12], AC064843.1 [ENSG00000286621.1], CHRND [ENSG00000135902.10], ASPSCR1 [ENSG00000169696.16], RAD9A [ENSG00000172613.8], lnc-RTN4R-l [lnc-RTN4R-l], lnc-MRPLl l-1 [lnc-MRPLl l-1], SSH3 [ENSG00000172830.13], XLOC_000637 [XLOC_000637], AP000873.2 [ENSG00000247137.9], lnc-TRPTl-4 [lnc-TRPTl-4], XLOC_027568 [XLOC_027568], LINC01503 [ENSG00000233901.6], RNASEH2C [ENSG00000172922.9], XLOC_000634 [XLOC_000634], MYO7A [ENSG00000137474.22], XLOC_000633 [XLOC_000633], Inc- BCL3-1 [lnc-BCL3-l], MTMR9LP [ENSG00000220785.7], AP5B1 [ENSG00000254470.3], lnc-EDFl-2 [lnc-EDFl-2], lnc-UNC93Bl-l [lnc-UNC93Bl-l], G0LGA8B [ENSG00000215252.il], MSH5 [ENSG00000204410.15], AP003119.1 [ENSG00000254632.2], GUSBP11 [ENSG00000228315.12], RPS6KB2 [ENSG00000175634.15], EME2 [ENSG00000197774.13], XLOC_028057 [XLOC_028057], FRMD8 [ENSG00000126391.14], lnc-OGFOD3-l [lnc-OGFOD3-l], XLOC_152482 [XLOC_152482], XLOC_028434 [XLOC_028434], ZNF276 [ENSG00000158805.12], AP000944.5 [ENSG00000285816.1], NRBP2 [ENSG00000185189.18], NDOR1 [ENSG00000188566.13], lnc-PHYHDl-1 [lnc-PHYHDl-1], lnc-RECQL4-3 [lnc-RECQL4-3], lnc-UAPlLl-4 [lnc-UAPlLl-4], MSH5-SAPCD1 [ENSG00000255152.8], lnc-P2RY6-l [Inc- P2RY6-1], RELT [ENSG00000054967.13], CPNE7 [ENSG00000178773.15], XLOC_028557 [XLOC_028557], XLOC_156663 [XLOC_156663], CORO6 [ENSG00000167549.18], RTEL1 [ENSG00000258366.8], MIR34AHG [ENSG00000228526.7], STPG3-AS1 [ENSG00000275549.1], lnc-WFIKKN2-4 [lnc-WFIKKN2-4], SYNGAP1 [ENSG00000197283.17], LRRC45 [ENSG00000169683.8], KIAA0895L [ENSG00000196123.13], PNKP [ENSG00000039650.12], lnc-EIFlAD-5 [lnc-EIFlAD-5], TM7SF2 [ENSG00000149809.15], NSUN5P2 [ENSG00000106133.18], lnc-POLR2L-l [Inc- POLR2L-1], Inc-PPP1R27-1 [Inc-PPP1R27-1], AC110285.2 [ENSG00000262877.5], Inc- LRRC32-5 [lnc-LRRC32-5], AC131009.4 [ENSG00000279283.1], BBS1 [ENSG00000174483.20], XLOC_061408 [XLOC_061408], lnc-SERPINHl-3 [Inc-SERPINHl- 3], AC027601.6 [ENSG00000287431.1], lnc-NFAMl-3 [lnc-NFAMl-3], EXD3 [ENSG00000187609.16], AC009022.1 [ENSG00000196696.12], MC1R [ENSG00000258839.3], PKD1P6 [ENSG00000250251.6], lnc-KLHL35-6 [lnc-KLHL35-6], Z97832.2 [ENSG00000272374.1], C19orf25 [ENSG00000119559.16], lnc-TMEM138-3 [Inc- TMEM138-3], AL031595.3 [ENSG00000280434.1], lnc-LRRC56-3 [lnc-LRRC56-3], Inc- STIP1-2 [lnc-STIPl-2], XLOC_095699 [XLOC_095699], SSSCA1-AS1 [ENSG00000260233.3], NPDC1 [ENSG00000107281.10], Inc-NR1D1-1 [Inc-NR1D1-1], Inc- RPL12-1 [lnc-RPL12-l], lnc-MRPL49-l [lnc-MRPL49-l], XLOC_061398 [XLOC_061398], TOB1-AS1 [ENSG00000229980.5], AC127502.1 [ENSG00000215302.8], XLOC_149046 [XLOC_ 149046], lnc-TRMT112-4 [Inc-TRMT 112-4], LINC02593 [ENSG00000223764.2], KLHL17 [ENSG00000187961.14], lnc-KLHL35-7 [lnc-KLHL35-7], lnc-TMEM258-2 [Inc- TMEM258-2], AP002495.1 [ENSG00000254469.7], XLOC_024025 [XLOC_024025], GPSM1 [ENSG00000160360.13], XLOC_152839 [XLOC_152839], LBHD1 [ENSG00000162194.12], GATD1 [ENSG00000177225.17], XLOC_149045 [XLOC_ 149045], LENG8-AS1 [ENSG00000226696.6], MAP4K2 [ENSG00000168067.12], Cllorf80 [ENSG00000173715.16], MAPK8IP3 [ENSG00000138834.12], XLOC_090526 [XLOC_090526], KIFC2 [ENSG00000167702.12], LRP5L [ENSG00000100068.13], SEC31B [ENSG00000075826.17], XLOC_024171 [XLOC_024171], PPP2R5B [ENSG00000068971.14], lnc-GIPC3-3 [lnc-GIPC3-3], AC020916.1 [ENSG00000267519.6], XLOC-156901 [XLOC_ 156901], AP006333.1 [ENSG00000256341.1], lnc-ZNF778-3 [Inc- ZNF778-3], lnc-LAMA5-l [lnc-LAMA5-l], lnc-TMEM106A-3 [lnc-TMEM106A-3], Inc- ACER3-1 [lnc-ACER3-l], RHPN1 [ENSG00000158106.14], XLOC_028558 [XLOC_028558], XLGC-088401 [XLOC_088401], BX255925.3 [ENSG00000284976.1], GUCY2EP [ENSG00000204529.4], XLOC_152506 [XLOC_ 152506], NOXA1 [ENSG00000188747.8], lnc-ARRDCl-2 [lnc-ARRDCl-2], XLOC_145191 [XLOC_145191], BSCL2 [ENSG00000168000.14], lnc-MACRODl-1 [lnc-MACRODl-1], AL162586.1 [ENSG00000225032.5], AP000944.7 [ENSG00000287917.1], AC091196.1 [ENSG00000285581.1], ZNRD2 [ENSG00000173465.8], XLOC_026268 [XLOC_026268], OSBPL7 [ENSG00000006025.12], lnc-SSH3-4 [lnc-SSH3-4], C9orfl06 [ENSG00000179082.3], AP000437.1 [ENSG00000279549.1], lnc-NCOA3-14 [lnc-NCOA3- 14], NADSYN1 [ENSG00000172890.13], XLGC_060204 [XLOC_060204], lnc-SHANK2-l [lnc-SHANK2-l], MEGF6 [ENSG00000162591.16], AC099811.1 [ENSG00000236194.3], ME3 [ENSG00000151376.16], XLOC_028655 [XLOC_028655], GDPD5 [ENSG00000158555.15], lnc-SPDYC-2 [lnc-SPDYC-2], AC008105.3 [ENSG00000267121.6], lnc-NCOA3-21 [lnc-NCOA3-21], lnc-FENl-6 [lnc-FENl-6], lnc-HYOUl-1 [lnc-HYOUl-1], AC102953.2 [ENSG00000273230.1], XLOC_095073 [XLOC_095073], LINC00235 [ENSG00000277142.1], AL355987.4 [ENSG00000273066.5], XLOC_152404 [XLOC_152404], lnc-CDK12-l [lnc-CDK12-l], XLGC_028004 [XLOC_028004], Inc- CCDC 154-2 [Inc-CCDC 154-2], lnc-CCDC87-l [lnc-CCDC87-l], INPP5E [ENSG00000148384.13], XLOC_021222 [XLOC_021222], AJM1 [ENSG00000232434.2], HSF4 [ENSG00000102878.16], LINC00313 [ENSG00000185186.10], lnc-UNC93Bl-7 [Inc- UNC93B1-7], lnc-PIDDl-2 [lnc-PIDDl-2], lnc-CSNKlG2-5 [lnc-CSNKlG2-5], Inc- UNC93B1-5 [lnc-UNC93Bl-5], AP006621.3 [ENSG00000255284.2], CCDC78 [ENSG00000162004.17], lnc-HAAO-7 [lnc-HAAO-7], EFEMP2 [ENSG00000172638.13], XLGC-000635 [XLOC_000635], XLOC_147952 [XLOC_147952], lnc-PKNOXl-1 [Inc- PKNOX1-1], lnc-LTBP3-9 [lnc-LTBP3-9], AC008895.1 [ENSG00000279948.1], Inc- TBC1D3H-7 [lnc-TBClD3H-7], lnc-TMEM250-3 [lnc-TMEM250-3], lnc-CDC42EP2-l [Inc- CDC42EP2-1], AC08774E1 [ENSG00000262580.5], XLOC_156972 [XLOC_156972], Inc-PC- 3 [lnc-PC-3], AC090589.3 [ENSG00000270060.1], XLOC_045084 [XLOC_045084], TIAF1 [ENSG00000221995.5], lnc-CYBA-4 [lnc-CYBA-4], lnc-SLCHA2-7 [lnc-SLCHA2-7], AC141586.1 [ENSG00000215154.6], AP003559.1 [ENSG00000256443.1], XLOC_095076 [XLOC_095076], PNPLA7 [ENSG00000130653.16], lnc-RNF166-5 [Inc-RNF 166-5], XLOC_023911 [XLOC_023911], AC092127.1 [ENSG00000260417.1], lnc-TRPTl-3 [Inc- TRPT1-3], XLOC_028195 [XLOC_028195], XLOC_080106 [XLOC_080106], XLOC_026739 [XLOC_026739], lnc-NUP98-l [lnc-NUP98-l], HDAC10 [ENSGOOOOO 100429.18], DRD4 [ENSG00000069696.7], lnc-DOC2B-3 [lnc-DOC2B-3], lnc-DOLK-1 [lnc-DOLK-1], CNIH2 [ENSG00000174871.i l], RGL3 [ENSG00000205517.12], GALT [ENSG00000213930.i l], AP001107.9 [ENSG00000255468.7], lnc-MKNK2-l [lnc-MKNK2-l], AL033543.1 [ENSG00000279175.1],

[0111] In some embodiments, the nuclear retention domain can promote accumulation of the trans-splicing molecule to nuclear speckles. In some embodiments, the nuclear retention domain is configured to promote accumulation of the trans-splicing molecule to nuclear speckles can be derived or isolated from a gene selected from the group consisting of: MALAT1

[NR_002819.4], MEG3[ENSG00000214548], XLOC_003526 [ENSG00000250657]. In some embodiments, the nuclear retention domain is configured to promote accumulation of the trans- splicing molecule to nuclear speckles via binding to a protein selected from the group consisting of: SRSF1 [ENSGOOOOO 136450], SRSF2 [ENSG00000161547], SRSF3 [ENSG00000112081], SRSF4 [ENSGOOOOO 116350], SFSF6 [ENSG00000124193], SFSF7 [ENSG00000115875], SRSF10 [ENSG00000188529], SRSF11 [ENSGOOOOO 116754], CLK1 [ENSG00000013441], CLK2 [ENSGOOOOO 176444].

[0112] In some embodiments, the nuclear retention domain can promote accumulation of the trans-splicing molecule in nuclear speckles via association to a protein. In some embodiments, this protein is selected from group consisting of: ADNP [ENSG00000101126], ANXA7 [ENSG00000138279], API5 [ENSG00000166181], AQR [ENSG00000021776], ATAD2 [ENSGOOOOO 156802], BAZ1B [ENSG00000009954], BCLAF1 [ENSG00000029363], BTAF1 [ENSG00000095564], CCAR1 [ENSG00000060339], CCAR2 [ENSG00000158941], CDC5L [ENSG00000096401], CDC73 [ENSG00000134371], CDK11B [ENSG00000248333], CDK12 [ENSG00000167258], CDKN2AIP [ENSGOOOOO 168564], CHD3 [ENSG00000170004], CHD4 [ENSG00000111642], CHTF18 [ENSG00000127586], CPSF1 [ENSG00000071894], CSTF3 [ENSG00000176102], CTR9 [ENSG00000198730], CUL3 [ENSG00000036257], CUL4B [ENSG00000158290], CWC22 [ENSG00000163510], CWF19L1 [ENSG00000095485], DDX23 [ENSG00000174243], DDX39A [ENSG00000123136], DDX42 [ENSG00000198231], DDX46 [ENSG00000145833], DHX16 [ENSG00000204560], DHX38 [ENSG00000140829], DNMT1 [ENSG00000130816], ELOA [ENS G00000011007], EWSR1 [ENSG00000182944], FAF1 [ENSG00000185104], FBXO22 [ENSG00000167196], FKBP5 [ENSG00000096060], FUBP1 [ENSG00000162613], FUBP3 [ENSG00000107164], GPATCH8

[ENSG00000186566], GPS1 [ENSG00000169727], GTF3C1 [ENSG00000077235], GTF3C4 [ENSG00000125484], GTF3C5 [ENSG00000148308], HCFC1 [ENSG00000172534], HELLS [ENSG00000119969], IK [ENSG00000113141], ILF2 [ENSG00000143621], INTS13 [ENSG00000064102], KDM1A [ENSG00000004487], KHDRBS1 [ENSG00000121774], KHSRP [ENSG00000088247], LIG1 [ENSG00000105486], MATR3 [ENSG00000280987], METTL1 [ENSG00000037897], MRE11 [ENSG00000020922], MSH2 [ENSG00000095002], MSH3 [ENSG00000113318], MSH6 [ENS G00000116062], NBN [ENSG00000104320], NCBP1 [ENSG00000136937], NONO [ENSG00000147140], PAF1 [ENSG00000006712], PDS5B [ENSG00000083642], POLDI [ENSG00000062822], POLR2A [ENSG00000181222], POLR2B [ENSG00000047315], PPM1G [ENSG00000115241], PPP1R10 [ENSG00000204569], PRPF19 [ENS G00000110107], PRPF3 [ENSG00000117360], PRPF31 [ENSG00000105618], PRPF40A [ENSG00000196504], PRPF4B [ENSG00000112739], PRPF6 [ENSG00000101161], PSPC1 [ENSG00000121390], PTBP2 [ENSG00000117569], PUS7 [ENS G00000091127], RAD21 [ENSG00000164754], RAD50 [ENSG00000113522], RALY [ENSG00000125970], RBM10 [ENSG00000182872], RBM12 [ENSG00000244462], RBM14 [ENSG00000239306], RBM17 [ENSG00000134453], RBM25 [ENSG00000119707], RBM26 [ENSG00000139746], RBM4 [ENSG00000173933], RBMX [ENSG00000147274], RFC1 [ENSG00000035928], RFC4 [ENSG00000163918], RNF20 [ENSG00000155827], RNF40 [ENSG00000103549], RNMT [ENSG00000101654], RPL35A [ENSG00000182899], RPRD1B [ENSG00000101413], RPRD2 [ENSG00000163125], SAMHD1 [ENSG00000101347], SART1 [ENSG00000175467], SART3 [ENSG00000075856], SBNO1 [ENSG00000139697], SF3A1 [ENSG00000099995], SF3B1 [ENSG00000115524], SF3B2 [ENSG00000087365], SFPQ [ENSG00000116560], SIN3A [ENSG00000169375], SLC4A1AP [ENSG00000163798], SMARCC1 [ENSG00000173473], SMU1 [ENSG00000122692], SON [ENSG00000159140], STAG2 [ENSG00000101972], SUGT1 [ENSG00000165416], SUPT5H [ENSG00000196235], SUPT6H [ENSG00000109111], SYMPK [ENSG00000125755], TARDBP [ENSG00000120948], TCERG1 [ENSGOOOOO 113649], THOC2 [ENSG00000125676], THOC5 [ENSG00000100296], TP53BP1 [ENSG00000067369], TRMT1 [ENSGOOOOO 104907], TRMT1L [ENSG00000121486], TSR1 [ENSG00000167721], UBR5 [ENSGOOOOO 104517], UHRF1 [ENSG00000276043], USP39 [ENSG00000168883], USP48 [ENSG00000090686], USP7 [ENSGOOOOO 187555], WAC [ENSG00000095787], WDHD1 [ENSGOOOOO 198554], WRNIP1 [ENSGOOOOO 124535], XPO5 [ENSG00000124571], XPO7 [ENSGOOOOO 130227], XPOT [ENSGOOOOO 184575], YLPM1 [ENSGOOOOO 119596], ZC3H11A [ENSG00000058673], ZC3H14 [ENSG00000100722], ZMYND8 [ENSGOOOOO 101040], ZNF326 [ENSGOOOOO 162664],

[0113] In some embodiments, the nuclear retention domain sequence(s) can be isolated or derived from a long non-coding RNA that is involved in transcriptional regulation. In some embodiments, the long non-coding RNA comprises Air, Alpha 250/ Alpha 280, ANRIL, Betaglobin transcripts, Beta-MHC antisense transcripts, CAR Intergenic 10, CCND1 associated ncRNAs, COLD AIR, COOLAIR, DHFR upstream transcripts, Emx2os, Evf2, fbpl+ promoter RNAs, GALlO-ncRNA, H19, H19 antisense, H19 upstream conserved 1 and 2, H19 ICR ncRNAs, HOTAIRM1, HOTTIP, Hoxal las, ICR1, Kcnqlotl, Khpsla, L1PA16, LINoCRb, MEG3, Mistral, Msxlas, Nespas, ncR-Upar, PHO5 IncRNA, PHO84 antisense, pRNA, PWR1, RTL, SRG1, TEA ncRNAs, TIRlaxut, TPOlaxut, Tsix, Xist, 7SK, B2 SINE RNA, GAS5, HOTAIR, Jpx, LXRBSV, PR antisense transcripts, VL30 RNAs, Adapt33, antiPegl l, Gtl2-as, HOXA3as, HOXA6as, lincl242, lincl257, lincl368, lincl547, lincl582, lincl609, lincl610, lincRNA-p21, lincRNA-RoR b, Malatl-as, MEG9, NDM29, NEAT1, PANDA, PCAT-1 , Rian, Satlll transcripts, SNHG3, SRA, Tmevpgl, TncRNA, TUG1, or another combination thereof.

[0114] In some embodiments, the nuclear retention domain sequence(s) can be isolated or derived from a long non-coding RNA that is involved in splicing regulation. In some embodiments, the long non-coding RNA comprises MIAT, LUST, Malatl, SAF, VL30 RNAs, Zeb2NAT, or any combination thereof.

[0115] In some embodiments, the nuclear retention domain sequence(s) can be directly adjacent to an antisense domain. In some embodiments, the nuclear retention domain sequence(s) can be directly adjacent to the exonic domain.

[0116] In some embodiments, the nuclear retention domain(s) can be adjacent to a 5’ end of a trans- splicing molecule. In some embodiments, the nuclear retention domain(s) are 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 110 nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, 150 nucleotides, 160 nucleotides, 170 nucleotides, 180 nucleotides, 190 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, more than 500 nucleotides, or any number of nucleotides in between distant from the 5’ end of the transsplicing molecule.

[0117] In some embodiments, the nuclear retention domain(s) can be adjacent to the 3’ end of the trans- splicing molecule. In some embodiments, the nuclear retention domain(s) are 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 110 nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, 150 nucleotides, 160 nucleotides, 170 nucleotides, 180 nucleotides, 190 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, more than 500 nucleotides, or any number of nucleotides in between distant from the 3’ end of the transsplicing molecule.

[0118] In some embodiments, the nuclear retention domain(s) can be 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 110 nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, 150 nucleotides, 160 nucleotides, 170 nucleotides, 180 nucleotides, 190 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, more than 500 nucleotides, or any number of nucleotides in between distant from the first nucleotide of the exonic domain or antisense domain in the 5’ direction.

[0119] In some embodiments, nuclear retention domain(s) can bel nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 110 nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, 150 nucleotides, 160 nucleotides, 170 nucleotides, 180 nucleotides, 190 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, more than 500 nucleotides, or any number of nucleotides in between distant from the last nucleotide of the exonic domain or antisense domain in the 3’ direction.

[0120] In some embodiments, the trans-splicing molecule may comprise a nuclear retention domain. In some embodiments, the trans-splicing molecule may comprise 2 or more nuclear retention domains. In some embodiments, the trans-splicing molecule comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 75, 100, 200, 300 or more nuclear retention domains. [0121] Compositions comprising localization sequences disclosed herein can include any sequences that promote nuclear or subnuclear localization of trans- splicing molecules. Nonlimiting examples of localization sequences can include sequences that promote localization of trans-splicing molecules to the cellular nucleus from the cytoplasm or to specific structures within the nucleus such as nuclear speckles or paraspeckles. In some embodiments, the localization sequences can also include sequences that promote association of the trans-splicing molecule with nuclear-localized proteins and protein complexes such as the spliceosome, transcriptional proteins, or splicing factors.

Stabilization domain

[0122] The composition provided herein can comprise one or more stabilization domains to prevent or attenuate degradation of the nucleic acid molecules and/or the trans-splicing molecules provided herein. The nucleic acid may comprise RNA, DNA, a DNA/RNA hybrid, and/or may comprise at least one of a nucleic acid analog, a chemically modified nucleic acid, or a chimera composed of two or more nucleic acids or nucleic acid analogs. In some embodiments, an RNA molecule provided herein comprises one or more stabilization domains to prevent degradation of the RNA molecule. A nucleic acid molecule comprising DNA may encode the one or more stabilization domains. The DNA molecule encoding one or more stabilization domains may be transcribed into an RNA molecule comprising one or more stabilization domains (e.g., a stabilization domain in an RNA trans-splicing molecule can be a complement of the stabilization domain in the DNA molecule decoding the RNA trans-splicing molecule). Degradation of nucleic acids may be caused by, e.g., the activity of exonucleases. The exonuclease may act in the 5’ to 3’ direction or the 3’ to 5’ direction. In some embodiments, the stabilization domain protects the 5’ end of the nucleic acid molecules and/or the trans- splicing molecule provided herein. In some embodiments, the stabilization domain protects the 3’ end of the nucleic acid molecules and/or the trans-splicing molecule provided herein. In some embodiments, the stabilization domain is in a 3’ domain of an RNA trans-splicing molecule and protects the 3’ end of the RNA trans-splicing molecule from degradation.

[0123] In some embodiments, the stabilization domain comprises RNA. In some embodiments, the stabilization domain comprises DNA. In some embodiments, the stabilization domain comprising DNA encodes a stabilization domain comprising RNA. In some embodiments, the DNA molecule is transcribed into a messenger RNA. [0124] In some embodiments of the compositions of the present disclosure, the stabilization domain is derived from a flavivirus. In some embodiments, the stabilization domain is an exonuclease-resistant RNA (“xrRNA”) that block 5 ’-3’ exonuclease activity and is derived or isolated from a viral genome selected from the group consisting of: Turnip yellow mosaic virus, Apoi virus, Aroa virus, Bagaza virus, Banzi virus, Bouboui virus, Bukalasa bat virus, Cacipacore virus, Carey Island virus, Dakar bat virus, Cowbone Ridge virus, Dengue virus, Edge Hill virus, Entebbe bat virus, Gadgets Gully virus, Ilheus virus, Israel turkey meningoencephalomyelitis virus, Japanese encephalitis virus, Jugra virus, Jutiapa virus, Kadam virus, Kunjin virus, Kedougou virus, Kokobera virus, Koutango virus, Kyasanur Forest disease virus, Langat virus, Louping ill virus, Meaban virus, Modoc virus, Montana myotis leukoencephalitis virus, Murray Valley encephalitis virus, Ntaya virus, Omsk hemorrhagic fever virus, Phnom Penh bat virus, Powassan virus, Rio Bravo virus, Royal Farm virus, Saboya virus, Saint Louis encephalitis virus, Sal Vieja virus, San Perlita virus, Saumarez Reef virus, Sepik virus, Tembusu virus, Tick-borne encephalitis virus, Tyuleniy virus, Uganda S virus, Wesselsbron virus, Usutu virus, West Nile virus, Yaounde virus, Yellow fever virus, Yokose virus, Zika virus.

[0125] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from Kunjin virus. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from Kunjin virus comprise or consist of: TTAGTGAGGATGTCAGACCACGGCCATGGCGTGCCACTCTGCGGAGAGTGCAGTCT GCGACAGTGCCCCAGGAGGACTGGG (SEQ ID NO: 1). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 1. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 1. The stabilization domain may be transcribed into an RNA molecule.

[0126] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from Cell fusing agent virus. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from Cell fusing agent virus comprise or consist of: ACAGGAGCAGGGCATGAAAATGTCGGGCATGACGAACCCGCTCCCCCGAGTCCCCT GGCAACAGGGT (SEQ ID NO: 2). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 2. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 2. The stabilization domain may be transcribed into an RNA molecule.

[0127] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from Flavivirus Tick-borne encephalitis virus. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from Flavivirus Tick-bome encephalitis virus comprise or consist of: CACAGATCATGGAATGATGCGGCAGCGCGCGAGAGCAACGGGGAAGTGGTGGCAC CCGACGCACCATCCATGAAGCAATACTTCGTGAGACCCCCCCTGACCAGCAAAGGG GGCAGACCGGTCAGGGGTGAGGAATGCCCCCAGAGTGCATTACGGCAGCACGCCA GTGAGAGTGGCGACGGGAAAATGGTCGATCCCGACGTAGGGCACTCTGAAAAATTT TGTGAGACC (SEQ ID NO: 3). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 3. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 3. The stabilization domain may be transcribed into an RNA molecule.

[0128] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from Murine leukemia virus. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from Murine leukemia virus comprise or consist of: TGGAAAAAATGCGAGTGAGGGCAACTCTGGGATTAGCTCAATGGGTGTGACGACCC TACCCTTCCGCATTTGTAAATAATTGAGCCAGTCATTTCCGTAGGGAAGAGAGTTAT TCGCTCCTCTCGAGATTGAGCGGCCTGCTCCTTGGAGCATGAGATGGGAGGCCCGA A (SEQ ID NO 4). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 4. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 4. The stabilization domain may be transcribed into an RNA molecule.

[0129] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number AF346759.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number AF346759.1 comprise or consist of:

GAAAGGCAAGGTACGGATTAGCCGTAGGGGCTTGAGAACCCCCCCTCCCCACTCAT TTTATTTCCTCTATGAGGAAGG (SEQ ID NO: 5). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 5. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 5. The stabilization domain may be transcribed into an RNA molecule.

[0130] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number AF346759.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number AF346759.1 comprise or consist of:

TTTGGGCAAGGTGCAGGTTAGCTGCAGGGGCTTGAAAAACCCCCCCCCCCATTCAA GACTTTTAGTGCATTAGTT (SEQ ID NO:6). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 6. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 6. The stabilization domain may be transcribed into an RNA molecule.

[0131] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_008604.2. The sequence may comprise a DNA sequence. The sequence may be comprise RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_008604.2 comprise or consist of:

ACCCCGTAAGGAAGGACAAGGCTGTCCTTGAGTACTAACGACACTCCGGCCCCAGT TCCCAGAGCCAGGGTTTTAGCTCC (SEQ ID NO: 7). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 7. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 7. The stabilization domain may be transcribed into an RNA molecule.

[0132] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_008604.2. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_008604.2 comprise or consist of:

CGCGCGCAAGGAAGGACATGGCTGTCCTTGGGTACGAACGACACCCCGCCCCCAGT TCTCAAGGTTAGAGTTATAACCTC (SEQ ID NO: 8). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 8. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 8. The stabilization domain may be transcribed into an RNA molecule.

[0133] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_008604.2. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_008604.2 comprise or consist of:

CCATCGCAAGGGAGGATTTTCCTCGGGTACTGACCATACCCCGACCCCAGTCCGAT AGGTCATGGAATGACCCC (SEQ ID NO: 9). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 9. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 9. The stabilization domain may be transcribed into an RNA molecule.

[0134] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_008604.2. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_008604.2 comprise or consist of:

CTCCCGTAAGGAAAGCGCAAGCTTTGAGCATTGACAACGCTCCGGCCCCAGTCCCC CAGGTTATGGGAGAATAACCC (SEQ ID NO: 10). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 10. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 10. The stabilization domain may be transcribed into an RNA molecule.

[0135] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number HE574574.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number HE574574.1 comprise or consist of:

CTAGCGCAAGGAAGGAAAGTCGCAGACTACCTTGGGTGTTGACGACACTCCGCCCC CAGTCACCTTGGCCGAAGGTTAAACGGCAT (SEQ ID NO: 11). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 11. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 11. The stabilization domain may be transcribed into an RNA molecule.

[0136] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number HE574574.1. The sequence may comprise a DNA sequence. The stabilization domain may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number HE574574.1 comprise or consist of:

CCACGCAATGGGAGGCATCATTTCGCCTCCGGGTGCTGACTACTCCCCGTCCCCGTC CCTAGGTCAAGTGAATGACCCCGTGATGTTGTGATGACATCATACCAGGCTTGGCAT CCTGGCAACTGCCACCCGCAAGGGGAGGGTTTTCTAACTCTCCGGGTGTTGACGAC ACCCCGGCCCCAGTCCCCAAGGTCTTGGGAAAAAGACCCCGAAGTGTTGCAAGGAC ACTAATCACCGAAAGGTGAGGGCGCACAGGATC (SEQ ID NO: 12). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 12. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 12. The stabilization domain may be transcribed into an RNA molecule.

[0137] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number HE574574.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number HE574574.1 comprise or consist of:

CACCCGCAAGGGGAGGGTTTTCTAACTCTCCGGGTGTTGACGACACCCCGGCCCCA GTCCCCAAGGTCTTGGGAAAAAGACCCC (SEQ ID NO: 13). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 13. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 13. The stabilization domain may be transcribed into an RNA molecule.

[0138] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_012671.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_012671.1 comprise or consist of:

TCAGCGCAAGGAAGGGAAGCTTGAGGCTACCTTAGGTGGTGACGACACCTCGCCCC CAGTCTCCCGGGTTGGGGATAATACAACCTC (SEQ ID NO: 14). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 14. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 14. The stabilization domain may be transcribed into an RNA molecule.

[0139] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_012671.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_012671.1 comprise or consist of:

CCCCCGCAAGGAGGGGTGTGTGTTTCACGCCCCTGGGAGTTAACGATTCTCCGGCCC CAGTTCCTAGGTCCAGGGAGGGCCCC (SEQ ID NO: 15). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 15. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 15. The stabilization domain may be transcribed into an RNA molecule.

[0140] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_012671.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_012671.1 comprise or consist of:

CCCGCGCAAGGAAGGACACGTAAATCACGTGTTCTTGGGAGTTGACGACTCTCCGC CCCCAGTCCCCAGGTCAGGGTATGACTCC (SEQ ID NO: 16). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 16. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 16. The stabilization domain may be transcribed into an RNA molecule.

[0141] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_012671.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_012671.1 comprise or consist of:

CCACCGCAAGGGAGAGGGATCCCCTCTCGGGTTTGGACGACACCCCGGCCCCAGTC CCCCAGGTCATGGGAAAAACTGACCCC (SEQ ID NO: 17). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 17. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 17. The stabilization domain may be transcribed into an RNA molecule.

[0142] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_021069.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_021069.1 comprise or consist of:

TCAGCGCAAGGAAGGAAAGCTGGACGCTACCTTAGGTGGTGACGACACCTCGCCCC CAGTCTCCCAGGTTGGGGATCGTACAACTTC (SEQ ID NO: 18). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 18. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 18. The stabilization domain may be transcribed into an RNA molecule.

[0143] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_021069.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_021069.1 comprise or consist of:

CCCCCGCAAGGAGGGACGTGTGCATCACGTTTCTGGGAGTTAACGGCTCTCCGGCC CCAGTTCCTAGGTCCAGGTAGGATCCC (SEQ ID NO: 19). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 19. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 19. The stabilization domain may be transcribed into an RNA molecule.

[0144] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_021069.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_021069.1 comprise or consist of:

CCCGCGCAAGGAAGGATGCGATGAAAACGCTTGTCCTTGGGAGTTGACGACTCTCC GCCCCCAGTCCCCAGGTCAGGGTATGACCCC (SEQ ID NO: 20). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 20. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 20. The stabilization domain may be transcribed into an RNA molecule.

[0145] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_021069.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_021069.1 comprise or consist of:

CCACCGCAAGGGAGAGGGATTCCCTCTCGGGTGTGGACGACACCCCGGCCCCAGTC CCCTAGGTCATGGGAAAAACTGACCCC (SEQ ID NO: 21). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 21. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 21. The stabilization domain may be transcribed into an RNA molecule.

[0146] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number KX652378.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number KX652378.1 comprise or consist of:

CTAGCGCAAGGAAGGAAAGTCGCAGACTACCTTGGGTGTTGACGACACTCCGCCCC CAGTCACCTTGGCCAAAGGTTAAATGGCAT (SEQ ID NO: 22). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 22. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 22. The stabilization domain may be transcribed into an RNA molecule.

[0147] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number KX652378.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number KX652378.1 comprise or consist of:

CCCACGCAAGGGGAGGCATCATATTGCCTCCGGGTGCTGACGACACCCCGTCCCCA GTCCCTAGGTCAAGTGAATGACCCC (SEQ ID NO: 23). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 23. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 23. The stabilization domain may be transcribed into an RNA molecule.

[0148] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number KX652378.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number KX652378.1 comprise or consist of:

CACCCGCAAGGGGGGAGTTTTCTAACTCCCCGGGTGTTGACGACACCCCGGCCCCA GTCCCCAAGGTCTTGGGAAAAAGACCCC (SEQ ID NO: 24). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 24. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 24. The stabilization domain may be transcribed into an RNA molecule.

[0149] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_001564.2. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_001564.2 comprise or consist of:

ACAGGAGCAGGGCATGAAAATGTCGGGCATGACGAACCCGCTCCCCCGAGTCCCCT GGCAACAGGGTGTGTTCC (SEQ ID NO: 25). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 25. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 25. The stabilization domain may be transcribed into an RNA molecule.

[0150] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_001564.2. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_001564.2 comprise or consist of:

TTCGAGCAGGGCACATTAGTGTCGGGCGTGACGCACCCGCTCCCCTCAGTCCCCTGT GCAACAGGGAGGGCACTT (SEQ ID NO: 26). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 26. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 26. The stabilization domain may be transcribed into an RNA molecule.

[0151] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_001564.2. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_001564.2 comprise or consist of:

ACGGGGCAACAGGGAGAAATCCCGGGGTAGCGAACCTCCTCCGTTAATGTGAAAA AGTATGGGGAAAGAACTCATCT (SEQ ID NO: 27). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 27. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 27. The stabilization domain may be transcribed into an RNA molecule.

[0152] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_012932.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_012932.1 comprise or consist of:

TCAGAGCAGGGCACAATAGTGTCGGGCCTGACGACCCCGCTCCCCCGAGTCGCCCA ACGGAGTTTGGCTCAACTCTAA (SEQ ID NO: 28). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 28. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 28. The stabilization domain may be transcribed into an RNA molecule.

[0153] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_012932.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_012932.1 comprise or consist of:

GCAGCGCAGGGCATGAAAATGTCGGGCCTGACGAACCCGCGCACCCGAGTCCCCCA GTTGGGGAAGGGATCCTTGCAT (SEQ ID NO: 29). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 29. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 29. The stabilization domain may be transcribed into an RNA molecule.

[0154] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_012932.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_012932.1 comprise or consist of:

GCGGAGCAACAGGGAGAAATCCCGGGGAATTGCGAACCCCCTCCGAAATGTGAAA AATTATGGGGAAAAGTACCCATCT (SEQ ID NO: 30). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 30. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 30. The stabilization domain may be transcribed into an RNA molecule.

[0155] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number KJ741266.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number KJ741266.1 comprise or consist of:

GAGCAGGGCACAACAGTGTCGGGCCTGACGACCCCGCTCCCCCGAGTCACCAACTG GAGTTTGGCTCAACTCCAA (SEQ ID NO: 31). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 31. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 31. The stabilization domain may be transcribed into an RNA molecule.

[0156] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_031327.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_031327.1 comprise or consist of:

GGAAGGCAACACACCAGTAATCTGGTGGGGTGAGTTGCGACACCCCCTTGTGAGCA ACCGACCTAGCCGGTCTATTGACCGGCTTG (SEQ ID NO: 32). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 32. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 32. The stabilization domain may be transcribed into an RNA molecule.

[0157] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_024299.2. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_024299.2 comprise or consist of:

ACAGGGCAACAGGGATCGCCAACATCAGATCCCGGGTGAGTGACGACACCCCCCAT GTGAATCGTCAACTTAGGAACACATTCAAATAGAGGA (SEQ ID NO: 33). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 33. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 33. The stabilization domain may be transcribed into an RNA molecule.

[0158] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_024299.2. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_024299.2 comprise or consist of:

ACAGGGCAACAGGGATCACCAACATCAGATCCCGGGTGAGTGACGACACCCCCCAT GTGAATCGTCGATACAAAAACACGATAGG (SEQ ID NO: 34). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 34. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 34. The stabilization domain may be transcribed into an RNA molecule.

[0159] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_024299.2. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_024299.2 comprise or consist of:

ACACCGCAAGGAAGAGAAATCTTGGGTGGTAACAACACCCCGGCCCCAGTTCTCGC GTGCCACGAGTCATTGGCACAA (SEQ ID NO: 35). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 35. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 35. The stabilization domain may be transcribed into an RNA molecule.

[0160] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_024299.2. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_024299.2 comprise or consist of:

AAACCGCAAGGAAGGAGCAATCCTTGGGTATTAACGACACCCCGGCCCCAGTTCCC GAAGTCAAGGGGACCCTTGACCC (SEQ ID NO: 36). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 36. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 36. The stabilization domain may be transcribed into an RNA molecule.

[0161] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_034242.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_034242.1 comprise or consist of:

CATTGCAGCAGAGATTTATCTCGGGGGAGTTACGCCCCTCCATTGCCAGTAGAGTTT GCATGTCTCTATAAACATGACGTT (SEQ ID NO: 37). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 37. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 37. The stabilization domain may be transcribed into an RNA molecule.

[0162] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_027817.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_027817.1 comprise or consist of: CCATTGCCAGTAGAGTTTGCATGTCTCTATAAACATGACGTTCTGACTGACTA (SEQ ID NO: 102). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 102. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 102. The stabilization domain may be transcribed into an RNA molecule.

[0163] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_027817.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_027817.1 comprise or consist of: AATATGGCAGCAGAGCTTGTCTCGGGGATTCACGCTCCCCCCATTGTGAGTGTGTCG AACTGGTTTCGAAGGACGTCTAGAA (SEQ ID NO: 38). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 38. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 38. The stabilization domain may be transcribed into an RNA molecule. [0164] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_027817.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_027817.1 comprise or consist of:

ATCCGGCAACAGAGAGTTTATGTCTCGGGGCCTCACGCACCCCCCGTTGTGAGTGA AGTCCTTTCTGGCCATTTAGTGGTCAGGAAGGG (SEQ ID NO: 39). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 39. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 39. The stabilization domain may be transcribed into an RNA molecule.

[0165] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_027817.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_027817.1 comprise or consist of:

GCAGGGCAACAAAGTTCTAACGAACTAGGGTGAGTAGCGTCACCCCCCGGTTGTGA AAACGATTGCGACTAGAACTAAAGTCGAGAGTCTC (SEQ ID NO: 40). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 40. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 40. The stabilization domain may be transcribed into an RNA molecule.

[0166] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_005064.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_005064.1 comprise or consist of:

TTAGAGCAGGGCACGAAAGTGTCGGGCATGACGCACCCGCTCCCCCGAGTCCCCTG AAAATAGGGTGGGCAATGCACTCCT (SEQ ID NO: 41). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 41. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 41. The stabilization domain may be transcribed into an RNA molecule.

[0167] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_005064.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_005064.1 comprise or consist of:

TTTGAGCAGGGCACGAAAGTGTCGGGCCTGACGCACCCGCTCCCCCGAGTCCCCTG GAAACAGGGTGGGCCTCGAAAAATCCACCGT (SEQ ID NO: 42). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 42. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 42. The stabilization domain may be transcribed into an RNA molecule.

[0168] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_027819.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_027819.1 comprise or consist of:

GCAGGGCAACAGGAAGAAATTCCGGGTGATTAGCCACACCCCCCGAAACGTGATTT ATATGATGACAAGAATCAGA (SEQ ID NO: 43). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 43. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 43. The stabilization domain may be transcribed into an RNA molecule. [0169] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_034017.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_034017.1 comprise or consist of:

TGGGGGCAGCCGGGGGAAACCCTGGGGCTTGGCGACCTCCCCCCACAAGCCATCAT GCGAAATTAAGGCAGCCGCGAG (SEQ ID NO: 44). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 44. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 44. The stabilization domain may be transcribed into an RNA molecule.

[0170] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_034204.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_034204.1 comprise or consist of:

TGGGGGCAGCCGGGAGTCAAACTCCCGGGGCCTGGCGACCCCCCCCTTCCGCCTCC AAAAATTAAGGCAGCCCCGAGGGAGCTCTCCTCGGTGTGA (SEQ ID NO:45). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 45. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 45. The stabilization domain may be transcribed into an RNA molecule.

[0171] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_020902.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_020902.1 comprise or consist of:

CAAGGGCAGGTTGAAAGGGGCTTGGCGACCCCCCCCTAACCAGCCACGCGCACTGT GCGTGCGC (SEQ ID NO: 46). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 46. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 46. The stabilization domain may be transcribed into an RNA molecule.

[0172] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_020902.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_020902.1 comprise or consist of:

CCGGGGCAGGTTGAAAGGGGTTTCACGCACCCCCCCCTACCAGTGCCGTCGTTGGT AACGCG (SEQ ID NO: 47). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 47. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 47. The stabilization domain may be transcribed into an RNA molecule.

[0173] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number MF438044.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number MF438044.1 comprise or consist of:

CAAGGGCAGGTTGAAAGGGGCTTGGCGACCCCCCCCTAACTAGCCACGCGCACTGA TGTGCGCGG (SEQ ID NO: 48). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 48. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 48. The stabilization domain may be transcribed into an RNA molecule.

[0174] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number MF438044.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number MF438044.1 comprise or consist of:

CCGGGGCAGGTTGAAAGGGGCATCACGCACCCCCCCCTACCAGTGCCGTCGTTGGT AACGCG (SEQ ID NO: 49). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 49. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 49. The stabilization domain may be transcribed into an RNA molecule.

[0175] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number AF070476.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number AF070476.1 comprise or consist of:

ACGGGCAAGGAGCGCAAGCTGGGGCTTTCCGACCCCCCCCCCCAGGACGATTCCCC GCTTGGTAAAAAGGGCCAGGCCA (SEQ ID NO: 50). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 50. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 50. The stabilization domain may be transcribed into an RNA molecule.

[0176] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_001837.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_001837.1 comprise or consist of:

CTCCAGGCAGCAGCAGACGCAAGTCTGGGGGAAACGATCGCTCCTCCCTCTGCAGA TCTCTAGCTCGGATAGAGCGG (SEQ ID NO: 51). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 51. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 51. The stabilization domain may be transcribed into an RNA molecule.

[0177] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number KF234530.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number KF234530.1 comprise or consist of:

ATGTGGCAAGGGGCCTGCTAACACAGGCCGGGGCTTCCTGACCCCCCCACCCCAAG GCTGTTCCCCGCTCGGTAAAAAGGGCCGGGCCA (SEQ ID NO: 52). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 52. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 52. The stabilization domain may be transcribed into an RNA molecule.

[0178] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number KT166442.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number KT166442.1 comprise or consist of:

ATGAGGCAAGGAGCCTGCTAACACAGGCTGGGGCTTCCTGACCCCCCCTCCCCAAG GCGGTTCCCCGCTCGGTAAAAAGGGCCGGG (SEQ ID NO: 53). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 53. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 53. The stabilization domain may be transcribed into an RNA molecule.

[0179] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_024377.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_024377.1 comprise or consist of:

GTGAGGCAAGGAGACATTCCAGGAATGTCTGGGGCTTTCCGACCCCCCCTCCCCAG GACGGTTCCCCGCTGAGTAAAAAGGGCT (SEQ ID NO: 54). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 54. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 54. The stabilization domain may be transcribed into an RNA molecule.

[0180] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number KF234529.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number KF234529.1 comprise or consist of:

AATGTGGCAAGGGGCCTGTCCAAGACAGGCCGGGGCTTTCCGACCCCCCACCCCCA GGACGGTTCCCCGCTCGGTAAAAAGGGCCGGGCTA (SEQ ID NO: 55). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 55. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 55. The stabilization domain may be transcribed into an RNA molecule.

[0181] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number AB008335.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number AB008335.1 comprise or consist of:

TTGCGGCAAGGTCGGCCGACTGATCATCGGCTGAGGAGGTTCCCGCCCTCCCCGCC CCAGGGGTCTCCCCGCTGGGTAAAAAGGGCCCGGCCT (SEQ ID NO: 56). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 56. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 56. The stabilization domain may be transcribed into an RNA molecule.

[0182] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_001710.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_001710.1 comprise or consist of:

TTGCGGCAAGGTCTGGTGACTGATCATCACCGGAGGAGGTTCCCGCCCTCCCCGCCC CAGGGGTCTCCCCGCTGGGTAAAAAGGGCCCGGCCT (SEQ ID NO: 57). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 57. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 57. The stabilization domain may be transcribed into an RNA molecule.

[0183] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number AB018667.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number AB018667.1 comprise or consist of:

TTGCGGCAAGGTCGGGCGACTGATCATCGCCTGAGGAGGTTCCCGCCCTCCCCGCC CCAGGGGTCTCCCCGCTGGGTAAAAAGGGCCCGGCCT (SEQ ID NO: 58). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 58. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 58. The stabilization domain may be transcribed into an RNA molecule.

[0184] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_027998.2. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_027998.2 comprise or consist of:

AGGCAGGAGGTGAAGTCAGCTGTACCCACGGCTGGCTGAAACCGGGGCTTGACGAC CCCCCCTATCCGAGTTGGGCAAGGTAACATCACGGGTGTGACGACCCC (SEQ ID NO: 59). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 59. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 59. The stabilization domain may be transcribed into an RNA molecule. In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number KC796093.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number KC796093.1 comprise or consist of:

TTCCGGCAAGGTGCGCCGGGGGGGCCTTCACGGGCCCTTCTAGCGCAGGGGTTTGA GACACCCCCCGCCCCACTCCTTCCCAGGGTTGGCAACCTGGGTC (SEQ ID NO: 60). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 60. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 60. The stabilization domain may be transcribed into an RNA molecule.

[0185] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number KC796084.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number KC796084.1 comprise or consist of: GTAAGGCAAGGTGTGCACTGGCAGCCTTAACGGGCTGTTGGGGCGCAGGGGCTTGA GCACCCCCCTTCCCCACTCCCAGCGGGGCTTGGCAACCCTG (SEQ ID NO: 61). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 61. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 61. The stabilization domain may be transcribed into an RNA molecule. [0186] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number KC796084.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number KC796084.1 comprise or consist of:

GTTAGCAAGGCGCTGTCATGGTGCTTCTAACGAGGCATGAGGCAGCGGGGGCGTGA GAACCCCCCTTCCCCACTCCGGCGTGTAGATTGGCAATCTTGCGCT (SEQ ID NO: 62). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 62. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 62. The stabilization domain may be transcribed into an RNA molecule.

[0187] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number KC796084.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number KC796084.1 comprise or consist of:

ACTTGGCAAGGCACCCCCTGGCTAGTGGCTGCACTTAACTGAGTGGCCGGCTAGTC GGTGGGTGGGGGCACGTGACTCCCCCTTCCCCACACCGGCGGCGGCCGTAAAACGC CCC (SEQ ID NO: 63). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 63. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 63. The stabilization domain may be transcribed into an RNA molecule.

[0188] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_038435.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_038435.1 comprise or consist of:

TCCTGGCAAGGGCTACTCTGAAAGCTGCTAACGTGGTGATCGGCGTAGCGGGGCGT GAGGAACCCCCCACCCCACTCCTGGGTCAGCTTGGTAACTGGCCCA (SEQ ID NO: 64). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 64. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 64. The stabilization domain may be transcribed into an RNA molecule.

[0189] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_038434.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_038434.1 comprise or consist of:

GCGGGCAGAGACTGGGCCTCGCTGGCGCGTGAGCTCCTGTGGGATCCGGCCTGTTC CATGCCACCTGTTCTCGATCAGGGGGAGGGGATCTTACCCCCTGAACTGT (SEQ ID NO: 65). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 65. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 65. The stabilization domain may be transcribed into an RNA molecule.

[0190] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number KC796079.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number KC796079.1 comprise or consist of:

GTAAGGCAAGGTGTGCACTGGCAGCCTTAACGGGCTGTTGGGACACAGGGGCTTGA GCACCCCCCTTCCCCACTCCCAGCGGGGCTTGGCAACCCCCC (SEQ ID NO: 66). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 66. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 66. The stabilization domain may be transcribed into an RNA molecule. [0191] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_021154.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_021154.1 comprise or consist of:

TTGGGGCAGGCACGCTTGCGTGGGGGAGTTGCGCCCCCCCCCAGCCAGCACTCGTC ATGATGTGTCGGATGCCAGTGATAGGCAGCCTC (SEQ ID NO: 67). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 67. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 67. The stabilization domain may be transcribed into an RNA molecule.

[0192] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_038433.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_038433.1 comprise or consist of:

GCCCGGCAAGGTTAACAGGGGGAGTAGTGCCCCCCCCGCCCCAACTCGGGTAGCGC GTACGCTC (SEQ ID NO: 68). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 68. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 68. The stabilization domain may be transcribed into an RNA molecule.

[0193] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_038433.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_038433.1 comprise or consist of:

TCCAAGGCAACAGGCTTCGGCCGGGGGAGTAGCGCCCCCCCCTTTGTGAGCTCGTA ACCCCCTTTTGGGGCT (SEQ ID NO: 69). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 69. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 69. The stabilization domain may be transcribed into an RNA molecule.

[0194] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_030291.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_030291.1 comprise or consist of:

ACATGGCAAGGGTGCTTCGGCATGGGGGAGTAGCGTCCCTCCCACCCCAGCGAGGC TGCAAGCCTAT (SEQ ID NO: 70). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 70. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 70. The stabilization domain may be transcribed into an RNA molecule.

[0195] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number HM047196.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number HM047196.1 comprise or consist of:

ACCTGGCAACAGCCCCGTCGGGGCGGGGGAGTAGCGCCCCCCCCAGTGTGAGCGAG GTGGGAAACCACCTA (SEQ ID NO: 71). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 71. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 71. The stabilization domain may be transcribed into an RNA molecule.

[0196] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_038437.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_038437.1 comprise or consist of:

CAAGGGCAAGGTGTCTTGCGAGACAGGGGCTTAACGCACCCCCCCCCCCAGTGAGG GGGGCTGATCCCCCA (SEQ ID NO: 72). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 72. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 72. The stabilization domain may be transcribed into an RNA molecule.

[0197] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_021154.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_021154.1 comprise or consist of:

ACCGGGCAAGGGCTCACGCGGAGTGTGACAAGCTCCCCCCCCCAGTCCATGGCCGT GGATCGGCTC (SEQ ID NO: 73). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 73. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 73. The stabilization domain may be transcribed into an RNA molecule.

[0198] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_025677.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_025677.1 comprise or consist of:

GAATAGGCAGGGAGGAGTCCAAGAACCGTCTCGGGGACTCTTTGGGGCTTGACGAA CCCCCCTACCCGAGTCTATATTCAGTGGCTGGAACC (SEQ ID NO: 74). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 74. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 74. The stabilization domain may be transcribed into an RNA molecule.

[0199] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number KY370101.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number KY370101.1 comprise or consist of:

AAAGGCAGGGAGAGGCTTAAGAACCGTCTCGGGAGCCTCTTGGGGCTTGACGAACC CCCCAACCCGAGTCAAGTCCTTCAACAGTACCGTTTCGAG (SEQ ID NO: 75). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 75. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 75. The stabilization domain may be transcribed into an RNA molecule.

[0200] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_038964.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_038964.1 comprise or consist of:

TGAAAGGGGCAAGTGGCCGTATAGGCTGGGGCGATCGCCGTACCCCCCCTTTACCA GGCGCCTCAACCCCATGTACCATGGGGTT (SEQ ID NO: 76). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 76. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 76. The stabilization domain may be transcribed into an RNA molecule.

[0201] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number KY370100.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number KY370100.1 comprise or consist of:

ACACGGCAAGGTGCTAGAAGCTGAAACCGACTCGGAGCTCTAGCAGGGGGACTGG CGACCCTCCCGCCCCAGCTGGCCTCTGGCAGAAACGACTCGTGCCATT (SEQ ID NO: 77). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 77. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 77. The stabilization domain may be transcribed into an RNA molecule.

[0202] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number MH282908.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number MH282908.1 comprise or consist of:

AAGAGGCAAGGGGAGGCAGCTAGCCTCCGGGGCCTGACGACCCCCCTTCCCCAGTC ATTGAAGGCAAGGGGCTGC (SEQ ID NO: 78). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 78. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 78. The stabilization domain may be transcribed into an RNA molecule.

[0203] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number MH282908.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number MH282908.1 comprise or consist of:

AAAGGGCAAGGCACCACACGGCTAGTGTGGTGAGGGATTGACAACCCCTCCTCCCC AGTCGGCATGAACTT (SEQ ID NO: 79). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 79. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 79. The stabilization domain may be transcribed into an RNA molecule.

[0204] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number MH282908.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number MH282908.1 comprise or consist of:

AAAGGGCAAGGCACCACATGGAAATGTGGTGAGGGTTTGACCTCCCCTCCCCCCCA GTCAACCATACATAAAACTTGAAAAACACATATTGGTACT (SEQ ID NO: 80). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 80. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 80. The stabilization domain may be transcribed into an RNA molecule.

[0205] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_001655.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_001655.1 comprise or consist of:

CAGCGGCAACAGGGGAGACCCCGGGCTTAACGACCCCGCCGATGTGAGTTTGGCGA CCATGGTGGATCAG (SEQ ID NO: 81). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 81. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 81. The stabilization domain may be transcribed into an RNA molecule.

[0206] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_031950.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_031950.1 comprise or consist of:

CAATGGCAACAGCACTCTCTTAGGTGCGGGGTATGGCGAACCCCCCAATGTGAGCT CCTCCCCGGATGGGGCG (SEQ ID NO: 82). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 82. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 82. The stabilization domain may be transcribed into an RNA molecule.

[0207] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_038430.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_038430.1 comprise or consist of:

TTCGGGCAAGGCATGCGAGATAAAAAGGGTCTCGTATGAGGGCGTGGCAACCCCTC CCCCCCAGCTGCGGCGGCACAAAAGCGTCTCGCGTGTCGTC (SEQ ID NO: 83). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 83. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 83. The stabilization domain may be transcribed into an RNA molecule.

[0208] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_040815.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_040815.1 comprise or consist of:

TAGCGGCAGGGAGCAGGGTAGACCAACCTGCAGGGGCTTGACGACCCCCCCGTCCC GAGTCAGCCAGGAGGCAGAAGCGACTCGC (SEQ ID NO: 84). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 84. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 84. The stabilization domain may be transcribed into an RNA molecule.

[0209] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number KY370094.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number KY370094.1 comprise or consist of:

TGAGGGCAGGATAGGCCAAGAATCGTCTCGAGGCTGATTGGGGCTTCACGCACCCC CCCATCCGAGTGCCTCTCATCTTCCAAAACCGTCTCGGGGGAGATGAGAC (SEQ ID NO: 85). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 85. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 85. The stabilization domain may be transcribed into an RNA molecule.

[0210] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number KX905133.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number KX905133.1 comprise or consist of:

AAAGGGCAGCGGTGCCGGACGGCATTGGGGCTTGGCGACCCCCCCCACGCGAGCTA CCCACCATTGGTGGGTTC (SEQ ID NO: 86). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 86. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 86. The stabilization domain may be transcribed into an RNA molecule.

[0211] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_021153.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_021153.1 comprise or consist of:

CAGGGGCAGAGGCCGGTTGATTATCATTGCAGCCGTAGGGGCTTGGCGACCCCCCC CCCTCGAGCCAGCCTTTCAACAAAACCGTCTCGGGTTGGAAGG (SEQ ID NO: 87). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 87. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 87. The stabilization domain may be transcribed into an RNA molecule.

[0212] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number NC_038428.1. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number NC_038428.1 comprise or consist of: ACACTGCAACAGGGGAAACCCGGGGATTTCCGATCCCCCAGATGTGAGGAGGCTGG TTGCCTAACAACCTG (SEQ ID NO: 88). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 88. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 88. The stabilization domain may be transcribed into an RNA molecule.

[0213] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number KJ412989. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number KJ412989 comprise or consist of: CTCCCGTAAGGAAAGCGCAAGCTTTGAGCATTGACAACGCTCCGGCCCCAGTCCCC CAGGTTATGGAGGAATAACCC (SEQ ID NO: 89). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 89. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 89. The stabilization domain may be transcribed into an RNA molecule. [0214] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number MN242370. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number MN242370 comprise or consist of:

AAGGGGCAGGATATGGACAGAACCGTCTCGGGTCCAGAAGGGGCTTGGCGACCCC CCCCATCCGAGCCACCCCTCTAGGAAGACCGTCTCGGCCTAGAGG (SEQ ID NO: 90). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 90. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 90. The stabilization domain may be transcribed into an RNA molecule.

[0215] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from a flavivirus with genome accession number MH824541. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from this flavivirus with genome accession number MH824541 comprise or consist of: AAGGGGCAGCCATGCCGCAGAACCGTCTCGGGCGGCAAGGGGCTTAGCGACCCCCC CCTGGCGAGCTGTATGAGTGTGATAAGGGCGACATAGC (SEQ ID NO: 91). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 91. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 91. The stabilization domain may be transcribed into an RNA molecule.

[0216] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 5’ to 3’ direction comprises or consists of sequences from Tamana Bat virus (AF346759.1). The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from Tamana Bat virus comprise or consist of: AF346759.1, pos: 10305-10380: TTTGGGCAAGGTGCAGGTTAGCTGCAGGGGCTTGAAAAACCCCCCCCCCCATTCAA GACTTTTAGTGCATTAGTT (SEQ ID NO: 6). The stabilization domain may be transcribed into an RNA molecule.

[0217] In some embodiments, the stabilization domain prevents or attenuates the activity of exonucleases that act in the 3’ to 5’ direction on RNA. In some instances, the prevention or attenuation of the activity of exonucleases increases the effectiveness of the trans-splicing molecule.

[0218] In some embodiments of the compositions of the present disclosure, the stabilization domain forms a tertiary structure. In some embodiments of the compositions of the present disclosure, the tertiary structure is a triplex.

[0219] In some embodiments, the Stabilization domain forms an RNA triplex that blocks 3’- 5’ exonuclease activity and is derived or isolated from a vertebrate gene or microbial genome selected from the group consisting of: MALAT1 [ENSG00000251562], NEAT1 [ENSG00000245532], Turnip yellow mosaic virus genome, Kaposi's sarcoma-associated herpesvirus genome, TER telomerase- associated RNA [ENSG00000270141], SAM-II bacterial riboswitch.

[0220] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 3’ to 5’ direction comprises or consists of sequences from the MALAT1 gene. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from the MALAT1 gene comprise or consist of: AAGCTGATCTCCAATGCTCTTCAGTAGGGTCATGAAGGTTTTTCTTTTCCTGAGAAA ACAACACGTATTGTTTTCTCAGGTTTTGCTTTTTGGCCTTTTTCTAGCTTAAAAAAAA AAAAAGCAAAAGATGCTGGTGGTTGGCACTCCTGGTTTCCAGGACGGGGTTCAAAT (SEQ ID NO: 92). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 92. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 92. The sequence may be transcribed into an RNA molecule.

[0221] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 3’ to 5’ direction comprises or consists of sequences from rhesus rhadinovirus. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from rhesus rhadinovirus comprise or consist of: CGTTTGTGTTGGTTTTTATGACCAGCTTGGTACAAAACCTGCTGGTGATTTTTTACCC AACAAATATTA (SEQ ID NO: 93). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 93. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 93. The stabilization domain may be transcribed into an RNA molecule.

[0222] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 3’ to 5’ direction comprises or consists of sequences from Equine Herpesvirus 2. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from Equine Herpesvirus 2 comprise or consist of: AAGAATATTTTTAAAGACTTTTTTCCCCAACCTCTGGGTTGGGTTTTTTCTCTTTAAA ATATTCAATA (SEQ ID NO: 94). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 94. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 94. The stabilization domain may be transcribed into an RNA molecule.

[0223] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 3’ to 5’ direction comprises or consists of sequences from Kaposi's sarcoma-associated herpesvirus. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from Kaposi's sarcoma-associated herpesvirus comprise or consist of: TGTTTTGTGTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACACCTCCAGTGACCAGA CGGCAAGGTTTTTATCCCAGTGTATATT (SEQ ID NO: 95). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 95. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 95. The stabilization domain may be transcribed into an RNA molecule.

[0224] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 3’ to 5’ direction comprises or consists of sequences from Plautia stali intestine virus. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from Plautia stali intestine virus comprise or consist of: ATTGGCAGTAGAGTTTTTCCCCAGGGAGCTTCACTGTCTGGGTTTTCTCTACT (SEQ ID NO: 96). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 96. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 96. The stabilization domain may be transcribed into an RNA molecule.

[0225] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 3’ to 5’ direction comprises or consists of sequences from Cotesia congregata bracovirus. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from Cotesia congregata bracovirus comprise or consist of: TTCATCAAGGAGGTTTTTTCCCAGCCTAGCTGGGTTTTCCTCCTTTGGGGACA (SEQ ID NO: 97). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 97. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 97. The stabilization domain may be transcribed into an RNA molecule.

[0226] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 3’ to 5’ direction comprises or consists of sequences from Cotesia sesamiae bracoviruses. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from Cotesia sesamiae bracoviruses comprise or consist of: TTTTTCGAGGAGGTTTTTTCCTAGCACCACTAGGTTTTCCTCCTCTGGGAAC (SEQ ID NO: 98). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 98. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 98. The stabilization domain may be transcribed into an RNA molecule.

[0227] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 3’ to 5’ direction comprises or consists of sequences from Acanthamoeba polyphaga mimivirus. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from Acanthamoeba polyphaga mimivirus comprise or consist of: ATTTACTGTTGGTTTTCTTCTCTGATTTTCATAAGAACTTTTCCCAACA (SEQ ID NO: 99). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 99. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 99. The stabilization domain may be transcribed into an RNA molecule.

[0228] In some embodiments, the stabilization domain forms a tertiary structure. In some embodiments of the compositions of the present disclosure, the tertiary structure is a pseudoknot.

[0229] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 3’ to 5’ direction comprises or consists of sequences that form a pseudoknot derived or isolated from the list consisting of: group 1 self-splicing introns from Azoarcus or Tetrahymena or Twort, drosophila sytl pre-mRNA, human CPEB3 ribozyme, E. coli RydC gene, prokaryotic plasmids I-complex or IncL/M or ColIB/P9, Mycobacterium bovis leuA mRNA, GlmS riboswitch ribozyme, Agrobacterium tumefa- ciens metA gene, L- and c-myc genes, Human interferon gamma mRNA, Ornithine decarboxylase antizyme, Prion mRNAs (human, cattle, yeast), Human and Tetrahymena telomerase, 16S rRNA, 16S rRNA, 18S V4 region, 23S rRNA, Ml RNA component of bacterial RNase P, Neurospora VS ribozyme, Pyrimidine nucleotide synthase ribozyme, Alcohol dehydrogenase ribozyme (l-ribox02), a ribozyme, an aptamer, foot and mouse disease virus genome, Mengovirus genome, paraechovirus 1 genome, Aichivirus genome, hepatoviridae genomes, HCV, Classical swine fever virus genome, Bovine Viral Diarrhea virus genome, Porcine teschovirus, Cricket paralysis virus-like virus genomes, Giardia lamblia virus genome, Tobacco etch virus genome, retroviridae genomes, Nidovirales genomes, Totiviridae genomes, Luteoviridae genomes, Myoviridae genomes, Listeria monocytogenes phage genome, Murine leukemia virus genome, Hepatitis C virus genome, Influenza A and B genomes, Turnip yellow mosaic virus genomes, Tobacco mosaic virus-like virus genomes, Bamboo mosaic virus genome, Strawberry chlorotic fleck- associated virus genome; potato yellow vein virus genome, Tomato bushy stunt virus genome, Turnip crinkle virus genome, Encephalomyocarditis virus genome, Enterovirus genomes, Dengue virus genome, yellow fever virus genome, Japanese encephalitis virus genome, tick-borne encephalitis virus genome, Cauliflower mosaic virus genome, Barley yellow dwarf virus genome, Bacteriophage QP genome, Avian leukosis virus genome, Peach latent mosaic viroid genome, Large pospiviroidae genome, Sat C satellite RNA of Turnip crinkle virus genome, Hepatitis delta virus genome, and Marek’s disease virus genome.

[0230] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 3’ to 5’ direction comprises or consists of sequences that a form pseudoknot from Murine leukemia virus. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from Murine leukemia virus comprise or consist of: GGGTCAGGAGCCCCCCCCCTGAACCCAGGATAACCCTCAAAGTCGGGGGGCAACCC (SEQ ID NO: 100). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 100. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 100. The stabilization domain may be transcribed into an RNA molecule.

[0231] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 3’ to 5’ direction comprises or consists of sequences that a form pseudoknot from the evopreQl riboswitch aptamer. The sequence may comprise a DNA sequence. The sequence may comprise an RNA sequence. In some embodiments, the sequences from the evopreQl riboswitch aptamer comprise or consist of: TTGACGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAA (SEQ ID NO: 101). In some embodiments, the stabilization domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence encoded by SEQ ID NO: 101. In some embodiments, the stabilization domain comprises a sequence encoded by SEQ ID NO: 101. The stabilization domain may be transcribed into an RNA sequence.

[0232] In some embodiments, the stabilization domain that protects the trans-splicing molecule from exonucleases that act in the 3’ to 5’ direction comprises or consists of sequences that form G-quadruplexes, sequences isolated or derived from ribosomal RNAs, sequences isolated or derived from ribozymes, or sequences isolated or derived from prion mRNAs. Cleavage domain

[0233] In some embodiments, the cleavage domain disclosed herein comprises a sequence or structure of any one or more molecules selected from the group consisting of: Hammerhead ribozyme, Hammerhead ribozyme, tRNA(gly), tRNA-Gly-CCC-1-1, tRNA-Gly-CCC-1-2, tRNA-Gly-CCC-3-1, tRNA-Gly-CCC-2-2, tRNA-Gly-CCC-2-1, tRNA-Gly-GCC-2-4, tRNA- Gly-GCC-2-5, tRNA-Gly-GCC-2-6, tRNA-Gly-GCC-2-1, tRNA-Gly-GCC-2-2, tRNA-Gly- GCC-1-1, tRNA-Gly-GCC-1-3, tRNA-Gly-GCC-1-4, tRNA-Gly-GCC-1-5, tRNA-Gly-GCC-3- 1, tRNA-Gly-GCC-4-1, tRNA-Gly-GCC-5-1, tRNA-Gly-TCC-3-1, tRNA-Gly-TCC-1-1, tRNA- Gly-TCC-2-1, tRNA-Gly-TCC-2-2, tRNA-Gly-TCC-2-3, tRNA-Gly-TCC-2-4, tRNA-Gly-TCC- 2-5, tRNA-Gly-TCC-2-6, tRNA-Gly-TCC-4-1, rice tRNA, MALAT1 mascRNA, 7QR4_2|Chain B|RNA CPEB3 ribozyme|Homo sapiens (9606), 7QR3_2|Chains C, D|chimpanzee CPEB3 ribozyme|Pan troglodytes (9598), 4R4P_l|Chain A|VS ribozyme RNA|NEUROSPORA (5140), 4R4P_l|Chain A|VS ribozyme RNA|NEUROSPORA (5140) [tandem dimer], 4R4V_l|Chain A|VS ribozyme RNA|NEUROSPORA (5140), 4R4V_l|Chain A|VS ribozyme RNA|NEUROSPORA (5140) [tandem dimer], histone 3'UTR with SLBP and HDE Hl-4 , histone 3'UTR with SLBP and HDE H2BC21D , histone 3'UTR with SLBP and HDE H2AC20, histone 3'UTR with SLBP and HDE H2BC13 , histone 3'UTR with SLBP and HDE H2AC25, Pri-miR-16-1, Pri-miR-30a, Artificial microprocessor substrate, eIF4H exon 5, Pri-miR-7, targeted pseudouridylation snoRNA-1, targeted pseudouridylation snoRNA-2, scaRNA-9, scaRNA-2, 5K7C_l|Chain A|RNA 47-MER| synthetic construct (32630), 5T5A_l|Chain A|DNA/RNA (71-MER)|metagenome (256318), 4OJI_l|Chain A|RNA (52-MER)|null, 4RGE_l|Chains A, B, C|env22 twister ribozyme|Synthetic (32630), 6JQ5_l|Chains A, B|RNA (82-MER) | synthetic construct (32630), 6JQ5_l|Chains A, B|RNA (82-MER)|synthetic construct (32630) DIMER, Pri-miR-31(51+51), Pri-miR-31(51+51)/Pre-miR-HBV, Pri-miR-

3 l(38+40)/Pre-miR-HBV, Pri-miR-31(30+3 l)/Pre-miR-HBV, Pri-miR-31(22+2 l)/Pre-miR- HBV. In some embodiments, the cleavage domain comprises at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97.5%, about 98%, about 99%, or about 100% identity with a sequence in the aforementioned molecules.

Engineered Cl snUNA

[0234] The composition provided herein can comprise a nucleic acid sequence encoding an engineered U1 snRNA (enzyme staple molecule or “ESM”). The nucleic acid may comprise RNA, DNA, a DNA/RNA hybrid, and/or comprises at least one of a nucleic acid analog, a chemically modified nucleic acid, or a chimera composed of two or more nucleic acids or nucleic acid analogs. A nucleic acid comprising DNA may encode the ESM. The DNA may be transcribed into an RNA, e.g., engineered small nuclear RNA (snRNA). In some embodiments, the ESM comprises an engineered snRNA. The engineered snRNA (e.g., esnRNA) can recruit members of the spliceosome. In some embodiments, the engineered snRNA is configured to promote RNA splicing. In some embodiments, the engineered snRNA is configured to promote RNA splicing of the trans-splicing RNA molecule or portions thereof to form a full length transsplicing RNA molecule. In some embodiments, the engineered snRNA is configured to promote RNA splicing of the exonic domains to replace a portion of the target RNA. The engineered snRNA may interact with a nucleic acid sequence, or a transcribed copy of the nucleic acid sequence, to enhance a trans-splicing of the nucleic acid sequences. In this manner, the engineered snRNA may promote an association of the exonic domain with a target RNA, thereby resulting in a trans-splicing of the exonic domain to the target RNA. The engineered snRNA may promote an association of a portion of a trans-splicing RNA molecule with another portion of a trans-splicing RNA molecule, thereby resulting in full length trans-splicing RNA molecule.

[0235] In some embodiments, an engineered snRNA can interact with the Intronic Domain to increase the trans-splicing efficiency of the trans-splicing nucleic acid molecule. In some embodiments, the engineered snRNA domain comprise a sequence derived or isolated from a human small nuclear RNA gene. In some embodiments, the human small nuclear RNA gene comprises of Ul, U2, U4, U5, U6, U7, Ul l, and U12 snRNA. In some embodiments, there may be an engineered snRNA sequence that is configured to promote trans-splicing. In some embodiment, the engineered snRNA can be derived or isolated from the human Ul snRNA gene. In some embodiments, the sequences of the engineered snRNA can be derived or isolated from a Ul snRNA variant. In some embodiments of the compositions of the disclosure, the Ul snRNA variant is selected from the list consisting of (name followed by genomic location in brackets according to UCSC human genome assembly 2006): tUl.l [chrl: 16713367- 16712967], tU1.2 [chrl:16866030-16865630], vUl.l [chrl: 142438700- 142438300], vU1.2 [chrl:142464813-142464413], vU1.4 [chrl:143022739-143022339], vU1.5 [chrl: 143202968- 143202568], vU1.7 [chrl: 144680790- 144680390], vU1.8 [chrl: 145022927- 145022527], vU1.9 [chrl:145977791-145977391], vUl.10 [chrl:146301289-146300889], vUl.l l [chrl: 146327427- 146327027], vU1.15 [chrl: 146871696- 146871296], vU1.16 [chrl: 147033726- 147033326], vUl.17 [chrl: 147460893- 147460493], vU1.18 [chrl: 147490845- 147490445], vU1.19 [chrl:147780880-147780480], tU1.3 [chrl:16939762-16940162], tU1.4 [chrl: 17095226- 17095626], vU1.3 [chrl: 142478876- 142479276], vU1.6 [chrl: 144094114- 144094514], vU1.12 [chrl:146341486-146341886], vU1.13 [chrl: 146460770- 146461170], vU1.14 [chrl: 146608089- 146608489], vU1.20 [chrl: 147872535- 147872935].

[0236] In some embodiments, the ESM comprises or consists of the following sequence: CGAGCTCTCTgcaggggagataccaTGATCAcgaaggtggttttcccagggcgaggcttatccattgcactccggatgtgct gacccctgcgatttccccaaatgtgggaaactcgactgcataatttgtggtagtCACCTTCGTGATCATGGTATCTCC CCCG (SEQ ID NO: 124). In some embodiments, the ESM comprises or consists of the following sequence: CGAGCTCTCTgcaggggagataccaTGATCAcgaaggtggttttcccagggcgaggcttatccattgcactccggatgtgct gacccctgcgatttccccaaatgtgggaaactcgactgcataatttgtggtagtgggggactgcgttcgcgctttcccctg (SEQ ID NO: 125).

Ribozymes

[0237] Ribozymes are noncoding RNAs that, similarly to enzymes, catalyze specific biochemical transformations. Some ribozymes can catalyze or perform RNA splicing. Ribozymes can perform cleavage or ligation of RNA and DNA, and can catalyze peptide bond formation. Within ribosomes, ribozymes may function as a part of the large subunit ribosomal RNA to link amino acids during protein synthesis.

[0238] In some embodiments, the trans-splicing nucleic acid molecule of the present disclosure comprises (a) an exonic domain (e.g., one that encodes a therapeutic sequence); b) an intronic domain configured to promote ribonucleic acid (RNA) trans-splicing; c) an antisense domain configured to bind to a target RNA molecule; d) a sequence or structure derived or isolated from a ribozyme, wherein the ribozyme is selected from the group consisting of wherein the ribozyme is selected from the group consisting of: a VS ribozyme, a twister ribozyme, a twister sister ribozyme, a lantern ribozyme, a pistol ribozyme, a hairpin ribozyme, a leadzyme, a hatchet ribozyme, a GIRI branching ribozyme, a glmS ribozyme, a Class I intron, a Class II intron, a RNAse P, a CoTC ribozyme, a Hoylinc ribozyme, a Varkud satellite ribozyme, a mammalian CPEB3 ribozyme, and a riboswitch. In some embodiments, trans-splicing nucleic acid molecule further comprises a G-quadruplex and/or a pseudoknot. In some embodiments, trans-splicing nucleic acid molecule further comprises a poly(A). In some embodiments, trans- splicing nucleic acid molecule comprises from 5’ to 3’: the G-quadruplex and/or a pseudoknot, the sequence or structure derived or isolated from a ribozyme, and the poly (A). In some embodiments, the trans-splicing nucleic acid molecule comprises from 5’ to 3’: the sequence or structure derived or isolated from a ribozyme, the G-quadruplex and/or a pseudoknot, and the poly (A). In some embodiments, the trans-splicing nucleic acid molecule comprises from 5’ to 3’: the G-quadruplex and/or a pseudoknot and the sequence or structure derived or isolated from a ribozyme, and the trans-splicing nucleic acid molecule does not comprises a 3’ poly(A) tail. In some embodiments, the trans-splicing nucleic acid molecule comprises from 5’ to 3’: the sequence or structure derived or isolated from a ribozyme and the G-quadruplex and/or a pseudoknot, and the trans-splicing nucleic acid molecule does not comprises a 3’ poly(A) tail.

[0239] In some embodiments, the trans-splicing nucleic acid molecule of the present disclosure comprises (a) an exonic domain (e.g., one that encodes a therapeutic sequence); (b) an intronic domain configured to promote ribonucleic acid (RNA) trans-splicing; (c) an antisense domain configured to bind to a target RNA molecule; (d) a sequence or structure derived or isolated from a ribozyme; (e) a 3’ domain, wherein the 3’ domain does not comprise a poly(A). In some embodiments, the 3’ domain comprises the sequence or structure derived or isolated from a ribozyme. In some embodiments, the 3’ domain does not comprise the sequence or structure derived or isolated from the ribozyme. In some embodiments, the ribozyme is selected from the group consisting of a hammerhead ribozyme, an HDV ribozyme, a twister ribozyme, a twister sister ribozyme, a lantern ribozyme, a pistol ribozyme, a hairpin ribozyme, a VS ribozyme, a leadzyme, a hatchet ribozyme, a GIRI branching ribozyme, a glmS ribozyme, a Class I intron, a Class II intron, a RNAse P, a CoTC ribozyme, a Hoylinc ribozyme, a Varkud satellite ribozyme, a mammalian CPEB3 ribozyme, and a riboswitch. In some embodiments, the 3’ domain comprises a cleavage domain. In some embodiments, the cleavage domain comprises the sequence or structure derived or isolated from the ribozyme. In some embodiments, the cleavage domain does not comprise the sequence or structure derived or isolated from the ribozyme. In some embodiments, the stabilization domain comprises the sequence or structure derived or isolated from the ribozyme. In some embodiments, the stabilization domain does not comprise the sequence or structure derived or isolated from the ribozyme. In some embodiments, the stabilization domain comprises a G-quadruplex. In some embodiments, the stabilization domain comprises a pseudoknot. In some embodiments, the 3’ domain comprises a nuclear retention domain. In some embodiments, the nuclear retention domain comprises the sequence or structure derived or isolated from the ribozyme. In some embodiments, the nuclear retention domain does not comprise the sequence or structure derived or isolated from the ribozyme. In some embodiments, the 3’ domain comprises from 5’ to 3’: the G-quadruplex and/or the pseudoknot and the sequence or structure derived or isolated from the ribozyme. In some embodiments, the 3’ domain comprises from 5’ to 3’: the sequence or structure derived or isolated from the ribozyme and the G-quadruplex and/or the pseudoknot. In some embodiments, the 3’ domain comprises, from 5’ to 3’: a sequence or structure derived or isolated from a ribozyme, and a G-quadruplex. In some embodiments, the 3’ domain comprises, from 5’ to 3’: a G-quadruplex and a sequence or structure derived or isolated from a ribozyme. In some embodiments, the 3’ domain comprises, from 5’ to 3’: a sequence or structure derived or isolated from a ribozyme, and a pseudoknot. In some embodiments, the 3’ domain comprises, from 5’ to 3’: a pseudoknot and a sequence or structure derived or isolated from a ribozyme.

[0240] In some embodiments, the ribozyme is a twister ribozyme. In some embodiments, the ribozyme is a twister ribozyme comprising the sequence set forth in SEQ ID NO: 115. In some embodiments, the ribozyme is an env22 twister ribozyme. In some embodiments, the ribozyme is an env22 twister ribozyme comprising the sequence set forth in SEQ ID NO: 116. In some embodiments, the ribozyme is a twister sister ribozyme. In some embodiments, the ribozyme is a twister sister ribozyme comprising the sequence set forth in SEQ ID NO: 114. Twister sister ribozymes are described, for example, in Zheng et al, Structure-based insights into self-cleavage by a four-way junctional twister- sister ribozyme, Nat Commun (2017) Oct 30;8(l): 1180, which is herein incorporated by reference in its entirety. In some embodiments, the ribozyme is a hammerhead ribozyme. In some embodiments, the ribozyme is a hammerhead ribozyme comprising the sequence set forth in SEQ ID NO: 123. Hammerhead ribozymes are described, for example, in Scott et al, (2013) Prog Mol Bio Trans Sci. 120:1-23, which is hereby incorporated by reference in its entirety. In some embodiments, the ribozyme is a Rzb hammerhead ribozyme. In some embodiments, the ribozyme is a self-cleaving hammerhead ribozyme. In some embodiments, the ribozyme is a Class I intron. In some embodiments, the ribozyme is a Class II intron. In some embodiments, the ribozyme is an RNAse P. In some embodiments, the ribozyme is a peptidyl transferase 23SrRNA. In some embodiments, the ribozyme is a GIRI branching ribozyme. In some embodiments, the ribozyme is a leadzyme. In some embodiments, the ribozyme is a hairpin ribozyme. In some embodiments, the ribozyme is a hepatitis delta virus (HDV) ribozyme. In some embodiments, the ribozyme is a VS ribozyme. In some embodiments, the ribozyme is a mutant VS ribozyme. In some embodiments, the mutant VS ribozyme comprises a G638A mutation. In some embodiments, the mutant VS ribozyme comprises a A756G mutation. In some embodiments, the ribozyme is a VS ribozyme comprising a G638A mutation. In some embodiments, the ribozyme is a VS ribozyme comprising a A756G mutation. In some embodiments, the ribozyme is a pistol ribozyme. In some embodiments, the ribozyme is a hatchet ribozyme. In some embodiments, the ribozyme is a tandem dimer hatchet ribozyme. In some embodiments, the ribozyme comprises one copy of a hatchet ribozyme. In some embodiments, the ribozyme is a CPEB3 ribozyme derived from a mammal (See, Chadalavada DM, et al., The human HDV-like CPEB3 ribozyme is intrinsically fast-reacting; Biochemistry 2010, 49, 25, 5321-5330; and Chen C et al, Inhibition of Cpeb3 ribozyme elevates CPEB3 protein expression and poly adenylation of its target mRNAs and enhances object location memory, eLife 2024, DOI: 10.7554/eLife.90116, each of which is hereby incorporated by reference in its entirety). In some embodiments, the ribozyme is a human CPEB3 ribozyme. In some embodiments, the ribozyme comprises the sequence set forth in SEQ ID NO: 121. In some embodiments, the ribozyme is a chimpanzee CPEB3 ribozyme. In some embodiments, the ribozyme is a CPEB3 ribozyme derived from Pan troglodytes. In some embodiments, the ribozyme comprises the sequence set forth in SEQ ID NO: 122. In some embodiments, the ribozyme is a CoTC ribozyme. In some embodiments, the ribozyme is a glmS ribozyme. In some embodiments, the ribozyme is a ribozyme derived from or encoded by a IncRNA. In some embodiments, the ribozyme is a Hovlinc ribozyme (see, for example, Chen et al, Hovlink is a recently evolved class of ribozyme found in human IncRNA, Nat Chem Biol 2021, 17:601-607, which is hereby incorporated by reference in its entirety). In some embodiments, the ribozyme is a lantern ribozyme (see, for example, Zhou et al, Human lantern ribozymes: smallest known self-cleaving ribozymes, ELife reviewed preprint posted September 11, 2023, which is hereby incorporated by reference in its entirety). In some embodiments, the ribozyme is a self-alkylating ribozyme (see, for example, Krochmal et al, Structural basis for substrate binding and catalysis by a self-alkylating ribozyme, Nat Chem Biol 2022 18:376-384, which is hereby incorporated by reference in its entirety). In some embodiments, the ribozyme is a Varkud satellite ribozyme.

[0241] In some embodiments, the ribozyme is a prokaryotic ribozyme. In some embodiments, the ribozyme is derived or isolated from a prokaryotic organism. In some embodiments, the ribozyme is derived or isolated from bacteria or archaea. In some embodiments, the ribozyme is a ribozyme derived from T. thermophila. In some embodiments, the ribozyme is a eukaryotic ribozyme. In some embodiments, the ribozyme is derived or isolated from a eukaryotic organism. In some embodiments, the ribozyme is derived or isolated from a mammalian organism. In some embodiments, the ribozyme is derived or isolated from a human. In some embodiments, the ribozyme derived or isolated from a human is a CPEB3 ribozyme. In some embodiments, the ribozyme is derived or isolated from a chimpanzee (Pan troglodytes'). In some embodiments, the ribozyme derived or isolated from a chimpanzee is a CPEB3 ribozyme.

[0242] In some embodiments, the ribozyme is a self-cleaving ribozyme. In some embodiments, the self-cleaving ribozyme is a hammerhead ribozyme. In some embodiments, the ribozyme cleaves the target RNA molecule. In some embodiments, the ribozyme does not cleave the target RNA molecule. In some embodiments, the ribozyme acts as a steric blocker to the target RNA molecule. In some embodiments, the ribozyme acts as a steric blocking group to the target RNA molecule. In some embodiments, the ribozyme acting as a steric blocking group to the target RNA molecule improves or aids nuclear retention of the target RNA molecule. In some embodiments, the ribozyme acting as a steric blocking group to the target RNA molecule prevents or inhibits cleavage of the target RNA molecule by the spliceosome. In some embodiments, the ribozyme is a self-alkylating ribozyme.

[0243] In some embodiments, the ribozyme is regulated by the presence of one or more metal ions. In some embodiments, the secondary structure of the ribozyme is regulated or changed by the presence or absence of one or more metal ions. In some embodiments, the tertiary structure of the ribozyme is regulated or changed by the presence or absence of one or more metal ions. In some embodiments, the catalytic function of the ribozyme is regulated or changed by the presence or absence of one or more metal ions. In some embodiments, the one or more metal ions are Mg²⁺ cations. In some embodiments, the ribozyme is not regulated by the presence or absence of one or more metal ions. In some embodiments, the catalytic function of the ribozyme occurs by acid-base catalysis. Mechanisms for ribozyme catalysis are described, for example, in Lilley, Mechanisms of RNA Catalysis, Philos Trans R Soc Lond B Biol Sci. 2011 Oct 27; 366(1580):2910-2917, and by Ren et al, Structure-based mechanistic insights into catalysis by small self-cleaving ribozymes, Curr Opin Chem Biol 2017 Dec:41:71-83, each of which is hereby incorporated by reference in its entirety. In some embodiments, regulated by or changed means that the ribozyme undergoes a conformational change when bound to the one or more metal ions. In some embodiments, regulated by or changed means that the ribozyme undergoes a conformational change when bound to the one or more metal ion cofactors such that the ribozyme’s ability to catalyze a biochemical reaction is increased.

[0244] In some embodiments, the ribozyme comprises a pseudoknot. In some embodiments, the ribozyme comprises a first pseudoknot and a second pseudoknot. In some embodiments, the ribozyme comprises one or more pseudoknots. In some embodiments, the ribozyme comprises two or more pseudoknots. In some embodiments, the ribozyme is a twister ribozyme having or comprising a secondary structure of three stems joined by internal and terminal loops. In some embodiments, the ribozyme is a twister ribozyme comprising two pseudoknot structures. In some embodiments, the two pseudoknots of the twister ribozyme provide tertiary structure contacts that are critical for catalytic activity (See, for example, Roth et.al., A widespread selfcleaving ribozyme class is revealed by bioinformatics. Nature Chemical Biology 10, 56-60 (2014), which is herein incorporated by reference in its entirety). In some embodiments, the ribozyme is a twister sister ribozyme comprising a four-way junctional pre-catalytic structure (see, for example, Zheng et al., Structure-based insights into self-cleavage by a four- way junctional twister- sister ribozyme, Nat Comm 2017 8(1180), which is hereby incorporated by reference in its entirety).

[0245] In some embodiments, the ribozyme increases the trans-splicing efficiency of the trans-splicing molecule. In some embodiments, the ribozyme increases the trans-splicing efficiency of the trans-splicing molecule by at least about 20%, at least about 25%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 550%, at least about 600%, at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, at least about 1000%, or more.

[0246] In some embodiments, the G-quadruplex and/or the pseudoknot increases the trans- splicing efficiency of the trans-splicing molecule. In some embodiments, the G-quadruplex and/or the pseudoknot increases the trans-splicing efficiency by at least about 20%, at least about 25%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 550%, at least about 600%, at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, at least about 1000%, or more.

[0247] In some embodiments, the ribozyme comprises a nucleic acid sequence selected from SEQ ID NOs: 114-123. In some embodiments, the ribozyme comprises a nucleic acid sequence with at least about 80%, at least about 85%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or higher percent sequence homology to any one of SEQ ID NOs: 114-123.

Table 1: Ribozyme Sequences

Nucieic acids

Trans- splicing nucleic acid molecule

[0248] In some embodiments, the present disclosure provides trans-splicing nucleic acid molecules comprising one or more exonic domains. The nucleic acid may comprise RNA, DNA, a DNA/RNA hybrid, and/or comprises at least one of a nucleic acid analog, a chemically modified nucleic acid, or a chimera composed of two or more nucleic acids or nucleic acid analogs. In some embodiments, the trans-splicing nucleic acid molecule may be provided by reconstitution of two or more exonic domains. In some embodiments, the trans-splicing nucleic acid molecule comprises a trans-splicing RNA molecule. In some embodiments, the nucleic acid molecules provided herein encodes one or more trans-splicing RNA molecules or portions thereof. In some embodiments, the trans-splicing RNA molecules or portions thereof comprises one or more exonic domains. In some embodiments, the trans-splicing RNA molecules may be reconstituted from one or more exonic domains. In some embodiments, the trans-splicing RNA molecule or a portion thereof comprises one or more antisense domains. In some embodiments, the one or more antisense domains facilitate an association the trans-splicing RNA and a target RNA.

[0249] In some embodiments, the trans-spicing RNA molecule comprises one or more exonic domains. In some embodiments, the trans-spicing RNA molecule comprises one or more antisense domains. In some embodiments, the trans-splicing RNA molecule comprises: (a) one or more intronic domains that promote trans-splicing, (b) one or more antisense domains that are reverse complementary to a target RNA, (c) one or more exonic domains, and (d) a 3’ domain that is configured to increase the safety and efficiency of the trans-splicing molecule. In some embodiments, the 3’ domain comprises a nuclear retention domain that is configured to promote the retention of the trans-splicing RNA molecule within the nuclei and thus increases trans- splicing activity or trans-splicing occurrence. In some embodiments, the 3’ domain further comprises a stabilization domain that prevents degradation of the trans-splicing RNA molecule by nucleases. In some embodiments, the 3’ domain further comprises a cleavage domain that results in cleavage of the trans- splicing RNA at a site within or adjacent to the cleavage domain. The composition provided herein can further comprise a nucleic acid encoding an engineered U1 snRNA. In some embodiments, the intronic domain of the trans-splicing RNA molecule comprises a sequence which binds to an engineered U 1 snRNA.

[0250] In some embodiments, the trans-splicing nucleic acid molecule is RNA, DNA, a DNA/RNA hybrid, and/or comprises at least one of a nucleic acid analog, a chemically modified nucleic acid, or a chimera composed of two or more nucleic acids or nucleic acid analogs. As used herein, the term “nucleic acid analog” refers to a compound having structural similarity to a canonical purine or pyrimidine base occurring in DNA or RNA. The nucleic acid analog may contain a modified sugar and/or a modified nucleobase, as compared to a purine or pyrimidine base occurring naturally in DNA or RNA. In some embodiments, the nucleic acid analog is a 2’- deoxyribonucleoside, 2’ -ribonucleoside, 2’ -deoxyribonucleotide or a 2 ’-ribonucleotide, wherein the nucleobase includes a modified base (such as, for example, xanthine, uridine, oxanine (oxanosine), 7-methlguanosine, dihydrouridine, 5-methylcytidine, C3 spacer, 5-methyl dC, 5- hydroxybutynl-2’ -deoxyuridine, 5-nitroindole, 5-methyl iso-deoxycytosine, iso-deoxyguanosine, deoxyuridine, iso-deoxycytidine, other 0-1 purine analogs, N-6-hydroxylaminopurine, nebularine, 7-deaza hypoxanthine, other 7-deazap urines, and 2-methyl purines). In some embodiments, the nucleic acid analog may be selected from the group consisting of inosine, 7- deaza-2’ -deoxyinosine, 2’ -aza-2’ -deoxyinosine, PNA-inosine, morpholino-inosine, LNA- inosine, phosphoramidate-inosine, 2’-O-methoxyethyl-inosine, and 2’-OMe-inosine. In other embodiments the nucleic acid analog is a nucleic acid mimic (such as, for example, artificial nucleic acids and xeno nucleic acids (XNA).

[0251] The nucleic acids of the present disclosure can be chemically modified from naturally occurring nucleic acids. In some embodiments, the modification may improve the stability of the nucleic acids. In some embodiments, the modification comprises N6- methyladenosine (m6A), N6,2'-O-dimethyladenosine (m6Am), 8-oxo-7,8-dihydroguanosine (8- oxoG), pseudouridine ( ), 5-methylcytidine (m5C), or N4-acetylcytidine (ac4C).

[0252] In some embodiments, the trans-splicing RNA molecule further comprises a 5’ untranslated region. In some embodiments, the 5’ untranslated region increases the stability of the trans-splicing nucleic acid molecule. In some embodiments, the 5’ untranslated region alters the localization of the trans-splicing nucleic acid molecule. In some embodiments, the 5’ untranslated region alters the processing of the trans-splicing nucleic acid molecule. [0253] In some embodiments, the trans-splicing RNA molecule further comprises a 3' untranslated region. In some embodiments, the 3' untranslated region increases the stability of the trans-splicing nucleic acid molecule. In some embodiments, the 3' untranslated region alters the localization of the trans-splicing nucleic acid molecule. In some embodiments, the 3' untranslated region alters the processing of the trans-splicing nucleic acid molecule.

[0254] In some embodiments, provided herein is a trans-splicing nucleic acid molecule configured to trans-splice with a target RNA, e.g., a pre-mRNA in a cell. In some embodiments, the trans-splicing nucleic acid molecule is configured to trans-splice with a target RNA involved in a disease or condition.

Sequences of Trans-splicing molecules

[0255] Also provided herein are nucleic acid sequences encoding the trans-splicing nucleic acid molecules disclosed herein for use in gene transfer and expression techniques described herein. It should be understood, although not always explicitly stated that the sequences provided herein can be used to provide the expression product as well as substantially identical sequences that produce a protein that has the same biological properties. These “biologically equivalent” or “biologically active” or “equivalent” polypeptides are encoded by equivalent polynucleotides as described herein. They may possess at least 60%, or alternatively, at least 65%, or alternatively, at least 70%, or alternatively, at least 75%, or alternatively, at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% or alternatively at least 98%, identical nucleic acid sequence to the reference nucleic acid sequence when compared using sequence identity methods run under default conditions. Specific sequences are provided as examples of particular embodiments. Additionally, an equivalent polynucleotide is one that hybridizes under stringent conditions to the reference polynucleotide or its complement.

[0256] The nucleic acid sequences (e.g., polynucleotide sequences) disclosed herein may be codon-optimized. Codon optimization refers to the fact that different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. It is also possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are rare in a particular cell type, such as through codon usage tables. Based on the genetic code, nucleic acid sequences encoding various exonic domains can be generated. In some embodiments, such a sequence is optimized for expression in a host or target cell, such as a host cell used to express the trans- splicing RNA molecule containing an exonic domain in which the disclosed methods are practiced (such as in a mammalian cell, e.g., a human cell).

[0257] Codon preferences and codon usage tables for a particular species can be used to engineer isolated nucleic acid molecules encoding an exonic domain (such as one encoding a protein having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to its corresponding wild-type protein) that takes advantage of the codon usage preferences of that particular species. For example, the exonic domains disclosed herein can be designed to have codons that are preferentially used by a particular organism of interest. In one example, an exonic domain nucleic acid sequence is optimized for expression in human cells, such as one having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to its corresponding wild-type or originating nucleic acid sequence. In some embodiments, an isolated trans-splicing nucleic acid molecule encoding at least one exonic domain (which can be part of a vector) includes at least one exonic domain that is codon optimized for expression in a eukaryotic cell, or at least one exonic domain codon optimized for expression in a human cell. In one embodiment, such a codon optimized exonic domain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to its corresponding wild-type or originating sequence.

[0258] In some embodiments, a eukaryotic cell codon optimized nucleic acid sequence encodes an exonic domain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to its corresponding wild-type or originating protein. In some embodiments, a variety of clones containing functionally equivalent nucleic acids may be routinely generated, such as nucleic acids which differ in sequence but which encode the same exonic domain protein sequence. Silent mutations in the exonic domain result from the degeneracy (i.e., redundancy) of the genetic code, whereby more than one codon can encode the same amino acid residue. Thus, for example, leucine can be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT or AAC; aspartic acid can be encoded by GAT or GAC; cysteine can be encoded by TGT or TGC; alanine can be encoded by GCT, GCC, GCA, or GCG; glutamine can be encoded by CAA or CAG; tyrosine can be encoded by TAT or TAC; and isoleucine can be encoded by ATT, ATC, or ATA. Tables showing the standard genetic code can be found in various sources (see, for example, Stryer, 1988, Biochemistry, 3.sup.rd Edition, W.H.5 Freeman and Co., NY).

Vectors

[0259] The present composition provides a vector to deliver any one of the compositions, nucleic acids or systems as described herein, or for use in any one of the methods as described herein. In some embodiments of the compositions and methods of the disclosure, a vector of the disclosure is a viral vector. In some embodiments, the viral vector comprises a sequence isolated or derived from a retrovirus. In some embodiments, the viral vector comprises a sequence isolated or derived from a lentivirus. In some embodiments, the viral vector comprises a sequence isolated or derived from an adenovirus. In some embodiments, the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV). In some embodiments, the viral vector comprises a sequence isolated or derived from a herpes simplex virus (HSV). In some embodiments, the viral vector is replication incompetent. In some embodiments, the viral vector is isolated or recombinant. In some embodiments, the viral vector is self-complementary.

[0260] In some embodiments, the vector is a viral vector. In some embodiments, the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, or a lentiviral vector. In some embodiments, the vector is a retroviral vector, an adenoviral/retroviral chimera vector, a herpes simplex viral I or II vector, a parvoviral vector, a reticuloendotheliosis viral vector, a polioviral vector, a papillomaviral vector, a vaccinia viral vector, or any hybrid or chimeric vector incorporating favorable aspects of two or more viral vectors. In some embodiments, the vector further comprises one or more expression control elements operably linked to the polynucleotide. In some embodiments, the vector further comprises one or more selectable markers.

[0261] In some embodiments of the compositions and methods of the disclosure, the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV). In some embodiments, the viral vector comprises an inverted terminal repeat sequence or a capsid sequence that is isolated or derived from an AAV of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12. In some embodiments, the viral vector is replication incompetent. In some embodiments, the viral vector is isolated or recombinant (rAAV). In some embodiments, the viral vector is self-complementary (scAAV). In some embodiments, the AAV vector has low toxicity. In some embodiments, the AAV vector does not incorporate into the host genome, thereby having a low probability of causing insertional mutagenesis. In some embodiments, the AAV vector can encode a range of total polynucleotides from .3 kb to 4.75 kb. In some embodiments, AAV vectors that may be used in any of the herein described compositions, systems, methods, and kits can include an AAV1 vector, a modified AAV1 vector, an AAV2 vector, a modified AAV2 vector, an AAV3 vector, a modified AAV3 vector, an AAV4 vector, a modified AAV4 vector, an AAV5 vector, a modified AAV5 vector, an AAV6 vector, a modified AAV6 vector, an AAV7 vector, a modified AAV7 vector, an AAV8 vector, an AAV9 vector, an AAV.rhlO vector, a modified AAV.rhlO vector, an AAV.rh32/33 vector, a modified AAV.rh32/33 vector, an AAV.rh43 vector, a modified AAV.rh43 vector, an AAV.rh74 vector, a modified AAV.rh74 vector, an AAV.rh64Rl vector, and a modified AAV.rh64Rl vector and any combinations or equivalents thereof.

[0262] In some embodiments, the lentiviral vector is an integrase-competent lentiviral vector (ICLV). In some embodiments, the lentiviral vector can refer to the transgene plasmid vector as well as the transgene plasmid vector in conjunction with related plasmids (e.g., a packaging plasmid, a rev expressing plasmid, an envelope plasmid) as well as a lentiviral-based particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism. In some embodiments, lentiviral vectors that may be used in any of the herein described compositions, systems, methods, and kits can include a human immunodeficiency virus (HIV) 1 vector, a modified human immunodeficiency virus (HIV) 1 vector, a human immunodeficiency virus (HIV) 2 vector, a modified human immunodeficiency virus (HIV) 2 vector, a sooty mngabey simian immunodeficiency virus (SIVSM) vector, a modified sooty mangabey simian immunodeficiency virus (SIVSM) vector, a African green monkey simian immunodeficiency virus (SIVAGM) vector, a modified African green monkey simian immunodeficiency virus (SIVAGM) vector, an equine infectious anemia virus (EIAV) vector, a modified equine infectious anemia virus (EIAV) vector, a feline immunodeficiency virus (FIV) vector, a modified feline immunodeficiency virus (FIV) vector, a Visna/maedi virus (VNV/VMV) vector, a modified Visna/maedi virus (VNV/VMV) vector, a caprine arthritisencephalitis virus (CAEV) vector, a modified caprine arthritis-encephalitis virus (CAEV) vector, a bovine immunodeficiency virus (BIV), or a modified bovine immunodeficiency virus (BIV).

[0263] In some embodiments, a vector of the disclosure is a non- viral vector. In some embodiments, the vector comprises or consists of a lipid nanoparticle, a micelle, a liposome or lipoplex, a polymersome, a polyplex, an exosome or a dendrimer. In some embodiments, the vector is an expression vector or recombinant expression system. As used herein, the term “recombinant expression system” refers to a genetic construct for the expression of certain genetic material formed by recombination.

[0264] In some embodiments, the liposome, lipoplex, or lipid nanoparticle can further comprise a non-cationic lipid, a PEG conjugated lipid, a sterol, or any combination thereof.

[0265] In some embodiments, the liposome, lipoplex, or lipid nanoparticle further comprises a non-cationic lipid, wherein the non-ionic lipid is selected from the group consisting of distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidy lethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane- 1 - carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl- ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl- phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1 -trans PE, l-stearoyl-2- oleoyl- phosphatidy ethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid, cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine and non-cationic lipids described, for example, in WO2017/099823 or US2018/0028664.

[0266] In some embodiments, the liposome, lipoplex, or lipid nanoparticle further comprises a conjugated lipid, wherein the conjugated lipid, wherein the conjugated-lipid is selected from the group consisting of PEG-diacylglycerol (DAG) (such as l-(monomethoxy- polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG- ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0- (w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N- (carbonyl-methoxypoly ethylene glycol 2000)- 1 ,2-distearoyl-sn-glycero-3 - phosphoethanolamine sodium salt.

[0267] In some embodiments, the liposome, lipoplex, or nanoparticle further comprises cholesterol or a cholesterol derivative.

[0268] In some embodiments, the liposome, lipoplex, or nanoparticle further comprises an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits aggregation of particles, and a sterol. The amount of the ionizable lipid, the non-cationic lipid, the conjugated lipid that inhibits aggregation of particles, and the sterol can be varied independently. In some embodiments, the lipid nanoparticle comprises an ionizable lipid in an amount from about 20 mol % to about 90 mol % of the total lipid present in the particle, a non-cationic lipid in an amount from about 5 mol % to about 30 mol % of the total lipid present in the particle, a conjugated lipid that inhibits aggregation of particles in an amount from about 0.5 mol % to about 20 mol % of the total lipid present in the particle, and a sterol in an amount from about 20 mol % to about 50 mol % of the total lipid present in the particle.

[0269] The ratio of total lipid to DNA vector can be varied as desired. For example, the total lipid to DNA vector (mass or weight) ratio can be from about 10: 1 to about 30: 1.

[0270] In some embodiments of the compositions and methods of the disclosure, an expression vector, viral vector or non- viral vector provided herein, includes without limitation, an expression control element. An “expression control element” as used herein refers to any sequence that regulates the expression of an exonic domain, such as a gene. Expression control elements include but are not limited to promoters, enhancers, microRNAs, post-transcriptional regulatory elements, polyadenylation signal sequences, 5’ or 3’ untranslated regions, and introns.

[0271] Expression control elements may be constitutive, inducible, repressible, or tissuespecific, for example. A “promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. In some embodiments, expression control by a promoter is tissue specific. Non-limiting examples of promoters include CMV, CBA, CAG, Cbh, EF-la, PGK, UBC, GUSB, UCOE, hAAT, TBG, Desmin, MCK, C5-12, NSE, Synapsin, PDGF, MecP2, CaMKII, mGluR2, NFL, NFH, np2, PPE, ENK, EAAT2, GFAP, MBP, Hl and U6 promoters. In some embodiments, the promoter is a sequence isolated or derived from a promoter capable of driving expression of a transfer RNA (tRNA). In some embodiments, the promoter is isolated or derived from an alanine tRNA promoter, an arginine tRNA promoter, an asparagine tRNA promoter, an aspartic acid tRNA promoter, a cysteine tRNA promoter, a glutamine tRNA promoter, a glutamic acid tRNA promoter, a glycine tRNA promoter, a histidine tRNA promoter, an isoleucine tRNA promoter, a leucine tRNA promoter, a lysine tRNA promoter, a methionine tRNA promoter, a phenylalanine tRNA promoter, a proline tRNA promoter, a serine tRNA promoter, a threonine tRNA promoter, a tryptophan tRNA promoter, a tyrosine tRNA promoter, or a valine tRNA promoter. In some embodiments, the promoter is isolated or derived from a valine tRNA promoter.

[0272] An “enhancer” is a region of DNA that can be bound by activating proteins to increase the likelihood or frequency of transcription. Non-limiting examples of enhancers and post-transcriptional regulatory elements include the CMV enhancer and WPRE.

[0273] In some embodiments of the compositions and methods of the disclosure, an expression vector, viral vector or non- viral vector provided herein, includes without limitation, vector elements such as an IRES or 2A peptide sites for configuration of “multicistronic” or “polycistronic” or “bicistronic” or tricistronic” constructs, i.e., having double or triple or multiple coding areas or exons, and as such will have the capability to express from mRNA two or more proteins from a single construct. Multicistronic vectors simultaneously express two or more separate proteins from the same mRNA. The two strategies most widely used for constructing multicistronic configurations are through the use of an IRES or a 2A self-cleaving site. An “IRES” refers to an internal ribosome entry site or portion thereof of viral, prokaryotic, or eukaryotic origin which are used within polycistronic vector constructs. In some embodiments, an IRES is an RNA element that allows for translation initiation in a capindependent manner. The term “self-cleaving peptides” or “sequences encoding self-cleaving peptides” or “2A self-cleaving site” refer to linking sequences which are used within vector constructs to incorporate sites to promote ribosomal skipping and thus to generate two polypeptides from a single promoter, such self-cleaving peptides include without limitation, T2A, and P2A peptides or sequences encoding the self-cleaving peptides.

[0274] In some embodiments of the compositions and methods of the present disclosure, a vector comprises or encodes a trans- splicing nucleic acid molecule of the disclosure. In some embodiments, the vector comprises or encodes at least one trans-splicing nucleic acid molecule of the disclosure. In some embodiments, the vector comprises or encodes one or more trans- splicing nucleic acid(s) of the disclosure. In some embodiments, the vector comprises or encodes two or more trans-splicing nucleic acid molecules of the disclosure.

Ce/is an Tissues

[0275] The present disclosure provides compositions, systems, nucleic acid molecules, and methods for use in a cell or a tissue. In some embodiments, the target RNA is in a cell. In some embodiments of the compositions and methods of the disclosure, a cell comprises a eukaryotic cell. In some embodiments, the cell comprises a mammalian cell. In some embodiments, the cell comprises a bovine, murine, feline, equine, porcine, canine, simian, or human cell. In some embodiments, the cell comprises a non-human mammalian cell such as a non-human primate cell.

[0276] In some embodiments, the cell comprises a somatic cell. In some embodiments, the cell comprises a germline cell. In some embodiments, the germline cell of the disclosure comprises not a human cell.

[0277] In some embodiments, the cell comprises a stem cell. In some embodiments, the cell comprises an embryonic stem cell. In some embodiments, the embryonic stem cell comprises not a human cell. In some embodiments, the cell comprises a multipotent stem cell or a pluripotent stem cell. In some embodiments, the cell comprises an adult stem cell. In some embodiments, the cell comprises an induced pluripotent stem cell (iPSC). In some embodiments, the cell of the disclosure comprises a hematopoietic stem cell (HSC).

[0278] In some embodiments, the cell comprises an immune cell. In some embodiments, the immune cell comprises a lymphocyte. In some embodiments, an immune cell comprises a T lymphocyte (also referred to herein as a T-cell). Examples of T-cells of the disclosure include, but are not limited to, naive T cells, effector T cells, helper T cells, memory T cells, regulatory T cells (Tregs) and Gamma delta T cells. In some embodiments, the immune cell comprises a B lymphocyte. In some embodiments, the immune cell comprises a natural killer cell. In some embodiments, the immune cell comprises an antigen-presenting cell.

[0279] In some embodiments, the cell is a muscle cell. In some embodiments, the muscle cell comprises a myoblast or a myocyte. In some embodiments, the muscle cell comprises a cardiac muscle cell, skeletal muscle cell or smooth muscle cell. In some embodiments, the muscle cell comprises a striated cell.

[0280] In some embodiments, the cell is a somatic cell. In some embodiments, the somatic cell comprises an epithelial cell. In some embodiments, the epithelial cell comprises a squamous cell epithelium, a cuboidal cell epithelium, a columnar cell epithelium, a stratified cell epithelium, a pseudostratified columnar cell epithelium or a transitional cell epithelium. In some embodiments, the epithelial cell forms a gland including, but not limited to, a pineal gland, a thymus gland, a pituitary gland, a thyroid gland, an adrenal gland, an apocrine gland, a holocrine gland, a merocrine gland, a serous gland, a mucous gland and a sebaceous gland. In some embodiments, the epithelial cell contacts an outer surface of an organ including, but not limited to, a lung, a spleen, a stomach, a pancreas, a bladder, an intestine, a kidney, a gallbladder, a liver, a larynx, or a pharynx. In some embodiments, the epithelial cell of the disclosure contacts an outer surface of a blood vessel or a vein.

[0281] In some embodiments, the cell provided herein is a nerve cell. In some embodiments, the nerve cell comprises a neuron. In some embodiments, the nerve cell comprises a neuroglial or a glial cell. In some embodiments, the glial comprises a glial cell of the central nervous system including, but not limited to, oligodendrocytes, astrocytes, ependymal cells, and microglia. In some embodiments, theglial of the disclosure is a glial cell of the peripheral nervous system including, but not limited to, Schwann cells and satellite cells.

[0282] In some embodiments, the cell provided herein comprises a liver cell. In some embodiments, the liver cell comprises a hepatocyte. In some embodiments, the liver cell comprises a hepatic stellate cell. In some embodiments, the liver cell comprises Kupffer cell. In some embodiments, the liver cell comprises a sinusoidal endothelial cell.

[0283] In some embodiments, the cell provided herein comprises a retinal cell. In some embodiments, the retinal cell comprises a photoreceptor cell. In some embodiments, the photoreceptor cell comprises a rod. In some embodiments, the retinal cell comprises cone. In some embodiments, the retinal cell comprises a bipolar cell. In some embodiments, the retinal cell comprises a ganglion cell. In some embodiments, the retinal cell comprises a horizontal cell. In some embodiments, the retinal cell comprises an amacrine cell.

[0284] In some embodiments, the cell provided herein comprises a heart cell. In some embodiments, the heart cell comprises a cardiomyocyte. In some embodiments, the heart cell comprises a cardiac pacemaker cell.

[0285] In some embodiments, the somatic cell comprises a primary cell. In some embodiments, the somatic cell comprises a cultured cell. In some embodiments, the somatic cell comprises a somatic cell in vivo, in vitro, ex vivo or in situ.

[0286] In some embodiments, the somatic cell comprises an autologous or allogeneic cell. Methods

[0287] In some aspects, described herein are methods for repairing a target RNA using a trans-splicing molecule (e.g., trans-splicing RNA molecule) using a new trans-splicing system. In some embodiments, the trans-splicing RNA molecule or a portion thereof comprises two or more exonic domains and one or more antisense domains. The antisense domains may facilitate association of the trans-splicing RNA and a target RNA, thus promoting a trans-splicing reaction and RNA repair process of the target RNA. The trans-splicing RNA molecule may then replace one or more exonic domains of a target RNA. The nucleic acid sequences can encode one or more 3’ domains which represses protein production in the absence of a trans-splicing event. The 3’ domain can alternatively or in addition promote nuclear retention and/or increase trans- splicing efficiency by localizing the trans-splicing RNA to the nucleus. In some embodiments, the trans-splicing system comprises any nucleic acid sequences encoding or comprising any one of the domains provided herein, or a combination thereof. With more regulatory domains, the trans-splicing efficacy may be much improved.

[0288] In certain embodiments, the methods comprise providing a trans-splicing RNA molecule comprising: one more exonic domains; an intronic domain; one or more antisense domains; and a 3’ domain. In certain embodiments, the methods comprise providing a trans- splicing RNA molecule further comprising any one of the domains provided herein. In certain embodiments, the methods comprise providing a trans-splicing RNA molecule comprising any one or more of a nuclear retention domain, a stabilization domain, a cleavage domain, and/or a sequence capable of binding to an engineered U1 snRNA.

[0289] The method provided herein may promote RNA trans-splicing in a manner that is sufficient to repair disease-causing RNA sequences in human cells to address disease. Indeed, low efficiency has been a major barrier to many nucleic acid editing approaches including RNA trans-splicing. The disclosure provides compositions and methods for specifically repairing RNA sequences within these RNA trans-splicing molecules with high efficiency. The trans- splicing RNA molecule implementations show utility in a variety of contexts including repairing of disease-causing sequences or insertion of engineered sequences into target RNAs. The engineered sequences can alter the translation or stability of target RNAs to increase or decrease protein production or target RNA levels.

[0290] In some embodiments, the methods comprise administering to a subject in need thereof, a therapeutically effective amount of a treatment comprising the compositions or systems described herein. In some embodiments, the subject is afflicted with, diagnosed, or suspected to have, a genetic disease. In some embodiments, the disease comprises myotonic dystrophy, Duchenne muscular dystrophy, Dravet syndrome, LCA10, Dystrophic Epidermolysis bullosa, retinitis pigmentosa, Otoferlin syndrome, hemophilia A, dysferlinopathy.

Definitions

[0291] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0292] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0293] As used herein, the term “coupled” may refer to a weak or strong interaction between two or more atoms or molecules. The interaction may be directly or indirectly mediated by one or more molecules.

[0294] “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence- specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi- stranded complex, a single selfhybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PC reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0295] Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about lOx SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC. Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about O.lx SSC;

[0296] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non- homologous” sequence shares less than 40% identity, less than 35% identity, less than 30% identity, less than 25% identity, orless than 20% identity with one of the sequences of the present invention.

[0297] ‘ ‘Nucleic acid”, “ nucleic acid molecules”, or “nucleic acid sequences” can refer toRNA, DNA, or a DNA/RNA hybrid. The nucleic acid can comprise at least one of a nucleic acid analog, a chemically modified nucleic acid, or a chimera composed of two or more nucleic acids or nucleic acid analogs. The nucleic acid can be artificial, such as synthesized, engineered, or modified. As used herein, the term “nucleic acid analog” refers to a compound having structural similarity to a canonical purine or pyrimidine base occurring in DNA or RNA.

[0298] A permuted tRNA, or permutated tRNA is the gene product of a permuted or permutated RNA gene. As used herein, a permuted tRNA can refer to a pre-tRNA gene product of a permuted RNA gene (e.g., a circular tRNA intermediate) or a final permuted tRNA. Permuted tRNA can be found in unicellular algae (e.g., red alga, green alga, or chlorarachniophyte alga ) or archaea (e.g., crenarchaea).

[0299] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

References:

Espinoza, C.A., Goodrich, J.A., and Kugel, J.F. (2007). Characterization of the structure, function, and mechanism of B2 RNA, an ncRNA repressor of RNA polymerase II transcription. RNA 13, 583-596. 10.1261/rna.310307.

Guo, C.J., Xu, G., and Chen, L.L. (2020). Mechanisms of Long Noncoding RNA Nuclear Retention. Trends Biochem Sci 45, 947-960. 10.1016/j.tibs.2020.07.001. Long, Y., Wang, X., Youmans, D.T., and Cech, T.R. (2017). How do IncRNAs regulate transcription? Sci Adv 3, eaao2110. 10.1126/sciadv.aao2110.

Lubelsky, Y., and Ulitsky, I. (2018). Sequences enriched in Alu repeats drive nuclear localization of long RNAs in human cells. Nature 555, 107-111. 10.1038/nature25757. Miyagawa, R., Tano, K., Mizuno, R., Nakamura, Y., Ijiri, K., Rakwal, R., Shibato, J., Masuo, Y., Mayeda, A., Hirose, T., and Akimitsu, N. (2012). Identification of cis- and trans-acting factors involved in the localization of MALAT-1 noncoding RNA to nuclear speckles. RNA 18, 738-751. 10.1261/ma.028639.111.

Shukla, C.J., McCorkindale, A.L., Gerhardinger, C., Korthauer, K.D., Cabili, M.N., Shechner, D.M., Irizarry, R.A., Maass, P.G., and Rinn, J.L. (2018). High-throughput identification of RNA nuclear enrichment sequences. EMBO J 37. 10.15252/embj.201798452.

Wilusz, J.E., JnBaptiste, C.K., Lu, L.Y., Kuhn, C.D., Joshua-Tor, L., and Sharp, P.A. (2012). A triple helix stabilizes the 3' ends of long noncoding RNAs that lack poly(A) tails. Genes Dev 26, 2392-2407. 10.1101/gad.204438.112.

EXAMPLES

[0300] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: Identifying a combination of 3’ domain subdomains

[0301] In this example, the 3’ domain of a trans-splicing RNA comprises three subdomains: a stabilization domain, a cleavage domain, and a nuclear retention domain. High throughput screening can be used to identify combinations of these heterologous sequences with the desired effect of increasing the safety and efficiency of trans-splicing RNAs. The high throughput screening can test various combinations of the stabilization domain, the cleavage domain, and the nuclear retention domain in a trans-splicing RNA, as well as factors affecting RNA processing and stability, such as adjacent RNA sequences (in spite of their known activities in different contexts), to identify trans-splicing RNAs with various desired effects, such as nuclear retention, stability, and/or ability to be cleaved in order to block translation of truncated mRNA.

[0302] A pool of trans-splicing RNAs was generated using Golden Gate DNA assembly where the 3’ domain contained a mixture of different sequences at each of the subdomain positions. The subdomains were positioned relative to each other within the 3’ domain from the 5’ to 3’ direction in the following manner: nuclear retention domain, stabilization domain, and cleavage domain.

[0303] The different sequences present in the stabilization domain of this screen were the following (136 sequences): MMLV pseudo knot (mpknot), KSHV PAN ENE, RRV ENE with A-tract, 2xRRV ENE A-tract, EHV2 ENE A-tract, 2xEHV ENE A-tract, KSHV PAN ENE (79b), KSHV PAN ENE + A tract, 5x KSHV PAN ENE (79b), PSIV ENE, PSIV ENE + A tract, 2xPSIV ENE, c.c.bracovirus ENE + A-tract, 2x c.c.bracovirus ENE + A-tract, c.s. bracocirus ene + A-tract, 2x c.s. bracocirus ene + A-tract, a.p. mimivirus ene (with 26b spacer), 2x a.p. mimivirus ene (with 26b spacer), KSHV + RRV ENE, MMTV pseudoknot, SRV-1 pseudoknot, Human telomerase pseudoknot, Bacteriophage T2 pseudoknot, ydaO_riboswitch_Bs, ydaO_riboswitch_Tl, ydaO_riboswitch_Tp, ydaO_riboswitch_Tt, ToxI_Pa, SAM-II_riboswitch, SAM-I_riboswitch, SAH_riboswitch, KUNV, CPEB3_rz_Rn, CPEB3_rz_Pt, CPEB3_rz_Oc, CPEB3_rz_Mm, CPEB3_rz_Md, CPEB3_rz_La, CPEB3_rz_Hs, CPEB3_rz_Cf, CPEB3_rz_Bt, IFNG_PK_S_scrofa, IFNG_PK_M_musculus, IFNG_PK_CJacchus, IFNG_PK_C_familiaris, IFNG_PK_B_taurus, IFNG_PK, PDV3, ApMV3, PNRSV3, FCiLV3, HJLV3, APLPV3, LiRMV3, EMoV3, AV-2_3, CVV3, CiLRV3, SpLV3, TAMV1, PMoV3, BCRV1, SNSV1, CCR5_PRF, CMMV_psiA, R2_rz_Da, SESV, MIDV, MVEV, drz_Cjap_l, drz_Agam_2_l, Tetrahymena self splicing intron (PDB 1U6B), Group I self-splicing intron P4-P6 domain mutant U131A (PDB 6D8L), SELF-SPLICING GROUP I INTRON WITH BOTH EXONS (e coli, PDB 1U6B, chains A-B), Cryo-EM structure of Tetrahymena ribozyme conformation 5 undergoing the second-step self-splicing (PDB 7yG8), Structure of the lariat form of a chimeric derivative of the Oceanobacillus iheyensis group II intron in the presence of NH4+ and MG2+. (PDB 5J01), The relaxed pre-Tet-Sl state of G264A mutated Tetrahymena group I intron with 6nt 375'-exon and 2-aminopurine nucleoside (PDB 8HD6), 3DEG_l|Chain A|A/L- tRNA|Escherichia coli (562), 3DEG_2|Chain B|P-tRNA|Escherichia coli (562), 3DEG_5|Chain E[auth G]|50S RNA helix 42-44|Escherichia coli (562), lZZN_l|Chain A[auth B] 1197- MER|null, 7QR4_2|Chain B|RNA CPEB3 ribozyme|Homo sapiens (9606), 7QR3_2|Chains C, D|chimpanzee CPEB3 ribozyme|Pan troglodytes (9598), The leader region of the repA mRNA of pMU720, plasmid Collb-P9 REPZ RNA, Mycobacterium bovis leuA mRNA PSEUDOKNOT, 4MEG_l|Chain A|glmS triple mutant ribozyme|null, 3B4A_2|Chain B|glmS ribozyme RNA|Thermoanaerobacter tengcongensis (119072), 3L3C_3|Chains C[auth P], F[auth Q], I[auth R], L[auth S]|GLMS RIBOZYME|null, lYMO_l|Chain A|Telomerase RNA P2b-P3 pseudoknot|null, 5KMZ_l|Chain A|Telomerase RNA pseudoknot|Tetrahymena thermophila (5911), 486D_3|Chain C|A-SITE TRNA OF 70S RIBOSOME|Saccharomyces cerevisiae (4932), 486D_5|Chain E|E-SITE TRNA OF 70S RIBOSOME|Thermus thermophilus (274), 486D_6|Chain F|PENULTIMATE STEM OF 16S RRNA IN THE 70S RIBOSOME|Thermus thermophilus (274), 486D_l|Chain A|P-SITE TRNA OF 70S RIBOSOME|Escherichia coli (562), 4P8Z_l|Chain A|Didymium iridis partial IGS, 18S rRNA gene, I-Dirl gene and partial ITS 1 |Didymium iridis (5793), 2NOQ_l|Chain A|CrPV IRES|null, triple hairpin 1, triple hairpin

D(*CP*GP*GP*GP*GP*CP*GP*GP*GP*CP*CP*TP*TP*GP*GP*GP*CP*GP*GP*GP*GP* T)-3')_|Homo sapiens (9606) [4X], 2M27_l|Chain A|DNA_(5'- D(*CP*GP*GP*GP*GP*CP*GP*GP*GP*CP*CP*TP*TP*GP*GP*GP*CP*GP*GP*GP*GP* T)-3')_|Homo sapiens (9606) [2X], GGGGCC hexanucleotide repeat (4x), GGGGCC hexanucleotide repeat (8x), TERRA (UUAGGG)4 Telomeric repeat-containing RNAs, TERRA (UUAGGG)4 Telomeric repeat-containing RNAs [2x], TERRA (UUAGGG)4 Telomeric repeatcontaining RNAs [6x], Nkx2-5 5' UTR G-quadruplex, immunoglobulin switch (S) region G- QUADRUPLEX, Ribox02 RIBOZYME, 6PRV_l|Chains A, B, C, D|23S rRNA|Escherichia coli (562), 2Z74_2|Chain B|glmS ribozyme RNA|, 2Z74_2|Chain B|glmS ribozyme RNA| + target.

[0304] The different sequences present in the cleavage subdomain of this screen were the following (63 sequences): Hammerhead ribozyme, Hammerhead ribozyme, tRNA(gly), tRNA- Gly-CCC-1-1, tRNA-Gly-CCC-1-2, tRNA-Gly-CCC-3-1, tRNA-Gly-CCC-2-2, tRNA-Gly- CCC-2-1, tRNA-Gly-GCC-2-4, tRNA-Gly-GCC-2-5, tRNA-Gly-GCC-2-6, tRNA-Gly-GCC-2- 1, tRNA-Gly-GCC-2-2, tRNA-Gly-GCC-1-1, tRNA-Gly-GCC-1-3, tRNA-Gly-GCC-1-4, tRNA-Gly-GCC-1-5, tRNA-Gly-GCC-3-1, tRNA-Gly-GCC-4-1, tRNA-Gly-GCC-5-1, tRNA- Gly-TCC-3-1, tRNA-Gly-TCC-1-1, tRNA-Gly-TCC-2-1, tRNA-Gly-TCC-2-2, tRNA-Gly-TCC- 2-3, tRNA-Gly-TCC-2-4, tRNA-Gly-TCC-2-5, tRNA-Gly-TCC-2-6, tRNA-Gly-TCC-4-1, rice tRNA, MALAT1 mascRNA, 7QR4_2|Chain B|RNA CPEB3 ribozyme|Homo sapiens (9606), 7QR3_2|Chains C, D|chimpanzee CPEB3 ribozyme|Pan troglodytes (9598), 4R4P_l|Chain A|VS ribozyme RNA|NEUROSPORA (5140), 4R4P_l|Chain A|VS ribozyme RNA|NEUROSPORA (5140) [tandem dimer], 4R4V_l|Chain A|VS ribozyme RNA|NEUROSPORA (5140), 4R4V_l|Chain A|VS ribozyme RNA|NEUROSPORA (5140) [tandem dimer], histone 3'UTR with SLBP and HDE Hl -4 , histone 3'UTR with SLBP and HDE H2BC21D , histone 3'UTR with SLBP and HDE H2AC20 , histone 3'UTR with SLBP and HDE H2BC13 , histone 3'UTR with SLBP and HDE H2AC25, Pri-miR-16-1, Pri-miR-30a, Artificial microprocessor substrate, eIF4H exon 5 , Pri-miR-7, targeted pseudouridylation snoRNA-1, targeted pseudouridylation snoRNA-2, scaRNA-9, scaRNA-2, 5K7C_l|Chain A|RNA 47- MER|synthetic construct (32630), 5T5A_l|Chain A|DNA/RNA (71-MER)|metagenome (256318), 4OJI_l|Chain A|RNA (52-MER)|null, 4RGE_l|Chains A, B, C|env22 twister ribozyme|Synthetic (32630), 6JQ5_l|Chains A, B|RNA (82-MER) | synthetic construct (32630), 6JQ5_l|Chains A, B|RNA (82-MER)|synthetic construct (32630) DIMER, Pri-miR-31(51+51), Pri-miR-31(51+5 l)/Pre-miR-HBV, Pri-miR-3 l(38+40)/Pre-miR-HBV, Pri-miR-31(30+31)/Pre- miR-HBV, Pri-miR-31(22+2 l)/Pre-miR-HBV.

[0305] The different sequences present in the nuclear retention subdomain of this screen were the following (13 sequences): MALATI-NucRet (5471-5951) Shukla et al. 2018, MALATI-NucRet (5971-6401) Shukla et al. 2018, MALATI-NucRet (6851-7241) Shukla et al. 2018, Malatl-NucRet (SpeckleF2) Wilusz et al, 2012, MALATI-NucRet (MALAT-E) Miyagawa et al. 2012, MALATI-NucRet (MALAT-M) Miyagawa et al. 2012, Jpx #9 IncRNA nuclear loc (Lubelsky 2018), Pvtl IncRNA nuclear loc (Lubelsky 2018), Nr2fl-asl IncRNA nuclear loc (Lubelsky 2018), Emx2os IncRNA nuclear loc (Lubelsky 2018) , Alusx IncRNA nuclear loc (Lubelsky 2018) , SIRLOIN IncRNA nuclear loc (Lubelsky 2018).

[0306] The DNA library that resulted contained 13*63*136=111,384 different 3’ domains. This library of trans-splicing RNAs was applied to cells and the identity of the 3’ domains that promote highest trans-splicing efficiency were determined using long-read sequencing techniques. These combinations of subdomains were utilized in subsequent followup experiments to identify high-quality 3’ domain designs that increased trans-splicing efficiency. Example 2: Assessing the RNA repair efficiency of the CEP290 gene in vitro

[0307] In order to further investigate the activity of the trans-splicing RNA molecules carrying an effective 3’ domain design, a trans-splicing RNA was applied via transfection to cells that express CEP290. RNA was extracted from these cells and CEP290 and the repaired version of CEP290 that was subject to a trans-splicing reaction were both reverse transcribed. The resulting RNA was subject to qPCR and digital PCR to assess the efficiency of the trans- splicing reaction. The trans-splicing molecule editing efficiency was calculated as the amount of trans-spliced CEP290 divided by the sum of trans-spliced CEP290 and un-trans-spliced CEP290.

Example 3: Assessing the relative repair efficiencies of various 3’ domain

[0308] A library of trans-splicing RNAs that target CEP290 intron 26 was created that contain various 3’ domains. Each 3’ domain had a set of unique barcodes associated. These barcodes were utilized to measure the relative RNA editing efficiency of each trans-splicing RNA 3’ domain design. HEK293 cells were transfected with the library and barcodes amplified from the edited CEP290 gene. Long-read sequencing using an Oxford Nanopore system revealed the relative RNA editing activities of the different 3’ domains (FIG. 3). 'C15 vl' refers to sequence SEQ ID NO: 111, 'C15 v2' refers to sequence SEQ ID NO: 109, ‘C15 v3’ refers to sequence SEQ ID NO: 107, ‘wt triplex vl’ refers to sequence SEQ ID NO: 113, ‘wt triplex v2’ refers to sequence SEQ ID NO: 104, 'C14 vl' refers to sequence SEQ ID NO: 108, 'C14 v2' refers to sequence SEQ ID NO: 106, ‘C15 v4’ refers to sequence SEQ ID NO: 103, ‘C14 v3’ refers to sequence SEQ ID NO: 112.

Example 4: Assessing the relative repair efficiencies of various 3’ domains

[0309] In this experiment, we compared the activity of thousands of different trans-splicing systems by conducting a pooled screen of activity. First, we assembled double-stranded DNA sequences encoding various trans-splicing molecule components including intronic domain and 3’ domains. Random barcodes were also incorporated into these Golden Gate assembly reactions. The outcome of these reactions was a randomized combination of parts with predefined positions for each part type. Each individual plasmid additionally carried a unique 33-nucleotide barcode.

[0310] Next, these libraries were subjected to long-read sequencing using an Oxford Nanopore sequencer to map barcodes to specific combinations of parts. The same libraries were then applied to HEK293 cells expressing the CEP290 gene. The trans-spliced CEP290 gene products were sequenced using the long-read sequencer and barcode counts were used to assess editing efficiency of each design as reported in the figure.

[0311] Each bar in FIG. 5 represents a specific combination of parts where the intronic domain was held constant while only the 3’ domain varied. The 3’ domain is composed of two parts (shown in Table 2), and the trans-splicing molecules comprise sequences set forth in SEQ ID NOs: 126-130 shown below. In summary, the combination of specific structured sequences with a twister ribozyme outperforms the twister ribozyme alone. For example, the presence of a TERRA G-quadruplex upstream of the twister ribozyme increases editing efficiency compared to the ribozyme alone. Other structured sequences, such as the CPEB3 ribozyme, also enhance editing efficiency when combined with the twister ribozyme. Additionally, the use of specific ribozymes promotes efficient trans-splicing.

Table 2.

[0312] The trans-splicing molecules in this Example comprise the following sequences:

[0313] ATGCCGCCCAATATCAACTGGAAGGAGATCATGAAGGTGGACCCGGACGATCTC

CCCCGCCAGGAGGAGCTAGCTGACAACCTCCTGATATCTTTATCAAAGGTTGAGGTGAACG

AACTCAAGTCGGAGAAGCAGGAGAATGTTATCCACTTGTTTCGTATCACCCAATCTTTGATG

AAGATGAAGGCTCAGGAGGTTGAGCTAGCCCTGGAGGAGGTGGAGAAGGCCGGTGAGGAG

CAGGCCAAGTTCGAGAACCAGCTCAAGACGAAGGTCATGAAGCTGGAGAACGAGCTTGAG

ATGGCTCAGCAGTCCGCAGGAGGCCGGGACACCCGGTTCCTGCGGAACGAGATCTGCCAGC

TGGAGAAACAGCTGGAGCAGAAGGACCGTGAGCTGGAGGACATGGAGAAGGAGCTGGAGA

AGGAGAAGAAGGTCAACGAGCAGCTCGCCCTCCGCAACGAGGAGGCCGAGAACGAGAACT

CCAAGTTGCGGAGGGAGAACAAGCGCCTCAAGAAGAAGAATGAACAGCTCTGTCAAGATA

TTATTGACTATCAGAAGCAGATCGATAGTCAAAAGGAGACTCTGCTGTCCAGGCGGGGGGA

AGACTCCGACTACAGAAGCCAATTGAGCAAGAAGAACTATGAATTGATACAaTACTTGGAC

GAGATCCAAACCCTCACTGAGGCCAATGAGAAGATAGAGGTACAAAATCAAGAAATGCGG

AAAAATCTTGAGGAAAGTGTGCAAGAGATGGAGAAGATGACTGATGAGTATAACCGCATG

AAAGCGATAGTACACCAGACCGATAACGTAATCGACCAGCTGAAGAAAGAGAACGACCAT

TACCAGTTGCAAGTGCAAGAATTGACAGACCTGTTGAAGTCTAAAAATGAAGAGGACGACC

CAATCATGGTTGCCGTTAACGCAAAGGTAGAAGAGTGGAAGCTGATACTGTCCAGTAAGGA TGATGAAATAATTGAGTACCAACAAATGCTTCACAATCTCCGAGAGAAACTGAAGAACGCG

CAGCTGGACGCAGATAAGAGTAACGTAATGGCACTGCAGCAAGGCATTCAAGAACGAGACT

CTCAAATCAAGATGTTGACAGAGCAAGTGGAGCAATATACAAAGGAAATGGAGAAAAACA

CATGCATAATTGAGGACTTGAAAAATGAATTGCAGCGGAATAAGGGAGCGTCCACGCTGAG

TCAACAAACGCATATGAAAATACAGTCCACACTTGATATTCTTAAGGAGAAAACCAAAGAG

GCAGAGAGAACAGCGGAGTTGGCCGAAGCGGATGCCCGAGAGAAAGATAAGGAACTGGTC

GAAGCTCTCAAGCGCCTGAAAGACTATGAGAGTGGCGTCTATGGACTGGAGGACGCGGTTG

TGGAAATTAAAAATTGTAAGAACCAAATAAAAATCCGAGACAGGGAGATAGAAATTCTTAC

AAAGGAGATTAATAAACTTGAGCTGAAAATCTCTGATTTTCTCGACGAAAATGAAGCCCTG

CGCGAGCGAGTCGGGCTaGAGCCAAAGACCATGATAGATCTGACTGAGTTCCGCAATAGTA

AACACCTGAAACAGCAGCAGTATAGAGCTGAGAACCAAATACTTCTCAAAGAGATCGAGTC

TCTCGAAGAAGAACGCCTTGATCTTAAGAAAAAGATTAGACAGATGGCCCAGGAAAGAGGC

AAACGCAGTGCCACCTCCGGCCTCACCACTGAAGACTTGAATTTGACCGAGAATATATCTCA

AGGGGATCGGATATCAGAACGCAAGCTCGACCTGCTGTCCTTGAAAAACATGTCAGAGGCG

CAGTCAAAAAATGAGTTCTTGTCTAGGGAATTGATCGAAAAAGAACGCGACCTaGAGCGCT

CTCGAACGGTCATAGCCAAGTTTCAAAACAAACTGAAGGAACTGGTGGAGGAAAATAAACA

ACTGGAGGAAGGAATGAAAGAAATACTTCAAGCTATCAAAGAGATGCAAAAGGATCCGGA

TGTTAAAGGCGGTGAAACGAGTCTCATAATACCATCACTTGAAAGGCTTGTCAACGCGATTG

AAAGCAAAAACGCTGAAGGGATTTTTGACGCGTCACTTCATCTGAAGGCGCAAGTTGATCA

ACTGACGGGAAGAAATGAGGAACTTCGGCAGGAGCTTAGGGAGTCAAGAAAAGAAGCGAT

TAACTATTCTCAACAACTTGCCAAAGCCAATTTGAAGATCGATCACCTGGAGAAGGAGACA

AGCTTGCTGCGCCAGTCGGAGGGGTCGAATGTCGTGTTCAAGGGCATCGACCTCCCCGACG

GCATAGCACCCTCGTCTGCCTCCATAATCAATTCCCAGAATGAGTATCTGATCCACCTTCTCC

AGGAGCTGGAGAACAAGGAGAAGAAGCTCAAGAATCTGGAGGACTCGCTGGAGGACTATA

ACCGCAAGTTTGCGGTTATACGTCACCAGCAGTCCTTACTCTACAAGGAGTATCTCTCCGAG

AAGGAGACTTGGAAGACCGAGTCTAAGACCATCAAGGAAGAGAAGCGCAAACTGGAGGAC

CAGGTCCAGCAGGATGCGATAAAGGTTAAGGAGTATAACAATCTTCTTAACGCTCTTCAGAT

GGATTCTGATGAGATGAAGAAGATTCTCGCTGAGAACAGCCGGAAGATCACGGTGTTACAG

GTGAATGAGAAGTCTCTCATTCGCCAGTACACCACCCTTGTGGAGCTGGAGAGACAGCTCC

GCAAGGAGAACGAGAAGCAGAAGAACGAGCTTCTGTCTATGGAGGCAGAAGTTTGTGAGA

AGATCGGCTGTTTGCAGCGCTTCAAGGGCAGTTCAGGTGGAAGCTCTGGAGTGTCAGGATG

GAGACTGTTCAAGAAGATCAGCGGAGCTVNNVNNVNNVNNVNNVNNVNNVNNVNNVNNV

NNGGGTCTAGTGGAGGTAGTAGCGAGATGGCTATCTTTAAAATCGCGGCACTGCAGAAGGT

GGTGGACAACTCAGTGTCACTCTCCGAGCTGGAGCTCGCCAACAAGCAATATAACGAGTTA

ACCGCCAAATATCGAGATATTCTGCAGAAGGACAACATGTTGGTCCAGCGGACCTCCAATC

TCGAGCATTTAGAGGTaagtccgaatacgatactcagcaTGAAggtgggaggtaattgaatcgtgggggtggtttcccccacgctatt ctcataatagtaagttctcacgatgtctgatggttttataaggggctttcccctttgctcggctcacattcttctaattccggccaccatgtgaagaaaaatgtg

GATAtgacctaggcccgaatcgacaaggctctatgctccacggtGATACCGCCTAACACTGCCAATGCCGGTCCCAA

GCCCGGATAAAAGTGGAGGGGGCGG (SEQ ID NO: 126)

[0314] ATGCCGCCCAATATCAACTGGAAGGAGATCATGAAGGTGGACCCGGACGATCTC

CCCCGCCAGGAGGAGCTAGCTGACAACCTCCTGATATCTTTATCAAAGGTTGAGGTGAACG

AACTCAAGTCGGAGAAGCAGGAGAATGTTATCCACTTGTTTCGTATCACCCAATCTTTGATG

AAGATGAAGGCTCAGGAGGTTGAGCTAGCCCTGGAGGAGGTGGAGAAGGCCGGTGAGGAG

CAGGCCAAGTTCGAGAACCAGCTCAAGACGAAGGTCATGAAGCTGGAGAACGAGCTTGAG

ATGGCTCAGCAGTCCGCAGGAGGCCGGGACACCCGGTTCCTGCGGAACGAGATCTGCCAGC

TGGAGAAACAGCTGGAGCAGAAGGACCGTGAGCTGGAGGACATGGAGAAGGAGCTGGAGA

AGGAGAAGAAGGTCAACGAGCAGCTCGCCCTCCGCAACGAGGAGGCCGAGAACGAGAACT

CCAAGTTGCGGAGGGAGAACAAGCGCCTCAAGAAGAAGAATGAACAGCTCTGTCAAGATA

TTATTGACTATCAGAAGCAGATCGATAGTCAAAAGGAGACTCTGCTGTCCAGGCGGGGGGA

AGACTCCGACTACAGAAGCCAATTGAGCAAGAAGAACTATGAATTGATACAaTACTTGGAC

GAGATCCAAACCCTCACTGAGGCCAATGAGAAGATAGAGGTACAAAATCAAGAAATGCGG

AAAAATCTTGAGGAAAGTGTGCAAGAGATGGAGAAGATGACTGATGAGTATAACCGCATG

AAAGCGATAGTACACCAGACCGATAACGTAATCGACCAGCTGAAGAAAGAGAACGACCAT

TACCAGTTGCAAGTGCAAGAATTGACAGACCTGTTGAAGTCTAAAAATGAAGAGGACGACC

CAATCATGGTTGCCGTTAACGCAAAGGTAGAAGAGTGGAAGCTGATACTGTCCAGTAAGGA

TGATGAAATAATTGAGTACCAACAAATGCTTCACAATCTCCGAGAGAAACTGAAGAACGCG

CAGCTGGACGCAGATAAGAGTAACGTAATGGCACTGCAGCAAGGCATTCAAGAACGAGACT

CTCAAATCAAGATGTTGACAGAGCAAGTGGAGCAATATACAAAGGAAATGGAGAAAAACA

CATGCATAATTGAGGACTTGAAAAATGAATTGCAGCGGAATAAGGGAGCGTCCACGCTGAG

TCAACAAACGCATATGAAAATACAGTCCACACTTGATATTCTTAAGGAGAAAACCAAAGAG

GCAGAGAGAACAGCGGAGTTGGCCGAAGCGGATGCCCGAGAGAAAGATAAGGAACTGGTC

GAAGCTCTCAAGCGCCTGAAAGACTATGAGAGTGGCGTCTATGGACTGGAGGACGCGGTTG

TGGAAATTAAAAATTGTAAGAACCAAATAAAAATCCGAGACAGGGAGATAGAAATTCTTAC

AAAGGAGATTAATAAACTTGAGCTGAAAATCTCTGATTTTCTCGACGAAAATGAAGCCCTG

CGCGAGCGAGTCGGGCTaGAGCCAAAGACCATGATAGATCTGACTGAGTTCCGCAATAGTA

AACACCTGAAACAGCAGCAGTATAGAGCTGAGAACCAAATACTTCTCAAAGAGATCGAGTC

TCTCGAAGAAGAACGCCTTGATCTTAAGAAAAAGATTAGACAGATGGCCCAGGAAAGAGGC

AAACGCAGTGCCACCTCCGGCCTCACCACTGAAGACTTGAATTTGACCGAGAATATATCTCA

AGGGGATCGGATATCAGAACGCAAGCTCGACCTGCTGTCCTTGAAAAACATGTCAGAGGCG

CAGTCAAAAAATGAGTTCTTGTCTAGGGAATTGATCGAAAAAGAACGCGACCTaGAGCGCT

CTCGAACGGTCATAGCCAAGTTTCAAAACAAACTGAAGGAACTGGTGGAGGAAAATAAACA

ACTGGAGGAAGGAATGAAAGAAATACTTCAAGCTATCAAAGAGATGCAAAAGGATCCGGA TGTTAAAGGCGGTGAAACGAGTCTCATAATACCATCACTTGAAAGGCTTGTCAACGCGATTG

AAAGCAAAAACGCTGAAGGGATTTTTGACGCGTCACTTCATCTGAAGGCGCAAGTTGATCA

ACTGACGGGAAGAAATGAGGAACTTCGGCAGGAGCTTAGGGAGTCAAGAAAAGAAGCGAT

TAACTATTCTCAACAACTTGCCAAAGCCAATTTGAAGATCGATCACCTGGAGAAGGAGACA

AGCTTGCTGCGCCAGTCGGAGGGGTCGAATGTCGTGTTCAAGGGCATCGACCTCCCCGACG

GCATAGCACCCTCGTCTGCCTCCATAATCAATTCCCAGAATGAGTATCTGATCCACCTTCTCC

AGGAGCTGGAGAACAAGGAGAAGAAGCTCAAGAATCTGGAGGACTCGCTGGAGGACTATA ACCGCAAGTTTGCGGTTATACGTCACCAGCAGTCCTTACTCTACAAGGAGTATCTCTCCGAG AAGGAGACTTGGAAGACCGAGTCTAAGACCATCAAGGAAGAGAAGCGCAAACTGGAGGAC

CAGGTCCAGCAGGATGCGATAAAGGTTAAGGAGTATAACAATCTTCTTAACGCTCTTCAGAT

GGATTCTGATGAGATGAAGAAGATTCTCGCTGAGAACAGCCGGAAGATCACGGTGTTACAG GTGAATGAGAAGTCTCTCATTCGCCAGTACACCACCCTTGTGGAGCTGGAGAGACAGCTCC GCAAGGAGAACGAGAAGCAGAAGAACGAGCTTCTGTCTATGGAGGCAGAAGTTTGTGAGA

AGATCGGCTGTTTGCAGCGCTTCAAGGGCAGTTCAGGTGGAAGCTCTGGAGTGTCAGGATG

GAGACTGTTCAAGAAGATCAGCGGAGCTVNNVNNVNNVNNVNNVNNVNNVNNVNNVNNV

NNGGGTCTAGTGGAGGTAGTAGCGAGATGGCTATCTTTAAAATCGCGGCACTGCAGAAGGT GGTGGACAACTCAGTGTCACTCTCCGAGCTGGAGCTCGCCAACAAGCAATATAACGAGTTA ACCGCCAAATATCGAGATATTCTGCAGAAGGACAACATGTTGGTCCAGCGGACCTCCAATC TCGAGCATTTAGAGGTaagtccgaatacgatactcagcaTGAAggtgggaggtaattgaatcgtgggggtggtttcccccacgctatt ctcataatagtaagttctcacgatgtctgatggttttataaggggctttcccctttgctcggctcacattcttctaattccggccaccatgtgaagaaaaatgtg

GATATTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGGATACCGCC TAACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGGGCGG (SEQ ID NO: 127)

[0315] ATGCCGCCCAATATCAACTGGAAGGAGATCATGAAGGTGGACCCGGACGATCTC

CCCCGCCAGGAGGAGCTAGCTGACAACCTCCTGATATCTTTATCAAAGGTTGAGGTGAACG

AACTCAAGTCGGAGAAGCAGGAGAATGTTATCCACTTGTTTCGTATCACCCAATCTTTGATG

AAGATGAAGGCTCAGGAGGTTGAGCTAGCCCTGGAGGAGGTGGAGAAGGCCGGTGAGGAG CAGGCCAAGTTCGAGAACCAGCTCAAGACGAAGGTCATGAAGCTGGAGAACGAGCTTGAG ATGGCTCAGCAGTCCGCAGGAGGCCGGGACACCCGGTTCCTGCGGAACGAGATCTGCCAGC

TGGAGAAACAGCTGGAGCAGAAGGACCGTGAGCTGGAGGACATGGAGAAGGAGCTGGAGA

AGGAGAAGAAGGTCAACGAGCAGCTCGCCCTCCGCAACGAGGAGGCCGAGAACGAGAACT

CCAAGTTGCGGAGGGAGAACAAGCGCCTCAAGAAGAAGAATGAACAGCTCTGTCAAGATA

TTATTGACTATCAGAAGCAGATCGATAGTCAAAAGGAGACTCTGCTGTCCAGGCGGGGGGA

AGACTCCGACTACAGAAGCCAATTGAGCAAGAAGAACTATGAATTGATACAaTACTTGGAC GAGATCCAAACCCTCACTGAGGCCAATGAGAAGATAGAGGTACAAAATCAAGAAATGCGG AAAAATCTTGAGGAAAGTGTGCAAGAGATGGAGAAGATGACTGATGAGTATAACCGCATG AAAGCGATAGTACACCAGACCGATAACGTAATCGACCAGCTGAAGAAAGAGAACGACCAT

TACCAGTTGCAAGTGCAAGAATTGACAGACCTGTTGAAGTCTAAAAATGAAGAGGACGACC

CAATCATGGTTGCCGTTAACGCAAAGGTAGAAGAGTGGAAGCTGATACTGTCCAGTAAGGA

TGATGAAATAATTGAGTACCAACAAATGCTTCACAATCTCCGAGAGAAACTGAAGAACGCG

CAGCTGGACGCAGATAAGAGTAACGTAATGGCACTGCAGCAAGGCATTCAAGAACGAGACT

CTCAAATCAAGATGTTGACAGAGCAAGTGGAGCAATATACAAAGGAAATGGAGAAAAACA

CATGCATAATTGAGGACTTGAAAAATGAATTGCAGCGGAATAAGGGAGCGTCCACGCTGAG

TCAACAAACGCATATGAAAATACAGTCCACACTTGATATTCTTAAGGAGAAAACCAAAGAG

GCAGAGAGAACAGCGGAGTTGGCCGAAGCGGATGCCCGAGAGAAAGATAAGGAACTGGTC

GAAGCTCTCAAGCGCCTGAAAGACTATGAGAGTGGCGTCTATGGACTGGAGGACGCGGTTG

TGGAAATTAAAAATTGTAAGAACCAAATAAAAATCCGAGACAGGGAGATAGAAATTCTTAC

AAAGGAGATTAATAAACTTGAGCTGAAAATCTCTGATTTTCTCGACGAAAATGAAGCCCTG

CGCGAGCGAGTCGGGCTaGAGCCAAAGACCATGATAGATCTGACTGAGTTCCGCAATAGTA

AACACCTGAAACAGCAGCAGTATAGAGCTGAGAACCAAATACTTCTCAAAGAGATCGAGTC

TCTCGAAGAAGAACGCCTTGATCTTAAGAAAAAGATTAGACAGATGGCCCAGGAAAGAGGC

AAACGCAGTGCCACCTCCGGCCTCACCACTGAAGACTTGAATTTGACCGAGAATATATCTCA

AGGGGATCGGATATCAGAACGCAAGCTCGACCTGCTGTCCTTGAAAAACATGTCAGAGGCG

CAGTCAAAAAATGAGTTCTTGTCTAGGGAATTGATCGAAAAAGAACGCGACCTaGAGCGCT

CTCGAACGGTCATAGCCAAGTTTCAAAACAAACTGAAGGAACTGGTGGAGGAAAATAAACA

ACTGGAGGAAGGAATGAAAGAAATACTTCAAGCTATCAAAGAGATGCAAAAGGATCCGGA

TGTTAAAGGCGGTGAAACGAGTCTCATAATACCATCACTTGAAAGGCTTGTCAACGCGATTG

AAAGCAAAAACGCTGAAGGGATTTTTGACGCGTCACTTCATCTGAAGGCGCAAGTTGATCA

ACTGACGGGAAGAAATGAGGAACTTCGGCAGGAGCTTAGGGAGTCAAGAAAAGAAGCGAT

TAACTATTCTCAACAACTTGCCAAAGCCAATTTGAAGATCGATCACCTGGAGAAGGAGACA

AGCTTGCTGCGCCAGTCGGAGGGGTCGAATGTCGTGTTCAAGGGCATCGACCTCCCCGACG

GCATAGCACCCTCGTCTGCCTCCATAATCAATTCCCAGAATGAGTATCTGATCCACCTTCTCC

AGGAGCTGGAGAACAAGGAGAAGAAGCTCAAGAATCTGGAGGACTCGCTGGAGGACTATA

ACCGCAAGTTTGCGGTTATACGTCACCAGCAGTCCTTACTCTACAAGGAGTATCTCTCCGAG

AAGGAGACTTGGAAGACCGAGTCTAAGACCATCAAGGAAGAGAAGCGCAAACTGGAGGAC

CAGGTCCAGCAGGATGCGATAAAGGTTAAGGAGTATAACAATCTTCTTAACGCTCTTCAGAT

GGATTCTGATGAGATGAAGAAGATTCTCGCTGAGAACAGCCGGAAGATCACGGTGTTACAG

GTGAATGAGAAGTCTCTCATTCGCCAGTACACCACCCTTGTGGAGCTGGAGAGACAGCTCC

GCAAGGAGAACGAGAAGCAGAAGAACGAGCTTCTGTCTATGGAGGCAGAAGTTTGTGAGA

AGATCGGCTGTTTGCAGCGCTTCAAGGGCAGTTCAGGTGGAAGCTCTGGAGTGTCAGGATG

GAGACTGTTCAAGAAGATCAGCGGAGCTVNNVNNVNNVNNVNNVNNVNNVNNVNNVNNV

NNGGGTCTAGTGGAGGTAGTAGCGAGATGGCTATCTTTAAAATCGCGGCACTGCAGAAGGT GGTGGACAACTCAGTGTCACTCTCCGAGCTGGAGCTCGCCAACAAGCAATATAACGAGTTA

ACCGCCAAATATCGAGATATTCTGCAGAAGGACAACATGTTGGTCCAGCGGACCTCCAATC

GATAGGGGGCCACAGCAGAAGCGTTCACGTCGCAGCCCCTGTCAGCCATTGCACTCCGGCT

GCGAATTCTGCTGATACCGCCTAACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGG

AGGGGGCGG (SEQ ID NO: 128)

[0316] ATGCCGCCCAATATCAACTGGAAGGAGATCATGAAGGTGGACCCGGACGATCTC

CCCCGCCAGGAGGAGCTAGCTGACAACCTCCTGATATCTTTATCAAAGGTTGAGGTGAACG

AACTCAAGTCGGAGAAGCAGGAGAATGTTATCCACTTGTTTCGTATCACCCAATCTTTGATG

AAGATGAAGGCTCAGGAGGTTGAGCTAGCCCTGGAGGAGGTGGAGAAGGCCGGTGAGGAG

CAGGCCAAGTTCGAGAACCAGCTCAAGACGAAGGTCATGAAGCTGGAGAACGAGCTTGAG

ATGGCTCAGCAGTCCGCAGGAGGCCGGGACACCCGGTTCCTGCGGAACGAGATCTGCCAGC

TGGAGAAACAGCTGGAGCAGAAGGACCGTGAGCTGGAGGACATGGAGAAGGAGCTGGAGA

AGGAGAAGAAGGTCAACGAGCAGCTCGCCCTCCGCAACGAGGAGGCCGAGAACGAGAACT

CCAAGTTGCGGAGGGAGAACAAGCGCCTCAAGAAGAAGAATGAACAGCTCTGTCAAGATA

TTATTGACTATCAGAAGCAGATCGATAGTCAAAAGGAGACTCTGCTGTCCAGGCGGGGGGA

AGACTCCGACTACAGAAGCCAATTGAGCAAGAAGAACTATGAATTGATACAaTACTTGGAC

GAGATCCAAACCCTCACTGAGGCCAATGAGAAGATAGAGGTACAAAATCAAGAAATGCGG

AAAAATCTTGAGGAAAGTGTGCAAGAGATGGAGAAGATGACTGATGAGTATAACCGCATG

AAAGCGATAGTACACCAGACCGATAACGTAATCGACCAGCTGAAGAAAGAGAACGACCAT

TACCAGTTGCAAGTGCAAGAATTGACAGACCTGTTGAAGTCTAAAAATGAAGAGGACGACC

CAATCATGGTTGCCGTTAACGCAAAGGTAGAAGAGTGGAAGCTGATACTGTCCAGTAAGGA

TGATGAAATAATTGAGTACCAACAAATGCTTCACAATCTCCGAGAGAAACTGAAGAACGCG

CAGCTGGACGCAGATAAGAGTAACGTAATGGCACTGCAGCAAGGCATTCAAGAACGAGACT

CTCAAATCAAGATGTTGACAGAGCAAGTGGAGCAATATACAAAGGAAATGGAGAAAAACA

CATGCATAATTGAGGACTTGAAAAATGAATTGCAGCGGAATAAGGGAGCGTCCACGCTGAG

TCAACAAACGCATATGAAAATACAGTCCACACTTGATATTCTTAAGGAGAAAACCAAAGAG

GCAGAGAGAACAGCGGAGTTGGCCGAAGCGGATGCCCGAGAGAAAGATAAGGAACTGGTC

GAAGCTCTCAAGCGCCTGAAAGACTATGAGAGTGGCGTCTATGGACTGGAGGACGCGGTTG

TGGAAATTAAAAATTGTAAGAACCAAATAAAAATCCGAGACAGGGAGATAGAAATTCTTAC

AAAGGAGATTAATAAACTTGAGCTGAAAATCTCTGATTTTCTCGACGAAAATGAAGCCCTG

CGCGAGCGAGTCGGGCTaGAGCCAAAGACCATGATAGATCTGACTGAGTTCCGCAATAGTA

AACACCTGAAACAGCAGCAGTATAGAGCTGAGAACCAAATACTTCTCAAAGAGATCGAGTC

TCTCGAAGAAGAACGCCTTGATCTTAAGAAAAAGATTAGACAGATGGCCCAGGAAAGAGGC

AAACGCAGTGCCACCTCCGGCCTCACCACTGAAGACTTGAATTTGACCGAGAATATATCTCA AGGGGATCGGATATCAGAACGCAAGCTCGACCTGCTGTCCTTGAAAAACATGTCAGAGGCG

CAGTCAAAAAATGAGTTCTTGTCTAGGGAATTGATCGAAAAAGAACGCGACCTaGAGCGCT

CTCGAACGGTCATAGCCAAGTTTCAAAACAAACTGAAGGAACTGGTGGAGGAAAATAAACA

ACTGGAGGAAGGAATGAAAGAAATACTTCAAGCTATCAAAGAGATGCAAAAGGATCCGGA

TGTTAAAGGCGGTGAAACGAGTCTCATAATACCATCACTTGAAAGGCTTGTCAACGCGATTG

AAAGCAAAAACGCTGAAGGGATTTTTGACGCGTCACTTCATCTGAAGGCGCAAGTTGATCA

ACTGACGGGAAGAAATGAGGAACTTCGGCAGGAGCTTAGGGAGTCAAGAAAAGAAGCGAT

TAACTATTCTCAACAACTTGCCAAAGCCAATTTGAAGATCGATCACCTGGAGAAGGAGACA

AGCTTGCTGCGCCAGTCGGAGGGGTCGAATGTCGTGTTCAAGGGCATCGACCTCCCCGACG

GCATAGCACCCTCGTCTGCCTCCATAATCAATTCCCAGAATGAGTATCTGATCCACCTTCTCC

AGGAGCTGGAGAACAAGGAGAAGAAGCTCAAGAATCTGGAGGACTCGCTGGAGGACTATA

ACCGCAAGTTTGCGGTTATACGTCACCAGCAGTCCTTACTCTACAAGGAGTATCTCTCCGAG

AAGGAGACTTGGAAGACCGAGTCTAAGACCATCAAGGAAGAGAAGCGCAAACTGGAGGAC

CAGGTCCAGCAGGATGCGATAAAGGTTAAGGAGTATAACAATCTTCTTAACGCTCTTCAGAT

GGATTCTGATGAGATGAAGAAGATTCTCGCTGAGAACAGCCGGAAGATCACGGTGTTACAG

GTGAATGAGAAGTCTCTCATTCGCCAGTACACCACCCTTGTGGAGCTGGAGAGACAGCTCC

GCAAGGAGAACGAGAAGCAGAAGAACGAGCTTCTGTCTATGGAGGCAGAAGTTTGTGAGA

AGATCGGCTGTTTGCAGCGCTTCAAGGGCAGTTCAGGTGGAAGCTCTGGAGTGTCAGGATG

GAGACTGTTCAAGAAGATCAGCGGAGCTVNNVNNVNNVNNVNNVNNVNNVNNVNNVNNV

NNGGGTCTAGTGGAGGTAGTAGCGAGATGGCTATCTTTAAAATCGCGGCACTGCAGAAGGT

GGTGGACAACTCAGTGTCACTCTCCGAGCTGGAGCTCGCCAACAAGCAATATAACGAGTTA

ACCGCCAAATATCGAGATATTCTGCAGAAGGACAACATGTTGGTCCAGCGGACCTCCAATC

GATACGGGTGCGCGGGCGGGCGGCGGCACCATGCAGGGAAGCTGCCAGGGGCCGTGGGCA

GCGGATAGCAGGGCAAGGCCCAGTCCCGTGCAAGCCGGGACCGCCCCGGGGCGCGGCGCT

CATTCCTGC (SEQ ID NO: 129)

[0317] ATGCCGCCCAATATCAACTGGAAGGAGATCATGAAGGTGGACCCGGACGATCTC

CCCCGCCAGGAGGAGCTAGCTGACAACCTCCTGATATCTTTATCAAAGGTTGAGGTGAACG

AACTCAAGTCGGAGAAGCAGGAGAATGTTATCCACTTGTTTCGTATCACCCAATCTTTGATG

AAGATGAAGGCTCAGGAGGTTGAGCTAGCCCTGGAGGAGGTGGAGAAGGCCGGTGAGGAG

CAGGCCAAGTTCGAGAACCAGCTCAAGACGAAGGTCATGAAGCTGGAGAACGAGCTTGAG

ATGGCTCAGCAGTCCGCAGGAGGCCGGGACACCCGGTTCCTGCGGAACGAGATCTGCCAGC

TGGAGAAACAGCTGGAGCAGAAGGACCGTGAGCTGGAGGACATGGAGAAGGAGCTGGAGA

AGGAGAAGAAGGTCAACGAGCAGCTCGCCCTCCGCAACGAGGAGGCCGAGAACGAGAACT

CCAAGTTGCGGAGGGAGAACAAGCGCCTCAAGAAGAAGAATGAACAGCTCTGTCAAGATA TTATTGACTATCAGAAGCAGATCGATAGTCAAAAGGAGACTCTGCTGTCCAGGCGGGGGGA

AGACTCCGACTACAGAAGCCAATTGAGCAAGAAGAACTATGAATTGATACAaTACTTGGAC

GAGATCCAAACCCTCACTGAGGCCAATGAGAAGATAGAGGTACAAAATCAAGAAATGCGG

AAAAATCTTGAGGAAAGTGTGCAAGAGATGGAGAAGATGACTGATGAGTATAACCGCATG

AAAGCGATAGTACACCAGACCGATAACGTAATCGACCAGCTGAAGAAAGAGAACGACCAT

TACCAGTTGCAAGTGCAAGAATTGACAGACCTGTTGAAGTCTAAAAATGAAGAGGACGACC

CAATCATGGTTGCCGTTAACGCAAAGGTAGAAGAGTGGAAGCTGATACTGTCCAGTAAGGA

TGATGAAATAATTGAGTACCAACAAATGCTTCACAATCTCCGAGAGAAACTGAAGAACGCG

CAGCTGGACGCAGATAAGAGTAACGTAATGGCACTGCAGCAAGGCATTCAAGAACGAGACT

CTCAAATCAAGATGTTGACAGAGCAAGTGGAGCAATATACAAAGGAAATGGAGAAAAACA

CATGCATAATTGAGGACTTGAAAAATGAATTGCAGCGGAATAAGGGAGCGTCCACGCTGAG

TCAACAAACGCATATGAAAATACAGTCCACACTTGATATTCTTAAGGAGAAAACCAAAGAG

GCAGAGAGAACAGCGGAGTTGGCCGAAGCGGATGCCCGAGAGAAAGATAAGGAACTGGTC

GAAGCTCTCAAGCGCCTGAAAGACTATGAGAGTGGCGTCTATGGACTGGAGGACGCGGTTG

TGGAAATTAAAAATTGTAAGAACCAAATAAAAATCCGAGACAGGGAGATAGAAATTCTTAC

AAAGGAGATTAATAAACTTGAGCTGAAAATCTCTGATTTTCTCGACGAAAATGAAGCCCTG

CGCGAGCGAGTCGGGCTaGAGCCAAAGACCATGATAGATCTGACTGAGTTCCGCAATAGTA

AACACCTGAAACAGCAGCAGTATAGAGCTGAGAACCAAATACTTCTCAAAGAGATCGAGTC

TCTCGAAGAAGAACGCCTTGATCTTAAGAAAAAGATTAGACAGATGGCCCAGGAAAGAGGC

AAACGCAGTGCCACCTCCGGCCTCACCACTGAAGACTTGAATTTGACCGAGAATATATCTCA

AGGGGATCGGATATCAGAACGCAAGCTCGACCTGCTGTCCTTGAAAAACATGTCAGAGGCG

CAGTCAAAAAATGAGTTCTTGTCTAGGGAATTGATCGAAAAAGAACGCGACCTaGAGCGCT

CTCGAACGGTCATAGCCAAGTTTCAAAACAAACTGAAGGAACTGGTGGAGGAAAATAAACA

ACTGGAGGAAGGAATGAAAGAAATACTTCAAGCTATCAAAGAGATGCAAAAGGATCCGGA

TGTTAAAGGCGGTGAAACGAGTCTCATAATACCATCACTTGAAAGGCTTGTCAACGCGATTG

AAAGCAAAAACGCTGAAGGGATTTTTGACGCGTCACTTCATCTGAAGGCGCAAGTTGATCA

ACTGACGGGAAGAAATGAGGAACTTCGGCAGGAGCTTAGGGAGTCAAGAAAAGAAGCGAT

TAACTATTCTCAACAACTTGCCAAAGCCAATTTGAAGATCGATCACCTGGAGAAGGAGACA

AGCTTGCTGCGCCAGTCGGAGGGGTCGAATGTCGTGTTCAAGGGCATCGACCTCCCCGACG

GCATAGCACCCTCGTCTGCCTCCATAATCAATTCCCAGAATGAGTATCTGATCCACCTTCTCC

AGGAGCTGGAGAACAAGGAGAAGAAGCTCAAGAATCTGGAGGACTCGCTGGAGGACTATA

ACCGCAAGTTTGCGGTTATACGTCACCAGCAGTCCTTACTCTACAAGGAGTATCTCTCCGAG

AAGGAGACTTGGAAGACCGAGTCTAAGACCATCAAGGAAGAGAAGCGCAAACTGGAGGAC

CAGGTCCAGCAGGATGCGATAAAGGTTAAGGAGTATAACAATCTTCTTAACGCTCTTCAGAT

GGATTCTGATGAGATGAAGAAGATTCTCGCTGAGAACAGCCGGAAGATCACGGTGTTACAG

GTGAATGAGAAGTCTCTCATTCGCCAGTACACCACCCTTGTGGAGCTGGAGAGACAGCTCC GCAAGGAGAACGAGAAGCAGAAGAACGAGCTTCTGTCTATGGAGGCAGAAGTTTGTGAGA AGATCGGCTGTTTGCAGCGCTTCAAGGGCAGTTCAGGTGGAAGCTCTGGAGTGTCAGGATG GAGACTGTTCAAGAAGATCAGCGGAGCTVNNVNNVNNVNNVNNVNNVNNVNNVNNVNNV NNGGGTCTAGTGGAGGTAGTAGCGAGATGGCTATCTTTAAAATCGCGGCACTGCAGAAGGT GGTGGACAACTCAGTGTCACTCTCCGAGCTGGAGCTCGCCAACAAGCAATATAACGAGTTA ACCGCCAAATATCGAGATATTCTGCAGAAGGACAACATGTTGGTCCAGCGGACCTCCAATC TCGAGCATTTAGAGGTaagtccgaatacgatactcagcaTGAAggtgggaggtaattgaatcgtgggggtggtttcccccacgctatt ctcataatagtaagttctcacgatgtctgatggttttataaggggctttcccctttgctcggctcacattcttctaattccggccaccatgtgaagaaaaatgtg GATAGCGGCCAGCTCCAGGCCGCCAAACAATATGGAGCACGATACCGCCTAACACTGCCAA TGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGGGCGG (SEQ ID NO: 130)

Example 5: Splicing efficiencies of trans-splicing systems targeting CEP290

[0318] A library of trans-splicing systems targeting intron 26 of CEP290 was constructed using Golden Gate assembly techniques. This library included unique 33-base barcodes that identified each trans-splicing system, along with intronic domains, antisense domains, and 3’ prime terminal domains, including ribozymes and triple helices. A constant exonic domain containing the first 26 exons of the CEP290 gene was incorporated. The assembly process resulted in a library containing 3,825 distinct trans-splicing systems, which were then amplified in bacterial cultures and characterized using long-read sequencing technologies to provide detailed information on the genetic constructs.

[0319] Using Oxford nanopore sequencing of the library, each unique barcode was mapped to specific combinations of the assembled domains, ensuring that every combination of intronic, antisense, and 3’ prime terminal domains could be unambiguously linked to at least one unique barcode. This mapping was utilized to assess the performance of each trans-splicing system within the library. The mixture of barcoded trans-splicing systems, encoded in DNA, was introduced into HEK293 cells engineered to overexpress the CEP290 gene via a promoter knock-in. After 48 hours post-transfection, RNA was extracted from these cells, and Polymerase Chain Reaction (PCR) was used to amplify both the trans-spliced RNA products and their corresponding barcodes.

[0320] As shown in FIG. 6A, the amplified barcodes were quantified and correlated back to their specific combinations of domains. The abundance of each barcode served as a proxy for RNA repair activity, allowing determination the relative trans-splicing activity of each system within the library. For each 3’ terminal domain, separate panels were generated to visualize the distribution of trans-splicing system activities. The dark gray distribution represented the activity levels of systems containing a specific 3’ prime terminal domain, while the light gray distribution reflected the activity levels across the entire library of trans- splicing systems. The quality of a trans-splicing system was assessed based on two key metrics: the shift in distribution and the area under the curve (AUC). Distributions shifted further to the right indicated higher trans-splicing activity, and a larger AUC signified greater overall representation of systems with the listed 3’ domain. Other types of ribozymes and structured sequences that were tested (data not shown) include: MALAT1 triple helix, Lantern Rz2, chimpanzee CPEB3 ribozyme, pistol ribozyme, human CPEB3 ribozyme, env22 twister ribozyme, and Hatchet ribozyme. As shown in FIGs. 6A-6B, ribozymes such as Twister-SNV, Twister sister, Twister WT (wild type), and Lantern outperformed other types of ribozymes. The TSMs comprise sequences set forth in Table 4 below.

Example 6: Splicing efficiencies of trans-splicing systems targeting SCN1A

[0321] Libraries of trans-splicing systems targeting introns 8, 11 and 17 of SCN1A were constructed using Golden Gate assembly techniques. The libraries included unique 33-base barcodes that identified each trans-splicing system, along with intronic domains, antisense domains, and 3’ prime terminal domains, including ribozymes and triple helices. A constant exonic domain containing either exons 1-8, 1-11, or 1-17 of SCN1A were incorporated depending on the respective intron targeted. The assembly process resulted in a library containing -200,000 distinct trans-splicing systems, which were then amplified in bacterial cultures and characterized using long-read sequencing technologies to provide detailed information on the genetic constructs.

[0322] Using Oxford nanopore sequencing of the library, each unique barcode was mapped to specific combinations of the assembled domains, ensuring that every combination of intronic, antisense, and 3’ prime terminal domains could be unambiguously linked to at least one unique barcode. This mapping was utilized to assess the performance of each trans-splicing system within the library. The mixture of barcoded trans-splicing systems, encoded in DNA, was introduced into HEK293 cells engineered to overexpress the SCN1A gene via a promoter knock- in. After 48 hours post-transfection, RNA was extracted from these cells, and Polymerase Chain Reaction (PCR) was used to amplify both the trans- spliced RNA products and their corresponding barcodes.

[0323] The amplified barcodes were quantified and correlated back to their specific combinations of domains. The abundance of each barcode served as a proxy for RNA repair activity, allowing determination of the relative trans- splicing activity of each system within the library. For each three 3’ terminal domains, separate curves of relative trans-splicing activity were generated that illustrate the relative trans-splicing activity across each intron among trans- splicing systems contain the annotated 3’ terminal domain (FIG. 7).

[0324] The top designs in FIG. 7, based on relative editing efficiency for each intron, were selected and constructed as individual plasmids. These selected designs were then subjected to digital PCR to measure the fraction of edited target SCN1A RNA. The results are presented in the accompanying bar graphs at the bottom. The components of the constructs P2594, P2585, P2566 and P2572 are shown in Table 3. The TSMs comprise sequences set forth in Table 4 below.

Table 3.

Table 4.

[0325] The present disclosure is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the present disclosure. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.

Claims

1. A trans- splicing nucleic acid molecule, comprising:

(a) an exonic domain;

(b) an intronic domain configured to promote ribonucleic acid (RNA) trans-splicing;

(c) an antisense domain configured to bind to a target RNA molecule; and

(d) a sequence or structure derived or isolated from a ribozyme, wherein the ribozyme is selected from the group consisting of: a VS ribozyme, a twister ribozyme, a twister sister ribozyme, a lantern ribozyme, a pistol ribozyme, a hairpin ribozyme, a leadzyme, a hatchet ribozyme, a GIRI branching ribozyme, a glmS ribozyme, a Class I intron, a Class II intron, a RNAse P, a CoTC ribozyme, a Hoylinc ribozyme, a Varkud satellite ribozyme, a CPEB3 ribozyme, and a riboswitch.

2. The trans-splicing nucleic acid molecule of claim 1, further comprising a G-quadruplex and/or a pseudoknot, and optionally a poly(A).

3. The trans-splicing nucleic acid molecule of claim 2, comprising: from 5’ to 3’: the G-quadruplex and/or the pseudoknot, and the sequence or structure derived or isolated from a ribozyme, and optionally the poly (A) at the 3’ end.

4. The trans-splicing nucleic acid molecule of claim 2, comprising: from 5’ to 3’: the sequence or structure derived or isolated from a ribozyme, the G- quadruplex and/or the pseudoknot, and optionally the poly(A) at the 3’ end.

5. The trans-splicing nucleic acid molecule of any one of claims 1-4, wherein the trans- splicing nucleic acid molecule does not comprise a hammerhead ribozyme or an HDV ribozyme.

6. A trans-splicing nucleic acid molecule, comprising:

(a) an exonic domain;

(c) an antisense domain configured to bind to a target RNA molecule; and d) a sequence or structure derived or isolated from a ribozyme, wherein the trans-splicing nucleic acid molecule does not comprise a 3’ poly(A).

7. The trans-splicing nucleic acid molecule of claim 6, wherein the sequence or structure derived or isolated from the ribozyme is in a 3’ domain of the trans-splicing nucleic acid molecule.

8. The trans-splicing nucleic acid molecule of claim 6, wherein the sequence or structure derived or isolated from the ribozyme is in a 5’ domain of the trans-splicing nucleic acid molecule.

9. The trans-splicing nucleic acid molecule of any one of claims 6-8, wherein the ribozyme is selected from the group consisting of a hammerhead ribozyme, an HDV ribozyme, a twister ribozyme, a twister sister ribozyme, a lantern ribozyme, a pistol ribozyme, a hairpin ribozyme, a VS ribozyme, a leadzyme, a hatchet ribozyme, a GIRI branching ribozyme, a glmS ribozyme, a Class I intron, a Class II intron, a RNAse P, a CoTC ribozyme, a Hoylinc ribozyme, a Varkud satellite ribozyme, a CPEB3 ribozyme, and a riboswitch.

10. The trans-splicing nucleic acid molecule of any one of claims 1-9, comprising a sequence or structure derived or isolated from a twister ribozyme or a twister sister ribozyme.

11. The trans-splicing nucleic acid molecule of any one of claims 1-10, comprising a sequence or structure derived or isolated from a lantern ribozyme.

12. The trans-splicing nucleic acid molecule of any one of claims 1-11, comprising a sequence or structure derived or isolated from a CPEB3 ribozyme, optionally wherein the CPEB3 ribozyme is a mammalian CPEB3 ribozyme, and optionally a human CPEB3 ribozyme or a Pan troglodytes CPEB3 ribozyme.

13. The trans-splicing nucleic acid molecule of any one of claims 1-12, comprising a sequence or structure derived or isolated from a VS ribozyme.

14. The trans-splicing nucleic acid molecule of any one of claims 1-13, comprising a sequence or structure derived or isolated from a hatchet ribozyme.

15. The trans-splicing nucleic acid molecule of any one of claims 1-14, wherein the ribozyme is a ribozyme derived or isolated from an eukaryote, optionally wherein the eukaryote is a mammal, and optionally wherein the mammal is a human or Pan troglodytes.

16. The trans-splicing nucleic acid molecule of any one of claims 1-14, wherein the ribozyme is a ribozyme derived or isolated from a virus.

17. The trans-splicing nucleic acid molecule of any one of claims 1-14, wherein the ribozyme is a ribozyme derived or isolated from a prokaryote, optionally wherein the prokaryote is a bacterium.

18. The trans-splicing nucleic acid molecule of any one of claims 1-17, wherein the ribozyme is a mutant ribozyme.

19. The trans-splicing nucleic acid molecule of any one of claims 1-17, wherein the ribozyme is an engineered ribozyme.

20. The trans-splicing nucleic acid molecule of any one of claims 1-19, wherein the ribozyme is derived from or encoded by a IncRNA.

21. The trans-splicing nucleic acid molecule of any one of claims 1-20, wherein the ribozyme is a self-cleaving ribozyme.

22. The trans-splicing nucleic acid molecule of any one of claims 1-20, wherein the ribozyme is a self-alkylating ribozyme.

23. The trans-splicing nucleic acid molecule of any one of claims 1-22, wherein the ribozyme comprises one or more pseudoknots.

24. The trans-splicing nucleic acid molecule of any one of claims 1-23, wherein the ribozyme is regulated by or requires one or more metal ion cofactors.

25. The trans-splicing nucleic acid molecule of claim 24, wherein the one or more metal ion cofactors comprise a divalent cation.

26. The trans-splicing nucleic acid molecule of claim 25, wherein the one or more metal ion cofactors comprise Mg²⁺.

27. The trans-splicing nucleic acid molecule of any one of claims 1-23, comprising a 3’ domain which comprises a cleavage domain, wherein the sequence or structure derived or isolated from the ribozyme is a first ribozyme sequence or structure, and optionally wherein the cleavage domain comprises: a second ribozyme sequence or structure that is the same or different from the first ribozyme sequence or structure; a microprocessor substrate; an RNAse P/Z substrate; or any combination thereof.

28. The trans-splicing nucleic acid molecule of claim 27, wherein the cleavage domain comprises the sequence or structure derived or isolated from the ribozyme.

29. The trans-splicing nucleic acid molecule of claim 27, wherein the cleavage domain does not comprise the sequence or structure derived or isolated from the ribozyme.

30. The trans-splicing nucleic acid molecule of any one of claims 1-29, comprising a 3’ domain which comprises a stabilization domain.

31. The trans-splicing nucleic acid molecule of claim 30, wherein the stabilization domain comprises the sequence or structure derived or isolated from the ribozyme.

32. The trans-splicing nucleic acid molecule of claim 30, wherein the stabilization domain does not comprise the sequence or structure derived or isolated from the ribozyme.

33. The trans-splicing nucleic acid molecule of claim 32, wherein the stabilization domain comprises a G-quadruplex and/or a pseudoknot.

34. The trans-splicing nucleic acid molecule of claim 33, wherein the stabilization domain comprises a G-quadruplex.

35. The trans-splicing nucleic acid molecule of claim 33, wherein the stabilization domain comprises a pseudoknot.

36. The trans-splicing nucleic acid molecule of any one of claims 1-35, comprising a 3’ domain which comprises a nuclear retention domain, optionally wherein the nuclear retention domain comprises a triple helix (optionally viral or human triple helices), a pseudoknot, a riboswitch, a G-quadruplex (optionally a telomerase G-quadruplex), an RNAse P RNA, a stemloop structure, a snoRNA, or any combination thereof.

37. The trans-splicing nucleic acid molecule of claim 36, wherein the nuclear retention domain comprises the sequence or structure derived or isolated from the ribozyme.

38. The trans-splicing nucleic acid molecule of claim 36, wherein the nuclear retention domain does not comprise the sequence or structure derived or isolated from the ribozyme.

39. The trans-splicing nucleic acid molecule of any one of claims 1-38, comprising a 3’ domain which comprises, from 5’ to 3’: the G-quadruplex and/or the pseudoknot and the sequence or structure derived or isolated from the ribozyme.

40. The trans-splicing nucleic acid molecule of any one of claims 1-38, comprising a 3’ domain which comprises, from 5’ to 3’: the sequence or structure derived or isolated from the ribozyme and the G-quadruplex and/or the pseudoknot.

41. A trans-splicing nucleic acid molecule comprising, from 5' to 3', the following operably linked domains:

(a) an exonic domain;

(b) an intronic domain comprising a 5' splice site, a branch site, and a polypyrimidine tract;

(c) an antisense domain configured to bind to a target RNA molecule; and

(d) a 3' domain comprising a sequence or structure derived or isolated from a ribozyme, wherein the trans-splicing nucleic acid molecule is configured to trans-splice the exonic domain to an exon of the target RNA molecule.

42. The trans-splicing nucleic acid molecule of claim 41, wherein the antisense domain is configured to hybridize to an intron of the target RNA molecule which is 5’ to the exon of the target RNA molecule.

43. A trans-splicing nucleic acid molecule comprising, from 5' to 3', the following operably linked domains:

(a) an antisense domain configured to bind to a target RNA molecule;

(b) an intronic domain comprising a branch site, a polypyrimidine tract, and a 3' splice site;

(c) an exonic domain; and

44. The trans-splicing nucleic acid molecule of claim 43, wherein the antisense domain is configured to hybridize to an intron of the target RNA molecule which is 3’ to the exon of the target RNA molecule.

45. The trans-splicing nucleic acid molecule of any one of claims 41-44, wherein the intronic domain comprises an intronic splicing enhancer sequence.

46. The trans-splicing nucleic acid molecule of claim 41 or claim 42, wherein the intronic domain comprises the 5' splice site, an intronic splicing enhancer sequence, the branch site, and the polypyrimidine tract.

47. The trans-splicing nucleic acid molecule of claim 43 or claim 44, wherein the intronic domain comprises an intronic splicing enhancer sequence, the branch site, the polypyrimidine tract, and the 3' splice site.

48. The trans-splicing nucleic acid molecule of any one of claims 41-47, wherein the exonic domain comprises an exonic splicing enhancer sequence.

49. The trans-splicing nucleic acid molecule of any one of claims 41-48, wherein the 3' domain further comprises a structured sequence which is 5' to the sequence or structure derived or isolated from the ribozyme.

50. The trans-splicing nucleic acid molecule of claim 49, wherein the structured sequence comprises a G-quadruplex, a pseudoknot, and/or a triple helix.

51. The trans-splicing nucleic acid molecule of any one of claims 41-50, further comprising a nuclear retention domain.

52. The trans-splicing nucleic acid molecule of claim 51, wherein the 3' domain comprises the nuclear retention domain which is 5' to the sequence or structure derived or isolated from the ribozyme.

53. The trans-splicing nucleic acid molecule of claim 52, wherein the 3' domain comprises, from 5' to 3', the nuclear retention domain, the structured sequence, and the sequence or structure derived or isolated from the ribozyme.

54. The trans-splicing nucleic acid molecule of any one of claims 41-53, wherein the 3' domain does not comprise any one or more of a triple helix, an RNase P cleavage site, a tRNA- like domain, and a poly(A) tail.

55. The trans-splicing nucleic acid molecule of any one of claims 41-54, which does not comprise any one or more of a triple helix, an RNase P cleavage site, a tRNA-like domain, and a poly(A) tail.

56. The trans-splicing nucleic acid molecule of any one of claims 41-55, wherein the ribozyme is selected from the group consisting of: a VS ribozyme, a twister ribozyme, a twister sister ribozyme, a lantern ribozyme, a pistol ribozyme, a hairpin ribozyme, a leadzyme, a hatchet ribozyme, a GIRI branching ribozyme, a glmS ribozyme, a Class I intron, a Class II intron, a RNAse P, a CoTC ribozyme, a Hoylinc ribozyme, a Varkud satellite ribozyme, a CPEB3 ribozyme, and a riboswitch.

57. The trans-splicing nucleic acid molecule of any one of claims 41-56, wherein the sequence or structure derived or isolated from the ribozyme has a cleavage activity.

58. The trans-splicing nucleic acid molecule of any one of claims 41-56, wherein the sequence or structure derived or isolated from the ribozyme does not have a cleavage activity.

59. A trans-splicing nucleic acid molecule comprising, from 5' to 3', the following operably linked domains:

(a) an exonic domain;

(c) an antisense domain configured to bind to a target RNA molecule; and

(d) a 3' domain comprising, from 5' to 3': (i) a G-quadruplex or a pseudoknot, and (ii) a sequence or structure derived or isolated from a twister ribozyme or a twister sister ribozyme, wherein the trans-splicing nucleic acid molecule is configured to trans-splice the exonic domain to an exon of the target RNA molecule.

60. The trans-splicing nucleic acid molecule of claim 59, which does not comprise any one or more of a triple helix, an RNase P cleavage site, a tRNA-like domain, and a poly(A) tail.

61. The trans-splicing nucleic acid molecule of any one of claims 1-60, wherein the sequence or structure derived or isolated from the ribozyme increases trans-splicing efficiency of the trans-splicing nucleic acid molecule.

62. The trans-splicing nucleic acid molecule of claim 61, wherein the increase in transsplicing efficiency is by at least about 10%, by at least about 20%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 100%, by at least about 200%, by at least about 300%, by at least about 400%, by at least about 500%, by at least about 600%, by at least about 700%, by at least about 800%, by at least about 900%, or by 1000%, compared to a reference trans-splicing nucleic acid molecule without the sequence or structure derived or isolated from the ribozyme.

63. The trans-splicing nucleic acid molecule of claim 61 or claim 62, further comprising a G- quadruplex and/or the pseudoknot that increases trans-splicing efficiency of the trans-splicing nucleic acid molecule.

64. The trans-splicing nucleic acid molecule of claim 63, wherein the increase in trans- splicing efficiency is by at least about 10%, by at least about 20%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 100%, by at least about 200%, by at least about 300%, by at least about 400%, by at least about 500%, by at least about 600%, by at least about 700%, by at least about 800%, by at least about 900%, or by 1000%, compared to a reference trans-splicing nucleic acid molecule without the sequence or structure derived or isolated from the ribozyme and the G-quadruplex and/or the pseudoknot.

65. The trans-splicing nucleic acid molecule of any one of claims 1-64, wherein the target RNA molecule is in a cell.

66. The trans-splicing nucleic acid molecule of any one of claims 1-64, wherein the target RNA molecule is a messenger RNA (mRNA) or a pre-mRNA.

67. The trans-splicing nucleic acid molecule of any one of claims 1-64, wherein the target RNA molecule comprises a mutation.

68. The trans-splicing nucleic acid molecule of claim 67, wherein the mutation is selected from the group consisting of a missense mutation, a nonsense mutation, a frameshift mutation, an insertion, a duplication, an inversion, a deletion, a splice site mutation, and a truncating mutation.

69. The trans-splicing nucleic acid molecule of claim 67, wherein the mutation is a diseasecausing mutation.

70. The trans-splicing nucleic acid molecule of any one of claims 1-69, wherein the transsplicing nucleic acid molecule is packaged in or encoded by a viral vector for delivery to a subject in need thereof.

71. The trans-splicing nucleic acid molecule of claim 70, wherein the viral vector is a herpes simplex virus (HSV) vector.

72. The trans-splicing nucleic acid molecule of claim 70, wherein the viral vector is an adeno-associated virus (AAV) vector.

73. The trans-splicing nucleic acid molecule of any one of claims 1-72, wherein the exonic domain comprises one or more functional exons of a gene associated with a disease or condition.

74. A composition comprising the viral vector of any one of claims 70-72 and a pharmaceutically acceptable carrier or excipient.

75. The trans-splicing nucleic acid molecule of any one of claims 1-69, wherein the trans- splicing nucleic acid molecule is packaged in or encoded by a nucleic acid in a lipid nanoparticle for delivery to a subject in need thereof.

76. A composition comprising the lipid nanoparticle of claim 75 and a pharmaceutically acceptable carrier or excipient.

77. The trans-splicing nucleic acid molecule of any one of claims 1-69, wherein the engineered nucleic acid is packaged in or encoded by a nucleic acid in a vesicle for delivery to a subject in need thereof.

78. A composition comprising the vesicle of claim 77 and a pharmaceutically acceptable carrier or excipient.

79. A cell comprising the trans-splicing nucleic acid molecule or the composition of any one of claims 1-78.

80. The trans-splicing nucleic acid molecule, the composition, or the cell of any one of claims 1-79 for use in treating a disease and/or correcting a genetic defect in a subject in need thereof.

81. Use of the trans-splicing nucleic acid molecule, the composition, or the cell of any one of claims 1-79 for treating a disease and/or correcting a genetic defect in a subject in need thereof.

82. Use of the trans-splicing nucleic acid molecule, the composition, or the cell of any one of claims 1-79 in the manufacture of a medicament for treating a disease and/or correcting a genetic defect in a subject in need thereof.

83. A method for treating a disease and/or correcting a genetic defect in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the trans- splicing nucleic acid molecule, the composition, or the cell of any one of claims 1-79.

84. A method for processing a cell comprising contacting the cell with the trans-splicing nucleic acid molecule or the composition of any one of claims 1-78.

85. The method of claim 84, wherein the method is performed in vitro and optionally the cell is a primary cell isolated from a subject, a cultured cell, or a transformed cell.