EP4689118A1 - Compositions for and methods of engineering the transcriptome - Google Patents

Abstract

Disclosed herein are CRISPR-free compositions for and methods of generating chimeric RNA molecules via trans-splicing and methods of treating and/or preventing a genetic disease or disorder using chimeric RNA molecules generated via trans-splicing.

Description

COMPOSITIONS FOR AND METHODS OF ENGINEERING THE TRANSCRIPTOME

I. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63/493,696 filed 31 March 2023, which is incorporated herein in its entirety.

II. STATEMENT REGARDING FEDERAL FUNDING

[0002] This invention was made with Government support under Federal Grant No. NS099371 awarded by the National Institute of Neurological Disorders & Stroke (NIH/NINDS). The Federal Government has certain rights to this invention.

III. REFERENCE TO THE SEQUENCE LISTING

[0003] The Sequence Listing submitted 29 March 2024 as an XML file named “23-2078- WO_Sequence Listing”, created on 29 March 2024 and having a size of 95 kilobytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

IV. BACKGROUND

[0004] In eukaryotic organisms, chromosomal DNA is transcribed into precursor RNA messages (pre-mRNA) which contain protein coding regions (exons) and intervening non-protein coding regions (introns). Prior to processing, these pre-mRNA molecules do not possess a sequence primed for translation by the ribosome, due to the retention of non-coding intronic sequences. Thus, prior to nuclear export, the exons of pre-mRNA transcripts are joined through a cellular mechanism known as splicing. This mechanism features dual transesterifications mediated by a large multi ribonucleoprotein structure, called the spliceosome. In the first transesterification, the branch point sequence of the intervening intron attacks the 5’ splice site, forming a lariat structure. This reaction frees the 5’ splice site to attack the 3’ splice site removing the intervening intron, joining the adjacent exons. Upon removal of all intronic sequences, the precursor message matures into a translation competent mature RNA transcript, which is trafficked to the ribosome where it is decoded to manufacture cellular proteins.

[0005] In mammalian cells, mutations in transcriptionally active regions of chromosomal DNA give rise to pre-mRNA bearing identical mutations. If the mutation is located in a non-coding region, then processing of the pre-mRNA may be altered or abolished. If the mutation is located in an exonic region of the pre-mRNA, then that mutation will be passed to the mature mRNA sequence. These mutations can contribute to inhibition of complete protein translation of the encoded protein (non-sense mutation) or modify the primary structure of the encoded protein in a counter-productive manner (missense mutation). Collectively, these genetically encoded mutations may function to contribute to pathogenesis in eukaryotes.

[0006] The field of gene therapy has aimed to correct such genetic abnormalities through adoptive gene transfer of recombinant nucleic acids bearing a sequence capable of producing the protein product of the mutated gene. This strategy, conventionally termed "classical gene therapy’’ has proven to be a safe and effective strategy for phenoty pic correction of genetic disorders, with several gene therapy products available on the market.

[0007] However, current approaches to gene therapy are limited due to toxicity arising from overexpression of transcripts, the inability to deliver large transcripts exceeding the packaging capacity of AAV vectors (i.e., considered to be the standard vector for gene therapy and having a packaging capacity of ~4.7 KB), the inability to correct and replace large stretches of disease causing mRNA, and the inability to correct and replace autosomal dominant inheritance with a single transcript and/or approach.

[0008] Thus, there remains an urgent need for a minimally invasive, definitive therapy to address the underlying cause of as well as the sequelae of symptoms associated with these various genetic diseases and disorders. Consequently, the present disclosure provides compositions for and methods of generating chimeric RNA molecules via trans-splicing (and without CRISPR) and treating and/or preventing a genetic disease and/or disorder, which can be used alone or in combination with other treatments.

V. BRIEF DESCRIPTION OF THE FIGURES

[0009] FIG. 1 (left side) shows a mechanism of 5’ trans-splicing while FIG. 1 (right side) shows the mechanism of 3’ trans-splicing. The schematic on the left side shows first the constructs that were transfected to demonstrate trans-splicing for 5’ replacement. The stop codon in the first half of the open reading frame blocks translation if the RNA species splices in cis. If the trans-splicing RNA successfully edits the RNA, then the open reading frame of EGFP is restored and fluorescent expression is restored. The schematic on the right side shows first the constructs that were transfected to demonstrate trans-splicing for 3’ replacement. The stop codon in the second half of the open reading frame blocks translation if the RNA species splices in cis. If the trans-splicing RNA successfully edits the RNA, the open reading frame of EGFP is restored and fluorescent expression is restored. GRAFT is Guide RNA Assisted Fragment Trans-splicing.

[0010] FIG. 2A - FIG. 2B show a panel of 5’-splicing motifs. These are flow cytometry data from co-transfection experiment of 5’ trans-splicing candidate molecules. In FIG. 2A, the percent of cells that express green fluorescence when trans-splicing RNA candidates were delivered to cells was measured. RNA structures 1-11 are plotted against the x axis, and percent GFP positive cells is plotted on the y axis. In FIG. 2B. the mean fluorescent intensity of cells when trans- splicing RNA candidates were delivered was measured. RNA structures 1-11 were plotted against the x axis, and mean fluorescent intensity is plotted on the y axis.

[0011] FIG. 3 A - FIG. 3D show a panel of 3’ trans-splicing motifs. Flow cytometry data from co-transfection experiment of 3‘ trans-splicing candidate molecules. In FIG. 3A, the percent of cells that express green fluorescence when trans-splicing RNA candidates were delivered to cells was measured. RNA structures 1-11 are plotted against the x axis, and percent GFP positive cells is plotted on the y axis. In FIG. 3B, the mean fluorescent intensity of cells when trans-splicing RNA candidates were delivered was measured. RNA structures 1-11 are plotted against the x axis, and mean fluorescent intensity is plotted on the y axis. The data in FIG. 3C and FIG. 3D were measured in the same way for a different target intron.

[0012] FIG. 4A - FIG. 4B show repeat validation of top 3’ Trans-splicing RNA candidates. Flow cytometry data from co-transfection experiment of 3’ trans-splicing candidate molecules. In FIG. 4A, the percent of cells that express green fluorescence when trans-splicing RNA candidates were delivered to cells was measured. RNA structures are plotted against the x axis, and percent GFP positive cells is plotted on the y axis. In FIG. 4B, the mean fluorescent intensity of cells when trans-splicing RNA candidates were delivered was measured. RNA structures are plotted against the x axis, and mean fluorescent intensity is plotted on the y axis.

[0013] FIG. 5A- FIG. 5B show repeat validation of top 3’ trans-splicing RNA candidates against new target. Flow cytometry data from co-transfection experiment of 3’ trans-splicing candidate molecules was measured. In FIG. 5A, the percent of cells that express green fluorescence w hen trans-splicing RNA candidates we are delivered to cells was measured. RNA structures are plotted against the x axis, and percent GFP positive cells is plotted on the y axis. In FIG. 5B, the mean fluorescent intensity of cells when trans-splicing RNA candidates were delivered was measured. RNA structures are plotted against the x axis, and mean fluorescent intensity is plotted on the y axis.

[0014] FIG. 6A - FIG. 6G provide plasmid maps for the constructs used in Example 1 and/or disclosed herein. FIG. 6A shows an exemplary 3’ Replacement Construct (Null). FIG. 6B the 3’ Replacement Construct for RYR2 (3-GRAFT-RYR2) while FIG. 6C shows the 5’ Replacement Construct for RYR2 (5-GRAFT-RYR2). FIG. 6D shows the 3’ Replacement Construct for LMNA (3-GRAFT-LMNA) while FIG. 6E shows the 3’ Replacement Construct for FXN (3-GRAFT-FXN). FIG. 6F shows the Split GFP Reporter Construct for LMNA while FIG. 6G shows Split GFP Reporter Construct for RYR2.

[0015] FIG. 7A show s the 5’ replacement construct for DMD in Example 2. FIG. 7B shows the 3’ replacement construct for DMD in Example 2.

VI. BRIEF SUMMARY

[0016] Disclosed herein is a nucleic acid molecule comprising an exogenous RNA to be transspliced to a targeted endogenous pre-mRNA; a 5’ hemi intron; one or more RNA targeting motifs; and one or more RNA structures. [0017] Disclosed herein is a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA.

[0018] Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron; one or more RNA targeting motifs; one or more RNA structures.

[0019] Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA.

[0020] Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or a viral vector comprising a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5' hemi intron; one or more RNA targeting motifs; one or more RNA structures.

[0021] Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or a viral vector comprising a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA.

[0022] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a disclosed non-viral vector or a disclosed viral vector or a pharmaceutical formulation thereof, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation.

[0023] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a disclosed non-viral vector or a disclosed viral vector or a pharmaceutical formulation thereof, wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0024] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a non-viral vector, a viral vector, an AAV particle, or a pharmaceutical formulation thereof comprising (i) one or more 5’ replacement constructs, (ii) one or more 3' replacement constructs, or (iii) one or more 5’ replacement constructs and/or one or more 3’ replacement constructs, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation, and/or wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0025] Disclosed herein is a method of inhibiting and/or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a disclosed non-viral vector or a disclosed viral vector or a pharmaceutical formulation thereof, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation.

[0026] Disclosed herein is a method of inhibiting and/or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a disclosed non-viral vector or a disclosed viral vector or a pharmaceutical formulation thereof, wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity⁷ of a missing, deficient, and/or mutant protein or enzyme.

[0027] Disclosed herein is method of inhibiting and/or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subj ect in need thereof a therapeutically effective amount of a non-viral vector, a viral vector, an AAV particle, or a pharmaceutical formulation thereof comprising (i) one or more 5‘ replacement constructs, (ii) one or more 3’ replacement constructs, or (iii) one or more 5’ replacement constructs and/or one or more 3’ replacement constructs, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation, and/or wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

VII. DETAILED DESCRIPTION

[0028] The present disclosure describes formulations, compounded compositions, kits, capsules, containers, and/or methods thereof. It is to be understood that the inventive aspects of which are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0029] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

A. Definitions

[0030] Before the present compounds, compositions, articles, systems, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0031] This disclosure describes inventive concepts with reference to specific examples. However, the intent is to cover all modifications, equivalents, and alternatives of the inventive concepts that are consistent with this disclosure.

[0032] As used in the specification and the appended claims, the singular forms "‘a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0033] The phrase “consisting essentially of’ limits the scope of a claim to the recited components in a composition or the recited steps in a method as well as those that do not materially affect the basic and novel characteristic or characteristics of the claimed composition or claimed method. The phrase “consisting of’ excludes any component, step, or element that is not recited in the claim. The phrase “comprising” is synonymous with “including”, “containing”, or “characterized by”, and is inclusive or open-ended. “Comprising” does not exclude additional, unrecited components or steps.

[0034] As used herein, when referring to any numerical value, the term “about” means a value falling within a range that is ± 10% of the stated value.

[0035] Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0036] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0037] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. In an aspect, a disclosed method can optionally comprise one or more additional steps, such as, for example, repeating an administering step or altering an administering step.

[0038] As used herein, “isolated” refers to a nucleic acid molecule or a nucleic acid sequence that has been substantially separated, produced apart from, or purified away from other biological components in the cell or tissue of an organism in which the component occurs, such as other cells, chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins.

[0039] As used herein, the term “subject” refers to the target of administration, e.g., a human being. The term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g, cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g, mouse, rabbit, rat, guinea pig, fruit fly. etc.). Thus, the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Alternatively, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not denote a particular age or sex, and thus, adult and child subjects, as well as fetuses, whether male or female, are intended to be covered. In an aspect, a subject can be a human patient. In an aspect, a subject can have a disease or disorder, be suspected of having a disease or disorder, or be at risk of developing a disease or disorder (e.g., a genetic disease or disorder). In an aspect, a subject can be treatment-naive. [0040] As used herein, a ‘'regulatory element” can refer to promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Regulatory' elements can include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulator}' sequences). %

[0041] As used herein, the term “diagnosed” means having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods. For example, '‘diagnosed with a disease or disorder” means having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition (such as a genetic disease or disorder) that can be treated by one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods. For example, “suspected of having a disease or disorder” can mean having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition (such as a genetic disease or disorder) that can likely be treated by one or more of by one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods. In an aspect, an examination can be physical, can involve various tests (e.g., blood tests, genotyping, biopsies, etc.) and assays (e.g., enzymatic assay), or a combination thereof.

[0042] A “patient” refers to a subject afflicted with a disease or disorder (e.g., a genetic disease or disorder). In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder. In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder and is seeking treatment or receiving treatment for a disease or disorder.

[0043] As used herein, the phrase “identified to be in need of treatment for a disease or disorder.” or the like, refers to selection of a subject based upon need for treatment of the disease or disorder. For example, a subject can be identified as having a need for treatment of a disease or disorder (e.g., a genetic disease or disorder) based upon an earlier diagnosis by a person of skill and thereafter subjected to treatment for the genetic disease or disorder. In an aspect, the identification can be performed by a person different from the person making the diagnosis. In an aspect, the administration can be performed by one who performed the diagnosis.

[0044] As used herein, “inhibit,” “inhibiting”, and “inhibition” mean to diminish or decrease an activity, level, response, condition, severity, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, level, response, condition, severity, disease, or other biological parameter. This can also include, for example, a 10% inhibition or reduction in the activity, level, response, condition, severity, disease, or other biological parameter as compared to the native or control level (e.g.. a subject not having a disease or disorder such as a genetic disease or disorder). Thus, in an aspect, the inhibition or reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of reduction in between as compared to native or control levels. In an aspect, the inhibition or reduction can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% as compared to native or control levels. In an aspect, the inhibition or reduction can be 0-25%, 25- 50%, 50-75%, or 75-100% as compared to native or control levels. In an aspect, a native or control level can be a pre-disease or pre-disorder level.

[0045] The words “treat’' or “treating” or “treatment” include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In an aspect, the terms cover any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the undesired physiological change, disease, pathological condition, or disorder from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the physiological change, disease, pathological condition, or disorder, i.e.. arresting its development; or (iii) relieving the physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease. For example, in an aspect, treating a disease or disorder can reduce the severity of an established a disease or disorder in a subject by l%-100% as compared to a control (such as, for example, an individual not having a genetic disease or disorder). In an aspect, treating can refer to a 1%. 2%, 3%, 4%, 5%, 6%, 7%. 8%, 9%. 10%. 20%. 30%. 40%. 50%. 60%. 70%. 80%. 90%. or 100% reduction in the severity- of a disease or disorder (such as a genetic disease or disorder). For example, treating a disease or disorder can reduce one or more symptoms of a disease or disorder in a subject by l%-100% as compared to a control (such as, for example, an individual not having a genetic disease or disorder). In an aspect, treating can refer to 1%. 2%, 3%. 4%, 5%, 6%. 7%, 8%. 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% reduction of one or more symptoms of an established a disease or disorder. It is understood that treatment does not necessarily refer to a cure or complete ablation or eradication of a disease or disorder. However, in an aspect, treatment can refer to a cure or complete ablation or eradication of a disease or disorder. [0046] As used herein, the term “prevent” or “preventing” or “prevention” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. In an aspect, preventing a disease or disorder having chromatin deregulation and/or chromatin dysregulation is intended. The words “prevent”, “preventing”, and “prevention” also refer to prophylactic or preventative measures for protecting or precluding a subject (e.g., an individual) not having a given a disease or disorder (such as a genetic disease or disorder) or related complication from progressing to that complication.

[0047] As used herein, the terms “administering” and “administration” refer to any method of providing one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, the following: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can also include hepatic intra-arterial administration or administration through the hepatic portal vein (HPV). Administration of a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical composition, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed small molecule, a disclosed endonuclease, a disclosed oligonucleotide, and/or a disclosed RNA therapeutic can comprise administration directly into the CNS or the PNS. Administration can be continuous or intermittent. Administration can comprise a combination of one or more route.

[0048] In an aspect, the skilled person can determine an efficacious dose, an efficacious schedule, and an efficacious route of administration for one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof to treat or prevent a disease or disorder (such as genetic disease or disorder). In an aspect, the skilled person can also alter, change, or modify an aspect of an administering step to improve efficacy of one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof.

[0049] By “determining the amount” is meant both an absolute quantification of a particular analyte (e.g.. an mRNA sequence containing a particular tag) or a determination of the relative abundance of a particular analyte (e.g., an amount as compared to a mRNA sequence including a different tag). The phrase includes both direct or indirect measurements of abundance (e.g., individual mRNA transcripts may be quantified or the amount of amplification of an mRNA sequence under certain conditions for a certain period may be used a surrogate for individual transcript quantification) or both.

[0050] As used herein, “modifying the method” can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof administered to a subject, or by changing the frequency of administration of one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof to a subject, by changing the duration of time one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination are administered to a subject, or by substituting for one or more of the disclosed components and/or reagents with a similar or equivalent component and/or reagent. The same applies to all disclosed therapeutic agents, immune modulators, immunosuppressive agents, proteosome inhibitors, etc.

[0051] As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. In an aspect, a pharmaceutical carrier employed can be a solid, liquid, or gas. In an aspect, examples of solid carriers can include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. In an aspect, examples of liquid carriers can include sugar syrup, peanut oil, olive oil, and water. In an aspect, examples of gaseous carriers can include carbon dioxide and nitrogen. In preparing a disclosed composition for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions: while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical earners are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly (orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0052] As used herein, the term “excipient” refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.). See, also, for reference, Remington’s Pharmaceutical Sciences, (1990) Mack Publishing Co., Easton, Pa., which is hereby incorporated by reference in its entirety.

[0053] As used herein, “concurrently” means (1) simultaneously in time, or (2) at different times during the course of a common treatment schedule.

[0054] The term “contacting” as used herein refers to bringing one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof together with a target area or intended target area in such a manner that the one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof exert an effect on the intended target or targeted area either directly or indirectly. A target area can comprise one or more cells, and in an aspect, one or more cells can be in a subject. A target area or intended target area can be one or more of a subject’s organs (e.g., lungs, heart, liver, kidney, brain, etc.). In an aspect, a target area or intended target area can be any cell or any organ infected by a disease or disorder (such as a genetic disease or disorder). In an aspect, a target area or intended target area can be any organ, tissue, or cells that are affected by a disease or disorder (such as a genetic disease or disorder).

[0055] As used herein, “determining” can refer to measuring or ascertaining the presence and severity of a disease or disorder, such as, for example, a genetic disease or disorder. Methods and techniques used to determine the presence and/or severity of a disease or disorder are typically known to the medical arts. For example, the art is familiar with the ways to identify and/or diagnose the presence, severity, or both of a disease or disorder (such as, for example, a genetic disease or disorder).

[0056] As used herein, “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired result such as, for example, the treatment and/or prevention of a disease or disorder (e.g., a genetic disease or disorder) or a suspected disease or disorder. As used herein, the terms “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired an effect on an undesired condition (e.g., a disease or disorder). For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. In an aspect, “therapeutically effective amount” means an amount of a disclosed nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation; that (i) treats the particular disease, condition, or disorder (e.g., a genetic disease or disorder), (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder e.g., a genetic disease or disorder), or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein (e.g., a genetic disease or disorder). The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations employed; the disclosed methods employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations employed; the duration of the treatment; drugs used in combination or coincidental with the disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations employed, and other like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, then the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, a single dose of the disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a '■prophylactically effective amount"; that is, an amount effective for prevention of a disease or condition, such as, for example, a disease or disorder due to a missing, deficient, and/or mutant protein or enzyme.

[0057] As used herein, “RNA therapeutics” can refer to the use of oligonucleotides to target RNA. RNA therapeutics can offer the promise of uniquely targeting the precise nucleic acids involved in a particular disease with greater specificity, improved potency, and decreased toxicity. This could be particularly powerful for genetic diseases where it is most advantageous to aim for the RNA as opposed to the protein. In an aspect, a therapeutic RNA can comprise one or more expression sequences. As known to the art, expression sequences can comprise an RNAi, shRNA, mRNA, non-coding RNA (ncRNA). an antisense such as an antisense RNA, miRNA. morpholino oligonucleotide, peptide-nucleic acid (PNA) or ssDNA (with natural, and modified nucleotides, including but not limited to, LNA, BNA, 2’-O-Me-RNA, 2’-MEO-RNA, 2’-F-RNA), or analog or conjugate thereof. In an aspect, a disclosed therapeutic RNA can comprise one or more long non-coding RNA (IncRNA), such as, for example, a long intergenic non-coding RNA (lincRNA), pre-transcript, pre-miRNA, pre-mRNA, competing endogenous RNA (ceRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), pseudo-gene, rRNA, or tRNA. In an aspect, ncRNA can be piwi-interacting RNA (piRNA), primary miRNA (pri-miRNA), or premature miRNA (pre-miRNA). In an aspect, a disclosed therapeutic RNA or an RNA therapeutic can comprise antisense oligonucleotides (ASOs) that inhibit mRNA translation, oligonucleotides that function via RNA interference (RNAi) pathway, RNA molecules that behave like enzymes (ribozy mes), RNA oligonucleotides that bind to proteins and other cellular molecules, and ASOs that bind to mRNA and form a structure that is recognized by RNase H resulting in cleavage of the mRNA target. In an aspect, RNA therapeutics can comprise RNAi and ASOs that inhibit mRNA translation. Generally speaking, as known to the art, RNAi operates sequence specifically and post-transcriptionally by activating ribonucleases which, along with other enzymes and complexes, coordinately degrade the RNA after the original RNA target has been cut into smaller pieces while antisense oligonucleotides bind to their target nucleic acid via Watson-Crick base pairing, and inhibit or alter gene expression via steric hindrance, splicing alterations, initiation of target degradation, or other events.

[0058] As used herein, “small molecule'’ can refer to any organic or inorganic material that is not a polymer. Small molecules exclude large macromolecules, such as large proteins (e.g., proteins with molecular weights over 2,000, 3,000, 4,000. 5,000, 6.000, 7,000. 8,000, 9.000, or 10,000), large nucleic acids (e.g., nucleic acids with molecular weights of over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000), or large polysaccharides (e.g., polysaccharides with a molecular weight of over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000. 8,000, 9.000, or 10,000). In an aspect, a “small molecule”, for example, can be a drug that can enter cells easily because it has a low molecular weight. In an aspect, a small molecule can be used in conjunction with a disclosed composition in a disclosed method.

[0059] In an aspect, the term “ex vivo” can refer generally to activities that take place outside an organism or subject such as experimentation, modification, differentiation, manipulation, and/or measurement done in or on living tissue in an artificial environment outside the organism. In an aspect, ex vivo experimentation, ex vivo modification, ex vivo differentiation, ex vivo manipulation, and/or ex vivo measurement can occur with a minimum alteration of the natural conditions. In an aspect, “ex vivo"’ can comprise living cells, tissues, or organs (e.g., cells in need of trans-splicing for one or more protein coding genes) taken from a subject in need thereof or a donor subject and cultured and/or maintained and/or perfused in a laboratory apparatus, usually under sterile conditions, and typically for a limited duration of time (e.g., a few hours or up to about 24 hours, up to about 48 hours, up to about 72 hours, up to about 96 hours, up to about 120 hours, up to about 144 hours, up to about 168 hours, or more depending on the circumstances and/or the desired characteristics. In an aspect, tissues, cells, or organs can be collected, frozen, and later thawed for ex vivo treatment.

[0060] As used herein, “operably linked” means that expression of a gene or a transgene is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5’ (upstream) or 3’ (downstream) of a gene under its control. The distance between the promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance can be accommodated without loss of promoter function.

[0061] As used herein, “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein must contain at least two amino acids and there is no limitation on the maximum number of amino acids that can comprise a protein's sequence. The term “peptide” can refer to a short chain of amino acids including, for example, natural peptides, recombinant peptides, synthetic peptides, or any combination thereof. Proteins and peptides can include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others.

[0062] "‘Nucleic acid” or “oligonucleotide” or “polynucleotide” as used herein means at least two nucleotides covalently linked together. The depiction of a single strand can also define the sequence of the complementary strand. Thus, a nucleic acid can encompass the complementary strand of a depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid can encompass substantially identical nucleic acids and complements thereof. A single strand can provide a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid can encompass a probe that hybridizes under stringent hybridization conditions. A nucleic acid can be single-stranded, or double-stranded, or can contain portions of both double-stranded and single-stranded sequence. The nucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods. Also as used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid construct,” “nucleotide sequence”, and “polynucleotide” can refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term can encompass RNA/DNA hybrids. When dsRNA is produced synthetically, less common bases, such as inosine, 5- methylcytosine, 6-methyladenine, hypoxanthine and others can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been show n to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modification to the phosphodiester backbone, or the 2'-hy droxy in the ribose sugar group of the RNA can also be made. A “synthetic” nucleic acid or polynucleotide, as used herein, refers to a nucleic acid or polynucleotide that is not found in nature but is constructed by the hand of man and therefore is not a product of nature.

[0063] A “polynucleotide” is a sequence of nucleotide bases, and may be RNA, DNA, or DNA- RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotides).

[0064] A “fragment” or “portion” of a nucleotide sequence can be understood to mean a nucleotide sequence of reduced length relative (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides) to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of, or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence. Such a nucleic acid fragment or portion according to the disclosure can be. where appropriate, included in a larger polynucleotide of which it is a constituent. In an aspect, a fragment or portion of a nucleotide sequence or nucleic acid sequence can comprise the sequence encoding an exon having one or more mutations.

[0065] A “fragment” or “portion” of an amino acid sequence can be understood to mean an amino acid sequence of reduced length relative (e.g., reduced by 1, 2. 3, 4, 5. 6, 7, 8. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or more amino acids) to a reference amino acid sequence and comprising, consisting essentially of, or consisting of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the reference amino acid sequence. Such an amino acid fragment or portion according to the disclosure can be, where appropriate, included in a larger amino acid sequence of which it is a constituent.

[0066] A “heterologous” or a “recombinant” nucleotide or amino acid sequence as used interchangeably herein can refer to a nucleotide or an amino acid sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleotide or amino acid sequence.

[0067] As used herein, the term “endogenous” can refer to a gene, protein, compound, or activity that is normally present in a host cell (e.g., a pre-mRNA). As used herein, an “exogenous” nucleic acid molecule, construct, or sequence (e.g., an RNA sequence to be trans-spliced) can refer to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to a host cell, but may be homologous to a nucleic acid molecule or portion of a nucleic acid molecule from the host cell.

[0068] Different nucleic acids or proteins having homology can be referred to as “homologues”. The term homologue includes homologous sequences from the same and other species and orthologous sequences from the same and other species. “Homology” refers to the level of similarity between two or more nucleic acid and/or amino acid sequences in terms of percent of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties among different nucleic acids or proteins. Thus, the disclosed compositions and disclosed methods can comprise homologues to the disclosed nucleotide sequences and/or disclosed polypeptide sequences. [0069] “Orthologous,” as used herein, can refer to homologous nucleotide sequences and/or amino acid sequences in different species that arose from a common ancestral gene during speciation. A homologue of a disclosed nucleotide sequence or a disclosed polypeptide can have substantial sequence identity (e.g., at least about 70%, 71%, 72%, 73%, 74%. 75%. 76%, 77%, 78%. 79%. 80%. 81%. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100%) to a disclosed nucleotide sequence or a disclosed polypeptide.

[0070] “Complement’' or “complementary” as used herein means a nucleic acid can mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. “Complementarity” refers to a property shared between two nucleic acid sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.

[0071] As used herein, “promoter” or “promoters” are known to the art. Depending on the level and tissue-specific expression desired, a variety of promoter elements can be used. A promoter can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the gene expression desired. A promoter can be native (endogenous) or foreign (exogenous) and can be a natural or a synthetic sequence. By foreign or exogenous, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

[0072] “Tissue-specific promoters” are known to the art and include, but are not limited to, neuron-specific promoters, muscle-specific promoters, liver-specific promoters, skeletal musclespecific promoters, and heart-specific promoters.

[0073] “Liver-specific promoters” are known to the art and include, but are not limited to, the thyroxin binding globulin (TBG) promoter, the al-microglobulin/bikunin enhancer/thyroid hormone-binding globulin promoter, the human albumin (hALB) promoter, the thyroid hormone- binding globulin promoter, the a- 1 -anti-trypsin promoter, the bovine albumin (bAlb) promoter, the murine albumin (mAlb) promoter, the human al -antitrypsin (hAAT) promoter, the ApoEhAAT promoter comprising the ApoE enhancer and the hAAT promoter, the transthyretin (TTR) promoter, the liver fatty acid binding protein promoter, the hepatitis B virus (HBV) promoter, the DC172 promoter comprising the hAAT promoter and the al -microglobulin enhancer, the DC 190 promoter comprising the human albumin promoter and the prothrombin enhancer, or any other natural or synthetic liver-specific promoter. In an aspect, a liver specific promoter can comprise about 845-bp and comprise the thyroid hormone-binding globulin promoter sequences (2382 to 13), two copies of al-microglobulin/bikunin enhancer sequences (22,804 through 22,704), and a 71-bp leader sequence as described by Ill CR, et al. (1997). [0074] Ubiquitous/constitutive promoters’’ are known to the art and include, but are not limited to, a CMV major immediate-early enhancer/chicken beta-actin promoter, a cytomegalovirus (CMV) major immediate-early promoter, an Elongation Factor 1-a (EFl -a) promoter, a simian vacuolating virus 40 (SV40) promoter, an AmpR promoter, a PyK promoter, a human ubiquitin C gene (Ubc) promoter, a MFG promoter, a human beta actin promoter, a CAG promoter, a EGR1 promoter, a FerH promoter, a FerL promoter, a GRP78 promoter, a GRP94 promoter, a HSP70 promoter, a P-kin promoter, a murine phosphoglycerate kinase (mPGK) or human PGK (hPGK) promoter, a ROSA promoter, human Ubiquitin B promoter, a Rous sarcoma vims promoter, or any other natural or synthetic ubiquitous/constitutive promoters.

[0075] As used herein, an “inducible promoter” refers to a promoter that can be regulated by positive or negative control. Factors that can regulate an inducible promoter include, but are not limited to, chemical agents (e.g., the metallothionein promoter or a hormone inducible promoter), temperature, and light.

[0076] As used herein, the term “serotype” is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serologic distinctiveness can be determined by the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences/antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequence differences of AAV serotypes).

[0077] As used herein, “tropism” refers to the specificity of an AAV capsid protein present in an AAV viral particle, for infecting a particular type of cell or tissue. The tropism of an AAV capsid for a particular type of cell or tissue may be determined by measuring the ability of AAV vector particles comprising the hybrid AAV capsid protein to infect or to transduce a particular type of cell or tissue, using standard assays that are well-known in the art such as those disclosed in the examples of the present application. As used herein, the term “liver tropism” or “hepatic tropism” refers to the tropism for liver or hepatic tissue and cells, including hepatocytes.

[0078] “Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as “substantially identical” or “essentially similar” when they are optimally aligned. For example, sequence similarity or identity can be determined by searching against databases such as FASTA, BLAST, etc., but hits should be retrieved and aligned pairwise to compare sequence identity. Two proteins or two protein domains, or two nucleic acid sequences can have “substantial sequence identity” if the percentage sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, preferably 90%, 95%, 98%, 99% or more. Such sequences are also referred to as “variants” herein, e.g.. other variants of a missing, deficient, and/or mutant protein or enzyme. It should be understood that sequence with substantial sequence identity do not necessarily have the same length and may differ in length. For example, sequences that have the same nucleotide sequence but of which one has additional nucleotides on the 3’- and/or 5'-side are 100% identical.

[0079] As used herein, “codon optimization” can refer to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing one or more codons or more of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. As contemplated herein, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database.” Many methods and software tools for codon optimization have been reported previously. (See, for example, genomes urv.es/ OPTIMIZER/) .

[0080] In an aspect, "RNA editing” can be a post-transcriptional modification where a precursor mRNA (pre-mRNA) nucleotide sequence is changed by base insertion, deletion, or modification. The extent of RNA editing varies from a few hundred bases, in mitochondrial DNA of trypanosomes, to a just single base, in nuclear genes of mammals. Even a single base change in the pre-mRNA can convert a codon for one amino acid into the codon for another amino acid or a stop codon. This type of re-coding can significantly affect the structure and function of a protein and may lead to the production of multiple variants of a protein from a single gene.

[0081] In an aspect, insertional and deletional RNA editing can involve the addition and deletion of specific nucleotides or sequences of nucleotides from pre-mRNA. In an aspect, substitutional RNA editing by base modifications is observed in higher eukaryotes, where the base is modified without changing the length of the pre-mRNA.

[0082] As used herein, “immune tolerance,” “immunological tolerance,” and “immunotolerance” refers to a state of unresponsiveness or blunted response of the immune system to substances (e.g., a disclosed nucleic acid molecule, a disclosed vector, a disclosed transgene product, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, etc.) that have the capacity to elicit an immune response in a subject. Immune tolerance is induced by prior exposure to a specific antigen. Immune tolerance can be determined in a subject by measuring antibodies against a particular antigen or by liver-restricted transgene expression with a viral vector (such as, for example, AAV). Low or absent antibody titers over time is an indicator of immune tolerance. For example, in some embodiments, immune tolerance can be established by having IgG antibody titers of less than or equal to about 12,000, 11,500, 11,000, 10,500, 10,000, 9,500, 9,000, 8,500, 8,000, 7,500, 7,000, 6,500, or 6,000 within following gene therapy (such as the administration of the transgene encoding, for example, a missing, deficient, and/or mutant protein or enzyme).

[0083] As known to the art, antibodies (Abs) can mitigate AAV infection through multiple mechanisms by binding to AAV capsids and blocking critical steps in transduction such as cell surface attachment and uptake, endosomal escape, productive trafficking to the nucleus, or uncoating as well as promoting AAV opsonization by phagocytic cells, thereby mediating their rapid clearance from the circulation. For example, in humans, serological studies reveal a high prevalence of NAbs in the worldwide population, with about 67% of people having antibodies against AAV1. 72% against AAV2, and approximately 40% against AAV serotypes 5 through 9. Vector immunogenicity represents a major challenge in re-administration of AAV vectors.

[0084] In an aspect, also disclosed herein are partial self-complementary parvovirus (e.g., a disclosed AAV) genomes, plasmid vectors encoding the parvovirus genomes, and parvovirus (e.g., a disclosed AAV) particles including such genomes. In an aspect, provided herein is a plasmid vector comprising a nucleotide sequence encoding a disclosed parvovirus genome such as for example, a disclosed AAV. In an aspect, provided herein is a partial self-complementary parvovirus genome including a payload construct, parvovirus ITRs flanking the payload construct, and a self-complementary region flanking one of the ITRs. A self-complementary region can comprise a nucleotide sequence that is complementary to the payload construct. A disclosed self- complementary region can have a length that is less the entire length of the pay load construct.

[0085] In an aspect, a disclosed self-complementary' region of a disclosed parvovirus genome can comprise a minimum length, while still having a length that is less the entire length of the payload construct. In an aspect, a disclosed self-complementary’ region can comprise at least 50 bases in length, at least 100 bases in length, at least 200 in length, at least 300 bases in length, at least 400 bases in length, at least 500 bases in length, at least 600 bases in length, at least 700 bases in length, at least 800 bases in length, at least 900 bases in length, or at least 1,000 bases in length.

[0086] In an aspect, a “self-complementary parvovirus genome” can be a single stranded polynucleotide having, in the 5’ to 3’ direction, a first parvovirus ITR sequence, a heterologous sequence (e g., pay load construct comprising, for example, a desired gene), a second parvovirus ITR sequence, a second heterologous sequence, wherein the second heterologous sequence is complementary' to the first heterologous sequence, and a third parvovirus ITR sequence. In contrast to a self-complementary genome, a “partial self-complementary genome” does not include three parvovirus ITRs and the second heterologous sequence that is complementary’ to the first heterologous sequence has a length that is less than the entire length of the first heterologous sequence (e.g., payload construct). Accordingly, a partial self-complementary⁷ genome is a single stranded polynucleotide having, in the 5’ to 3 ‘ direction or the 3’ to 5 ‘ direction, a first parvovirus ITR sequence, a heterologous sequence (e.g., payload construct), a second parvovirus ITR sequence, and a sei f-complementary region that is complementary to a portion of the heterologous sequence and has a length that is less than the entire length the heterologous sequence.

[0087] As used herein, “immune-modulating” refers to the ability of a disclosed nucleic acid molecules, a disclosed vector, a disclosed pharmaceutical formulation, or a disclosed agent to alter (modulate) one or more aspects of the immune system. The immune system functions to protect the organism from infection and from foreign antigens by cellular and humoral mechanisms involving lymphocytes, macrophages, and other antigen-presenting cells that regulate each other by means of multiple cell-cell interactions and by elaborating soluble factors, including lymphokines and antibodies, that have autocrine, paracrine, and endocrine effects on immune cells.

[0088] As used herein, “immune modulator” refers to an agent that is capable of adjusting a given immune response to a desired level (e.g., as in immunopotentiation, immunosuppression, or induction of immunologic tolerance). Examples of immune modulators include but are not limited to, a disclosed immune modulator can comprise aspirin, azathioprine, belimumab, betamethasone dipropionate, betamethasone valerate, bortezomib, bredinin, cyazathioprine, cyclophosphamide, cyclosporine, deoxy spergualin, didemnin B, fluocinolone acetonide, folinic acid, ibuprofen. IL6 inhibitors (such as sarilumab) indomethacin, inebilizumab, intravenous gamma globulin (IVIG), methotrexate, methylprednisolone, mycophenolate mofetil, naproxen, prednisolone, prednisone, prednisolone indomethacin, rapamycin, rituximab, sirolimus, sulindac, synthetic vaccine particles containing rapamycin (SVP-Rapamycin or ImmTOR), thalidomide, tocilizumab, tolmetin, triamcinolone acetonide. anti-CD3 antibodies, anti-CD4 antibodies, anti-CD19 antibodies, anti- CD20 antibodies, anti-CD22 antibodies, anti-CD40 antibodies, anti-FcRN antibodies, anti-IL6 antibodies, anti-IGFIR antibodies, an IL2 mutein, a BTK inhibitor, or a combination thereof. In an aspect, a disclosed immune modulator can comprise one or more Treg (regulatory T cells) infusions (e.g., antigen specific Treg cells to AAV). In an aspect, a disclosed immune modulator can be bortezomib or SVP-Rapamycin. In an aspect, an immune modulator can be administered by any suitable route of administration including, but not limited to, in utero, intra-CSF, intrathecally, intravenously, subcutaneously, transdermally, intradermally, intramuscularly, orally, transcutaneously, intraperitoneally (IP), or intravaginally. In an aspect, a disclosed immune modulator can be administered using a combination of routes. Administration can also include hepatic intra-arterial administration or administration through the hepatic portal vein (HPV). Administration of an immune modulator can be continuous or intermittent, and administration can comprise a combination of one or more routes. [0089] As used herein, the term “immunotolerant” refers to unresponsiveness to an antigen (e.g., a vector, a therapeutic protein, a transgene product, etc.). An immunotolerant promoter can reduce, ameliorate, or prevent transgene-induced immune responses that can be associated with gene therapy. Assays known in the art to measure immune responses, such as immunohistochemical detection of cytotoxic T cell responses, can be used to determine whether one or more promoters can confer immunotolerant properties.

[0090] As used herein, the term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.

[0091] As used herein, the term “in combination” in the context of the administration of other therapies (e.g., other agents) includes the use of more than one therapy (e.g., drug therapy). Administration “in combination with” one or more further therapeutic agents includes simultaneous (e.g., concurrent) and consecutive administration in any order. The use of the term “in combination” does not restrict the order in which therapies are administered to a subject. By way of non-limiting example, a first therapy (e.g., a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof) may be administered prior to (e.g.. 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks), concurrently, or after (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours. 12 hours. 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or longer) the administration of a second therapy (e.g., agent) to a subject having or diagnosed with a disease or disorder (such as a genetic disease or disorder).

[0092] Disclosed are the components to be used to prepare the disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations as well as the disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E. B-F, C- D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety' of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention.

B. Compositions for Transcriptome Engineering

1. Nucleic Acid Molecules

5’ Replacement Constructs 0093] Disclosed herein is a nucleic acid molecule, comprising an exogenous RNA to be transspliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures.

[0094] Disclosed herein is a nucleic acid molecule, comprising an exogenous RNA to be transspliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09.

[0095] Disclosed herein is a nucleic acid molecule, comprising an exogenous RNA to be trans- spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA.

[0096] Disclosed herein is a nucleic acid molecule, comprising an exogenous RNA to be trans- spliced to a targeted endogenous pre-mRNA; a 5 ’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA.

[0097] In an aspect, a disclosed targeted endogenous pre-mRNA can comprise one or more mutations. In an aspect of a disclosed targeted endogenous pre-mRNA, one or more disclosed mutations can be in the 5' portion of the pre-mRNA. In an aspect, one or more disclosed mutations in one or more exons can contribute to pathogenesis of one or more cells. [0098] In an aspect, the disclosed cells can be in a subject. In an aspect, a subject can be a human patient and can be male or female. In an aspect, a subject can have a genetic disease or disorder. In an aspect, a subject can be treatment-naive.

[0099] In an aspect, one or more disclosed mutations can inhibit translation of the encoded protein. In an aspect, one or more disclosed mutations can modify translation of the encoded protein. In an aspect, one or more disclosed mutations can generate an encoded protein having a non-sense mutation or a missense mutation.

[0100] In an aspect, a disclosed targeted endogenous pre-mRNA can comprise one or more mutations in one or more exons. In an aspect, a disclosed targeted endogenous pre-mRNA can comprise one or more mutations in one or more introns. In an aspect, one or more disclosed exonic mutations can contribute to pathogenesis in one or more cells. In an aspect, one or more disclosed intronic mutations can contribute to pathogenesis in one or more cells.

[0101] In an aspect, a disclosed targeted endogenous pre-mRNA can be a primary transcript of a protein coding gene. In an aspect of a disclosed targeted endogenous pre-mRNA, a disclosed protein coding gene can comprise one or more coding regions of ABCA1, ABCA12, ABCA13, ABCA2, ABCA3, ABCA4, ABCA5, ABCC1, ABCC2, ABCC6, ABCC8, ABCC9, ACAN, ADAMTS13, ADCY10, ADGRV1, AGL, AGRN, AHDC1, ALK, ALMS1, ALPK3, ALS2, ANAPC1, ANK1, ANK2, ANK3, ANKRD11, ANKRD26, ABC, APC2, APOB, ARFGEF2, ARHGAP31, ARHGEF10, ARHGEF18, ARID1A, ARID1B, ARID2, ASH1L, ASPM, ASXL1. ASXL2, ASXL3. ATM, ATP7A, ATP7B, ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, BLM, BPTF, BRCA1, BRCA2, BRD4, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD. CAMTAI, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP 152, CEP 164, CEP 250, CEP 290, CFAP43, C FAP 44, CFAP65, CFTRABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1, CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1, CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1, DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11, DNAH17, DNAH2, DNAH5, DNAH7, DNAH8, DNAH9, DNMBP, DNMT1, DOCK2, DOCKS, DOCK6, DOCK7, DOCK8, DSCAM, DSP, DST, DUOX2, DYNC1H1, DYNC2H1, DYSF, EIF2AK4, EP 300, EPG5, ERCC6, ERCC6L2, EXPH5, EYS. F5. F8. FANCA, FANCD2, FANCM, FAT1, FAT4, FBN1, FBN2, FLG, FLG2, FLNA, FLNB, FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2, FSIP2, FXN, FYCO1, GLI2, GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1, HERC2, HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R, IGF2R. IGSF1, INSR, INTSL IQSEC2. ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A, KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220, KIF14, KIF1A, KIF1B, KIF21A, KIF26B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1, LAMA1, LAMA2, LAMA3, LAMA4. LAMAS LAMB1, LAMB2. LAMC3, LCT, LMNA, LOXHD1. LPA. LRBA, LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC, LRRK1. LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD, MAGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1, MASTl. MBD5, MCM3AP, MED12, MED12L, MED13, MED13L, MED23, MEGF8, MET, MLH3, MPDZ. MSH6, MTOR MYH10, MYH11, MYH14, MYH2, MYH3, MYH6, MYH7, MYH7B, MYH8, MYH9, MYLK, MYO15A, MYO18B, MY03A, MYO5A, MY05B, MY07A, MY09A, NALCN, NBAS. NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF, NF1, NFASC, NHS, NIN, NIPBL, NLRP1, NOTCH1, NOTCH2, NOTCH3, NPHP4, NRXN1, NRXN3, NSD1, NSD2, NUP155, NUP188, NUP205, OBSCN, OBSL1, OTOF, OTOG, OTOGL, PARD3, PBRM1, PCDH15, PCLO, PCNT, PHIP, PI4KA, PIEZO1. PIEZO2, PIK3C2A, PIKFYVE, PKD1. PKD1L1, PKHD1, PLCE1, PLEC, PLEKHG2, PNPLA6, POGZ, POLA1, POLE. POLR1A, POLR2A, POLR3A, PRG4. PRKDC, PRPF8, PRR12, PRX, PTCHI, PTPN23, PTPRF, PTPRJ, PTPRQ, PXDN, QRICH2, RAB3GAP2, RAIL RALGAPA1, RANBP2, RB1CC1, RELN, RERE. REV3L, RIC1, RIMS1, RIMS2, RNF213. ROBOL ROBO2, ROBO3, ROS1, RP1, RP1L1, RTTN, RUSC2. RYR1, RYR2, SACS. SAMD9, SAMD9L, SBF2, SCAPER. SCN10A, SCN11A. SCN1A, SCN2A. SCN3A, SCN4A. SCN5A. SCN8A, SCN9A, SETBP1, SETD1A, SETD1B, SETD2, SETD5, SETX, SHANK2. SHANK3, SHROOM4, SI, SIPA1L3, SLIT2, SLX4, SMARCA2, SMARCA4, SMCHD1, SNRNP200, SON, SPEF2, SPEG, SPG11, SPTA1, SPTAN1, SPTB, SPTBN2, SPTBN4, SRCAP, STRC, SVIL, SYNE1, SYNGAP1, SYNJ1, SZT2, TAF1, TANC2, TCF20, TC0F1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B. TNXB, TOGARAMI, TONSL, TRIO, TRIOBP, TRIP 11, TRIP 12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, TTN, TUBGCP6, UBR1, UNC80, USH2A. USP9X, VC AN, VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62, WDR81. WNKL WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292. ZNF335. ZNF407. ZNF462. or ZNF469. In an aspect, a disclosed protein coding gene can comprise one or more coding regions of CFTR, MDX, DYSF/TTN, DMPK. COL7A1, K14. MAPI, FVIII, HTT, RHO, DNA-PKcs, SMN2, or CD40L. In an aspect, a disclosed protein coding gene can comprise one or more coding regions of FXN, LMNA, or RYR2. In an aspect, a disclosed protein coding gene can comprise a portion of a disclosed protein coding gene (such as, for example, Exon 1 or Exon 4, etc.)

[0102] In an aspect, a disclosed 3’ portion of the targeted endogenous pre-mRNA can be transspliced with the exogenous RNA. In an aspect, a disclosed RNA targeting motif can bind to the targeted endogenous pre-mRNA. In an aspect, a disclosed RNA targeting motif can bind to the 5’ end of the targeted endogenous pre-mRNA. In an aspect, a disclosed RNA targeting motif can be specific for an endogenous pre-mRNA having one or more mutations. In an aspect, a disclosed RNA targeting motif can be specific for an endogenous pre-mRNA having one or more exonic mutations. In an aspect, a disclosed RNA targeting motif can be specific for an endogenous pre- mRNA having one or more intronic mutations.

[0103] In an aspect, a disclosed RNA targeting motif can comprise an antisense oligonucleotide. In an aspect, a disclosed antisense oligonucleotide can comprise about 15 nucleotides to about 50 nucleotides. In an aspect, a disclosed antisense oligonucleotide can comprise about 30 nucleotides. In an aspect, a disclosed RNA targeting motif can be directed to the intron immediately 5’ to the exon of the targeted endogenous pre-mRNA with which it is to be spliced.

[0104] In an aspect, a disclosed 5’ hemi intron can comprise a 5’ splice site. In an aspect, a disclosed 5’ splice site can comprise a consensus 5' splice site. In an aspect, a disclosed consequence 5‘ splice site can comprise MAG | GURAGU (SEQ ID NO: 13), wherein | denotes the exon intron junction, wherein M = A or C, and wherein R = A or G. In an aspect, a disclosed 5’ hemi intron can be recognized by nuclear splicing components in a host cell. In an aspect, a disclosed 5’ hemi intron can be recognized by the spliceosome in a host cell. In an aspect, a disclosed 5’ hemi intron can facilitate the trans-splicing of the exogenous RNA to the exon immediately 3’ to the targeted intron in the endogenous pre-mRNA.

[0105] In an aspect, a disclosed exogenous RNA to be trans-spliced to the targeted endogenous pre-mRNA can comprise one or more exons of the protein coding gene. In an aspect, a disclosed exogenous RNA to be trans-spliced to the targeted endogenous pre-mRNA can comprise the primary sequence of the coding sequence of one or more exons having the one or more mutations. In an aspect, a disclosed exogenous RNA can be trans-spliced to a 3’ end of the targeted endogenous pre-mRNA.

[0106] In an aspect of a disclosed exogenous RNA, a disclosed protein coding gene can comprise one or more coding regions of ABCA1. ABCA12, ABCA13, ABCA2, ABCA3. ABCA4, ABCA5, ABCC1, ABCC2, ABCC6, ABCC8, ABCC9, ACAN. ADAMTS13, ADCY10, ADGRV1, AGL, AGRN, AHDC1, ALK, ALMS1, ALPK3, ALS2, ANAPC1, ANK1, ANK2, ANK3, ANKRD11, ANKRD26, APC, APC2. APOB, ARFGEF2, ARHGAP31, ARHGEF10, ARHGEF18, ARID1A, ARID1B, ARID2, ASH1L, ASPM, ASXL1. ASXL2, ASXL3, ATM, ATP7A, ATP7B, ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, BLM, BPTF, BRCA1, BRCA2, BRD4, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD, CAMTAI, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSRl, CEMIP2, CENPE, CENPF, CENPJ, CEP 152, CEP 164, CEP250, CEP290, CFAP43, CFAP44, CFAP65. CFTR/ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1, CNTNAP1, COL11A1, COL11A2, COL12A1. COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3. COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1. CPAMD8, CPLANEL CPS1, CPSF1, CRB1. CREBBP, CUBN. CUL7. CUX1. DCC, DCHS1, DEPDC5, DICER1, DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11, DNAH17, DNAH2, DNAH5. DNAH7, DNAH8, DNAH9, DNMBP, DNMT1, DOCK2, DOCK3, DOCK6, DOCK7, DOCK8, DSCAM, DSP, DST, DUOX2, DYNC1H1, DYNC2H1, DYSF, EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, EYS. F5, F8, FANCA. FANCD2, FANCM, FATE FAT4, FBN1, FBN2. FLG, FLG2, FLNA. FLNB, FLNC, FLT4, FMN2, FNR FRASE FREM1, FREM2, FSIP2, FXN, FYCO1. GLI2. GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1, HERC2, HFM1, HIVEPI, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R, IGF2R, IGSFL INSR, INTSE IQSEC2, ITGB4, ITPRE ITPR2, JMJD1C, KALRN, KANKE KAT6A, KAT6B, KDM3B, KDM5B, KDM5C, KDM6A. KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220, KIF14. KIF1A, KIF1B, KIF21A, KIF26B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNLI, LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMBE LAMB2, LAMC3, LCT, LMNA, LOXHD1, LPA, LRBA, LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2. LTBP2, LTBP4, LYST, MACFE MADD, MAGI2, MAP IB, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5. MCM3AP, MED12, MED12L, MED13, MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MYH11, MYH14, MYH2, MYH3, MYH6, MYH7, MYH7B, MYH8, MYH9, MYLK, MYOMA, MYO18B, MYO3A, MYO5A, MYO5B, MYO7A, MYO9A, NALCN, NBAS. NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIE NFE NFASC, NHS, NIN, NIPBL, NLRP1, NOTCH1, N0TCH2, NOTCH3, NPHP4, NRXN1, NRXN3, NSDE NSD2, NUP155, NUP188, NUP205, OBSCN, OBSL1, OTOF, OTOG, OTOGL, PARD3, PBRM1, PCDH15, PCLO, PCNT, PHIP, PI4KA, PIEZO 1, PIEZO2, PIK3C2A, PIKFYVE, PKDE PKD1LE PKHD1, PLCE1, PLEC. PLEKHG2, PNPLA6, POGZ, POLA1, POLE, POLR1A, POLR2A, POLR3A, PRG4, PRKDC, PRPF8, PRR12, PRX, PTCHL PTPN23, PTPRF, PTPRJ, PTPRQ, PXDN, QRICH2, RAB3GAP2, RAIL RALGAPA1, RANBP2, RB1CCL RELN, RERE, REV3L, RICE RIMSl, RIMS2, RNF213, ROBO1, ROBO2, ROBO3, ROS1, RPL RP1L1, RTTN, RUSC2, RYR1, RYR2, SACS, SAMD9, SAMD9L, SBF2, SCAPER SCN10A, SCN11A, SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A. SETBP L SETD1A, SETD1B, SETD2, SETD5, SETX, SHANK2. SHANK3, SHR00M4, SI. SIPA1L3, SLIT2, SLX4, SMARCA2, SMARCA4, SMCHD1. SNRNP200, SON, SPEF2, SPEG, SPG11, SPTA1, SPTAN1. SPTB, SPTBN2, SPTBN4, SRCAP, STRC, SVIL, SYNE1, SYNGAP1, SYNJ1, SZT2, TAFR TANC2, TCF20. TCOFL TDRD9. TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, TH0C2, TMEM94, TN TNIK. TNR. TNRC6B, TNXB, TOGARAMI, TONS!, TRIO, TRIOBP, TRIP11, TRIP12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, TTN, TUBGCP6, UBR1, UNC80, USH2A, USP9X, VCAN, VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62, WDR81, WNK1. WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292, ZNF335, ZNF407, ZNF462, or ZNF469. In an aspect, a disclosed protein coding gene can comprise one or more coding regions of CFTR, MDX, DYSF/TTN. DMPK, COL7A1, K14, MAPT, FVIII, HTT, RHO, DNA-PKcs, SMN2, or CD40L. In an aspect, a disclosed protein coding gene can comprise one or more coding regions of FXN^LMNA, or RYR2.

[0107] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode LMNA/C (SEQ ID NO:20) or a portion thereof. LMNA/C is known to the art (e.g., Gene ID 4000) and this nucleotide sequence can comprise nucleotides 4974 - 62517 in Accession No. NG008692.2. The nuclear lamina consists of a two-dimensional matrix of proteins located next to the inner nuclear membrane. The lamin family of proteins make up the matrix and are highly conserved in evolution. During mitosis, the lamina matrix is reversibly disassembled as the lamin proteins are phosphorylated. Lamin proteins are involved in nuclear stability, chromatin structure and gene expression. Vertebrate lamins consist of two ty pes, A and B. Alternative splicing results in multiple transcript variants. Mutations in this gene lead to several diseases: Emery-Dreifuss muscular dystrophy, familial partial lipodystrophy, limb girdle muscular dystrophy, dilated cardiomyopathy, Charcot-Marie-Tooth disease, and Hutchinson-Gilford progeria syndrome.

[0108] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode DP71 or a portion thereof. DP71 is known to the art (e.g., Gene ID 13405).

[0109] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode CFTR (SEQ ID NO: 19) or a portion thereof. CFTR is known to the art (e.g., Gene ID 1080) and this nucleotide sequence can comprise nucleotides 19180 - 207882 in Accession No. NG016465.4. This gene encodes a member of the ATP-binding cassette (ABC) transporter superfamily. The encoded protein functions as a chloride channel, making it unique among members of this protein family, and controls ion and water secretion and absorption in epithelial tissues. Channel activation is mediated by cycles of regulatory domain phosphorylation, ATP-binding by the nucleotide-binding domains, and ATP hydrolysis. Mutations in this gene cause cystic fibrosis, the most common lethal genetic disorder in populations of Northern European descent. The most frequently occurring mutation in cystic fibrosis, DeltaF508, results in impaired folding and trafficking of the encoded protein. Multiple pseudogenes have been identified in the human genome.

[0110] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode DMPK (SEQ ID NO:21) or a portion thereof. DMPK is known to the art (e.g., Gene ID 1760) and this nucleotide sequence can comprise nucleotides 5068 - 17841 in Accession No. NG009784.1. DMPK is a serine-threonine kinase that is closely related to other kinases that interact with members of the Rho family of small GTPases. Substrates for this enzyme include myogenin, the beta-subunit of the L-type calcium channels, and phosphol emman. The 3’ untranslated region of this gene contains 5-38 copies of a CTG trinucleotide repeat. Expansion of this unstable motif to 50-5.000 copies causes myotonic dystrophy type I, which increases in severity with increasing repeat element copy number. Repeat expansion is associated with condensation of local chromatin structure that disrupts the expression of genes in this region. Several alternatively spliced transcript variants of this gene have been described, but the full-length nature of some of these variants has not been determined.

[0111] In an aspect, a disclosed gene can be DMD (dystrophin) (SEQ ID NO: 17). DMD is known to the art (e.g., Gene ID 1756) and this nucleotide sequence can comprise nucleotides 5001 - 2225382 in Accession No. NG012232. 1. DMD spans a genomic range of greater than 2 Mb and encodes a large protein containing an N-terminal actin-binding domain and multiple spectrin repeats. The encoded protein forms a component of the dystrophin-glycoprotein complex (DGC), which bridges the inner cytoskeleton and the extracellular matrix. Deletions, duplications, and point mutations at this gene locus may cause Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or cardiomyopathy. Alternative promoter usage and alternative splicing result in numerous distinct transcript variants and protein isoforms for this gene.

[0112] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode LRRK2 (SEQ ID NO: 18) or a portion thereof. LRRK2 is known to the art (e.g., Gene ID 120892) and this nucleotide sequence can comprise nucleotides 5001 - 149275 in Accession No. NG011709.1. LRRK2 is a member of the leucine-rich repeat kinase family and encodes a protein with an repeat region, a leucine-rich repeat (LRR) domain, a kinase domain, a DFG-like motif, a RAS domain, a GTPase domain, a MLK-like domain, and a WD40 domain. The protein is present largely in the cytoplasm but also associates with the mitochondrial outer membrane. Mutations in this gene have been associated with Parkinson’s disease.

[0113] In an aspect, a disclosed exogenous RNA to be trans-spliced can further comprise a UTR. [0114] In an aspect, one or more disclosed RNA structures can bind to one or more RNA binding proteins. In an aspect, one or more disclosed RNA structures can bind to one or more doublestranded RNA binding proteins (dsRBP). In an aspect, dsRBPs are known to the skilled person in the art and include, but are not limited to. AD ARI. ADAR2, DICER, NF AR. PACT. PKR, RHA RNaselll, Stauffen, TRBP, TSEN, or any combination thereof.

[0115] In an aspect, one or more disclosed RNA structures can comprise the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09. In an aspect, one or more disclosed RNA structures can improve and/or can enhance trans-splicing efficiency. In an aspect, one or more disclosed RNA structures can stabilize the pre-mRNA. In an aspect, one or more disclosed RNA structures can localize the RNA to the nucleus. In an aspect, one or more disclosed RNA structures can stabilize the interaction between the targeted endogenous pre-mRNA molecule and the exogenous RNA to be trans-spliced. In an aspect, a disclosed nucleic acid molecule can lack a CRISPR-associated protein.

[0116] In an aspect, a disclosed resulting chimeric RNA transcript can comprise the 5’ portion of the targeted endogenous pre-mRNA and the 3 ’ portion of the exogenous RNA. In an aspect, a disclosed resulting chimeric RNA transcript can comprise the 3’ portion of the targeted endogenous pre-mRNA and the 5’ portion of the exogenous RNA.

[0117] In an aspect, a disclosed targeted endogenous pre-mRNA and a disclosed exogenous RNA can encode the same protein coding gene. In an aspect, a disclosed targeted endogenous pre- mRNA and a disclosed exogenous RNA can comprise one or more exons of the same protein coding gene.

[0118] In an aspect, a disclosed nucleic acid molecule can be packaged into a viral vector. In an aspect, a disclosed viral vector can comprise an AAV vector. In an aspect, a disclosed nucleic acid molecule can be packaged into a non-viral carrier. In an aspect, a disclosed nucleic acid molecule can be incorporated into a plasmid. In an aspect, a disclosed nucleic acid molecule can be incorporated into lipid nanoparticles.

[0119] In an aspect, a disclosed nucleic acid molecule can further comprise a polyadenylation sequence. In an aspect, a disclosed nucleic acid molecule can further comprise a sequence for a promoter. In an aspect, a disclosed nucleic acid molecule can further comprise a spacer region. In an aspect, a disclosed spacer region can separate the 5^? splice region from the one or more RNA structures. In an aspect, a disclosed spacer region can comprise any known spacer. In an aspect, a disclosed spacer region can comprise a consensus splicing motif (e.g., such as U1 or U2). In an aspect, a disclosed spacer region can comprise a limited number of consensus splicing motifs (e.g., such as U1 or U2).

[0120] In an aspect, a disclosed nucleic acid molecule can further comprise one or more nuclear localization signals (NLS). NLS are known to the skilled person in the art. In an aspect, a disclosed NLS can comprise any NLS known to the art. As known to the art (see, e.g., Lu J, et al. (2021) Cell Commun Signal. 19:60, which is incorporated herein by reference for its teachings of NLS), nuclear localization signals (NLS) are generally short peptides that act as a signal fragment that mediates the transport of proteins from the cytoplasm into the nucleus.

[0121] In an aspect, a disclosed nucleic acid molecule can further comprise one or more nuclear retention elements (NRE). NRE are known to the skilled person in the art. In an aspect, a disclosed NRE can comprise SIRLOIN (SEQ ID NO: 15) or BORG (SEQ ID NO: 16). [0122] In an aspect, a disclosed nucleic acid molecule can further comprise one or more Flavivirus genetic elements. In an aspect, Flavivirus genetic elements can comprise one or more Flavivirus 3’ untranslated region (3’ UTR), one or more subgenomic Flavivirus RNA (sfRNA) elements, one or more Flavivirus XRN1 -resistant RNA (xrRNA) elements, one or more Flavivirus dumbbell (DB) RNA elements, one or more Flavivirus 3’ stem loop (3’ SL) elements, or any combination thereof. (See WO 2022/182835 for a description of Flavivirus gene elements).

[0123] In an aspect, a disclosed exogenous RNA can induce a splice event. In an aspect, a disclosed 5’ hemi intron can be recognized by nuclear splicing components within a host cell.

[0124] In an aspect, a disclosed promoter for the 5’ replacement construct can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the expression desired. A promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-ty pe host into which the transcriptional initiation region is introduced. In an aspect, a disclosed promoter can be a promoter/enhancer. In an aspect, a disclosed promoter for the disclosed nucleic acid molecule can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter/enhancer. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can generally be obtained from a non-coding region upstream of a transcription initiation site of a gene of interest. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can be used for constitutive and efficient expression of a disclosed protein coding gene. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be a CMV promoter or a CMV promoter/enhancer. CMV promoters and CMV promoters/enhancers are well known to the art. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be any eukaryotic RNA polymerase II promoter.

[0125] Disclosed herein is an expression cassette comprising an exogenous RNA to be transspliced to a targeted endogenous pre-mRNA; a 5 ’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures. Disclosed herein is an expression cassette comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09. Disclosed herein is an expression cassette comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA. Disclosed herein is an expression cassette comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09, wherein the nucleic acid molecule enables the 5‘ replacement of the targeted endogenous pre-mRNA.

[0126] In an aspect, expression of a disclosed protein coding gene can be restored and/or returned to a wild-type, normal, or control expression level. In an aspect, a disclosed nucleic acid molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, a disclosed nucleic acid molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra). In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi- systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity.

[0127] In an aspect, restoring the activity and/or functionality of a missing, deficient, and/or mutant protein or enzyme can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%. 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and/or mutant protein or enzy me). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is similar to that of a wild-type or control level.

[0128] In an aspect, a disclosed 5’ replacement construct can be particularly useful in a method of treating a subject having an autosomal dominant genetic disease or disorder (such as, for example, progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert’s disease, hereditary haemorrhagic telangiectasia, hereditary elliptosis, hereditary spherocytosis, Huntington’s disease, idiopathic hypoparathyroidism, intestinal polyposis, marble bone disease. Marfan’s syndrome, neurofibromatosis, polycystic kidney disease (adult), protein C deficiency, osteogenesis imperfecta. Treacher Collins syndrome, tuberous sclerosis, Von Willebrand’s disease, etc.)

3’ Replacement Constructs

[0129] Disclosed herein is a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA. Disclosed herein is a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3' hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA. Disclosed herein is a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3 ’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre- mRNA, wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA. Disclosed herein is a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA.

[0130] In an aspect, a disclosed targeted endogenous pre-mRNA can comprise one or more mutations in one or more exons. In an aspect, one or more disclosed mutations can be in the 3’ portion of the one or more exons of the pre-mRNA. In an aspect, a disclosed targeted endogenous pre-mRNA can comprise one or more mutations in one or more introns. In an aspect, one or more disclosed mutations in one or more exons can contribute to pathogenesis of one or more cells. In an aspect, disclosed cells can be in a subject. In an aspect, a subject can be a human patient and can be male or female. In an aspect, a subject can have a genetic disease or disorder. In an aspect, a subject can be treatment-naive. In an aspect, the one or more disclosed mutations can inhibit translation of the encoded protein. In an aspect, the one or more disclosed mutations can modify translation of the encoded protein. In an aspect, during and/or following translation the one or more disclosed mutations can generate a protein having a non-sense mutation or a missense mutation. In an aspect, the one or more disclosed exonic mutations can contribute to pathogenesis in one or more cells. In an aspect, the one or more disclosed intronic mutations can contribute to pathogenesis in one or more cells. In an aspect a disclosed targeted endogenous pre-mRNA can encode a protein coding gene.

[0131] In an aspect of a disclosed targeted endogenous pre-mRNA, a disclosed protein coding gene can comprise one or more coding regions of ABCA1, ABCA12, ABCA13. ABCA2, ABCA3, ABCA4, ABCA5, ABCC1. ABCC2, ABCC6, ABCC8, ABCC9, ACAN, ADAMTS13, ADCY10, ADGRV1, AGL, AGRN, AHDC1. ALK, ALMS1, ALPK3, ALS2, ANAPC1, ANK1, ANK2, ANK3, ANKRD11, ANKRD26, APC, APC2, APOB, ARFGEF2, ARHGAP31, ARHGEF10, ARHGEF18, ARID1A, ARID1B, ARID2, ASH1L, ASPM, ASXL1, ASXL2, ASXL3, ATM, ATP7A, ATP7B, ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, BLM, BPTF, BRCA1, BRCA2, BRD4, BRWD3, C2CD3, C3, C5, CACNAIA, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F. CACNA1G, CACNAIH, CACNA1S, CAD, CAMTAI, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP 152, CEP 164, CEP 250, CEP 290, CFAP43, CFAP44, CFAP65, CFTR/ABCC7, CHD1. CHD2. CHD3, CHD4, CHD7, CHD8. CIG CIT, CLIP!, CLTC, CNOT1. CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, COL2A1, COL3A1, COE4A1, COL4A2, COL4A3, COE4A4, COL4A5, COL4A6, COE5A1, COL5A2, COL6A3, COL7A1, CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC. DCHS1, DEPDC5, DICER1, DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11, DNAH17, DNAH2, DNAH5, DNAH7, DNAH8, DNAH9, DNMBP, DNMT1, DOCK2, DOCK3, DOCK6, DOCK7, DOCK8, DSC AM, DSP, DST, DUOX2, DYNC1H1, DYNC2H1, DYSF, EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, EYS, F5, F8, FANCA, FANCD2, FANCM, FAT1, FAT4, FBN1, FBN2. FLG, FLG2, FLNA. FLNB. FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2, FSIP2, FXN, FYCO1, GLI2. GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1, HERC2, HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R IGF2R, IGSF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C KALRN, KANK1, KAT6A, KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220. KIF14, KIF1A, KIF1B, KIF21A, KIF26B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1, LAMAl, LAMA2, LAMA3, LAMA4, LAMAS, LAMB1, LAMB2, LAMC3, LCT, LMNA, LOXHD1, LPA, ERBA, LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD, MAGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1. MAST1, MBD5, MCM3AP, MED12, MED12L, MED13, MED13L, MED23, MEGF8, MET MLH3, MPDZ, MSH6. MTOR, MYH10, MYH11, MYH14, MYH2, MYH3, MYH6, MYH7, MYH7B, MYH8, MYH9, MYLK, MYO15A, MYO18B, MYO3A, MYO 5 A, MYO5B, MYO7A, MYO9A, NALCN, NBAS, NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF, NF1, NFASC, NHS, NIN, NIPBL, NLRP1, NOTCH1, NOTCH2, NOTCH3, NPHP4. NRXN1, NRXN3, NSD1, NSD2, NUP155, NUP188, NUP205. OBSCN, OBSL1, OTOF, OTOG, OTOGL, PARD3, PBRM1, PCDH15. PCLO. PCNT, PHIP, PI4KA, PIEZO1, PIEZO2, PIK3C2A, PIKFYVE, PKD1, PKD1L1, PKHD1, PLCE1, PLEC, PLEKHG2. PNPLA6, POGZ, POLA1, POLE, POLR1A, POLR2A, POLR3A. PRG4. PRKDC, PRPF8, PRR12, PRX, PTC Hl . PTPN23. FTP RE PTPRJ, PTPRQ, PXDN, QRICH2, RAB3GAP2, RAIL RALGAPA1, RANBP2. RB1CC1, RELN, RERE, REV3L, RIC1, RIMS1, RIMS2, RNF213, ROBO1, ROBO2, ROBO3, ROS1, RP1, RP1L1, RTTN, RUSC2, RYR1, RYR2, SACS, SAMD9, SAMD9L, SBF2, SCAPER, SCN10A, SCN11A, SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SETBP1, SETD1A, SETD1B, SETD2, SETD5, SETX, SHANK2, SHANK3, SHROOM4, SI. SIPA1L3, SLIT2, SLX4, SMARCA2. SMARCA4, SMCHD1. SNRNP200, SON, SPEF2. SPEG. SPG11, SPTA1, SPTAN1, SPTB, SPTBN2, SPTBN4, SRCAP, STRC. SVIL, SYNE1, SYNGAP1. SYNJ1, SZT2, TAF1, TANC2, TCF20, TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG. THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B. TNXB, TOGARAM1, TONSL, TRIO. TRIOBP. TRIP11. TRIP12. TRPM1. TRPM6. TRPM7. TRRAP. TSC2. TTC37. TIN, TUBGCP6, UBR1, UNC80, USH2A, USP9X. VCAN. PPS13A. VPS13B, VPS13C, VPS13D. VWF, WDFY3, WDR19, WDR62, WDR81, WNK1, WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292, ZNF335, ZNF407, ZNF462. or ZNF469. In an aspect, a disclosed protein coding gene can comprise one or more coding regions of CFTR, MDX, DYSF/TTN. DMPK, COL7A1, KI 4, MAPT, FVIIL HTT, RHO, DNA-PKcs, SMN2, or CD40L. In an aspect, a disclosed protein coding gene can comprise one or more coding regions of FXN, LMNA, or RYR2. In an aspect, a disclosed protein coding gene can comprise a portion of a disclosed protein coding gene (such as, for example. Exon 1 or Exon 4, etc.)

[0132] In an aspect, a disclosed 5’ portion of the targeted endogenous pre-mRNA can be transspliced with the exogenous RNA.

[0133] In an aspect, a disclosed RNA targeting motif can bind to the targeted endogenous pre- mRNA. In an aspect, a disclosed RNA targeting motif can bind to the 3' end of the targeted endogenous pre-mRNA. In an aspect, a disclosed RNA targeting motif can be specific for an endogenous pre-mRNA having one or more mutations. In an aspect, a disclosed RNA targeting motif can be specific for an endogenous pre-mRNA having one or more exonic mutations. In an aspect, a disclosed RNA targeting motif can be specific for an endogenous pre-mRNA having one or more intronic mutations. In an aspect, a disclosed RNA targeting motif can comprise an antisense oligonucleotide.

[0134] In an aspect, a disclosed antisense oligonucleotide can comprise about 15 nucleotides to about 50 nucleotides. In an aspect, a disclosed antisense oligonucleotide can comprise about 30 nucleotides. In an aspect, a disclosed RNA targeting motif can be directed to the intron immediately 3’ to the exon of the targeted endogenous pre-mRNA with which it is to be spliced. [0135] In an aspect, a disclosed 3’ hemi intron can comprise (i) a 3‘ splice region comprising a branch point, (ii) a poly pyrimidine tract, and (iii) a 3 ’ splice acceptor site. In an aspect, a disclosed branch point can comprise the sequence of SEQ ID NO: 10. In an aspect, a disclosed 3’ splice acceptor site can comprise the sequence YAG, where Y is a pyrimidine (SEQ ID NO: 11). In an aspect, a disclosed 3 ’ hemi intron can be recognized by nuclear splicing components in a host cell. In an aspect, a disclosed 3‘ hemi intron can be recognized by the spliceosome in a host cell. In an aspect, a disclosed 3^? hemi intron can facilitate the trans-splicing of the exogenous RNA to the exon immediately 5’ to the targeted intron in the endogenous pre-mRNA.

[0136] In an aspect, a disclosed exogenous RNA to be trans-spliced to the targeted endogenous pre-mRNA can comprise one or more exons of the protein coding gene. In an aspect, a disclosed exogenous RNA to be trans-spliced to the targeted endogenous pre-mRNA can comprise the primary sequence of the coding sequence of one or more exons having the one or more mutations. In an aspect, a disclosed exogenous RNA can be trans-spliced to a 5' end of the targeted endogenous pre-mRNA.

[0137] In an aspect of a disclosed exogenous RNA, a disclosed protein coding gene can comprise one or more coding regions of ABCA1, ABCA12, ABCA13, ABCA2, ABCA3. ABCA4, ABCA5, ABCC1. ABCC2, ABCC6. ABCC8, ABCC9, ACAN. ADAMTS13, ADCY10, ADGRV1, AGL. AGRN, AHDC1, ALK, ALMS1, ALPK3, ALS2, ANAPC1, ANK1, ANK2, ANK3, ANKRD11, ANKRD26, APC, APC2. APOB, ARFGEF2, ARHGAP31, ARHGEF10, ARHGEF18, ARID1A, ARID1B, ARID2, ASH1L, ASPM, ASXL1. ASXL2, ASXL3, ATM, ATP7A, ATP7B, ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, BLM, BPTF, BRCA1, BRCA2. BRD4, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD, CAMTAI, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSRl, CEMIP2, CENPE, CENPF, CENPJ, CEP 152, CEP 164, CEP250. CEP290, CFAP43, CFAP44, CFAP65, CFTR/ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT. CLIP1, CLTC, CNOT1, CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1, CPAMD8, CPLANE1, CPS1, CPSF1, CRB1. CREBBP, CUBN, CUL7, CUX1. DCC, DCHS1, DEPDC5, DICER1, DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11, DNAH17, DNAH2, DNAH5, DNAH7, DNAH8, DNAH9, DNMBP, DNMT1, DOCK2, DOCK3, DOCK6, DOCK7, DOCK8, DSCAM, DSP, DST, DUOX2, DYNC1H1, DYNC2H1, DYSF, EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, EYS. F5, F8, FANCA, FANCD2, FANCM, FAT1. FAT4, FBN1, FBN2. FLG, FLG2, FLNA, FLNB. FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2, FSIP2, FXN, FYC01, GLI2. GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1, HERC2, HFM1, HIVEPL HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R, IGF2R, IGSF1, INSR, INTSI, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A, KAT6B. KDM3B. KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220, K1F14. KIF1A, KIF1B, KIF21A, KIF26B, KIF7. KMT2A. KMT2B, KMT2C, KMT2D, KMT2E, KNLI, LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMBL LAMB2, LAMC3, LCT, LMNA, LOXHD1, LPA, LRBA, LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC. LRRK1, ERRK2. LTBP2, LTBP4, LYST, MACFE MADD, MAGI2, MAP IB, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5. MCM3AP, MED12, MED12L, MED13, MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MYH11, MYH14, MYH2, MYH3, MYH6, MYH7, MYH7B, MYH8, MYH9, MYLK, MYOMA, MYO18B, MYO3A, MYO5A, MYO5B, MY 07 A, MYO9A, NALCN, NBAS. NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF, NFE NFASC. NHS, NIN, NIPBL, NLRP1, NOTCH!. NOTCH2, NOTCH3, NPHP4, NRXN1, NRXN3, NSD1, NSD2, NUP155, NUP188, NUP205, OBSCN. OBSL1, OTOF, OTOG, OTOGL, PARD3, PBRM1, PCDH15, PCLO, PCNT, PHIP, PI4KA, PIEZO 1, PIEZO2, PIK3C2A, PIKFYVE, PKD1, PKD1L1, PKHD1, PLCE1, PLEC. PLEKHG2, PNPLA6, POGZ, POLA1, POLE, POLR1A, POLR2A, POLR3A, PRG4, PRKDC, PRPF8, PRR12, PRX, PTCHL PTPN23, PTPRF PTPRJ, PTPRQ, PXDN, QRICH2, RAB3GAP2, RAH, RALGAPA1, RANBP2, RB1CC1, RELN, RERE, REV3L, RIC1, RIMSR RIMS2, RNF213, ROBO1, ROBO2, ROBO3, ROSE RPL RP1L1, RTTN, RUSC2, RYR1, RYR2, SACS, SAMD9, SAMD9L, SBF2, SCAPER SCN10A, SCN11A, SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A. SETBP 1, SETD1A, SETD1B, SETD2, SETD5, SETX, SHANK2, SHANK3, SHR00M4, SI, SIPA1L3, SLIT2, SLX4, SMARCA2, SMARCA4, SMC HD 1. SNRNP200, SON, SPEF2, SPEG, SPG11, SPTA1, SPTAN1. SPTB, SPTBN2, SPTBN4, SRCAP, STRC, SVIL, SYNE1, SYNGAP1, SYNJ1, SZT2, TAFR TANC2, TCF20. TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, THOC2, TMEM94, TNC, TNIK, TNR. TNRC6B, TNXB, TOGARAMI, TONSL, TRIO. TRIOBP, TRIP11. TRIP12. TRPM1, TRPM6, TRPM7, TRRAP, TSC2. TTC37, TTN, TUBGCP6. UBR1. UNC80, USH2A, USP9X, VCAN, VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62, WDR81, WNK1. WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292, ZNF335, ZNF407, ZNF462, or ZNF469. In an aspect, a disclosed protein coding gene can comprise one or more coding regions of CFTR. MDX, DYSF/TTN, DMPK, COL7A1, K14, MAPT, FVIII, HTT, RHO, DNA-PKcs, SMN2, or CD40L. In an aspect, a disclosed protein coding gene can comprise one or more coding regions of FXN, LMNA, or RYR2. [0138] In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode LMNA/C (SEQ ID NO:20) or a portion thereof. LMNA/C is discussed supra. In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode DP71 or a portion thereof. DP71 is discussed supra. In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode CFTR (SEQ ID NO: 19) or a portion thereof. CFTR is discussed supra. In an aspect, a disclosed nucleic acid sequence to be trans-spliced can encode DMPK (SEQ ID NO:21) or a portion thereof. DMPK is discussed supra. In an aspect, a disclosed gene can be DMD (dystrophin) (SEQ ID NO: 17). DMD is discussed supra.

[0139] In an aspect, a disclosed exogenous RNA to be trans-spliced can further comprise a UTR. [0140] In an aspect, one or more disclosed RNA structures can bind to one or more RNA binding proteins. In an aspect, one or more disclosed RNA structures can bind to one or more doublestranded RNA binding proteins (dsRBP). In an aspect, dsRBPs are known to the skilled person in the art and include, but are not limited to. AD ARI. ADAR2, DICER, NF AR, PACT. PKR, RHA RNaselll, Stauffen, TRBP, TSEN, or any combination thereof.

[0141] In an aspect, one or more disclosed RNA structures can bind to one or more RNA binding proteins. In an aspect, one or more disclosed RNA structures can bind to one or more doublestranded RNA binding proteins. In an aspect, one or more disclosed RNA structures can comprise the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09. In an aspect, one or more disclosed RNA structures can improve and/or can enhance trans-splicing efficiency. In an aspect, one or more disclosed RNA structures can stabilize the pre-mRNA. In an aspect, one or more disclosed RNA structures can localize the RNA to the nucleus. In an aspect, one or more disclosed RNA structures can stabilize the interaction between the targeted endogenous pre-mRNA molecule and the exogenous RNA to be trans-spliced.

[0142] In an aspect, a disclosed nucleic acid molecule can lack a CRISPR-associated protein.

[0143] In an aspect, a disclosed resulting chimeric RNA transcript can comprise the 5’ portion of the targeted endogenous pre-mRNA and the 3 ' portion of the exogenous RNA. In an aspect, a disclosed resulting chimeric RNA transcript can comprise the 3’ portion of the targeted endogenous pre-mRNA and the 5’ portion of the exogenous RNA. In an aspect, a disclosed targeted endogenous pre-mRNA and a disclosed exogenous RNA can encode the same protein coding gene. In an aspect, a disclosed targeted endogenous pre-mRNA and a disclosed exogenous RNA can comprise one or more exons of the same protein coding gene.

[0144] In an aspect, a disclosed nucleic acid molecule can be packaged into a viral vector. In an aspect, a disclosed viral vector can comprise an AAV vector. In an aspect, a disclosed nucleic acid molecule can be packaged into a non-viral carrier. In an aspect, a disclosed nucleic acid molecule can be incorporated into a plasmid. In an aspect, a disclosed nucleic acid molecule can be incorporated into lipid nanoparticles.

[0145] In an aspect, a disclosed nucleic acid molecule can further comprise a polyadenylation sequence. In an aspect, a disclosed nucleic acid molecule can further comprise a sequence for a promoter. In an aspect, a disclosed 3’ hemi intron can be recognized by nuclear splicing components within a host cell. In an aspect, a disclosed exogenous RNA can induce a splice event. In an aspect, a disclosed nucleic acid molecule can further comprise a spacer region. In an aspect, a disclosed spacer region can separate the 5' splice region from the one or more RNA structures. In an aspect, a disclosed spacer region can comprise any known spacer. In an aspect, a disclosed spacer region can comprise a consensus splicing motif (e.g., such as U1 or U2). In an aspect, a disclosed spacer region can comprise a limited number of consensus splicing motifs (e.g., such as U1 or U2). In an aspect, a disclosed nucleic acid molecule can further comprise a nuclear localization signal (NLS). In an aspect, a disclosed nucleic acid molecule can further comprise one or more nuclear retention elements (NRE). NRE are known to the skilled person in the art. In an aspect, a disclosed NRE can comprise SIRLOIN (SEQ ID NO: 15) or BORG (SEQ ID NO: 16).

[0146] In an aspect, a disclosed nucleic acid molecule can further comprise one or more Flavivirus genetic elements. In an aspect, Flavivirus genetic elements can comprise one or more Flavivirus 3’ untranslated region (3' UTR), one or more subgenomic Flavivirus RNA (sfRNA) elements, one or more Flavivirus XRN1 -resistant RNA (xrRNA) elements, one or more Flavivirus dumbbell (DB) RNA elements, one or more Flavivirus 3’ stem loop (3’ SL) elements, or any combination thereof. (See WO 2022/182835 for a description of Flavivirus gene elements).

[0147] In an aspect, a disclosed nucleic acid molecule can comprise the sequence for one or more regulatory elements (e.g., Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulator Element (WPRE), triplex from MALAT1, the PRE of Hepatitis B virus (HPRE), and an iron response element). For example, a disclosed regulatory’ element can comprise a promoter operably linked to a disclosed nucleic acid molecule, wherein the promoter drives the expression of a disclosed variant capsid protein, a disclosed encoded polypeptide, a disclosed encoded therapeutic agent, or both.

[0148] In an aspect, a disclosed promoter for the 3‘ replacement construct can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the expression desired. A promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. In an aspect, a disclosed promoter can be a promoter/enhancer. In an aspect, a disclosed promoter for the disclosed nucleic acid molecule can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter/enhancer. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can generally be obtained from a non-coding region upstream of a transcription initiation site of a gene of interest. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can be used for constitutive and efficient expression of a disclosed gene. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be a CMV promoter or a CMV promoter/enhancer. CMV promoters and CMV promoters/enhancers are well known to the art. In an aspect, a disclosed promoter for the one or more disclosed guide RNA sequences can be any eukaryotic RNA polymerase II promoter.

[0149] Disclosed herein is an expression cassette comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA. Disclosed herein is an expression cassette comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA. Disclosed herein is an expression cassette comprising one or more RNA structures; one or more RNA targeting motifs; a 3 ’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre- mRNA, wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA. Disclosed herein is an expression cassette comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced: and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA.

[0150] In an aspect, expression of a disclosed protein coding gene can be restored and/or returned to a wild-type, normal, or control expression level. In an aspect, a disclosed nucleic acid molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, a disclosed nucleic acid molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra). In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzy me dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi - systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity⁷. In an aspect, restoring the activity⁷ and/or functionality⁷ of a missing, deficient, and/or mutant protein or enzy me can comprise a 10%, 20%, 30%, 40%, 50%, 60%. 70%. 80%. 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration can be 10- 20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and/or mutant protein or enzyme). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity⁷, and/or functionality⁷ is similar to that of a wild-type or control level.

[01511 In an aspect, a disclosed 3’ replacement construct can be particularly useful in a method of treating a subject having an autosomal dominant genetic disease or disorder (such as, for example, progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert’s disease, hereditary haemorrhagic telangiectasia, hereditary⁷ elliptosis, hereditary⁷ spherocytosis, Huntington’s disease, idiopathic hypoparathyroidism, intestinal polyposis, marble bone disease, Marfan’s syndrome, neurofibromatosis, polycystic kidney disease (adult), protein C deficiency, osteogenesis imperfecta, Treacher Collins syndrome, tuberous sclerosis, Von Willebrand’s disease, etc.).

2. Transcriptome Engineering Systems

[0152] Disclosed herein is a transcriptome engineering system comprising one or more disclosed 3’ replacement construct, one or more disclosed 5’ replacement constructs, or any combination thereof. Disclosed herein is a transcriptome engineering system comprising one or more of (i) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, (ii) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09, (iii) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre- mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA, and (iv) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA. [0153] Disclosed herein is a transcriptome engineering system comprising one or more (i) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3 ’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, (ii) a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3^? hemi intron linked to the exogenous RNA to be trans- spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, (iii) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3 ’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA, and (iv) a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre- mRNA, wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA.

[0154] Disclosed herein is a transcriptome engineering system comprising one or more of (i) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, (ii) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09, (iii) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre- mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA, (iv) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA, (v) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, (vi) a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3^? hemi intron linked to the exogenous RNA to be trans- spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, (vii) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3 ’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3‘ replacement of the targeted endogenous pre-mRNA, and (viii) a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre- mRNA, wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA.

3. Vectors

[0155] Disclosed herein is a vector comprising a disclosed nucleic acid molecule. In an aspect, a disclosed vector can be a non-viral vector or a viral vector. Disclosed herein is a non-viral vector comprising a disclosed nucleic acid molecule. Disclosed herein is a non-viral vector comprising one or more disclosed nucleic acid molecules. Disclosed herein is a viral vector comprising a disclosed nucleic acid molecule. Disclosed herein is a viral vector comprising one or more disclosed nucleic acid molecules. Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures. Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5^? hemi intron linked to the exogenous RNA to be trans- spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09.

[0156] Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA. Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA. Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA. Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3‘ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA. Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced: and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA. Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA.

[0157] Disclosed herein is a non-viral or viral vector comprising one or more 5’ replacement constructs. Disclosed herein is a non-viral or viral vector comprising one or more 3’ replacement constructs. Disclosed herein is a non-viral or viral vector comprising one or more 5’ replacement constructs and/or one or more 3’ replacement constructs. In an aspect, a disclosed 5’ replacement construct to be used in combination with one or more other replacement constructs can comprise any disclosed 5’ replacement construct. In an aspect, a disclosed 5’ replacement construct can comprise (i) nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, (ii) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09, (iii) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables the 5‘ replacement of the targeted endogenous pre-mRNA, or (iv) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre- mRNA, or (v) any combination thereof.

[0158] In an aspect, a disclosed 3’ replacement construct to be used in combination with one or more other replacement constructs can comprise any disclosed 3’ replacement construct. In an aspect, a disclosed 3’ replacement construct can comprise (i) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, (ii) a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09: one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced: and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, (iiii) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3^? replacement of the targeted endogenous pre-mRNA, (iv) a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans- spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3‘ replacement of the targeted endogenous pre-mRNA. or (v) any combination thereof.

[0159] In an aspect, a disclosed vector can be formulated for administration via one or more routes. Such methods are well known to those skilled in the art and include, but are not limited to, the following routes: oral administration, transdermal administration, administration byinhalation, nasal administration, topical administration, in utero administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can also include hepatic intraarterial administration or administration through the hepatic portal vein (HPV). Administration of a disclosed therapeutic agent, a disclosed pharmaceutical composition, or a combination thereof can comprise administration directly into the CNS (e.g., intraparenchymal, intracerebroventricular, intrathecal cisternal, intrathecal (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or the PNS. Administration can be continuous or intermittent. Administration can comprise administering a viral vector and/or generated optimized viral vector. Administration of a disclosed vector can be continuous or intermittent. [0160] In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x IO¹⁰ vg/kg to about 2 x 10¹⁴.vg/kg. In an aspect, for example, a disclosed vector can be administered at a dose of about 1 x 10¹¹ to about 8 x 10¹³ vg/kg or about 1 x 10¹² to about 8 x 10¹³ vg/kg. In an aspect, a disclosed vector can be administered at a dose of about 1 x 10¹³ to about 6 x 10¹³ vg/kg. In an aspect, a disclosed vector can be administered at a dose of at least about 1 x IO¹⁰, at least about 5 x IO¹⁰, at least about 1 x

10¹¹, at least about 5 x 10¹¹, at least about 1 x 10¹², at least about 5 x 10¹², at least about 1 x 10^1?, at least about 5 x 10¹³, or at least about 1 x 10¹⁴ vg/kg. In an aspect, a disclosed vector can be administered at a dose of no more than about 1 x IO¹⁰, no more than about 5 x IO¹⁰, no more than about 1 x 10¹¹, no more than about 5 x 10¹¹, no more than about 1 x 10¹², no more than about 5 x

10¹², no more than about 1 x 10¹³, no more than about 5 x 10¹³, or no more than about 1 x 10¹⁴ vg/kg. In an aspect, a disclosed vector can be administered at a dose of about 1 x 10¹² vg/kg. In an aspect, a disclosed vector can be administered at a dose of about 1 x 10¹¹ vg/kg. In an aspect, a disclosed vector can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results.

[0161] In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10¹² vg per subject total to about 1 x 10¹⁷ vg per subject total. In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10¹² vg per subject total, about 1 x 10¹³ vg per subject total, about 1 x 10¹⁴ vg per subject total, about 1 x 10¹⁵ vg per subject total, about 1 x 10¹⁶ vg per subject total, or about 1 x 10¹⁷ vg per subject total. In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can be by delivered retrograde ureteral infusion and/or renal arterial administration and can comprise a range of about 1 x 10¹² vg per subj ect total to about 1 x 10¹⁷ vg per subj ect total.

[0162] In an aspect, a therapeutically effective amount of a disclosed AAV particle can comprise about 1 x 10⁶ DRP/mL to about 1 x 10¹⁴ DRP/mL. In an aspect, a disclosed pharmaceutical formulation can comprise about 1 x 10⁶ DRP/mL, 1 x 10⁷ DRP/mL, 1 x 10⁸ DRP/mL, 1 x 10⁹ DRP/mL, 1 x IO¹⁰ DRP/mL, 1 x 10¹¹ DRP/mL, 1 x 10¹² DRP/mL, 1 x 10¹³ DRP/mL, or 1 x 10¹⁴ DRP/mL. In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed vector can comprise a range determined by a skilled person.

[0163] In an aspect, a disclosed non-viral vector can be a polymer-based vector, a peptide-based vector, a lipid nanoparticle, a solid lipid nanoparticle, or a cationic lipid-based vector. In an aspect, a disclosed vector can comprise exosomes, extracellular vesicles, and virus like particles.

[0164] In an aspect, a disclosed viral vector can be an adenovirus vector, an AAV vector, a herpes simplex virus vector, a retrovirus vector, a lentivirus vector, and alphavirus vector, a Flavivirus vector, a rhabdovirus vector, a measles virus vector, a Newcastle disease viral vector, a poxvirus vector, or a picomavirus vector. In an aspect, a disclosed nucleic acid sequence can have a coding sequence that is less than about 4.5 kilobases.

[0165] In an aspect, a disclosed AAV vector can include naturally isolated serotypes including, but not limited to, AAVL AAV2, AAV3 (including 3a and 3b), AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, AAV1 1, AAV12, AAV13, AAVrh39, AAVrh43, AAVcy.7 as well as bovine AAV, caprine AAV, canine AAV, equine AAV, ovine AAV, avian AAV, primate AAV, non-primate AAV, and any other virus classified by the International Committee on Taxonomy of Viruses (ICTV) as an AAV. In an aspect, an AAV capsid can be a chimera either created by capsid evolution or by rational capsid engineering from a naturally isolated AAV variants to capture desirable serotype features such as enhanced or specific tissue tropism and/or a host immune response escape. Naturally isolated AAV variants include, but not limited to, AAV-DJ, AAV-HAEL AAV-HAE2, AAVM4L AAV-1829, AAV2 Y/F, AAV2 T/V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A-String (e.g., AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep, and AAV9.47-AS, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, and AAVcc.81. In an aspect, a disclosed AAV vector can be AAV-Rh74 or a related variant (e.g., capsid variants like RHM4-1). In an aspect, a disclosed AAV vector can be AAV.cc47. In an aspect, a disclosed AAV vector can be AAV.cc81. In an aspect, a disclosed AAV vector can be a self-complementary AAV.

[0166] In an aspect, a disclosed vector can comprise one or more ITRs (such as, for example, ITRs from AAV2).

[0167] In an aspect, a disclosed vector can further comprise one or more nuclear localization signals (NLS). NLS are known to the skilled person in the art. In an aspect, a disclosed NLS can comprise any NLS known to the art. As known to the art (see, e.g., Lu J, et al. (2021) Cell Commun Signal. 19:60, which is incorporated herein by reference for its teachings of NLS), nuclear localization signals (NLS) are generally short peptides that act as a signal fragment that mediates the transport of proteins from the cytoplasm into the nucleus.

[0168] In an aspect, a disclosed vector can further comprise one or more nuclear retention elements (NRE). NRE are known to the skilled person in the art. In an aspect, a disclosed NRE can comprise SIRLOIN (SEQ ID NO: 15) or BORG (SEQ ID NO: 16).

[0169] In an aspect, a disclosed vector can further comprise one or more Flavivirus genetic elements. In an aspect, Flavivirus genetic elements can comprise one or more Flavivirus 3’ untranslated region (3‘ UTR), one or more subgenomic Flavivirus RNA (siRNA) elements, one or more Flavivirus XRN 1 -resistant RNA (xrRNA) elements, one or more Flavivirus dumbbell (DB) RNA elements, one or more Flavivirus 3’ stem loop (3’ SL) elements, or any combination thereof. (See WO 2022/182835 for a description of Flavivirus gene elements).

[0170] In an aspect, a disclosed vector can further comprise a nucleic acid sequence encoding a therapeutic protein, a therapeutic agent, and/or a therapeutic RNA. In an aspect, a disclosed therapeutic protein can comprise a polypeptide and/or a glycopeptide. In an aspect, a disclosed therapeutic agent can comprise an oligonucleotide therapeutic agent. In an aspect, a disclosed oligonucleotide therapeutic agent can be a single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA. non-coding RNA (ncRNA), an antisense molecule, miRNA, a morpholino, a peptide-nucleic acid (PNA). or an analog or conjugate thereof. In an aspect, a disclosed therapeutic agent can be an ASO or an RNAi. In an aspect, a disclosed therapeutic agent can comprise a CRISPR-based endonuclease (e.g., Cas9). In an aspect, a disclosed CRISPR-based endonuclease can be derived from a CRISPR/Cas ty pe I, type II, or ty pe III system.

In an aspect, a disclosed therapeutic RNA can comprise ribosomal RNA (rRNA). transfer RNA (tRNA), heterogeneous nuclear RNA (hnRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), micro RNA (miRNA), Pi wi -interacting RNA (piRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), singe guide RNA (sgRNA), non-coding RNA (ncRNA), long non-coding RNA (IncRNA), 7SL, Xist, short enhancer RNA (eRNA), circular RNA, intergenic RNA. or any combination thereof. In an aspect, a disclosed RNA can comprise IncRNA, siRNA, shRNA, sgRNA, circular RNA, snoRNA, miRNA, or any combination thereof. In an aspect, a disclosed encoded RNA can comprise a functional non-coding RNA element.

[0171] In an aspect, a disclosed vector can comprise one or more promoters operably linked to a disclosed nucleic acid molecule (e.g., a 5' replacement construct and/or 3’ replacement construct), a disclosed transgene, a disclosed sequence to be trans-spliced, and/or a disclosed nucleic acid sequence. In an aspect of a disclosed vector, a disclosed nucleic acid molecule can be operably linked to one or more transcription regulatory' elements. In an aspect, the one or more transcription regulatory elements (e.g.. Woodchuck Hepatitis Virus (WHY) Posttranscri phonal Regulator Element (WPRE), triplex from MALAT1, the PRE of Hepatitis B virus (HPRE), and an iron response element) can increase the transcription and/or expression of a disclosed nucleic acid molecule (e.g., a 5’ replacement construct and/or 3’ replacement construct), a disclosed transgene, a disclosed sequence to be trans-spliced. and/or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and/or a disclosed therapeutic RNA). In an aspect, a disclosed promoter can be positioned 5’ (upstream) or 3’ (downstream) of a disclosed nucleic acid molecule (e.g., a 5’ replacement construct and/or 3’ replacement construct), a disclosed transgene, a disclosed sequence to be trans-spliced, and/or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and/or a disclosed therapeutic RNA) under its control. The distance between a disclosed promoter and a disclosed nucleic acid molecule (e.g., a 5’ replacement construct and/or 3’ replacement construct), a disclosed transgene, a disclosed sequence to be trans- spliced, and/or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and/or a disclosed therapeutic RNA) can be approximately the same as the distance between that promoter and to the disclosed nucleic acid molecule (e.g., a 5‘ replacement construct and/or 3^? replacement construct), the disclosed transgene, the disclosed sequence to be trans-spliced, and/or the disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and/or a disclosed therapeutic RNA) under its control. As is known in the art. variation in this distance can be accommodated without loss of promoter function.

[0172] In an aspect of a disclosed vector, a disclosed promoter can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the expression desired. A disclosed promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. In an aspect, a disclosed promoter can be a promoter/enhancer. In an aspect, a disclosed promoter for a disclosed nucleic acid molecule (e.g., a 5’ replacement construct and/or 3’ replacement construct), a disclosed transgene, a disclosed sequence to be trans-spliced. and/or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and/or a disclosed therapeutic RNA) can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter/enhancer. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can generally be obtained from anon-coding region upstream of a transcription initiation site of a gene of interest. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can be used for constitutive and efficient expression of a disclosed protein coding gene. In an aspect, a disclosed promoter for a disclosed nucleic acid molecule (e.g., a 5’ replacement construct and/or 3’ replacement construct), a disclosed transgene, a disclosed sequence to be trans-spliced, and/or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and/or a disclosed therapeutic RNA) can be a CMV promoter or a CMV promoter/enhancer. CMV promoters and CMV promoters/ enhancers are well known to the art. In an aspect, a disclosed promoter for a disclosed nucleic acid molecule (e.g., a 5’ replacement construct and/or 3‘ replacement construct), a disclosed transgene, a disclosed sequence to be transspliced, and/or a disclosed nucleic acid sequence (e.g.. encoding a disclosed therapeutic protein and/or a disclosed therapeutic RNA) can be any eukaryotic RNA polymerase II promoter.

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

[0173] Disclosed herein is an AAV particle comprising a disclosed nucleic acid molecule (e.g., a 5’ replacement construct and/or 3' replacement construct), a disclosed transgene, a disclosed sequence to be trans-spliced, and/or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and/or a disclosed therapeutic RNA) under its control.

[0174] In an aspect, a disclosed vector or a disclosed AAV particle can be particularly useful in a method of treating a subject having an autosomal dominant genetic disease or disorder (such as, for example, progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert’s disease, hereditary haemorrhagic telangiectasia, hereditary elliptosis, hereditary spherocytosis, Huntington’s disease, idiopathic hypoparathyroidism, intestinal polyposis, marble bone disease, Marfan’s syndrome, neurofibromatosis, polycystic kidney disease (adult), protein C deficiency, osteogenesis imperfecta. Treacher Collins syndrome, tuberous sclerosis, Von Willebrand’s disease, etc.)

4. Pharmaceutical Formulations

[0175] Disclosed herein is a pharmaceutical formulation comprising a disclosed nucleic acid molecule. Disclosed herein is a pharmaceutical formulation comprising a disclosed nucleic acid molecule and a pharmaceutically acceptable carrier. Disclosed herein is a pharmaceutical formulation comprising a disclosed vector. Disclosed herein is a pharmaceutical formulation comprising a disclosed vector and a pharmaceutically acceptable carrier. Disclosed herein is a pharmaceutical formulation comprising a disclosed AAV particle. Disclosed herein is a pharmaceutical formulation comprising a disclosed AAV particle and a pharmaceutically acceptable carrier.

[0176] Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or a viral vector comprising a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5' hemi intron linked to the exogenous RNA to be trans- spliced; one or more RNA targeting motifs; one or more RNA structures. Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or a viral vector comprising a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre- mRNA; a 5' hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09. Disclosed herein is a pharmaceutical formulation comprising a non- viral vector or a viral vector comprising a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA. Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or a viral vector comprising a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans- spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA. Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or a viral vector comprising a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA. Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or viral vector comprising a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA. Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or a viral vector comprising a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans- spliced to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA. Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or a viral vector comprising a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre- mRNA, wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA. [0177] In an aspect, a disclosed pharmaceutical formulation can comprise (i) one or more active agents, (ii) biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In an aspect, a disclosed composition can comprise one or more immune modulators. In an aspect, a disclosed composition can comprise one or more proteasome inhibitors. In an aspect, a disclosed composition can comprise one or more immunosuppressives or immunosuppressive agents. In an aspect, an immunosuppressive agent can be anti -thymocyte globulin (ATG), cyclosporine (CSP), my cophenolate mofetil (MMF), or a combination thereof. In an aspect, a disclosed formulation can comprise an anaplerotic agent (such as, for example, C7 compounds like triheptanoin or MCT).

[0178] In an aspect, a disclosed formulation can comprise an RNA therapeutic. An RNA therapeutic can comprise RNA-mediated interference (RNAi) and/or antisense oligonucleotides (ASO). In an aspect, a disclosed RNA therapeutic can be directed at any protein or enzyme that is overexpressed or is overactive due to a missing, deficient, and/or mutant protein or enzyme. In an aspect, a disclosed RNA therapeutic can comprise therapy delivered via LNPs. In an aspect, a disclosed formulation can comprise an enzy me or enzy me precursor for enzy me replacement therapy (ERT).

[0179] In an aspect, a disclosed formulation can comprise a disclosed small molecule. In an aspect, a disclosed small molecule can assist in restoring the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0180] In an aspect, any disclosed pharmaceutical formulation can comprise one or more excipients and/or pharmaceutically acceptable carriers. Excipients and/or pharmaceutically acceptable carriers are known to the art and are discussed supra.

[0181] In an aspect, a therapeutically effective amount of a disclosed pharmaceutical formulation can comprise a range of about 1 x 10¹⁰ vg/kg to about 2 x 10¹⁴vg/kg of a disclosed vector and/or a disclosed AAV particle. In an aspect, for example, a dose of a disclosed pharmaceutical formulation can comprise about 1 x 10ⁿto about 8 x 10¹³ vg/kg or about 1 x 10¹² to about 8 x 10¹³ vg/kg. In an aspect, a dose of a disclosed pharmaceutical formulation can comprise about 1 x 10¹³ to about 6 x 10¹³ vg/kg. In an aspect, a dose of a disclosed pharmaceutical formulation can comprise at least about 1 x 10¹⁰, at least about 5 x 10ⁱⁿ. at least about 1 x 10¹¹, at least about 5 x 10¹¹, at least about 1 x 10¹², at least about 5 x 10¹², at least about 1 x 10¹³, at least about 5 x 10¹³, or at least about l x 10¹⁴ vg/kg. In an aspect, a dose of a disclosed pharmaceutical formulation can comprise no more than about 1 x 10¹⁰, no more than about 5 x 10¹⁰, no more than about 1 x 10¹¹, no more than about 5 x 10¹¹, no more than about 1 x 10¹², no more than about 5 x 10¹², no more than about 1 x 10¹³, no more than about 5 x 10¹³, or no more than about 1 x 10¹⁴ vg/kg. In an aspect, a dose of a disclosed pharmaceutical formulation can comprise about 1 x 10¹² vg/kg. In an aspect, a dose of a disclosed pharmaceutical formulation can comprise about 1 x 10¹¹ vg/kg. In an aspect, a dose of a disclosed pharmaceutical formulation can comprise a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results.

[0182] In an aspect, a therapeutically effective amount of a disclosed pharmaceutical formulation can comprise a range of about 1 x 10¹² vg per subject total to about 1 x 10¹⁷ vg per subject total. In an aspect, a therapeutically effective amount of a disclosed pharmaceutical formulation can comprise a range of about 1 x 10¹² vg per subject total, about 1 x 10¹³ vg per subject total, about 1 x 10¹⁴ vg per subject total, about 1 x 10¹⁵ vg per subject total, about 1 x 10¹⁶ vg per subject total, or about 1 x 10¹⁷ vg per subject total. In an aspect, a therapeutically effective amount of a disclosed pharmaceutical formulation can comprise about 1 x 10⁶ DRP/mL to about 1 x 10¹⁴ DRP/mL. In an aspect, a therapeutically effective amount of a disclosed pharmaceutical formulation can comprise about 1 x 10⁶ DRP/mL, 1 x 10⁷ DRP/mL, 1 x 10⁸ DRP/mL, 1 x 10⁹ DRP/mL, 1 x IO¹⁰ DRP/mL, 1 x 10¹¹ DRP/mL, 1 x 10¹² DRP/mL, 1 x 10¹³ DRP/mL, or 1 x 10¹⁴ DRP/mL.

[0183] In an aspect, a therapeutically effective amount of a disclosed pharmaceutical formulation can comprise a range determined by a skilled person.

[0184] In an aspect, a disclosed pharmaceutical formulation can be used to restore and/or return expression of a disclosed protein coding gene to a wild-type, normal, or control expression level. In an aspect, a disclosed pharmaceutical formulation can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, a disclosed nucleic acid molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra). In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzy me dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi-systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity. In an aspect, restoring the activity and/or functionality of a missing, deficient, and/or mutant protein or enzyme can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and/or mutant protein or enzyme). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is similar to that of a wild-type or control level.

[0185] In an aspect, a disclosed pharmaceutical formulation can be particularly useful in a method of treating a subject having an autosomal dominant genetic disease or disorder (such as, for example, progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary haemorrhagic telangiectasia, hereditary elliptosis, hereditary spherocytosis, Huntington’s disease, idiopathic hypoparathyroidism, intestinal polyposis, marble bone disease, Marfan’s syndrome, neurofibromatosis, polycystic kidney disease (adult), protein C deficiency, osteogenesis imperfecta, Treacher Collins syndrome, tuberous sclerosis, Von Willebrand’s disease, etc.)

5. Plasmids

[0186] Disclosed herein is a plasmid comprising one or more disclosed nucleic acid molecules. Disclosed herein is a plasmid comprising one or more disclosed vectors. Disclosed here are plasmids used in methods of making a disclosed composition such as, for example, a disclosed nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. Plasmids and using plasmids are known to the art. Disclosed herein is a plasmid comprising the sequence set forth in any one of SEQ ID NO:25 - SEQ ID NO:33 or a fragment thereof. Disclosed herein is a plasmid comprising a sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%. 89%. 90%. 91%. 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99% identity to the sequence set forth in any one of SEQ ID NO:25 - SEQ ID NO:33 or a fragment thereof. Disclosed herein is a plasmid comprising a sequence having at least 40%-60%, at least 60%-80%, at least 80%-90%, or at least 90%-100% identity to the sequence set forth in any one of SEQ ID NO:25 - SEQ ID NO: 33 or a fragment thereof.

6. Cells

[0187] Disclosed herein are cells comprising a disclosed nucleic acid molecule, a disclosed vector, and/or a disclosed plasmid. Disclosed herein are cells transduced by one or more disclosed viral vectors. Disclosed herein are cells transfected with one or more disclosed nucleic acid molecules. Techniques to achieve transfection and transduction are known to the art and using transfected or transduced cells are known to the art. In an aspect, disclosed herein are human cells lines transduced by one or more disclosed viral vectors or transfected with one or more disclosed nucleic acids, one or more disclosed non-viral vectors, or one or more disclosed plasmids. In an aspect, disclosed herein are human cells lines having one or more genetic diseases or genetic disorders contacted with one or more nucleic acid molecules, one or more disclosed vectors, and/or one or more disclosed pharmaceutical formulations. Disclosed herein are cells obtained for a subject treated with one or more disclosed nucleic acid molecules, one or more disclosed vectors, one or more disclosed plasmids, and/or one or more disclosed pharmaceutical formulations.

7. Animals

[0188] Disclosed herein are animals treated with one or more disclosed nucleic acid molecules, one or more disclosed replacement constructs, one or more disclosed vectors, one or more disclosed AAV particles, one or more disclosed pharmaceutical formulations, and/or one or more disclosed plasmids . Transgenic animals are known to the art as are the techniques to generate transgenic animals.

8. Libraries

[0189] Disclosed herein is a library of one or more disclosed nucleic acid molecules. Disclosed herein is a library of one or more disclosed 5' replacement constructs. Disclosed herein is a library of one or more disclosed 3’ replacement constructs. Disclosed herein is a library of one or more disclosed 5’ replacement constructs and/or disclosed 3’ replacement constructs. Disclosed herein is a library of one or more disclosed vectors. Disclosed herein is a library of one or more disclosed vectors comprising one or more disclosed 5’ replacement constructs, one or more disclosed 3’ constructs, or any combination thereof. Disclosed herein is a library of one or more disclosed AAV particles comprising one or more disclosed 5’ replacement constructs, one or more disclosed 3’ constructs, or any combination thereof. Disclosed herein is a library of one or more disclosed plasmids.

9. Kits

[0190] Disclosed herein is a kit comprising one or more disclosed nucleic acid molecules, disclosed vectors or disclosed AAV particles, disclosed pharmaceutical formulations, or any combination thereof. Disclosed herein is a kit comprising one or more disclosed nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof. In an aspect, a kit can comprise a disclosed nucleic acid molecule, a disclosed vector or disclosed AAV particle, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, or a combination thereof, and one or more agents. “Agents” and “Therapeutic Agents” are known to the art and are described supra.

[0191] In an aspect, the one or more agents can treat, prevent, inhibit, and/or ameliorate one or more comorbidities in a subject. In an aspect, one or more active agents can treat, inhibit, prevent, and/or ameliorate cellular and/or metabolic complications related to a missing, deficient, and/or mutant protein or enzyme.

[0192] In an aspect, a disclosed kit can comprise at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose (such as, for example, treating a subject diagnosed with or suspected of having a genetic disease or genetic disorder). Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. In an aspect, a kit for use in a disclosed method can comprise one or more containers holding a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed RNA therapeutic, or a combination thereof, and a label or package insert with instructions for use. In an aspect, suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers can be formed from a variety of materials such as glass or plastic. The container can hold a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof, and can have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert can indicate that a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, a disclosed RNA therapeutic agent, or a combination thereof can be used for treating, preventing, inhibiting, and/or ameliorating a disease or disorder or complications and/or symptoms associated with a disease or disorder. A disclosed kit can comprise additional components necessary for administration such as, for example, other buffers, diluents, filters, needles, and syringes. In an aspect, a disclosed kit can be used in any disclosed method. In an aspect, a disclosed kit can be used to generate one or more chimeric RNA molecules. In an aspect, a disclosed kit can be used to treat a genetic disease or genetic disorder. In an aspect, a disclosed kit can be used to inhibit and/or minimize disease progression.

C. Methods of Generating a Chimeric RNA Molecule

[0193] Disclosed herein is a method of generating a chimeric RNA molecule in a cell, the method comprising contacting an endogenous pre-mRNA in a cell with a disclosed 5’ replacement construct, wherein the resulting chimeric RNA transcript comprises the 3’ portion of the targeted endogenous pre-mRNA and the 5’ portion of the exogenous RNA.

[0194] Disclosed herein is a method of generating a chimeric RNA molecule, the method comprising contacting one or more cells with a disclosed 5 ’ replacement construct, wherein the resulting chimeric RNA transcript comprise the 3’ portion of the targeted endogenous pre-mRNA and the 5’ portion of the exogenous RNA.

[0195] Disclosed herein is a method of generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5^? hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the resulting chimeric RNA transcript comprise the 3’ portion of the targeted endogenous pre-mRNA and the 5’ portion of the exogenous RNA.

[0196] Disclosed herein is a method of generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule, compnsing an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09, wherein the resulting chimeric RNA transcript comprise the 3’ portion of the targeted endogenous pre-mRNA and the 5’ portion of the exogenous RNA.

[0197] Disclosed herein is a method of generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5^? hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA transcript comprise the 3' portion of the targeted endogenous pre-mRNA and the 5’ portion of the exogenous RNA.

[0198] Disclosed herein is a method of generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule, comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA transcript comprise the 3’ portion of the targeted endogenous pre- mRNA and the 5’ portion of the exogenous RNA. [0199] Disclosed herein is a method of generating a chimeric RNA molecule in a cell, the method comprising contacting an endogenous pre-mRNA in a cell with a disclosed 3’ replacement construct, wherein the resulting chimeric RNA transcript comprises the 5’ portion of the targeted endogenous pre-mRNA and the 3‘ portion of the exogenous RNA.

[0200] Disclosed herein is a method of generating a chimeric RNA molecule, the method comprising contacting one or more cells with a disclosed 3’ replacement construct, wherein the resulting chimeric RNA transcript can comprise the 5 ’ portion of the targeted endogenous pre- mRNA and the 3’ portion of the exogenous RNA.

[0201] Disclosed herein is a method of generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre- mRNA, wherein the resulting chimeric RNA transcript can comprise the 5’ portion of the targeted endogenous pre-mRNA and the 3’ portion of the exogenous RNA.

[0202] Disclosed herein is a method of generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3^? hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the resulting chimeric RNA transcript can comprise the 5’ portion of the targeted endogenous pre- mRNA and the 3’ portion of the exogenous RNA.

[0203] Disclosed herein is a method of generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre- mRNA, wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA transcript can comprise the 5’ portion of the targeted endogenous pre-mRNA and the 3’ portion of the exogenous RNA.

[0204] Disclosed herein is a method of generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA transcript can comprise the 5’ portion of the targeted endogenous pre- mRNA and the 3’ portion of the exogenous RNA.

[0205] In an aspect, the one or more cells can be in a subject. In an aspect, a subject can be diagnosed with or can be suspected of having a genetic disease or disorder. In an aspect, a disease or disorder can comprise any disease or disorder caused by a disclosed gene or a missing, deficient, and/or mutant gene. In an aspect, a subject can be a subject in need of treatment of a disclosed disease or disorder (e.g., a genetic disease or disorder). Genetic diseases and disorders are discussed extensively herein.

[0206] In an aspect, a disclosed method of generating a chimeric RNA molecule can further comprise identifying a subject in need of generating a chimeric RNA molecule.

[0207] In an aspect of a disclosed method of generating a chimeric RNA molecule, a disclosed vector or a disclosed nucleic acid molecule can be formulated for administration via one or more routes. Such methods are well known to those skilled in the art and include, but are not limited to, the following routes: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can also include hepatic intraarterial administration or administration through the hepatic portal vein (HPV). Administration of a disclosed therapeutic agent, a disclosed pharmaceutical composition, or a combination thereof can comprise administration directly into the CNS (e.g., intraparenchymal, intracerebroventricular, intrathecal cisternal, intrathecal (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or the PNS. Administration can be continuous or intermittent.

[0208] In an aspect, administration can be performed by one or more ex vivo methods such as, for example, an ex vivo perfusion protocol. In an aspect, an ex vivo perfusion protocol can be employed with a one or more cells, tissues, and/or organs affected by a genetic disease or disorder obtained for a subject. In an aspect, one or more cells and/or one or more tissues and/or one or more organs can be obtained from the subject in need thereof, can be subjected to an ex vivo perfusion and/or treatment and/or contact protocol, and can be returned to the subject in need thereof, wherein the one or more cells generate a chimeric RNA transcript.

[0209] In an aspect, a disclosed method of generating a chimeric RNA molecule in cells can comprise validating the trans-splicing event and/or the generation of the chimeric RNA molecule. Validation of the trans-splicing event and/or generation of the chimeric RNA molecule can be accomplished using methods and techniques known to the art (e.g., sequencing, northern blots, FISH, PCR, RNA-Seq, 3’ RACE, 5’ RACE, etc ).

[0210] In an aspect, a disclosed method of generating a chimeric RNA molecule can comprise preparing a disclosed 5’ replacement construct, a disclosed 3‘ replacement construct, a disclosed non-viral vector or disclosed viral vector, a disclosed nucleic acid molecule, a disclosed pharmaceutical formulation, or any combination thereof.

[0211] In an aspect, a disclosed method can be performed ex vivo. In an aspect, administration can be performed by one or more ex vivo methods such as. for example, an ex vivo perfusion protocol. In an aspect, an ex vivo perfusion protocol can be employed with a one or more cells, tissues, and/or organs affected by a genetic disease or disorder obtained for a subj ect. In an aspect, one or more cells and/or one or more tissues and/or one or more organs can be obtained from the subject in need thereof, can be subjected to an ex vivo perfusion and/or treatment and/or contact protocol, and can be returned to the subject in need thereof, wherein the one or more cells generate a chimeric RNA transcript.

[0212] In an aspect, a disclosed method can restore the activity⁷ and/or functionality⁷ of a missing, deficient, and/or mutant protein or enzy me can comprise a 10%, 20%, 30%, 40%. 50%, 60%, 70%. 80%. 90%. 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration can be 10-20%, 20- 30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and/or mutant protein or enzyme). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is similar to that of a wild-type or control level, e and/or return the expression level of one or more protein coding genes to a wild-type, normal, or control expression level.

[0213] In an aspect, a disclosed method can restore one or more aspects of cellular homeostasis and/or cellular functionality⁷ and/or metabolic dysregulation. In an aspect, a disclosed method can restore the functionality and/or structural integrity⁷ of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).

[0214] In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality⁷ can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy⁷ pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity⁷; (v) correcting enzy me dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi-systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity.

[0215] In an aspect, a disclosed chimeric RNA can be particularly useful in a method of treating a subject having an autosomal dominant genetic disease or disorder (such as, for example, progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert’s disease, hereditary haemorrhagic telangiectasia, hereditary elliptosis, hereditary spherocytosis, Huntington’s disease, idiopathic hypoparathyroidism, intestinal polyposis, marble bone disease, Marfan’s syndrome, neurofibromatosis, polycystic kidney disease (adult), protein C deficiency, osteogenesis imperfecta, Treacher Collins syndrome, tuberous sclerosis, Von Willebrand’s disease, etc.)

D. Methods of Treating a Genetic Disease or Genetic Disorder

[0216] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a disclosed non-viral vector or a disclosed viral vector or a pharmaceutical formulation thereof, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality⁷ and/or metabolic dysregulation.

[0217] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a disclosed non-viral vector or a disclosed viral vector or a pharmaceutical formulation thereof, wherein the resulting chimeric RNA molecule can restore the functionality⁷ and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).

[0218] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a non-viral or viral vector comprising a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5 ’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation, and/or wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity’ of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).

[0219] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subj ect in need thereof a therapeutically effective amount of a non- viral or viral vector comprising a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation, and/or wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).

[0220] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subj ect in need thereof a therapeutically effective amount of a non-viral or viral vector comprising a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5 ’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation, and/or wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).

[0221] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a non-viral or viral vector comprising a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO: 01 - SEQ ID NO: 09, wherein the nucleic acid molecule enables the 5‘ replacement of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation, and/or wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity’ of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).

[0222] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a non-viral or viral vector comprising a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality’ and/or metabolic dysregulation, and/or wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).

[0223] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a non-viral or viral vector comprising a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation, and/or wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity’ of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra). .

[0224] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a non-viral or viral vector comprising a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality’ and/or metabolic dysregulation, and/or wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).

[0225] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a non-viral or viral vector comprising a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3^? replacement of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation, and/or wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).

[0226] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a non-viral vector, a viral vector, an AAV particle, or a pharmaceutical formulation thereof comprising (i) one or more 5^? replacement constructs, (ii) one or more 3’ replacement constructs, or (iii) one or more 5’ replacement constructs and/or one or more 3’ replacement constructs, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation, and/or wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).

[0227] In an aspect of a disclosed method of treating, a disclosed 5’ replacement construct to be used in combination with one or more other replacement constructs can comprise any disclosed 5‘ replacement construct. In an aspect, a disclosed 5’ replacement construct can comprise (i) nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, (ii) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5' hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09, (iii) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre- mRNA; a 5‘ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA, or (iv) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09. wherein the nucleic acid molecule enables the 5' replacement of the targeted endogenous pre-mRNA, or (v) any combination thereof.

[0228] In an aspect of a disclosed method of treating, a disclosed 3’ replacement construct to be used in combination with one or more other replacement constructs can comprise any disclosed 3’ replacement construct. In an aspect, a disclosed 3’ replacement construct can comprise (i) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3 ’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, (ii) a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans- spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, (iiii) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA, (iv) a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO: 01 - SEQ ID NO: 091 one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre- mRNA, or (v) any combination thereof.

[0229] In an aspect of a disclosed method of treating, a subject can have a genetic disease or disorder related to and/or caused by one or more disclosed genes including those disclosed in Section VII(B)(1). In an aspect of a disclosed method of treating, a subject can have a genetic disease or disorder related to and/or caused by one or more disclosed genes including those disclosed in Section VII(B)( 1 ).

[0230] In an aspect of a disclosed method of treating, expression of a disclosed protein coding gene can be restored and/or returned to a wild-type, normal, or control expression level. In an aspect, a disclosed method of treating a genetic disease or disorder can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme. In an aspect, a disclosed method of treating can comprise restoring one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, restoring one or more aspect of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation comprises restoring the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme. In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzy me dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi-systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity. In an aspect, restoring the activity⁷ and/or functionality of a missing, deficient, and/or mutant protein or enzyme (e.g., encoded by a protein coding gene described supra) can comprise a 10%, 20%, 30%, 40%, 50%. 60%. 70%. 80%. 90%. 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and/or mutant protein or enzyme). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality⁷ is similar to that of a wild-type or control level.

[0231] In an aspect, a disclosed method of treating can further comprise monitoring the subject’s metabolic and/or physiologic improvement following the administering step and/or following the administering steps. In an aspect, a clinician can measure and/or determine the subject’s metabolic and/or physiologic status over time to identify one or more improvements and/or one or more diminishments. In an aspect of a disclosed method, a clinician can use the subject’s metabolic and/or physiologic status and/or the trend of the subject’s metabolic and/or physiological status and/or trend to make a treatment decision and/or to modify an aspect of a disclosed method and/or to continue treating the subject and/or continue to administer a disclosed AAV particle, a disclosed vector, a disclosed nucleic acid molecule, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, and/or a disclosed immune modulator, or any combination thereof. In an aspect, metabolic and/or physiologic data can inform the clinician and a treatment plan.

[0232] In an aspect of a disclosed method of treating, techniques to monitor, measure, and/or assess the restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example, representative regulated variables and sensors relating to systemic homeostasis are provided below.

[0233] In an aspect, a disclosed method of treating can further comprise subjecting the subject to one or more invasive or non-invasive diagnostic assessments. Diagnostic assessments are known to the art. In an aspect, a disclosed non-invasive diagnostic assessment can comprise x-rays, computerized tomography (CT) scans, magnetic resonance imaging (MRI) scans, ultrasounds, positron emission tomography (PET) scans, or any combination thereof. In an aspect, a disclosed invasive diagnostic assessment can comprise a tissue biopsy or exploratory surgery.

[0234] In an aspect, a disclosed method of treating can be used to repair diseased and/or dysfunctional cell types in the one or more diseased and/or disordered cells, tissues, and/or organs. [0235] In an aspect, a disclosed method of treating can be used to improve and/or can be used to enhance the quality of the subject's life when compared to a pre-treatment level. In an aspect, a disclosed method of treating can be used to improve the subject’s quality of life by at least 50% when compared to the subject’s pre-treatment quality of life.

[0236] In an aspect, a disclosed method of treating can be used to improve and/or can be used to enhance the uality of the subject's life when compared to a pre-treatment level. In an aspect, a disclosed method of treating can be used to improve the subject’s quality of life by at least 50% when compared to the subject’s pre-treatment quality of life. [0237] In an aspect, a disclosed method of treating can be used to diminish and/or decrease one or more symptoms associated with and/or related to the subject’s genetic disease and/or genetic disorder. In an aspect, a disclosed method of treating can be used to prevent an undesired physiological change, disease, pathological condition, or disorder from occurring in the subject. In an aspect, a disclosed method of treating can be used to inhibit a physiological change, disease, pathological condition, or disorder, i.e., arresting its development, in the subject. In an aspect, a disclosed method of treating can be used to relieve a physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease, in the subject.

[0238] In an aspect of a disclosed method of treating, administering to the subject can comprise contacting one or more cells with one or more disclosed nucleic acid molecules, disclosed vectors or disclosed AAV particles, disclosed pharmaceutical formulations, or any combination thereof.

[0239] In an aspect of a disclosed method of treating, administering of a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof can comprise one or more routes. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can also include hepatic intra-arterial administration or administration through the hepatic portal vein (HPV). Administration of a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination can comprise administration directly into the CNS (e.g., intraparenchymal, intracerebroventricular, intrathecal cisternal, intrathecal (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or the PNS. Administration can be continuous or intermittent.

[0240] In an aspect, a disclosed method of treating can employ multiple routes of administration to the subject. In an aspect, a disclosed method of treating can employ a first route of administration that can be the same or different as a second and/or subsequent routes of administration.

[0241] In an aspect of a disclosed method of treating a genetic disease or disorder, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a range of about 1 x 10¹⁰ vg/kg to about 2 x 10¹⁴ vg/kg. In an aspect, for example, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a dose of about 1 x 10¹¹ vg/kg to about 8 x 10¹³ vg/kg or about 1 x 10¹² vg/kg to about 8 x 10¹³ vg/kg. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a dose of about 1 x 10¹³ vg/kg to about 6 x 10¹³ vg/kg. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a dose of at least about 1 x IO¹⁰ vg/kg. at least about 5 x IO¹⁰ vg/kg, at least about 1 x 10¹¹ vg/kg, at least about 5 x 10¹¹ vg/kg, at least about 1 x 10¹² vg/kg, at least about 5 x 10¹² vg/kg, at least about 1 x 10¹³ vg/kg, at least about 5 x 10¹³ vg/kg, or at least about 1 x 10¹⁴ vg/kg. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a dose of no more than about 1 x IO¹⁰ vg/kg, no more than about 5 x IO¹⁰ vg/kg, no more than about 1 x 10¹¹ vg/kg, no more than about 5 x 10¹¹ vg/kg, no more than about 1 x 10¹² vg/kg, no more than about 5 x 10¹² vg/kg, no more than about 1 x 10¹³ vg/kg, no more than about 5 x 10¹³, or no more than about 1 x 10¹⁴ vg/kg. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a dose of about 1 x 10¹² vg/kg or about 1 x 10¹¹ vg/kg. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results.

[0242] In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a range of about 1 x 10¹² vg per subject total to about 1 x 10¹⁷ vg per subject total. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a range of about 1 x 10¹² vg per subject total, about 1 x 10¹³ vg per subject total, about 1 x 10¹⁴ vg per subject total, about 1 x 10¹⁵ vg per subject total, about 1 x 10¹⁶ vg per subject total, or about 1 x 10¹⁷ vg per subject total.

[0243] In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise about 1 x 10⁶ DRP/mL to about 1 x 10¹⁴ DRP/mL. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise about 1 x 10⁶ DRP/mL, 1 x 10⁷ DRP/mL, 1 x 10⁸ DRP/mL. 1 x 10⁹ DRP/mL, 1 x IO¹⁰ DRP/mL, 1 x 10¹¹ DRP/mL, 1 x 10¹² DRP/mL, 1 x 10¹³ DRP/mL, or 1 x 10¹⁴ DRP/mL. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a range determined by a skilled person.

[0244] In an aspect, a disclosed method of treating a genetic disease or disorder can further comprise administering to the subj ect a therapeutically effective amount of a therapeutic agent. A therapeutic agent can be any disclosed agent that effects a desired clinical outcome.

[0245] In an aspect, a disclosed method of treating a genetic disease or disorder can further comprise monitoring the subject for adverse effects. In an aspect, in the absence of adverse effects, the method can further comprise continuing to treat the subject. In an aspect, in the presence of adverse effects, the method can further comprise modifying the treating step. Methods of monitoring a subject’s well-being can include both subjective and objective criteria (and are discussed supra). Such methods are known to the skilled person.

[0246] In an aspect, a disclosed method of treating can further comprise administering to the subject a therapeutically effective amount of an agent that can correct one or more aspects of a dysregulated metabolic or enzymatic pathway. In an aspect, such an agent can comprise an enzyme for enzyme replacement therapy. In an aspect, a disclosed enzyme can replace any enzyme in a dysregulated or dysfunctional metabolic or enzymatic pathway. In an aspect, a disclosed method of treating can comprise replacing one or more enzymes in a dysregulated or dysfunctional metabolic pathway.

[0247] In an aspect, a disclosed method of treating a genetic disease or disorder can further comprise administering one or more immune modulators. In an aspect, a disclosed immune modulator can be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or a combination thereof. In an aspect, a disclosed immune modulator can be bortezomib or SVP- Rapamycin. In an aspect, a disclosed immune modulator can be Tacrolimus. In an aspect, a disclosed immune modulator such as methotrexate can be administered at a transient low to high dose. In an aspect, a disclosed immune modulator can be administered at a dose of about 0. 1 mg/kg body weight to about 0.6 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.4 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for 3 to 5 or greater cycles, with up to three days per cycle. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for a minimum of 3 cycles, with three days per cycle. In an aspect, a person skilled in the art can determine the appropriate number of cycles. In an aspect, a disclosed immune modulator can be administered as many times as necessary’ to achieve a desired clinical effect.

[0248] In an aspect, a disclosed immune modulator can be administered orally about one hour before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered orally about one hour or a few days before a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before or a few days before a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof.

[0249] In an aspect, a disclosed method of treating a genetic disease or disorder can further comprise administering one or more proteasome inhibitors (e.g., bortezomib, carfilzomib, marizomib, ixazomib, and oprozomib). In an aspect, a proteasome inhibitor can be an agent that acts on plasma cells (e.g., daratumumab). In an aspect, an agent that acts on a plasma cell can be melphalan hydrochloride, melphalan, pamidronate disodium, carmustine, carfilzomib, carmustine, cyclophosphamide, daratumumab, doxorubicin hydrochloride liposome, doxorubicin hydrochloride liposome, elotuzumab, melphalan hydrochloride, panobinostat, ixazomib citrate, carfilzomib, lenalidomide, melphalan, melphalan hydrochloride, plerixafor, ixazomib citrate, pamidronate disodium, panobinostat, plerixafor, pomalidomide, pomalidomide, lenalidomide, selinexor, thalidomide, thalidomide, bortezomib, selinexor, zoledronic acid, or zoledronic acid.

[0250] In an aspect, a disclosed method of treating stability can further comprise administering one or more proteasome inhibitors or agents that act on plasma cells prior to administering a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof. In an aspect, a disclosed method of treating can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells concurrently with administering a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination of thereof. In an aspect, a disclosed method of treating can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells subsequent to administering a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof. In an aspect, a disclosed method of treating can further comprise administering one or more proteasome inhibitors more than 1 time. In an aspect, a disclosed method of treating can comprise administering one or more proteasome inhibitors repeatedly over time.

[0251] In an aspect, a disclosed method of treating a genetic disease or disorder can further comprise administering one or more immunosuppressive agents. In an aspect, an immunosuppressive agent can be, but is not limited to, azathioprine, methotrexate, sirolimus, antithymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF). steroids, or a combination thereof. In an aspect, a disclosed method of treating can comprise administering one or more immunosuppressive agents more than 1 time. In an aspect, a disclosed method can comprise administering one or more one or more immunosuppressive agents repeatedly over time. In an aspect, a disclosed method of treating can comprise administering a compound that targets or alters antigen presentation or humoral or cell mediated or innate immune responses.

[0252] In an aspect, a disclosed method of treating a genetic disease or disorder can further comprise administering a compound that exerts a therapeutic effect against B cells and/or a compound that targets or alters antigen presentation or humoral or cell mediated immune response. In an aspect, a disclosed compound can be rituximab, methotrexate, intravenous gamma globulin, anti CD4 antibody, anti CD2, an anti-FcRN antibody, a BTK inhibitor, an anti-IGFIR antibody, a CD19 antibody (e.g., inebilizumab), an anti-IL6 antibody (e.g., tocilizumab), an antibody to CD40, an IL2 mutein, or a combination thereof. Also disclosed herein are Treg infusions that can be administered as a way to help with immune tolerance (e.g., antigen specific Treg cells to AAV). [0253] In an aspect, a disclosed method of treating can further comprise administering lipid nanoparticles (LNPs). In an aspect, LNPs can be organ-targeted. In an aspect, LNPs can be liver- targeted or testes-targeted. For example, in an aspect, mRNA therapy with LNP encapsulation for systemic delivery to a subject has the potential to restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0254] In an aspect, a disclosed method of treating a genetic disease or disorder can further comprise treating a subject that has developed or is likely to develop neutralizing antibodies (ABs) to a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof. In an aspect, treating a subject that has developed or is likely to develop neutralizing antibodies can comprise plasmapheresis and immunosuppression. In an aspect, a disclosed method can comprise using immunosuppression to decrease the T cell, B cell, and /or plasma cell population, decrease the innate immune response, inflammatory response, and antibody levels in general. In an aspect, a disclosed method can comprise administering an IgG-degrading agent that depletes pre-existing neutralizing antibodies. In an aspect, a disclosed method can comprise administering to the subject IdeS or IdeZ, rapamycin, and/or SVP-Rapamycin. In an aspect, a disclosed method of treating can comprise administering Tacrolimus. In an aspect, a disclosed IgG-degrading agent is bacteria-derived IdeS or IdeZ.

[0255] In an aspect, a disclosed method of treating can comprise repeating a disclosed administering step such as, for example, repeating the administering of a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed immunosuppressive agent, a disclosed compound that exerts a therapeutic effect against B cells and/or a disclosed compound that targets or alters antigen presentation or humoral or cell mediated immune response. [0256] In an aspect, a disclosed method of treating can comprise modifying one or more of the disclosed steps. For example, modifying one or more of steps of a disclosed method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof administered to a subject, or by changing the frequency of administration of one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof to a subject, or by changing the duration of time one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof are administered to a subject.

[0257] In an aspect, a disclosed method of treating can be altered by changing the amount of one or more disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject, or by changing the frequency of administration of one or more of the disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject.

[0258] In as aspect, a disclosed method of treating can comprise concurrent administration of one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, , one or more disclosed therapeutic agents, one or more disclosed immune modulators, one or more disclosed proteasome inhibitors, one or more disclosed immunosuppressive agents, one or more disclosed compounds that exert therapeutic effect against B cells, one or more disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response, or any combination thereof. In an aspect, a disclosed immune modulator can be administered prior to or after the administration of a disclosed therapeutic agent.

[0259] In an aspect, a disclosed method of treating a genetic disease or disorder can further comprise generating a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof. [0260] In an aspect, a disclosed method of treating can further comprise gene editing one or more relevant genes (such as, for example, a missing, deficient, and/or mutant protein or enzyme), wherein editing includes but is not limited to single gene knockout, loss of function screening of multiple genes at one, gene knockin. or a combination thereof.

[0261] In an aspect, a disclosed method of treating a genetic disease or disorder can further comprise administering to the subject an oligonucleotide therapeutic agent. A disclosed oligonucleotide therapeutic agent can comprise a single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), an antisense molecule, miRNA, a morpholino, a peptide-nucleic acid (PNA). or an analog or conjugate thereof. In an aspect, a disclosed oligonucleotide therapeutic agent can be an ASO or an RNAi. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise one or more modifications at any position applicable. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise a CRISPR- based endonuclease. In an aspect, a disclosed endonuclease can be Cas9. In an aspect, a disclosed Cas9 can be from Staphylococcus aureus or Streptococcus pyogenes. In an aspect, a disclosed method of treating can comprise administering the subject a disclosed RNA therapeutic.

[0262] In an aspect, a disclosed method of treating a genetic disease or disorder can further comprise generating and/or validating one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof. In an aspect, a disclosed method of treating can inhibit and/or minimize one or more aspects of disease progression in the subject (e.g., a genetic disease or disorder described supra).

[0263] In an aspect, a disclosed method of treating can slow and/or diminish one or more aspects of disease progression in the subject (e.g., a genetic disease or genetic disorder described supra).

[0264] In an aspect, a disclosed method can be particularly useful in treating a subject having an autosomal dominant genetic disease or disorder (such as, for example, progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome. Gilbert’s disease, hereditary haemorrhagic telangiectasia, hereditary elliptosis. hereditary spherocytosis, Huntington’s disease, idiopathic hypoparathyroidism, intestinal polyposis, marble bone disease, Marfan’s syndrome, neurofibromatosis, polycystic kidney disease (adult), protein C deficiency, osteogenesis imperfecta. Treacher Collins syndrome, tuberous sclerosis, Von Willebrand’s disease, etc.)

E. Methods of Inhibiting and/or Minimizing Disease Progression

[0265] Disclosed herein is a method of inhibiting and/or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a disclosed non-viral vector or a disclosed viral vector or a pharmaceutical formulation thereof, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation.

[0266] Disclosed herein is a method of inhibiting and/or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a disclosed non-viral vector or a disclosed viral vector or a pharmaceutical formulation thereof, wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzy me (such as those, for example, encoded by one of the genes provided supra).

[0267] Disclosed herein is method of inhibiting and/or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a non-viral or viral vector comprising a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be transspliced; one or more RNA targeting motifs; one or more RNA structures, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation, and/or wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).

[0268] Disclosed herein is method of inhibiting and/or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a non-viral or viral vector comprising a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans- spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality’ and/or metabolic dysregulation, and/or wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).

[0269] Disclosed herein is method of inhibiting and/or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a non-viral or viral vector comprising a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be transspliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables the 5 ’ replacement of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. and/or wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).

[0270] Disclosed herein is method of inhibiting and/or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a non-viral or viral vector comprising a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be transspliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation, and/or wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).

[0271] Disclosed herein is method of inhibiting and/or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a non-viral or viral vector comprising a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation, and/or wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzy me (such as those, for example, encoded by one of the genes provided supra).

[0272] Disclosed herein is method of inhibiting and/or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a non-viral or viral vector comprising a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation, and/or wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).

[0273] Disclosed herein is method of inhibiting and/or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a non-viral or viral vector comprising a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality' and/or metabolic dysregulation, and/or wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).

[0274] Disclosed herein is method of inhibiting and/or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of a non-viral or viral vector comprising a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA. wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality’ and/or metabolic dysregulation, and/or wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).

[0275] Disclosed herein is method of inhibiting and/or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering to a subj ect in need thereof a therapeutically effective amount of a non-viral vector, a viral vector, an AAV particle, or a pharmaceutical formulation thereof comprising (i) one or more 5‘ replacement constructs, (ii) one or more 3’ replacement constructs, or (iii) one or more 5’ replacement constructs and/or one or more 3’ replacement constructs, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation, and/or wherein the resulting chimeric RNA molecule can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).

[0276] In an aspect of a disclosed method of inhibiting and/or minimizing disease progression, a disclosed 5^? replacement construct to be used in combination with one or more other replacement constructs can comprise any disclosed 5' replacement construct. In an aspect, a disclosed 5’ replacement construct can comprise (i) nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, (ii) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09, (iii) anucleic acid molecule comprising an exogenous RNA to be trans- spliced to a targeted endogenous pre-mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA, or (iv) anucleic acid molecule comprising an exogenous RNA to be trans-spliced to a targeted endogenous pre- mRNA; a 5’ hemi intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09, wherein the nucleic acid molecule enables the 5’ replacement of the targeted endogenous pre-mRNA, or (v) any combination thereof.

[0277] In an aspect of a disclosed method of inhibiting and/or minimizing disease progression, a disclosed 3^? replacement construct to be used in combination with one or more other replacement constructs can comprise any disclosed 3’ replacement construct. In an aspect, a disclosed 3’ replacement construct can comprise (i) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, (ii) a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, (iiii) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre- mRNA, (iv) a nucleic acid molecule comprising one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs; a 3’ hemi intron linked to the exogenous RNA to be trans-spliced; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3’ replacement of the targeted endogenous pre-mRNA, or (v) any combination thereof.

[0278] In an aspect of a disclosed method of inhibiting and/or minimizing disease progression, a subject can have a genetic disease or disorder related to and/or caused by one or more disclosed genes including those disclosed in Section VII(B)(1). In an aspect of a disclosed method of inhibiting and/or minimizing disease progression, a subject can have a genetic disease or disorder related to and/or caused by one or more disclosed genes including those disclosed in Section VII(B)(1).

[0279] In an aspect of a disclosed method of inhibiting and/or minimizing disease progression, expression of a disclosed protein coding gene can be restored and/or returned to a wild-ty pe, normal, or control expression level. In an aspect, a disclosed method of inhibiting and/or minimizing disease progression of a genetic disease or disorder can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme. In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can comprise restoring one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, restoring one or more aspect of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation comprises restoring the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme. In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality⁷ and/or structural integrity⁷; (iv) improving, enhancing, restoring, and/or preserving organelle functionality⁷ and/or structural integrity: (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi- systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity. In an aspect, restoring the activity and/or functionality of a missing, deficient, and/or mutant protein or enzyme (e.g., encoded by a protein coding gene described supra) can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pretreatment level. In an aspect, the amount of restoration can be 10-20%, 20-30%, 30-40%, 40- 50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and/or mutant protein or enzyme). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is similar to that of a wild-type or control level.

[0280] In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can further comprise monitoring the subject’s metabolic and/or physiologic improvement following the administering step and/or following the administering steps. In an aspect, a clinician can measure and/or determine the subject’s metabolic and/or physiologic status over time to identify one or more improvements and/or one or more diminishments. In an aspect of a disclosed method, a clinician can use the subject’s metabolic and/or physiologic status and/or the trend of the subject’s metabolic and/or physiological status and/or trend to make a treatment decision and/or to modify an aspect of a disclosed method and/or to continue treating the subject and/or continue to administer a disclosed AAV particle, a disclosed vector, a disclosed nucleic acid molecule, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, and/or a disclosed immune modulator, or any combination thereof. In an aspect, metabolic and/or physiologic data can inform the clinician and a treatment plan.

[0281] In an aspect of a disclosed method of inhibiting and/or minimizing disease progression, techniques to monitor, measure, and/or assess the restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example, representative regulated variables and sensors relating to systemic homeostasis are provided supra. [0282] In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can further comprise subjecting the subject to one or more invasive or non-invasive diagnostic assessments. Diagnostic assessments are known to the art. In an aspect, a disclosed non-invasive diagnostic assessment can comprise x-rays, computerized tomography (CT) scans, magnetic resonance imaging (MRI) scans, ultrasounds, positron emission tomography (PET) scans, or any combination thereof. In an aspect, a disclosed invasive diagnostic assessment can comprise a tissue biopsy or exploratory surgery.

[0283] In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can be used to repair diseased and/or dysfunctional cell types in the one or more diseased and/or disordered cells, tissues, and/or organs.

[0284] In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can be used to improve and/or can be used to enhance the quality of the subject’s life when compared to a pre-treatment level. In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can be used to improve the subject’s quality of life by at least 50% when compared to the subject’s pre-treatment quality’ of life. In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can be used to improve and/or can be used to enhance the quality of the subject’s life when compared to a pre-treatment level. In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can be used to improve the subject’s quality of life by at least 50% when compared to the subject’s pre-treatment quality of life.

[0285] In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can be used to diminish and/or decrease one or more symptoms associated with and/or related to the subject’s genetic disease and/or genetic disorder. In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can be used to prevent an undesired physiological change, disease, pathological condition, or disorder from occurring in the subject. In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can be used to inhibit a physiological change, disease, pathological condition, or disorder, i.e., arresting its development, in the subject. In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can be used to relieve a physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease, in the subject.

[0286] In an aspect of a disclosed method of inhibiting and/or minimizing disease progression, administering to the subject can comprise contacting one or more cells with one or more disclosed nucleic acid molecules, disclosed vectors or disclosed AAV particles, disclosed pharmaceutical formulations, or any combination thereof.

[0287] In an aspect of a disclosed method of inhibiting and/or minimizing disease progression, administering of a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof can comprise one or more routes. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can also include hepatic intraarterial administration or administration through the hepatic portal vein (HPV). Administration of a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination can comprise administration directly into the CNS (e.g., intraparenchymal, intracerebroventricular, intrathecal cisternal, intrathecal (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or the PNS. Administration can be continuous or intermittent.

[0288] In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can employ multiple routes of administration to the subject. In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can employ a first route of administration that can be the same or different as a second and/or subsequent routes of administration.

[0289] In an aspect of a disclosed method of inhibiting and/or minimizing disease progression of a genetic disease or disorder, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a range of about 1 x IO¹⁰ vg/kg to about 2 x 10¹⁴ vg/kg. In an aspect, for example, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a dose of about 1 x 10¹¹ vg/kg to about 8 x 10¹³ vg/kg or about 1 x 10¹² vg/kg to about 8 x 10¹³ vg/kg. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a dose of about 1 x 10¹³ vg/kg to about 6 x 10¹³ vg/kg. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a dose of at least about 1 x IO¹⁰ vg/kg, at least about 5 x IO¹⁰ vg/kg, at least about 1 x 10¹¹ vg/kg, at least about 5 x 10¹¹ vg/kg, at least about 1 x 10¹² vg/kg, at least about 5 x 10¹² vg/kg, at least about 1 x 10¹³ vg/kg, at least about 5 x 10¹³ vg/kg. or at least about 1 x 10¹⁴ vg/kg. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a dose of no more than about 1 x IO¹⁰ vg/kg, no more than about 5 x IO¹⁰ vg/kg, no more than about 1 x 10¹¹ vg/kg, no more than about 5 x 10¹¹ vg/kg, no more than about 1 x 10¹² vg/kg, no more than about 5 x IO’² vg/kg, no more than about 1 x 10¹³ vg/kg, no more than about 5 x 10¹³, or no more than about 1 x 10¹⁴ vg/kg. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a dose of about 1 x 10¹² vg/kg or about 1 x 10¹¹ vg/kg. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7. 8, 9 or 10 doses) as needed for the desired therapeutic results. [0290] In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a range of about 1 x 10¹² vg per subject total to about 1 x 10¹⁷ vg per subject total. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a range of about 1 x 10¹² vg per subject total, about 1 x 10¹³ vg per subject total, about 1 x 10¹⁴ vg per subject total, about 1 x 10¹⁵ vg per subject total, about 1 x 10¹⁶ vg per subject total, or about 1 x 10¹⁷ vg per subject total.

[0291] In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise about 1 x 10⁶ DRP/mL to about 1 x IO¹⁴ DRP/mL. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise about 1 x 10⁶ DRP/mL, 1 x 10⁷ DRP/mL, 1 x 10⁸ DRP/mL, 1 x 10⁹ DRP/mL, 1 x IO¹⁰ DRP/mL, 1 x 10¹¹ DRP/mL, 1 x 10¹² DRP/mL, 1 x 10¹³ DRP/mL, or 1 x 10¹⁴ DRP/mL. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a range determined by a skilled person.

[0292] In an aspect, a disclosed method of inhibiting and/or minimizing disease progression of a genetic disease or disorder can further comprise administering to the subject a therapeutically effective amount of a therapeutic agent. A therapeutic agent can be any disclosed agent that effects a desired clinical outcome.

[0293] In an aspect, a disclosed method of inhibiting and/or minimizing disease progression of a genetic disease or disorder can further comprise monitoring the subject for adverse effects. In an aspect, in the absence of adverse effects, the method can further comprise continuing to treat the subject. In an aspect, in the presence of adverse effects, the method can further comprise modifying the treating step. Methods of monitoring a subject's well-being can include both subjective and objective criteria (and are discussed supra). Such methods are known to the skilled person.

[0294] In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can further comprise administering to the subject a therapeutically effective amount of an agent that can correct one or more aspects of a dysregulated metabolic or enzymatic pathway. In an aspect, such an agent can comprise an enzyme for enzyme replacement therapy. In an aspect, a disclosed enzyme can replace any enzyme in a dysregulated or dysfunctional metabolic or enzymatic pathway. In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can comprise replacing one or more enzymes in a dysregulated or dysfunctional metabolic pathway.

[0295] In an aspect, a disclosed method of inhibiting and/or minimizing disease progression of a genetic disease or disorder can further comprise administering one or more immune modulators. In an aspect, a disclosed immune modulator can be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or a combination thereof. In an aspect, a disclosed immune modulator can be bortezomib or SVP-Rapamycin. In an aspect, a disclosed immune modulator can be Tacrolimus. In an aspect, a disclosed immune modulator such as methotrexate can be administered at a transient low to high dose. In an aspect, a disclosed immune modulator can be administered at a dose of about 0. 1 mg/kg body weight to about 0.6 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.4 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for 3 to 5 or greater cycles, with up to three days per cycle. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for a minimum of 3 cycles, with three days per cycle. In an aspect, a person skilled in the art can determine the appropriate number of cycles. In an aspect, a disclosed immune modulator can be administered as many times as necessary to achieve a desired clinical effect.

[0296] In an aspect, a disclosed immune modulator can be administered orally about one hour before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered orally about one hour or a few days before a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before or a few days before a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof.

[0297] In an aspect, a disclosed method of inhibiting and/or minimizing disease progression of a genetic disease or disorder can further comprise administering one or more proteasome inhibitors (e.g.. bortezomib, carfilzomib. marizomib. ixazomib, and oprozomib). In an aspect, a proteasome inhibitor can be an agent that acts on plasma cells (e.g., daratumumab). In an aspect, an agent that acts on a plasma cell can be melphalan hydrochloride, melphalan, pamidronate disodium, carmustine, carfilzomib, carmustine, cyclophosphamide, daratumumab, doxorubicin hydrochloride liposome, doxorubicin hydrochloride liposome, elotuzumab, melphalan hydrochloride, panobinostat, ixazomib citrate, carfilzomib, lenalidomide, melphalan, melphalan hydrochloride, plerixafor, ixazomib citrate, pamidronate disodium, panobinostat, plerixafor, pomalidomide, pomalidomide, lenalidomide, selinexor, thalidomide, thalidomide, bortezomib, selinexor, zoledronic acid, or zoledronic acid. [0298] In an aspect a disclosed method of inhibiting and/or minimizing disease progression stability can further comprise administering one or more proteasome inhibitors or agents that act on plasma cells prior to administering a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof. In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells concurrently with administering a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination of thereof. In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells subsequent to administering a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof. In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can further comprise administering one or more proteasome inhibitors more than 1 time. In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can comprise administering one or more proteasome inhibitors repeatedly over time.

[0299] In an aspect, a disclosed method of inhibiting and/or minimizing disease progression of a genetic disease or disorder can further comprise administering one or more immunosuppressive agents. In an aspect, an immunosuppressive agent can be, but is not limited to, azathioprine, methotrexate, sirolimus, anti-thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), steroids, or a combination thereof. In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can comprise administering one or more immunosuppressive agents more than 1 time. In an aspect, a disclosed method can comprise administering one or more one or more immunosuppressive agents repeatedly over time. In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can comprise administering a compound that targets or alters antigen presentation or humoral or cell mediated or innate immune responses.

[0300] In an aspect, a disclosed method of inhibiting and/or minimizing disease progression of a genetic disease or disorder can further comprise administering a compound that exerts a therapeutic effect against B cells and/or a compound that targets or alters antigen presentation or humoral or cell mediated immune response. In an aspect, a disclosed compound can be rituximab, methotrexate, intravenous gamma globulin, anti CD4 antibody, anti CD2, an anti-FcRN antibody, a BTK inhibitor, an anti-IGFIR antibody, a CD19 antibody (e.g., inebilizumab), an anti-IL6 antibody (e.g., tocilizumab), an antibody to CD40, an IL2 mutein, or a combination thereof. Also disclosed herein are Treg infusions that can be administered as a way to help with immune tolerance (e.g., antigen specific Treg cells to AAV).

[0301] In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can further comprise administering lipid nanoparticles (LNPs). In an aspect. LNPs can be organ- targeted. In an aspect. LNPs can be liver-targeted or testes-targeted. For example, in an aspect, mRNA therapy with LNP encapsulation for systemic delivery to a subject has the potential to restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0302] In an aspect, a disclosed method of inhibiting and/or minimizing disease progression of a genetic disease or disorder can further comprise treating a subject that has developed or is likely to develop neutralizing antibodies (ABs) to a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof. In an aspect, treating a subject that has developed or is likely to develop neutralizing antibodies can comprise plasmapheresis and immunosuppression. In an aspect, a disclosed method can comprise using immunosuppression to decrease the T cell, B cell, and /or plasma cell population, decrease the innate immune response, inflammatory response, and antibody levels in general. In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can comprise administering an IgG-degrading agent that depletes pre-existing neutralizing antibodies. In an aspect, a disclosed method can comprise administering to the subject IdeS or IdeZ, rapamycin, and/or SVP-Rapamycin. In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can comprise administering Tacrolimus. In an aspect, a disclosed IgG-degrading agent is bacteria-derived IdeS or IdeZ.

[0303] In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can comprise repeating a disclosed administering step such as, for example, repeating the administering of a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed immunosuppressive agent, a disclosed compound that exerts a therapeutic effect against B cells and/or a disclosed compound that targets or alters antigen presentation or humoral or cell mediated immune response.

[0304] In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can comprise modifying one or more of the disclosed steps. For example, modifying one or more of steps of a disclosed method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof administered to a subject, or by changing the frequency of administration of one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof to a subject, or by changing the duration of time one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof are administered to a subject.

[0305] In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can be altered by changing the amount of one or more disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject, or by changing the frequency of administration of one or more of the disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject.

[0306] In as aspect, a disclosed method of inhibiting and/or minimizing disease progression can comprise concurrent administration of one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, , one or more disclosed therapeutic agents, one or more disclosed immune modulators, one or more disclosed proteasome inhibitors, one or more disclosed immunosuppressive agents, one or more disclosed compounds that exert therapeutic effect against B cells, one or more disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response, or any combination thereof. In an aspect, a disclosed immune modulator can be administered prior to or after the administration of a disclosed therapeutic agent. In an aspect, a disclosed method of inhibiting and/or minimizing disease progression of a genetic disease or disorder can further comprise generating a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof.

[0307] In an aspect, a disclosed method of inhibiting and/or minimizing disease progression can further comprise gene editing one or more relevant genes (such as, for example, those listed below in Table 1). Table 1 - Larges Gene with Identification of Affected Chromosomes and # of Mutations

[0308] In an aspect, a disclosed method can be particularly useful in treating a subject having an autosomal dominant genetic disease or disorder (such as, for example, progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert’s disease, hereditary haemorrhagic telangiectasia, hereditary elliptosis, hereditary spherocytosis, Huntington’s disease, idiopathic hypoparathyroidism, intestinal polyposis, marble bone disease, Marfan’s syndrome, neurofibromatosis, polycystic kidney disease (adult), protein C deficiency, osteogenesis imperfecta. Treacher Collins syndrome, tuberous sclerosis, Von Willebrand’s disease, etc.)

VIII. EXAMPLES

[0309] As there are pathogenic mutations in more than 500 genes exceeding the packaging capacity of AAV, several efforts have been aimed at circumventing this barrier to expression of large genes. While these techniques differ, the general approach remains broadly similar between strategies. Briefly, a dual AAV vector approach is taken; wherein the DNA sequence encoding the protein of interest is split and packaged into separate vectors. Upon co-infection of target cells by the two vectors, the genomes of the two vectors recombine with each other via inverted repeat sequences or overlapping complementary sequences forming a single genome bearing the reconstituted DNA sequence expressing the full protein of interest. While this strategy is feasible the efficiency of recombination between genomes has limited the viability of its widespread adoption.

[0310] Separately, other efforts to effect phenotypic correction of genes ineligible for classical AAV mediated gene therapy have inspired an approach involving the manipulation of endogenous messenger RNA, the conduit between DNA and protein. The RNA editing strategy known as spliceosome mediated RNA trans-splicing (SMART) has been developed as a strategy to introduce large precise modifications to the primary structure of RNA transcripts independent of target transcript length. The aim of this approach is to hijack the cellular RNA processing machinery for incorporation of a desired sequence into an endogenous transcript. In this strategy, a recombinant RNA molecule is introduced to the cell comprising 3 essential components: an RNA targeting motif, a hemi intron sequence, and the primary sequence of the desired RNA to be joined to an endogenous RNA transcript. The RNA targeting motif is comprised of a stretch of oligonucleotides anti-sense to an intron of an endogenous target pre-mRNA. Upon Watson-Crick base pairing of the RNA targeting motif with the endogenous intron, the hemi intron is then recognized by the spliceosome. Depending on the desired splicing reaction either a 5 ’ hemi intron facilitates the splicing of the trans-splicing molecule to the exon immediately 3’ to the targeted intron, or a 3 ’ hemi intron linked to the exogenous RNA to be trans-spliced facilitates the splicing of the trans-splicing molecule to the exon immediately 5’ to the targeted intron. This produces a mature chimeric RNA transcript comprised of the recombinant 5’ start of a transcript joined to an endogenous 3’ sequence or endogenous 5’ start of a transcript joined to a recombinant 3’ sequence, respectively. The utility of this strategy is that a single AAV vector needs only to package a genome capable of producing a trans-splicing RNA molecule containing the sequence for part of a gene, obviating the need to deliver the full-length protein coding sequence to a cell. As the entire recombinant RNA region of a chimeric RNA product may be specified by the user, the trans-splicing RNA may contain the wild type sequence of a target RNA, an inactivating mutation in the target RNA, or a modified RNA sequence encoding a novel protein. However, similar to the split- AAV vector approach, the low specificity and efficiency of RNA targeting by anti-sense RNA sequences has precluded the widespread use of this technology in research and clinical settings. Although promising, expressing a CRISPR protein indefinitely (e.g., long term expression) as a therapeutic presents challenges arising from immunogenicity and off-target effects. Moreover, CRISPR-based approaches often require two constructs to be delivered (namely the CRISPR effector protein and the RNA trans-splicing construct), thereby leading to high dose requirements and low efficiency of correction. Disclosed herein is a system for transcriptome engineering that does not need the CRISPR system. There are many endogenous RNA binding ribonucleoproteins in human cells, and the system disclosed herein exploits several RNA structures that interact with known human ribonucleoproteins for enabling trans-splicing. As detailed herein, by incorporating these RNA structures into trans-splicing RNA, effective trans-splicing of many targeted endogenous pre-mRNAs was achieved. This allowed for the rewriting of large stretches of mRNA.

EXAMPLE 1 Validation of Trans-Splicing Efficiency

[0311] To assay trans-splicing efficiency, a green fluorescence-based screening system was designed and built. The first step was to construct a reporter in which the green fluorescent protein (EGFP) open reading frame was split into two halves. An intron was inserted between the two halves. Accordingly, if this construct spliced in cis, then it would make a mature RNA that encoded EGFP and, upon translation, would express the green fluorophore.

[0312] Next, we abolished fluorescent expression by introducing a premature stop codon into the first “exon’^? of the EGFP open reading frame (FIG. 1 (left side)). If this construct spliced in cis, then there would be no green fluorescence expression because the premature stop codon in the first half of the EGFP open reading frame stopped translation of the full-length protein.

[0313] By delivering a trans-splicing RNA that encoded the correct open reading frame for the first half of EGFP followed by a hemi-intron and guide RNA sequence, green fluorescence expression could be restored. Thus, green fluorescence was used as an indirect readout of trans- splicing efficiency (FIG. 1). This reporter system was the workhorse of our downstream assays. [0314] The first trans-splicing RNA delivered to these cells (hereinafter - SMaRT or Spliceosome mediated RNA trans-splicing) had only the first half of EGFP, a hemi-intron and 30 bp antisense targeting domain. Sequences were then cloned into a trans-splicing RNA vector to ascertain whether trans-splicing efficiency could be enhanced. These new constructs were transfected into HEK293 cells with the split GFP reporter and 48 hours later green fluorescence intensity (percent GFP% and mean fluorescence intensity) was assayed by flow cytometry as a proxy for trans- splicing efficiency (FIG. 2A and FIG. 2B). Through this process, three (3) RNA structures that yielded significantly enhanced trans-splicing efficiency (RNA Structure 4. 5 and 7). In FIG. 2A - FIG. 2B, the RNA targeting motif for RYR2 was set forth in SEQ ID NO:23.

[0315] Whether any member of this panel of RNA sequences enhanced the 3’ trans-splicing approach was next examined. To do this, the same split GFP reporter was used, but for a slight modification in that the stop codon was moved from the first half of the open reading frame to the second half (FIG. 1 (right side)). This construct, on its own, does not express green fluorescence when delivered to HEK293 cells because cis splicing retains the premature stop codon. To restore EGFP expression, a trans-splicing RNA that completed the open reading frame of EGFP upon proper splicing was used. Like the 5' editing approach, a trans-splicing RNA was used and contained a 30 bp anti-sense targeting motif, followed by a hemi-intron, and the second half of the EGFP open reading frame (this construct was SMaRT). The same panel of RNA structures was then introduced into this RNA molecule with the intent of improving trans-splicing efficiency. [0316] The new constructs were co-transfected into HEK293 cells with the split GFP reporter. Then, 48 hrs. later, green fluorescence intensity (percent GFP% and mean fluorescence intensity) was assayed by flow' cytometry (as a proxy for trans-splicing efficiency). (FIG. 3A - FIG. 3B). Through this process, 4 RNA structures that yielded significantly enhanced trans-splicing efficiency (RNA Structure 1, 2, 10, and 11) were identified. This panel w as repeated on a second intron target intron (FIG. 3C - FIG. 3D) and a similar trend in fold change of editing efficiency was observed. In FIG. 3A - FIG. 3B, the RNA targeting motif for RYR2 is in SEQ ID NO:23 while the RNA targeting motif for LMNA in FIG. 3C - FIG. 3D was is in SEQ ID NO:22.

[0317] Following these results, the trans-splicing assay for the top hits (RNA Structures 1 and 2) were repeated. A second control RNA structure (i.e., the direct repeat from Ruminococcus flavefaciens XPD3002 (RfxCasl3d)) was also used. The rationale for this control was to ensure that the boost in RNA editing was unique to these RNA structures and not an artifact of including additional sequence length/complexity in the trans-splicing RNA. Indeed, only RNA structures 1 and 2 appeared to boost editing efficiency, while the Cast 3 direct repeat had a slightly detrimental effect of trans-splicing efficiency (FIG. 4A - FIG. 4B).

[0318] Finally, a new target intron was cloned into the split GFP reporter and assayed for editing efficiency with RNA structure 2 and several guide RNA candidates. At this new target, editing was observed in excess of 50% GFP positive cells (FIG. 5A - FIG. 5B). In FIG. 5A - FIG. 5B, the RNA targeting motif for FXN was set forth in SEQ ID NO:24. In Table 2, Structures 4 and 5 contain a cloning site within them for where guide RNA is inserted this is the bold sequence.

Table 2 - List of RNA Structures

Table 3 - List of Plasmids Used in Examples

EXAMPLE 2 Trans-Splicing of a Mutations in DMD

[0319] DMD (dystrophin) is known to the art (e.g., Gene ID 1756) and this nucleotide sequence can comprise nucleotides 5001 - 2225382 in Accession No. NG012232.1. DMD spans a genomic range of greater than 2 Mb and encodes a large protein containing an N-terminal actin-binding domain and multiple spectrin repeats. The encoded protein (SEQ ID NO: 17) forms a component of the dystrophin-gly coprotein complex (DGC), which bridges the inner cytoskeleton and the extracellular matrix. Deletions, duplications, and point mutations at this gene locus may cause Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or cardiomyopathy. Currently, about 1750 pathogenic mutations of DMD have been reported. Detailed below are constructs to address over 1000 of those pathogenic mutations. Here, 5’ replacement constructs for DMD are generated using a plasmid based on the schematic presented in FIG. 7A. The 5’ replacement construct is directed at Exons 1-23 of DMD. (SEQ ID NO:33). The 5’ replacement construct redresses one or more of the 618 mutations identified below in Table 4.

Table 4 - List of Mutations in Exons 1-23 of DMD

Ill

[0320] An AAV vector based on the plasmid construct show in in FIG. 7A is delivered to a subject having one or more 5’ mutations in DMD (e.g., Table 4). Following administration of a therapeutically effective amount of the AAV vector (e.g., about 1 x 10¹⁰ vg/kg to about 2 x 10¹⁴.vg/kg), one or more subject’s cells, tissues, and/or organs generate a chimeric RNA molecule encoding a corrected and/or restored DMD. Confirmation of the generation of the chimeric RNA molecule is performed by obtaining a sample from the subject and confirming the expression level of the corrected and/or restored DMD (by comparing the post-treatment level of operative/functional DMD to the subject’s pre-treatment level of operative/functional DMD). The subject experiences an inhibition and/or minimization of DMD disease progression. The subject’s quality of life improves. Here, 3’ replacement constructs for DMD are generated using a plasmid based on the schematic presented in FIG. 7B. The 3‘ replacement construct is directed at Exons 53-79 of DMD. (SEQ ID NO:32). The 3’ replacement construct redresses one or more of the 446 mutations identified below in Table 5.

Table 5 - List of Mutations in Exons 53-79 of DMD

[0321] An AAV vector based on the plasmid construct show in in FIG. 7B is delivered to a subject having one or more 3’ mutations (e.g., Table 5). Following administration of a therapeutically effective amount of the AAV vector (e.g., about 1 x IO¹⁰ vg/kg to about 2 x 10¹⁴.vg/kg), one or more subject’s cells, tissues, and/or organs generate a chimeric RNA molecule encoding a corrected and/or restored DMD. Confirmation of the generation of the chimeric RNA molecule is performed by obtaining a sample from the subject and confirming the expression level of the corrected and/or restored DMD (by comparing the post-treatment level of operative/functional DMD to the subject’s pre-treatment level of operative/functional DMD). The subject experiences an inhibition and/or minimization of DMD disease progression. The subject’s quality of life improves.

SUMMARY OF EXAMPLES

[0322] The compositions and methods disclosed herein are superior to previously disclosed compositions and methods such as SMART. The advantages conferred by the disclosed system are numerous. These surprising and unexpected advantages include (i) the lack of need for an additional effector protein (e g., CRISPR-based to enable trans-splicing); (ii) the ability to modify the effector RNA structure to bind endogenous splicing machinery and enable either 3’ or 5' end replacement of pre-spliced mRNA; (iii) the ability to deliver the construct in a single AAV vector or as a trans-splicing RNA fragment using a non- viral delivery system (e.g., LNP); (iv) the ability to edit large stretches of mRNA constituting multiple exons; (v) the ability⁷ to correct mRNA in a single "knockdow n and replace’' approach, of particular utility⁷ in autosomal dominant inheritance disorders using a single trans-splicing construct without having to provide two distinct constructs for knockdown and replacement; and (vi) the ability to prevent over-expression of any gene, correction is based on the trans-splicing fragment provided, which is dependent on endogenous mutant transcript levels (hence the corrected mRNA will never be expressed at levels higher than the endogenous disease transcript). While comprehensive, this list of advantages is not exhaustive.

[0323] Overall, described herein is a platform for efficient RNA editing that does not rely on CRISPR-based targeting and outperforms simple antisense base pairing. This enables efficient rewrite of large stretches of RNA, which has implications for human health and basic biology. As a therapeutic, this would allow for rewriting of genes that exceed the packaging capacity of AAV, correct dominant negative mutations, all while maintaining expression of the target transcript at endogenous levels.

Claims

IX. CLAIMS What is claimed is:

1. A nucleic acid molecule, comprising: one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:09; one or more RNA targeting motifs: a 3 ’ hemi intron; and an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA.

2. A nucleic acid molecule, comprising: an exogenous RNA to be trans-spliced to a targeted endogenous pre-mRNA; a 5 ’ hemi intron; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO: 09.

3. The nucleic acid molecule of Claim 1 or Claim 2, wherein the targeted endogenous pre-mRNA comprises one or more mutations in one or more exons.

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

5. The nucleic acid molecule of Claim 3, wherein the one or more mutations are in the 5’ portion of the targeted endogenous pre-mRNA.

6. The nucleic acid molecule of Claim 1, wherein the targeted endogenous pre-mRNA encodes a protein coding gene.

7. The nucleic acid molecule of Claim 6, wherein the protein coding gene comprises one or more coding regions of ABCA1, ABCA12, ABCA13, ABCA2, ABCA3, ABCA4, ABCA5, ABCC1, ABCC2, ABCC6, ABCC8, ABCC9,ACAN, ADAMTS13, ADCY10, ADGRV1, AGL, AGRN, AHDC1, ALK, ALMS1, AI.PK3. AI.S2. ANAPC1, ANK1, ANK2. ANK3, ANKRD11, ANKRD26. AFC, APC2, APOB. ARFGEF2, ARHGAP31, ARHGEF10. ARHGEF18, ARID1A, ARID1B, ARID2. ASH IL, ASPM, ASXL1,ASXL2, ASXL3, ATM,ATP7A,ATP7B, APR, ATRX. BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, RI.M. BPTF, BRCA1, BRCA2, BRD4, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E. CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD, CAMTAI, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP 152, CEP 164, CEP 250, CEP 290, CFAP43, CFAP44, CFAP65, CFTRABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1, CNTNAP1. COL11A1. COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, C0I.2A 1. C0L3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1, CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1, DIP2B. DLC1, DMD. DMXI.2. DNAH1, DNAH11, DNAH17. DNAH2. DNAH5, DNAH7, DNAH8. DNAH9. DNMBP. DNMT1, DOCK2. DOCKS, DOCK6, DOCK7, DOCK8, DSCAM, DSP, DST, DUOX2, DYNC1H1, DYNC2H1, DYSF, EIF2AK4, EP 300, EPG5, ERCC6, ERCC6L2, EXPH5, EYS, F5, F8, FANCA, FANCD2, FANCM, FAT1, FAT4. FBN1, FBN2, FLG, FLG2, FLNA, FLNB, FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2. FSIP2. FXN. FYCO1, GLI2. GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1, HERC2, HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R, IGF2R, IGSF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A, KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220, KIF14, KIF1A, KIF1B, KIF21A, KIF26B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1, LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMC3, LCT, LMNA, LOXHD1, LPA, LRBA, LRP1, LRP2, LRP4. LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD, MAGI2, MAP IB, MAP3K1, MAPK8IP3, MAPKBP1. MAST1, MBD5, MCM3AP, MED 12, MED12L, MED 13, MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MYH1LMYH14, MYH2, MYH3, MYH6, MYH7, MYH7B, MYH8, MYH9, MYLK, MYOMA, MYO18B, MYO3A, MYO5A, MYO5B, MYO7A, MYO9A, NALCN, NBAS, NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF. NEXMIF, NF1, NFASC, NHS, NIN, NIPBL, NLRP1, N0TCH1, N0TCH2, N0TCH3, NPHP4, NRXN1, NRXN3, NSD1, NSD2, NUP155, NUP188, NUP205, OBSCN, OBSL1, OTOF, OTOG, OTOGL, PARD3, PBRM1, PCDH15, PCLO, PCNT, PHIP, PI4KA, PIEZO1, PIEZO2, PIK3C2A, PIKFYVE, PKD1, PKD1L1, PKHD1, PLCE1, PLEC, PLEKHG2, PNPLA6, POGZ, POLA1, POLE, POLR1A. POLR2A, POLR3A. PRG4. PRKDC. PRPF8, PRR12. PRX, PTCHI, PTPN23, PTPRF, PTPRJ, PTPRQ, PXDN, QRICH2, RAB3GAP2, RAH, RALGAPA1, RANBP2, RB1CC1, RELN, RERE, REV3L, RIC1, RTMS1, RTMS2, RNF213, ROBO1, ROBO2, ROBO3, ROS1, RP1, RP1L1, RTTN, RUSC2, RYR1, RYR2, SACS, SAMD9, SAMD9L. SBF2, SCAPER, SCN10A, SCN11A, SCN1A. SCN2A, SCN3A. SCN4A, SCN5A, SCN8A, SCN9A, SETBP1, SETD1A, SETD1B, SETD2, SETD5, SETX, SHANK2, SHANK3, SHROOM4, SI, SIPA1L3, SLIT2, SLX4, SMARCA2, SMARCA4, SMC HD 1, SNRNP200, SON, SPEF2, SPEG, SPG11, SPTA1, SPTAN1, SPTB, SPTBN2, SPTBN4, SRCAP, STRC. SVIL, SYNE1, SYNGAP1, SYNJ1, SZT2, TAF1, TANC2, TCF20, TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX! 4. TEX15, TG, TH0C2, TMEM94, TNC, TNIK, TNR, TNRC6B, TNXB, TOGARAM 1, TONSL, TRIO, TRIOBP, TRIP11, TRIP12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, TIN, TUBGCP6, UBR1, UNC80, USH2A, USP9X, VCAN, VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62. WDR81, WNK1. WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292, ZNF335, ZNF407, ZNF462, or ZNF469.

8. The nucleic acid molecule of any one of Claims 3 - 7, wherein the one or more mutations in one or more exons contribute to pathogenesis in one or more cells.

9. The nucleic acid molecule of Claim 8, wherein the one or more cells are in a subject.

10. The nucleic acid molecule of Claim 1, wherein a 5’ portion of the targeted endogenous pre- mRNA is trans-spliced with the exogenous RNA.

11. The nucleic acid molecule of Claim 2, wherein a 3’ portion of the targeted endogenous pre- mRNA is trans-spliced with the exogenous RNA.

12. The nucleic acid molecule of Claim 1 or Claim 2, wherein the RNA targeting motif binds to the targeted endogenous pre-mRNA.

13. The nucleic acid molecule of Claim 12, wherein the RNA targeting motif comprises an antisense oligonucleotide.

14. The nucleic acid molecule of Claim 13, wherein the antisense oligonucleotide comprises about

15 nucleotides to about 50 nucleotides.

15. The nucleic acid molecule of Claim 1, wherein the RNA targeting motif binds to the 3’ end of the targeted endogenous pre-mRNA,

16. The nucleic acid molecule of Claim 2, wherein the RNA targeting motifs binds to the 5’ end of the targeted endogenous pre-mRNA.

17. The nucleic acid molecule of Claim 3, wherein the RNA targeting motif is specific for an endogenous pre-mRNA having one or more mutations.

18. The nucleic acid molecule of Claim 1, wherein the RNA targeting motif is directed to the intron immediately 3^? to the exon of the targeted endogenous pre-mRNA with which it is to be spliced.

19. The nucleic acid molecule of Claim 2, wherein the RNA targeting motif is directed to the intron immediately 5’ to the exon of the targeted endogenous pre-mRNA with which it is to be spliced.

20. The nucleic acid molecule of Claim 1, wherein the 3’ hemi intron is linked to the exogenous

RNA to be trans-spliced with the endogenous mRNA.

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

22. The nucleic acid molecule of Claim 1, wherein the 3’ hemi intron comprises (i) a 3’ splice region comprising a branch point, (ii) a polypyrimidine tract, and (iii) a 3’ splice acceptor site.

23. The nucleic acid molecule of Claim 2, wherein the 5' hemi intron is linked to the exogenous

RNA to be trans-spliced with the endogenous mRNA.

24. The nucleic acid molecule of Claim 2, wherein the 5’ hemi intron is recognized by nuclear splicing components in a host cell.

25. The nucleic acid molecule of Claim 2, wherein the 5’ hemi intron comprises a 5’ splice site.

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

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

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

28. A viral vector, comprising: the nucleic acid molecule of any one of Claims 1 - 27.

29. A pharmaceutical formulation, comprising: the vector of Claim 28; and one or more pharmaceutically acceptable carriers.

30. A method of generating a chimeric RNA molecule, the method comprising: contacting a targeted endogenous pre-mRNA in one or more cells with the nucleic acid molecule of any one of Claim 1 - 27 or the vector of Claim 28. wherein the resulting chimeric RNA molecule comprises a trans-spliced nucleic acid sequence.

31. The method of Claim 30, wherein the one or more cells are in a subject.

32. The method of Claim 31, wherein the subject has been diagnosed with or is suspected of having a genetic disease or a genetic disorder.

33. A method of treating a genetic disease or a genetic disorder, the method comprising: generating a chimeric RNA molecule in one or more cells by administering to a subject in need thereof a therapeutically effective amount of the vector of Claim 28 or the pharmaceutical formulation of Claim 29; wherein the resulting chimeric RNA molecule comprises a trans-spliced nucleic acid sequence; and wherein the resulting chimeric RNA molecule restores one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation.

34. The method of Claim 33, wherein restoring one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation comprises restoring the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

35. The method of Claim 33. wherein the therapeutically effective amount of the vector comprises about l x 10¹⁰ vg to about 2 x 10¹⁴ vg.

36. The method of Claim 33, further comprising administering to the subject a therapeutically effective amount of one or more therapeutic agents.

37. The method of Claim 33, further comprising administering to the subject a therapeutically effective amount of one or more immune modulators.

38. The method of any one of Claims 33 - 37, further comprising repeating the administering step.

39. The method of any one of Claims 33 - 38, further comprising monitoring the subject for adverse effects.

40. The method of any one of Claims 33 - 39, wherein restoring one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysfunction comprises (i) correcting cell starvation in one or more cells; (ii) correcting, preventing, reducing, and/or ameliorating autophagy⁷; (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi-systemic manifestations of the genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof.