Compositions and methods for manipulating transcriptomes

Abstract

Disclosed herein are CRISPR-free compositions and methods for producing chimeric RNA molecules via trans-splicing, and methods for treating and / or preventing genetic diseases or disorders using the chimeric RNA molecules produced via trans-splicing. Disclosed herein are nucleic acid molecules comprising exogenous RNA to be trans-spliced ​​into a target endogenous pre-mRNA, a 5' hemi-intron, one or more RNA targeting motifs, and one or more RNA structures.

Description

[Technical Field]

[0001] I. Cross-references to related applications This application claims priority under U.S. Provisional Application No. 63 / 493,696, filed on 31 March 2023, which is incorporated herein by reference in its entirety.

[0002] II. Statement on Federal Funding This invention was made with government support under federal grant number NS099371, awarded by the National Institute of Neurological Disorders and Stroke (NIH / NINDS). The federal government has certain rights to this invention.

[0003] III. References to Sequence Lists The sequence listing, created on March 29, 2024, and submitted on March 29, 2024, as an XML file named "23-2078-WO_Sequence Listing" with a size of 95 kilobytes, is incorporated herein by reference in accordance with 37 C. FR §1.52(e)(5). [Background technology]

[0004] IV. Background In eukaryotes, chromosomal DNA is transcribed into a precursor RNA message (pre-mRNA) containing protein-coding regions (exons) and intervening non-protein-coding regions (introns). Before processing, these pre-mRNA molecules lack the primed sequences for ribosome translation in order to retain the non-coding intron sequences. Therefore, before nuclear export, the exons of the pre-mRNA transcript are ligated via a cellular mechanism known as splicing. This mechanism is characterized by a double transesterification mediated by a large multiribonucleoprotein structure called a spliceosome. In the first transesterification, the branching point sequence of the intervening intron attacks the 5' splice site, forming a lariat structure. This reaction frees the 5' splice site, which then attacks the 3' splice site, thereby removing the intervening intron and ligating the adjacent exons. Once all intron sequences are removed, the precursor message matures into a translation-ready mature RNA transcript, which is transported to ribosomes where it is decoded to produce cellular proteins.

[0005] In mammalian cells, mutations in the transcriptionally active region of chromosomal DNA produce pre-mRNAs that carry the same mutation. If the mutation is located in a non-coding region, pre-mRNA processing may be altered or abolished. If the mutation is located in the exon region of the pre-mRNA, the mutation is carried over into the mature mRNA sequence. These mutations can either contribute to the complete inhibition of protein translation of the encoded protein (nonsense mutations) or alter the primary structure of the encoded protein in a counterproductive manner (missense mutations). In summary, these genetically encoded mutations can function to contribute to the pathogenesis of eukaryotic organisms.

[0006] The field of gene therapy aims to correct such genetic abnormalities by adoptive gene introduction of recombinant nucleic acids that have sequences capable of producing the protein products of mutated genes. This strategy, traditionally called "classical gene therapy," has proven to be a safe and effective strategy for phenotypic correction of genetic disorders, and several gene therapy products are available on the market.

[0007] However, current approaches to gene therapy are limited by the toxicity resulting from transcript overexpression, the inability to deliver transcripts larger than the packaging capacity of AAV vectors (i.e., the standard vector for gene therapy has a packaging capacity of approximately 4.7 KB), the inability to modify and replace large stretches of disease-causing mRNA, and the inability to modify and replace autosomal dominant inheritance with a single transcript and / or approach.

[0008] Therefore, there is an urgent need for minimally invasive curative treatments to address the underlying causes and complications of the symptoms associated with these various genetic disorders and disorders. Accordingly, this disclosure provides compositions and methods for generating chimeric RNA molecules via trans-splicing (and without CRISPR), which can be used alone or in combination with other procedures, to treat and / or prevent genetic disorders and / or disorders. [Brief explanation of the drawing]

[0009] V. Brief explanation of the drawing [Figure 1]Figure 1 (left) shows the mechanism of 5' trans-splicing, and Figure 1 (right) shows the mechanism of 3' trans-splicing. The schematic diagram on the left first shows a transfected construct to demonstrate trans-splicing for 5' substitution. When an RNA species is spliced ​​in cis, a stop codon in the first half of the open reading frame blocks translation. If the trans-splicing RNA successfully edits the RNA, the open reading frame of EGFP is restored and fluorescence expression is restored. The schematic diagram on the right first shows a transfected construct to demonstrate trans-splicing for 3' substitution. When an RNA species is spliced ​​in cis, a stop codon in the second half of the open reading frame blocks translation. If the trans-splicing RNA successfully edits the RNA, the open reading frame of EGFP is restored and fluorescence expression is restored. GRAFT is guide RNA auxiliary fragment trans-splicing.

[0010] [Figure 2A] Figures 2A and 2B show panels of 5'-splicing motifs. These are flow cytometry data from simultaneous transfection experiments of candidate 5'-trans-splicing molecules. In Figure 2A, the percentage of cells expressing green fluorescence upon delivery of candidate trans-splicing RNAs was measured. RNA structures 1-11 are plotted against the x-axis, and the percentage of GFP-positive cells is plotted on the y-axis. In Figure 2B, the average fluorescence intensity of cells upon delivery of candidate trans-splicing RNAs was measured. RNA structures 1-11 are plotted against the x-axis, and the average fluorescence intensity is plotted on the y-axis. [Figure 2B]Figures 2A and 2B show panels of 5'-splicing motifs. These are flow cytometry data from simultaneous transfection experiments of candidate 5'-trans-splicing molecules. In Figure 2A, the percentage of cells expressing green fluorescence upon delivery of candidate trans-splicing RNAs was measured. RNA structures 1-11 are plotted against the x-axis, and the percentage of GFP-positive cells is plotted on the y-axis. In Figure 2B, the average fluorescence intensity of cells upon delivery of candidate trans-splicing RNAs was measured. RNA structures 1-11 are plotted against the x-axis, and the average fluorescence intensity is plotted on the y-axis.

[0011] [Figure 3A] Figures 3A–3D show panels of 3' trans-splicing motifs. Flow cytometry data from simultaneous transfection experiments of candidate 3' trans-splicing molecules. In Figure 3A, the percentage of cells expressing green fluorescence upon delivery of candidate trans-splicing RNAs was measured. RNA structures 1–11 are plotted against the x-axis, and the percentage of GFP-positive cells is plotted against the y-axis. In Figure 3B, the average fluorescence intensity of cells upon delivery of candidate trans-splicing RNAs was measured. RNA structures 1–11 are plotted against the x-axis, and the average fluorescence intensity is plotted against the y-axis. The data in Figures 3C and 3D were measured in the same manner for different target introns. [Figure 3B]Figures 3A–3D show panels of 3' trans-splicing motifs. Flow cytometry data from simultaneous transfection experiments of candidate 3' trans-splicing molecules. In Figure 3A, the percentage of cells expressing green fluorescence upon delivery of candidate trans-splicing RNAs was measured. RNA structures 1–11 are plotted against the x-axis, and the percentage of GFP-positive cells is plotted against the y-axis. In Figure 3B, the average fluorescence intensity of cells upon delivery of candidate trans-splicing RNAs was measured. RNA structures 1–11 are plotted against the x-axis, and the average fluorescence intensity is plotted against the y-axis. The data in Figures 3C and 3D were measured in the same manner for different target introns. [Figure 3C] Figures 3A–3D show panels of 3' trans-splicing motifs. Flow cytometry data from simultaneous transfection experiments of candidate 3' trans-splicing molecules. In Figure 3A, the percentage of cells expressing green fluorescence upon delivery of candidate trans-splicing RNAs was measured. RNA structures 1–11 are plotted against the x-axis, and the percentage of GFP-positive cells is plotted against the y-axis. In Figure 3B, the average fluorescence intensity of cells upon delivery of candidate trans-splicing RNAs was measured. RNA structures 1–11 are plotted against the x-axis, and the average fluorescence intensity is plotted against the y-axis. The data in Figures 3C and 3D were measured in the same manner for different target introns. [Figure 3D]Figures 3A–3D show panels of 3' trans-splicing motifs. Flow cytometry data from simultaneous transfection experiments of candidate 3' trans-splicing molecules. In Figure 3A, the percentage of cells expressing green fluorescence upon delivery of candidate trans-splicing RNAs was measured. RNA structures 1–11 are plotted against the x-axis, and the percentage of GFP-positive cells is plotted against the y-axis. In Figure 3B, the average fluorescence intensity of cells upon delivery of candidate trans-splicing RNAs was measured. RNA structures 1–11 are plotted against the x-axis, and the average fluorescence intensity is plotted against the y-axis. The data in Figures 3C and 3D were measured in the same manner for different target introns.

[0012] [Figure 4A-B] Figures 4A and 4B show repeated validation of the top 3' trans-splicing RNA candidates. Flow cytometry data from simultaneous transfection experiments of 3' trans-splicing candidate molecules. In Figure 4A, the percentage of cells expressing green fluorescence after delivery of the trans-splicing RNA candidate was measured. RNA structures are plotted on the x-axis, and the percentage of GFP-positive cells is plotted on the y-axis. In Figure 4B, the average fluorescence intensity of cells after delivery of the trans-splicing RNA candidate was measured. RNA structures are plotted on the x-axis, and the average fluorescence intensity is plotted on the y-axis.

[0013] [Figure 5A-B]Figures 5A and 5B show repeated validation of top 3' trans-splicing RNA candidates for a new target. Flow cytometry data were measured from simultaneous transfection experiments of 3' trans-splicing candidate molecules. In Figure 5A, the percentage of cells expressing green fluorescence after delivery of the trans-splicing RNA candidate was measured. RNA structures are plotted on the x-axis, and the percentage of GFP-positive cells is plotted on the y-axis. In Figure 5B, the average fluorescence intensity of cells after delivery of the trans-splicing RNA candidate was measured. RNA structures are plotted on the x-axis, and the average fluorescence intensity is plotted on the y-axis.

[0014] [Figure 6A] Figures 6A to 6G provide plasmid maps of the constructs used in Example 1 and / or the constructs disclosed herein. Figure 6A shows an exemplary 3' substitution construct (null). Figure 6B shows the 3' substitution construct of RYR2(3-GRAFT-RYR2), and Figure 6C shows the 5' substitution construct of RYR2(5-GRAFT-RYR2). Figure 6D shows the 3' substitution construct of LMNA(3-GRAFT-LMNA), and Figure 6E shows the 3' substitution construct of FXN(3-GRAFT-FXN). Figure 6F shows the split GFP reporter construct for LMNA, and Figure 6G shows the split GFP reporter construct for RYR2. [Figure 6B] Figures 6A to 6G provide plasmid maps of the constructs used in Example 1 and / or the constructs disclosed herein. Figure 6A shows an exemplary 3' substitution construct (null). Figure 6B shows the 3' substitution construct of RYR2(3-GRAFT-RYR2), and Figure 6C shows the 5' substitution construct of RYR2(5-GRAFT-RYR2). Figure 6D shows the 3' substitution construct of LMNA(3-GRAFT-LMNA), and Figure 6E shows the 3' substitution construct of FXN(3-GRAFT-FXN). Figure 6F shows the split GFP reporter construct for LMNA, and Figure 6G shows the split GFP reporter construct for RYR2. [Figure 6C]Figures 6A to 6G provide plasmid maps of the constructs used in Example 1 and / or the constructs disclosed herein. Figure 6A shows an exemplary 3' substitution construct (null). Figure 6B shows the 3' substitution construct of RYR2(3-GRAFT-RYR2), and Figure 6C shows the 5' substitution construct of RYR2(5-GRAFT-RYR2). Figure 6D shows the 3' substitution construct of LMNA(3-GRAFT-LMNA), and Figure 6E shows the 3' substitution construct of FXN(3-GRAFT-FXN). Figure 6F shows the split GFP reporter construct for LMNA, and Figure 6G shows the split GFP reporter construct for RYR2. [Figure 6D] Figures 6A to 6G provide plasmid maps of the constructs used in Example 1 and / or the constructs disclosed herein. Figure 6A shows an exemplary 3' substitution construct (null). Figure 6B shows the 3' substitution construct of RYR2(3-GRAFT-RYR2), and Figure 6C shows the 5' substitution construct of RYR2(5-GRAFT-RYR2). Figure 6D shows the 3' substitution construct of LMNA(3-GRAFT-LMNA), and Figure 6E shows the 3' substitution construct of FXN(3-GRAFT-FXN). Figure 6F shows the split GFP reporter construct for LMNA, and Figure 6G shows the split GFP reporter construct for RYR2. [Figure 6E] Figures 6A to 6G provide plasmid maps of the constructs used in Example 1 and / or the constructs disclosed herein. Figure 6A shows an exemplary 3' substitution construct (null). Figure 6B shows the 3' substitution construct of RYR2(3-GRAFT-RYR2), and Figure 6C shows the 5' substitution construct of RYR2(5-GRAFT-RYR2). Figure 6D shows the 3' substitution construct of LMNA(3-GRAFT-LMNA), and Figure 6E shows the 3' substitution construct of FXN(3-GRAFT-FXN). Figure 6F shows the split GFP reporter construct for LMNA, and Figure 6G shows the split GFP reporter construct for RYR2. [Figure 6F]Figures 6A to 6G provide plasmid maps of the constructs used in Example 1 and / or the constructs disclosed herein. Figure 6A shows an exemplary 3' substitution construct (null). Figure 6B shows the 3' substitution construct of RYR2(3-GRAFT-RYR2), and Figure 6C shows the 5' substitution construct of RYR2(5-GRAFT-RYR2). Figure 6D shows the 3' substitution construct of LMNA(3-GRAFT-LMNA), and Figure 6E shows the 3' substitution construct of FXN(3-GRAFT-FXN). Figure 6F shows the split GFP reporter construct for LMNA, and Figure 6G shows the split GFP reporter construct for RYR2. [Figure 6G] Figures 6A to 6G provide plasmid maps of the constructs used in Example 1 and / or the constructs disclosed herein. Figure 6A shows an exemplary 3' substitution construct (null). Figure 6B shows the 3' substitution construct of RYR2(3-GRAFT-RYR2), and Figure 6C shows the 5' substitution construct of RYR2(5-GRAFT-RYR2). Figure 6D shows the 3' substitution construct of LMNA(3-GRAFT-LMNA), and Figure 6E shows the 3' substitution construct of FXN(3-GRAFT-FXN). Figure 6F shows the split GFP reporter construct for LMNA, and Figure 6G shows the split GFP reporter construct for RYR2.

[0015] [Figure 7A] Figure 7A shows the 5' substitution construct of DMD in Example 2. [Figure 7B] Figure 7B shows the 3' substitution construct of DMD in Example 2. [Overview of the project]

[0016] VI. Brief Summary Disclosed herein are nucleic acid molecules comprising an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron, one or more RNA targeting motifs, and one or more RNA structures.

[0017] Disclosed herein are nucleic acid molecules comprising one or more RNA structures, one or more RNA targeting motifs, a 3' hemiintron ligated to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​to a target endogenous pre-mRNA.

[0018] Disclosed herein are non-viral or viral vectors containing nucleic acid molecules comprising exogenous RNA, a 5' hemyintron, one or more RNA targeting motifs, and one or more RNA structures, which are to be transspliced ​​into an endogenous pre-mRNA to be targeted.

[0019] Disclosed herein are non-viral or viral vectors containing nucleic acid molecules comprising one or more RNA structures, one or more RNA targeting motifs, a 3' hemyintron ligated to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​to a target endogenous pre-mRNA.

[0020] Disclosed herein are pharmaceutical formulations comprising a non-viral vector or a viral vector containing a nucleic acid molecule comprising an exogenous RNA, a 5' hemyintron, one or more RNA targeting motifs, and one or more RNA structures, which will be transspliced ​​into an endogenous pre-mRNA to be targeted.

[0021] Disclosed herein are pharmaceutical formulations comprising a non-viral vector or a viral vector containing a nucleic acid molecule comprising one or more RNA structures, one or more RNA targeting motifs, a 3' hemyintron ligated to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​to a target endogenous pre-mRNA.

[0022] Disclosed herein are methods for treating genetic disorders or disorders, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a disclosed non-viral vector or a disclosed viral vector or a pharmaceutical formulation thereof to a subject in need thereof, the resulting chimeric RNA molecule being able to restore one or more characteristics of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation.

[0023] Disclosed herein are methods for treating genetic disorders or disorders, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a disclosed non-viral vector or a disclosed viral vector or a pharmaceutical formulation thereof to a subject in need thereof, the resulting chimeric RNA molecule being able to restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme.

[0024] Disclosed herein are methods for treating a genetic disorder or impairment, the methods comprising generating a chimeric RNA molecule in one or more cells by administering to a subject in need of such treatment a therapeutically effective amount of a non-viral vector, viral vector, AAV particles, or a pharmaceutical formulation thereof, comprising (i) one or more 5' substitution constructs, (ii) one or more 3' substitution constructs, or (iii) one or more 5' substitution constructs and / or one or more 3' substitution constructs, the resulting chimeric RNA molecule can restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme.

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

[0026] Disclosed herein are methods for inhibiting and / or minimizing disease progression, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of the disclosed non-viral vector or the disclosed viral vector or a pharmaceutical formulation thereof to a subject in need thereof, the resulting chimeric RNA molecule being able to restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme.

[0027] Disclosed herein are methods for inhibiting and / or minimizing disease progression, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral vector, viral vector, AAV particles, or a pharmaceutical formulation thereof to a subject requiring such action, comprising (i) one or more 5' substitution constructs, (ii) one or more 3' substitution constructs, or (iii) one or more 5' substitution constructs and / or one or more 3' substitution constructs, the resulting chimeric RNA molecule can restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme. [Modes for carrying out the invention]

[0028] V II. Detailed explanation This disclosure describes formulations, compound compositions, kits, capsules, containers, and / or methods thereof. It should be understood that aspects of the present invention are not limited to specific synthesis methods or specific reagents unless otherwise specified, and are therefore naturally subject to change. It should also be understood that the terms used herein are for the purpose of describing specific aspects only and are not intended to limit them. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention, but the methods and materials described herein are illustrative.

[0029] All publications referenced herein are incorporated herein by reference to disclose and explain the relevant methods and / or materials from which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an acknowledgment that the present invention has no prior rights to such publications by prior invention.

[0030] A.Definition Before the compounds, compositions, articles, systems, apparatus, and / or methods of the present invention are disclosed and described, it should be understood that, unless otherwise specified, they are not limited to specific synthesis methods or specific reagents, and are therefore naturally subject to change. It should also be understood that the terms used herein are intended to describe only specific embodiments and are not intended to limit them. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention, but exemplary methods and materials are described herein.

[0031] This disclosure illustrates the concept of the present invention with reference to specific examples. However, the intent is to cover all modifications, equivalents, and substitutions of the concept of the present invention that are consistent with this disclosure.

[0032] As used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless otherwise explicitly indicated by the context.

[0033] The phrase "consisting essentially of" limits the scope of the claim to the enumerated components in the composition or the enumerated steps in the method, and does not substantially affect the basic and novel features or characteristics of the composition or method described in the claim. The phrase "consisting of" excludes any components, steps, or elements not described in the claims. The phrase "comprising" is synonymous with "including," "containing," or "characterized by," and is comprehensive or open-ended. "Comprising" does not exclude additional components or steps not enumerated.

[0034] Where used herein, when referring to any number, the term "approximately" means a value that falls within ±10% of the stated value.

[0035] A range may be expressed herein as "about" one particular value and / or "about" another particular value. Where such a range is expressed, further embodiments include from one particular value and / or other particular values. Similarly, where a value is expressed as an approximation using the preceding term "about," it will be understood that a particular value forms further embodiments. It will be further understood that each endpoint of a range is important both in relation to the other endpoint and independently of the other endpoint. It will also be understood that there are several values ​​disclosed herein, and each value is disclosed herein not only as the value itself but also "about" its particular value. For example, if the value "10" is disclosed, "about 10" is also disclosed. It will also be understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0036] References in this specification and the final claims to parts by weight of a particular element or component in a composition indicate a weight relationship between that element or component and any other element or component in the composition or article in which parts by weight are represented. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight of component Y, X and Y exist in a weight ratio of 2:5, and such a ratio exists whether or not additional components are present in the compound.

[0037] As used herein, the terms “optional” or “optionally” mean that the events or circumstances described therein may or may not occur, and that the description includes both cases in which such events or circumstances occur and cases in which they do not occur. In one embodiment, the disclosed method may optionally include one or more additional steps, such as repeating or modifying the administration step.

[0038] As used herein, “isolated” refers to nucleic acid molecules or nucleic acid sequences that are substantially separated from other biological components in the cells or tissues of the organism in which the component resides, such as other cells, chromosomes, and extrachromosomal DNA and RNA, as well as proteins, and are produced separately from or purified from other biological components. “Isolated” nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in host cells, as well as chemically synthesized nucleic acids and proteins.

[0039] As used herein, the term “subject” refers to the target of administration, e.g., humans. 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., mice, rabbits, rats, guinea pigs, fruit flies, etc.). Thus, the subjects of the methods disclosed herein may be vertebrates such as mammals, fish, birds, reptiles, or amphibians. Alternatively, the subjects of the methods disclosed herein may be humans, non-human primates, horses, pigs, rabbits, dogs, sheep, goats, cattle, cats, guinea pigs, or rodents. The term is not intended to indicate a specific age or sex, and is therefore intended to encompass adult and child subjects, as well as fetuses (whether male or female). In one embodiment, the subject may be a human patient. In one embodiment, the subject may have a disease or disability, be suspected of having a disease or disability, or be at risk of developing a disease or disability (e.g., a genetic disorder or disability). In another embodiment, the subject may be treatment-inexperienced.

[0040] As used herein, “regulatory element” may refer to promoters, enhancers, internal ribosome entry sites (IRESs), and other expression regulatory elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences). Regulatory elements may include those that direct the constitutive expression of nucleotide sequences in many types of host cells, and those that direct the expression of nucleotide sequences only in specific host cells (e.g., tissue-specific regulatory elements).

[0041] As used herein, the term “diagnosed” means that a person skilled in the art, for example, a physician, has been subjected to examination 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 combinations thereof, or by one or more of the disclosed methods. For example, “diagnosed with a disease or disorder” means that a person skilled in the art, for example, a physician, has been subjected to examination and found to have a condition (such as a genetic disorder or disorder) that can be treated by one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or combinations thereof, or by one or more of the disclosed methods. For example, “suspected of having a disease or disorder” may mean that a person skilled in the art, for example, a physician, has been subjected to examination and found to have a condition (such as a genetic disorder or disorder) that can be treated by one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or combinations thereof, or by one or more of the disclosed methods. In one embodiment, the examination may be a physical examination and may include various tests (e.g., blood tests, gene typing, biopsies, etc.) and assays (e.g., enzyme assays), or a combination thereof.

[0042] "Patient" refers to an individual suffering from a disease or disorder (e.g., a genetic disorder or disorder). In one aspect, patient may refer to an individual who has been diagnosed with or is suspected of having a disease or disorder. In another aspect, patient may refer to an individual who has been diagnosed with or is suspected of having a disease or disorder and is seeking or receiving treatment for the disease or disorder.

[0043] Where used herein, phrases such as "identified as requiring treatment for a disease or disorder" refer to the selection of a subject based on the need for treatment for a disease or disorder. For example, a subject may be identified as requiring treatment for a disease or disorder (e.g., a genetic disorder or disorder) based on a prior diagnosis by a person skilled in the art, and subsequently be subjected to treatment for the genetic disorder or disorder. In one embodiment, the identification may be performed by a person other than the one performing the diagnosis. In one embodiment, the administration may be performed by the person performing the diagnosis.

[0044] As used herein, “inhibit,” “inhibiting,” and “inhibition” mean reducing or decreasing activity, level, response, symptom, severity, disease, or other biological parameter. This may include, but is not limited to, the complete disappearance of activity, level, response, symptom, severity, disease, or other biological parameter. This may also include, for example, a 10% inhibition or reduction of activity, level, response, symptom, severity, disease, or other biological parameter compared to native or control levels (e.g., a subject without disease or disorder, such as a genetic disorder or disorder). Thus, in one embodiment, inhibition or reduction may be a reduction of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between, compared to native or control levels. In one embodiment, inhibition or reduction may be 10–20%, 20–30%, 30–40%, 40–50%, 50–60%, 60–70%, 70–80%, 80–90%, or 90–100% compared to native or control levels. In one embodiment, inhibition or reduction may be 0–25%, 25–50%, 50–75%, or 75–100% compared to native or control levels. In one embodiment, native or control levels may be pre-disease or pre-disability levels.

[0045] The terms “treat,” “treating,” or “treatment” include palliative treatment, i.e., treatment designed to alleviate symptoms rather than cure a disease, pathological symptom, or disorder; preventive treatment, i.e., treatment aimed at minimizing, or partially or completely inhibiting, the onset of an associated disease, pathological symptom, or disorder; and supportive treatment, i.e., treatment used to complement another specific treatment aimed at improving an associated disease, pathological symptom, or disorder. In one embodiment, the term encompasses any treatment of an object, including mammals (e.g., humans), including (i) preventing the development of an undesirable physiological change, disease, pathological symptom, or disorder in an object that may be predisposed to a disease but has not yet been diagnosed with the disease; (ii) inhibiting a physiological change, disease, pathological symptom, or disorder, i.e., halting its progression; or (iii) reducing a physiological change, disease, pathological symptom, or disorder, i.e., causing disease regression. For example, in one embodiment, treatment of a disease or disorder can reduce the severity of an established disease or disorder in a subject by 1% to 100% compared to a control (e.g., an individual without a genetic disorder or disorder). In one embodiment, treatment can refer to a reduction of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in the severity of a disease or disorder (e.g., a genetic disorder or disorder). For example, treatment of a disease or disorder can reduce one or more symptoms of a disease or disorder in a subject by 1% to 100% compared to a control (e.g., an individual without a genetic disorder or disorder). In one embodiment, treatment can refer to a reduction of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of one or more symptoms of an established disease or disorder. It is understood that treatment does not necessarily mean a cure, complete disappearance, or eradication of the disease or disorder.However, in one embodiment, treatment may refer to the cure, complete disappearance, or eradication of a disease or disorder.

[0046] As used herein, the terms “prevent,” “preventing,” and “prevention” mean preventing, avoiding, eliminating, stopping, or hindering something from happening, particularly through a preceding action. Where “reduce,” “inhibit,” or “prevent” are used herein, it is understood that the use of the other two terms is also expressly disclosed unless otherwise stated. In one embodiment, the intention is to prevent a disease or disorder having chromatin deregulation and / or chromatin dysregulation. The terms “prevent,” “preventing,” and “prevention” also refer to prophylactic or preventive measures to protect or prevent a given disease or disorder (e.g., a genetic disorder or disorder) or associated complications from progressing to those complications in an object (e.g., an individual) that does not have such a disorder or associated complications.

[0047] As used herein, the terms “administering” and “administration” refer to any method of delivering one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or combinations thereof. Such methods are well known to those skilled in the art and include: oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, intrauterine administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraotoral administration, otological administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including, but not limited to, injectable forms such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration may also include hepatic arterial administration or administration via the hepatic portal vein (HPV). The administration of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical compositions, disclosed therapeutic agents, disclosed immunomodulators, disclosed proteasome inhibitors, disclosed small molecules, disclosed endonucleases, disclosed oligonucleotides, and / or disclosed RNA therapeutic agents may include direct administration to the CNS or PNS. Administration may be continuous or intermittent. Administration may include a combination of one or more routes.

[0048] In one embodiment, a person skilled in the art can determine an effective dose, effective schedule, and effective route of administration for one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or combinations thereof, in order to treat or prevent a disease or disorder (e.g., a genetic disorder or disorder). In one embodiment, a person skilled in the art can also change, alter, or modify one aspect of the administration process in order to improve the effectiveness of one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or combinations thereof.

[0049] "To determine quantity" means both the absolute quantification of a particular analyte (e.g., mRNA sequences containing a particular tag) or the determination of the relative abundance of a particular analyte (e.g., the amount compared to mRNA sequences containing different tags). This phrase includes both direct and indirect measurements of abundance (e.g., quantifying individual mRNA transcripts, or using the amplification of mRNA sequences over a period of time under certain conditions as a substitute for quantifying individual transcripts) or both.

[0050] As used herein, “modifying a method” may include altering or changing one or more features or aspects of one or more steps in the disclosed method. For example, in one embodiment, the method may be modified by changing the amount of one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or combinations thereof administered to a subject, or by changing the frequency of administration of one or more of them to a subject, or by changing the duration of administration of one or more of them to a subject, or by substituting one or more of the disclosed components and / or reagents with similar or equivalent components and / or reagents. The same applies to all disclosed therapeutic agents, immunomodulators, immunosuppressants, proteosome inhibitors, etc.

[0051] As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions immediately before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. In one embodiment, the pharmaceutical carrier used may be solid, liquid, or gaseous. In one embodiment, examples of solid carriers include lactose, clay, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. In one embodiment, examples of liquid carriers may include sugar syrup, peanut oil, olive oil, and water. In one embodiment, examples of gaseous carriers may include carbon dioxide and nitrogen. Any convenient pharmaceutical medium may be used when preparing compositions of the disclosed oral dosage forms. For example, oral liquid formulations such as suspensions, elixirs, and solutions can be formed using water, glycols, oils, alcohols, flavorings, preservatives, and colorants. On the other hand, oral solid formulations such as powders, capsules, and tablets can be formed using carriers such as starch, sugars, microcrystalline cellulose, diluents, granulators, lubricants, binders, and disintegrants. For ease of administration, tablets and capsules are preferred oral dosing units in which solid pharmaceutical carriers are used. If necessary, tablets may be coated by standard aqueous or non-aqueous techniques. Adequate fluidity can be maintained, for example, by the use of coating materials (such as lecithin), maintaining the required particle size in the case of dispersions, and using surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of microbial action can be ensured by including various antimicrobial and antifungal agents such as parabens, chlorobutanol, phenol, and sorbic acid. It may also be desirable to include isotonic agents such as sugars and sodium chloride.Long-term absorption of injectable pharmaceutical forms can be achieved by including activators such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are prepared by forming a microcapsule matrix of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoester), and poly(anhydride). The drug release rate can be controlled depending on the drug-to-polymer ratio and the properties of the specific polymer used. Depot injectable formulations are also prepared by encapsulating the drug in liposomes or microemulsions that conform to body tissues. Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating a sterilizer in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use. Suitable inert carriers may include sugars such as lactose. Preferably, at least 95% (by weight) of the active ingredient particles have an effective particle size in the range of 0.01 to 10 micrometers.

[0052] As used herein, the term “excipient” refers to inert substances commonly used as diluents, vehicles, preservatives, binders, or stabilizers, and includes, but is not limited to, proteins (e.g., serum albumin), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylates), surfactants (e.g., SDS, polysorbates, nonionic surfactants), sugars (e.g., sucrose, maltose, trehalose), and polyols (e.g., mannitol, sorbitol). See also, for reference, Remington's Pharmaceutical Sciences, (1990), Mack Publishing Co., Easton, Pa., which is incorporated herein by reference in its entirety.

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

[0054] As used herein, the term “to bring into contact” means bringing one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or combinations thereof together with a target region or intended target region to exert an effect directly or indirectly on the intended target or targeted region. The target region may contain one or more cells, and in one embodiment, one or more cells may be present within the subject. The target region or intended target region may be one or more organs of the subject (e.g., lungs, heart, liver, kidneys, brain, etc.). In one embodiment, the target region or intended target region may be any cell or any organ infected with a disease or disorder (such as a genetic disorder or disorder). In one embodiment, the target region or intended target region may be any organ, tissue, or cell affected by a disease or disorder (such as a genetic disorder or disorder).

[0055] As used herein, “determining” may mean measuring or confirming the presence and severity of a disease or disorder, such as a genetic disorder or disorder. Methods and techniques used to determine the presence and / or severity of a disease or disorder are typically known in the medical field. For example, the art is familiar with methods for identifying and / or diagnosing the presence, severity, or both of a disease or disorder (e.g., a genetic disorder or disorder).

[0056] As used herein, “effective dose” and “effective amount” may mean, for example, an amount sufficient to achieve a desired outcome, such as the treatment and / or prevention of a disease or disorder (e.g., a genetic disorder or disorder) or a suspected disease or disorder. As used herein, the terms “effective dose” and “effective amount” may mean an amount sufficient to achieve a desired effect against an undesirable symptom (e.g., a disease or disorder). For example, “therapeutic effective dose” means an amount sufficient to achieve a desired therapeutic outcome or to have an effect on an undesirable symptom, but insufficient to cause generally harmful side effects. In one embodiment, “therapeutic effective dose” means an amount of a disclosed nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation that (i) treats a particular disease, condition, or disorder (e.g., a genetic disorder or disorder), (ii) reduces, improves, or eliminates one or more symptoms of a particular disease, condition, or disorder (e.g., a genetic disorder or disorder), or (iii) delays the onset of one or more symptoms of a particular disease, condition, or disorder (e.g., a genetic disorder or disorder) as described herein. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disorder being treated and its severity, the disclosed nucleic acid molecule, disclosed vector, disclosed pharmaceutical formulation used, the disclosed method of use, the patient's age, weight, overall health, sex, and diet, administration time, route of administration, elimination rate of the disclosed nucleic acid molecule, disclosed vector, or disclosed pharmaceutical formulation used, duration of treatment, drugs used in combination with or concurrently with the disclosed nucleic acid molecule, disclosed vector, or disclosed pharmaceutical formulation, and other similar factors well known in the medical field. For example, it is well within the art of the art to start the dose of the disclosed nucleic acid molecule, disclosed vector, or disclosed pharmaceutical formulation at a level lower than the level required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. If desired, an effective daily dose can be divided into multiple doses for the purpose of administration. As a result, a single dose of the disclosed nucleic acid molecule, disclosed vector, or disclosed pharmaceutical formulation may contain such an amount or a fraction of such a dose to constitute a daily dose.Dosage may be adjusted by the individual physician in the event of any contraindications. Dosage may vary and may be administered as one or more doses per day, and may be administered over a day or several days. Guidelines for appropriate dosages of a given class of medicines can be found in the literature. Furthermore, in various aspects, the formulation may be administered in a “preventive effective dose,” that is, a dose effective in preventing a disease or symptom, such as a disease or disorder caused by a deficient, incomplete, and / or variant protein or enzyme.

[0057] As used herein, “RNA therapeutic agents” may refer to the use of oligonucleotides for targeting RNA. RNA therapeutic agents may offer the possibility of uniquely targeting the exact nucleic acids involved in a particular disease with higher specificity, improved potency, and reduced toxicity. This may be particularly potent for genetic diseases where targeting RNA rather than proteins is most advantageous. In one embodiment, therapeutic RNA may include one or more expression sequences. As known in the Art, expression sequences may include RNAi, shRNA, mRNA, non-coding RNA (ncRNA), antisense (e.g., antisense RNA), miRNA, morpholino oligonucleotides, peptide nucleic acids (PNA) or ssDNA (having native and modified nucleotides, including but not limited to LNA, BNA, 2'-O-Me-RNA, 2'-MEO-RNA, 2'-F-RNA), or analogues or conjugates thereof. In one embodiment, the disclosed therapeutic RNA may comprise one or more long non-coding RNAs (lncRNAs), such as long intergenic non-coding RNAs (lincRNAs), pre-transcripts, pre-miRNAs, pre-mRNAs, competitive endogenous RNAs (ceRNAs), micronuclear RNAs (snRNAs), micronucleolar RNAs (snoRNAs), pseudogenes, rRNAs, or tRNAs. In one embodiment, the ncRNA may be a piwi-interacting RNA (piRNA), a primary miRNA (pri-miRNA), or an early miRNA (pre-miRNA). In one embodiment, the disclosed therapeutic RNA or RNA therapeutic agent may comprise an antisense oligonucleotide (ASO) that inhibits mRNA translation, an oligonucleotide that functions via the RNA interference (RNAi) pathway, an enzyme-like RNA molecule (ribozyme), an RNA oligonucleotide that binds to proteins and other cellular molecules, and an ASO that binds to mRNA and forms a structure recognized by RNase H, resulting in cleavage of the mRNA target. In one embodiment, the RNA therapeutic agent may include RNAi and ASO that inhibit mRNA translation.Generally speaking, as is known in the art, RNAi functions sequence-specifically and post-transcriptionally by activating ribonucleases, which, in conjunction with other enzymes and complexes, degrade the RNA after the original RNA target has been cleaved into smaller fragments. Antisense oligonucleotides, on the other hand, bind to target nucleic acids via Watson-Crick base pairing and inhibit or alter gene expression through steric hindrance, splicing changes, initiation of targeted degradation, or other events.

[0058] As used herein, “small molecule” may refer to any organic or inorganic material that is not a polymer. Small molecules exclude large polymers, such as large proteins (e.g., proteins with molecular weights greater than 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 greater than 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 molecular weights greater than 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000 or 10,000). In one embodiment, the “small molecule” may be, for example, a drug that has a low molecular weight and can therefore easily enter cells. In one embodiment, the small molecule may be used in conjunction with the composition disclosed in the disclosed method.

[0059] In one embodiment, the term “ex vivo” can generally refer to activity performed outside of an organism or subject, e.g., experiment, modification, differentiation, manipulation, and / or measurement performed within or on a living tissue in an artificial environment outside of an organism. In one embodiment, ex vivo experiments, ex vivo modifications, ex vivo differentiation, ex vivo manipulations, and / or ex vivo measurements can be performed with minimal alteration of natural conditions. In one embodiment, “ex vivo” may include living cells, tissues, or organs (e.g., cells requiring trans-splicing of one or more protein-coding genes) taken from a subject or donor subject requiring it. These are cultured and / or maintained and / or perfused in laboratory equipment, usually under sterile conditions, and typically for a limited period (e.g., several 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 beyond, depending on the circumstances and / or desired characteristics). In one embodiment, tissues, cells, or organs can be collected, frozen, and then thawed for ex vivo treatment.

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

[0061] As used herein, “peptide,” “polypeptide,” and “protein” are interchangeable and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein sequence. The term “peptide” can refer to short chains of amino acids, including, for example, native peptides, recombinant peptides, synthetic peptides, or any combination thereof. Proteins and peptides may include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins.

[0062] As used herein, “nucleic acid,” “oligonucleotide,” or “polynucleotide” means at least two nucleotides covalently linked to one another. A single-stranded description can also define a complementary strand sequence. Thus, a nucleic acid can encompass the complementary strand of a single-stranded sequence as illustrated. Many variants of a nucleic acid can be used for the same purposes as a given nucleic acid. Thus, a nucleic acid can encompass substantially identical nucleic acids and their complementary strands. A single strand can provide a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid can encompass probes that hybridize under stringent hybridization conditions. A nucleic acid can be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA, both genomic DNA and cDNA, RNA, or hybrids, where nucleic acids may include combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or recombinant methods. Also, as used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid construct,” “nucleotide sequence,” and “polynucleotide” may refer to RNA or DNA that is linear or branched, single-stranded or double-stranded, or a hybrid thereof. This term may encompass RNA / DNA hybrids. When dsRNA is produced synthetically, less common bases such as inosine, 5-methylcytosine, 6-methyladenine, and hypoxanthine may also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides containing uridine and cytidine C-5 propine analogs have been shown to bind to RNA with high affinity and act as potent antisense inhibitors of gene expression. Other modifications are also possible, such as modifications to the phosphodiester backbone or to the 2'-hydroxyl group in the ribose sugar group of RNA.As used herein, “synthetic” nucleic acids or polynucleotides refer to nucleic acids or polynucleotides that are not found in nature but are constructed by human hands and are therefore not natural products.

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

[0064] A “fragment” or “part” of a nucleotide sequence can be understood to mean a nucleotide sequence that is shorter in length than a reference nucleic acid or nucleotide sequence (e.g., a reduction of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or more) and contains, essentially consists of, or comprises a sequence of consecutive nucleotides that are identical or nearly identical to the reference nucleic acid or nucleotide sequence (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). Such nucleic acid fragments or portions according to this disclosure may, where appropriate, be contained within a larger polynucleotide of which they are components. In one embodiment, a nucleotide sequence or a fragment or portion of a nucleic acid sequence may include a sequence encoding an exon having one or more mutations.

[0065] A "fragment" or "part" of an amino acid sequence is an amino acid sequence that is shortened in length compared to a reference amino acid sequence (for example, 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), and is shorter than the reference amino acid sequence. It can be understood that this means an amino acid sequence containing, essentially being, or consisting of an amino acid sequence of identical or nearly 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) consecutive amino acid sequences. Such amino acid fragments or portions according to this disclosure may, where appropriate, be included in a larger amino acid sequence of which they are components.

[0066] The terms “heterogeneous” or “recombinant” nucleotide or amino acid sequence used interchangeably herein may refer to a nucleotide or amino acid sequence that is not naturally associated with the host cell into which it is introduced, and this includes multiple copies of a naturally occurring nucleotide or amino acid sequence that are not naturally occurring.

[0067] As used herein, the term “endogenous” may refer to genes, proteins, compounds, or activities that are normally present in a host cell (e.g., pre-mRNA). As used herein, “exogenous” nucleic acid molecules, constructs, or sequences (e.g., RNA sequences to be trans-spliced) may refer to nucleic acid molecules or parts thereof that are not specific to a host cell but may be homologous to a nucleic acid molecule or part thereof derived from a host cell.

[0068] Different nucleic acids or proteins that exhibit homology can be called “homologous.” The term homologous includes homologous sequences from the same and other species, as well as orthologous sequences from the same and other species. “Homologous” refers to the level of similarity between two or more nucleic acids and / or amino acid sequences in terms of a percentage of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties between different nucleic acids or proteins. Thus, the disclosed compositions and disclosed methods may include homologous to the disclosed nucleotide sequences and / or disclosed polypeptide sequences.

[0069] As used herein, “orthologus” may refer to homologous nucleotide and / or amino acid sequences of different species arising from a common ancestral gene during speciation. Homologous counterparts of the disclosed nucleotide sequences or disclosed polypeptides may have substantial sequence identity with the disclosed nucleotide sequences or disclosed polypeptides (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%).

[0070] As used herein, “complementary strand” or “complementary” means that nucleic acids may represent Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule. “Complementarity” refers to a property shared between two nucleic acid sequences such that when the two nucleic acid sequences are aligned antiparallel to each other, the nucleotide bases at each position are complementary.

[0071] As used herein, “promoter” or “multiple promoters” are known in the art. Various promoter elements can be used depending on the desired level and tissue-specific expression. Promoters can be tissue-specific or ubiquitous, and can be constitutive or inducible, depending on the desired pattern of gene expression. Promoters can be native (endogenous) or exogenous (exogenous), and can be natural or synthetic sequences. Exogenous or exogenous means that the transcription initiation region is not present in the wild-type host into which it is introduced.

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

[0073] "Liver-specific promoters" are known in the art and include, but are not limited to, the thyroxine-binding globulin (TBG) promoter, the α1-microglobulin / bicinin enhancer / thyroid hormone-binding globulin promoter, the human albumin (hALB) promoter, the thyroid hormone-binding globulin promoter, the α1-antitrypsin promoter, the bovine albumin (bAlb) promoter, the mouse albumin (mAlb) promoter, the human α1-antitrypsin (hAAT) promoter, the ApoEhAAT promoter including 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 including the hAAT promoter and the α1-microglobulin enhancer, the DC190 promoter including the human albumin promoter and the prothrombin enhancer, or other natural or synthetic liver-specific promoters. In one embodiment, the liver-specific promoter may comprise approximately 845 bp and include a thyroid hormone-binding globulin promoter sequence (2382 to 13), two copies of the α1-microglobulin / bicinine enhancer sequence (22,804 to 22,704), and a 71 bp leader sequence described in Ill CR, et al. (1997).

[0074] "Ubiquitous / constitutive promoters" are known in the art and include, but are not limited to, the CMV major pre-early enhancer / chicken β-actin promoter, the cytomegalovirus (CMV) major pre-early promoter, the elongation factor 1-α (EF1-α) promoter, the monkey vacuolated virus 40 (SV40) promoter, the AmpR promoter, the PγK promoter, the human ubiquitin C gene (Ubc) promoter, the MFG promoter, the human β-actin promoter, the CAG promoter, the EGR1 promoter, the FerH promoter, the FerL promoter, the GRP78 promoter, the GRP94 promoter, the HSP70 promoter, the β-kin promoter, the mouse phosphoglycerate kinase (mPGK) or human PGK (hPGK) promoter, the ROSA promoter, the human ubiquitin B promoter, the Roussarcoma virus promoter, or other natural or synthetic ubiquitous / constitutive promoters.

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

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

[0077] As used herein, “tropism” refers to the specificity of the AAV capsid protein present in an AAV virus particle for infecting a particular type of cell or tissue. The tropism of an AAV capsid to a particular type of cell or tissue can be determined by measuring the ability of an AAV vector particle containing a hybrid AAV capsid protein to infect or transduce a particular type of cell or tissue using a standard assay well known in the art (e.g., one disclosed in the examples of this application). As used herein, the terms “hepatotropism” or “hepatic tropism” refer to tropism to the liver or liver tissue and hepatocytes (including hepatocytes).

[0078] "Sequence identity" and "sequence similarity" can be determined by the alignment of two peptide or nucleotide sequences using a global or local alignment algorithm. The sequences may then be referred to as "substantially identical" or "essentially similar" if they are optimally aligned. For example, sequence similarity or sequence identity can be determined by performing a search against databases such as FASTA and BLAST, but to compare sequence identity, hits must be obtained and aligned pairwise. Two proteins or two protein domains, or two nucleic acid sequences, may have "substantially identical" sequence identity if the sequence identity percentage is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or greater, preferably 90%, 95%, 98%, 99%, or greater. Such sequences are also referred herein as "variants," e.g., other variants of a protein or enzyme that are missing, incomplete, and / or mutant. It should be understood that sequences with substantial sequence identity do not necessarily need to have the same length; they can be of different lengths. For example, sequences that have the same nucleotide sequence but one of them has an additional nucleotide at the 3' and / or 5' end are 100% identical.

[0079] As used herein, “codon optimization” may refer to the process of modifying a nucleic acid sequence to enhance expression in a target host cell by replacing one or more codons in the native sequence with codons that are more or most frequently used in the host cell’s gene, while maintaining the native amino acid sequence. Different species exhibit specific biases to specific codons of specific amino acids. As intended herein, genes can be tuned for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the “Codon Usage Database.” Many methods and software tools for codon optimization have been previously reported (see, for example, genomes.urv.es / OPTIMIZER / ).

[0080] In one embodiment, "RNA editing" can be a post-transcriptional modification in which the sequence of precursor mRNA (pre-mRNA) nucleotides is altered by base insertion, deletion, or modification. The degree of RNA editing varies from hundreds of bases in mitochondrial DNA of trypanosomes to just a single base in mammalian nuclear genes. Even a single base change in pre-mRNA can convert one amino acid codon to another amino acid codon or stop codon. This type of recoding can significantly affect the structure and function of proteins and can lead to the production of multiple variants of a protein from a single gene.

[0081] In one embodiment, insertion and deletion RNA editing may involve the addition and deletion of specific nucleotides or sequences of nucleotides from pre-mRNA. In one embodiment, substitution RNA editing by base modification has been observed in higher eukaryotes, in which bases are modified without altering the length of pre-mRNA.

[0082] As used herein, “immunological tolerance,” “immunological tolerance,” and “immunological tolerance” refer to a state of unresponsiveness or blunt response of the immune system to a substance (e.g., disclosed nucleic acid molecules, disclosed vectors, disclosed transgene products, disclosed pharmaceutical formulations, disclosed therapeutic agents, etc.) that has the ability to induce 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 specific antigen or by hepatic restriction transgene expression induced by a viral vector (e.g., AAV). Low or absent antibody titers over time are indicators of immune tolerance. For example, in some embodiments, immune tolerance may be established in subsequent gene therapy (e.g., administration of a transgene encoding a deficient, incomplete, and / or mutant protein or enzyme) by having an IgG antibody titer of less than or equal to about 12,000, less than or equal to about 11,500, less than or equal to about 11,000, less than or equal to about 10,500, less than or equal to about 10,000, less than or equal to about 9,500, less than or equal to about 9,000, less than or equal to about 8,500, less than or equal to about 8,000, less than or equal to about 7,500, less than or equal to about 7,000, less than or equal to about 6,500, or less than or equal to about 6,000.

[0083] As is known in the art, antibodies (Ab) can mitigate AAV infection through multiple mechanisms by binding to AAV capsids and blocking key steps in transduction, such as cell surface adhesion and uptake, escape from endosomes, effective transport to the nucleus, or decoupling, as well as by promoting AAV opsonization by phagocytic cells, thereby mediating their rapid clearance from circulation. For example, in humans, serological studies have revealed high prevalence of NAb in populations worldwide, with approximately 67% of people having antibodies against AAV1, 72% against AAV2, and approximately 40% against AAV serotypes 5-9. Vector immunogenicity is a major challenge in the re-administration of AAV vectors.

[0084] In one embodiment, a partially self-complementary parvovirus (e.g., the disclosed AAV) genome, a plasmid vector encoding a parvovirus genome, and a parvovirus (e.g., the disclosed AAV) particle containing such a genome are also disclosed herein. In one embodiment, a plasmid vector containing a disclosed parvovirus genome, e.g., a nucleotide sequence encoding the disclosed AAV, is provided herein. In one embodiment, a partially self-complementary parvovirus genome comprising a payload construct, a parvovirus ITR adjacent to the payload construct, and a self-complementary region adjacent to one of the ITRs is provided herein. The self-complementary region may contain a nucleotide sequence complementary to the payload construct. The disclosed self-complementary region may have a length shorter than the full length of the payload construct.

[0085] In one embodiment, the disclosed self-complementary region of the disclosed parvovirus genome may include a minimum length, while still having a length shorter than the total length of the payload construct. In one embodiment, the disclosed self-complementary region may include lengths of at least 50 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, at least 900 nucleotides, or at least 1,000 nucleotides.

[0086] In one embodiment, the “self-complementary parvovirus genome” may be a single-stranded polynucleotide having, in the 5' to 3' direction, a first parvovirus ITR sequence, a heterologous sequence (e.g., a payload construct containing the desired gene), a second parvovirus ITR sequence, a second heterologous sequence complementary to the first heterologous sequence, and a third parvovirus ITR sequence. In contrast to a self-complementary genome, a “partially self-complementary genome” does not contain three parvovirus ITRs, and the second heterologous sequence complementary to the first heterologous sequence is shorter in length than the full length of the first heterologous sequence (e.g., a payload construct). Therefore, a partially self-complementary genome is a single-stranded polynucleotide having a first parvovirus ITR sequence, a heterologous sequence (e.g., a payload construct), a second parvovirus ITR sequence, and a self-complementary region that is complementary to a portion of the heterologous sequence and shorter in length than the entire heterologous sequence, in the 5'-to-3' or 3'-to-5' direction.

[0087] As used herein, “immunomodulatory” means the ability of a disclosed nucleic acid molecule, disclosed vector, disclosed pharmaceutical formulation, or disclosed agonist to alter (modulate) one or more characteristics of the immune system. The immune system functions to protect an organism from infection and foreign antigens by cellular and humoral mechanisms, including lymphocytes, macrophages, and other antigen-presenting cells that modulate each other through multiple intercellular interactions, as well as by producing soluble factors, including lymphokines and antibodies, which have autocrine, paracrine, and endocrine effects on immune cells.

[0088] As used herein, “immunomodulator” refers to an agonist capable of modulating a given immune response to a desired level (e.g., immunoenhancing, immunosuppressing, or inducing immune tolerance). Examples of immunomodulators, but not limited to, disclosed immunomodulators include aspirin, azathioprine, belimumab, betamethasone dipropionate, betamethasone valerate, bortezomib, brezinin, ciazathioprine, cyclophosphamide, cyclosporine, deoxysperguarine, didemnin B, fluocinolone acetonide, holinic acid, ibuprofen, IL-6 inhibitors (e.g., sarilumab), indomethacin, inebilizumab, intravenous gamma globulin (IVIG), methotrexate, methylprednisolone, mycophenolol The disclosed immunomodulator may include tomofetil, 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 antibody, anti-CD4 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD40 antibody, anti-FcRN antibody, anti-IL6 antibody, anti-IGF1R antibody, IL2 mutein, BTK inhibitors, or combinations thereof. In one embodiment, the disclosed immunomodulator may include the injection of one or more Tregs (regulatory T cells) (e.g., antigen-specific Treg cells against AAV). In one embodiment, the disclosed immunomodulator may be bortezomib or SVP-rapamycin. In one embodiment, the immunomodulator may be administered by any suitable route of administration, including but not limited to intrauterine, intra-cutaneous, intrathecal, intravenous, subcutaneous, percutaneous, intradermal, intramuscular, oral, percutaneous, intraperitoneal (IP), or vaginal. In one embodiment, the disclosed immunomodulator may be administered using a combination of routes. Administration may also include intra-arterial administration or administration via the hepatic portal vein (HPV). Administration of the immunomodulator may be continuous or intermittent, and administration may include one or more combinations of routes.

[0089] As used herein, the term “immune tolerance” refers to non-responsiveness to an antigen (e.g., a vector, therapeutic protein, or transgene product). Immune tolerance promoters can reduce, improve, or prevent transgene-induced immune responses that may be associated with gene therapy. One or more promoters can be used to determine whether they can confer immune tolerance properties using assays known in the art for measuring immune responses, such as immunohistochemical detection of cytotoxic T cell responses.

[0090] As used herein, the term “packaging instructions” is used to mean the instructions that are typically included in the market packaging of a therapeutic product, which include information relating to the indications, usage, dosage, administration, contraindications, and / or warnings for the use of such therapeutic product.

[0091] As used herein, the term “in combination” in the context of the administration of other therapies (e.g., other agonists) includes the use of one or more therapies (e.g., drug therapies). Administration “in combination” with one or more further therapies includes simultaneous (e.g., concurrent) and sequential administration in any order. The use of the term “in combination” does not limit the order in which these therapies are administered to a subject. As a 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 the administration of a second therapy (e.g., an agonist) to a subject having or diagnosed with a disease or disorder (e.g., a genetic disorder or disorder) (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, 9 hours). It may be administered at 6 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks, together with or after (for example, 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 more).

[0092] Disclosed herein are components used to prepare disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations, as well as disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations used within the methods disclosed herein. Where these and other materials are disclosed herein, and combinations, subsets, interactions, groups, etc., of these materials are disclosed, it is understood that each specific reference to various individual and collective combinations and permutations of these compounds may not be expressly disclosed, but each is specifically assumed and described herein. For example, where a particular compound is disclosed and discussed, and several modifications that can be made to multiple molecules containing that compound are discussed, all possible combinations and permutations of compounds and modifications are specifically assumed unless the opposite is explicitly indicated. Therefore, where groups of molecules A, B, and C, and groups of molecules D, E, and F, and AD as an example of a combined molecule, are disclosed, even if each is not described individually, each is assumed individually and collectively, i.e., combinations AE, AF, BD, BE, BF, CD, CE, and CF are considered disclosed. Similarly, any subsets or combinations of these are also disclosed. Therefore, it is considered that, for example, the subgroups AE, BF, and CE are disclosed. This concept applies to all aspects of this application, including but not limited to the steps of a method for producing and using the compositions of the present invention. Therefore, where there are various additional steps that can be performed, it is understood that each of these additional steps can be performed in any particular embodiment or combination of embodiments of the method of the present invention.

[0093] B. Compositions for transcriptome operations 1. Nucleic acid molecules 5' Replacement construct Disclosed herein are nucleic acid molecules comprising an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures.

[0094] Disclosed herein are nucleic acid molecules comprising an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemyintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures containing one of the sequences of SEQ ID NOs: 01 to 09.

[0095] Disclosed herein is a nucleic acid molecule comprising an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures, wherein the nucleic acid molecule enables the 5' substitution of the target endogenous pre-mRNA.

[0096] Disclosed herein is a nucleic acid molecule comprising an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures containing one of the sequences of SEQ ID NOs. 01 to SEQ ID NOs. 09, wherein the nucleic acid molecule enables the 5' substitution of the target endogenous pre-mRNA.

[0097] In one embodiment, the disclosed targeted endogenous pre-mRNA may contain one or more mutations. In one embodiment of the disclosed targeted endogenous pre-mRNA, one or more disclosed mutations may be present in the 5' portion of the pre-mRNA. In one embodiment, one or more disclosed mutations in one or more exons may contribute to the pathogenesis of one or more cells.

[0098] In one embodiment, the disclosed cells may be present within the subject. In one embodiment, the subject may be a human patient and may be male or female. In one embodiment, the subject may have a genetic disorder or disability. In one embodiment, the subject may be treatment-naive.

[0099] In one embodiment, one or more disclosed mutations may inhibit the translation of the encoded protein. In one embodiment, one or more disclosed mutations may modify the translation of the encoded protein. In one embodiment, one or more disclosed mutations may generate an encoded protein having a nonsense mutation or a missense mutation.

[0100] In one embodiment, the disclosed targeted endogenous pre-mRNA may contain one or more mutations in one or more exons. In one embodiment, the disclosed targeted endogenous pre-mRNA may contain one or more mutations in one or more introns. In one embodiment, one or more disclosed exon mutations may contribute to the pathogenesis in one or more cells. In one embodiment, one or more disclosed intron mutations may contribute to the pathogenesis in one or more cells.

[0101] In one embodiment, the disclosed target endogenous pre-mRNA may be the primary transcript of a protein-coding gene. In one embodiment of the disclosed target endogenous pre-mRNA, the disclosed protein-coding gene may be 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, BA Z2B, BCOR, BCORL1, BDP1, BLM, BPTF, BRCA1, BRCA2, BRD4, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA 1G, CACNA1H, CACNA1S, CAD, CAMTA1, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENP J, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44, CFAP65, CFTR / ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1, C NTNAP1, 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、DNA H9, DNMBP, DNMT1, DOCK2, DOCK3, DOCK6, DOCK7, DOCK8, DSCAM, DSP, DST, DUOX 2, 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, HERC 2. HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172 IGF1R, IGF2R, IGSF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, K ALRN、KANK1、KAT6A、KAT6B、KDM3B、KDM5B、KDM5C、KDM6A、KDM6B、KDR、KIAA0 586. KIAA1109, KIAA1549, KIDINS220, KIF14, KIF1A, KIF1B, KIF21A, KIF26B KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1, LAMA1, LAMA2, LAMA3, LAM A4, LAMA5, LAMB1, LAMB2, LAMC3, LCT, LMNA, LOXHD1, LPA, LRBA, LRP1, LRP2, L RP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD AGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5, MCM3AP, MED12, MED1 2L, MED13, MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MYH1 1. MYH14, MYH2, MYH3, MYH6, MYH7, MYH7B, MYH8, MYH9, MYLK, MYO15A, MYO18B.MYO3A, MYO5A, MYO5B, MYO7A, MYO9A, NALCN, NBAS, NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF, NF1, NFASC, NHS, NIN, NIPBL, NLRP1, NOTCH1, NO TCH2、NOTCH3、NPHP4、NRXN1、NRXN3、NSD1、NSD2、NUP155、NUP188、NUP205、O BSCN、OBSL1、OTOF、OTOG、OTOGL、PARD3、PBRM1、PCDH15、PCLO、PCNT、PHIP、PI 4KA、PIEZO1、PIEZO2、PIK3C2A、PIKFYVE、PKD1、PKD1L1、PKHD1、PLCE1、PLEC 、PLEKHG2、PNPLA6、POGZ、POLA1、POLE、POLR1A、POLR2A、POLR3A、PRG4、PRKDC 、PRPF8、PRR12、PRX、PTCH1、PTPN23、PTPRF、PTPRJ、PTPRQ、PXDN、QRICH2、RA B3GAP2、RAI1、RALGAPA1、RANBP2、RB1CC1、RELN、RERE、REV3L、RIC1、RIMS1、R IMS2、RNF213、ROBO1、ROBO2、ROBO3、ROS1、RP1、RP1L1、RTTN、RUSC2、RYR1、R YR2、SACS、SAMD9、SAMD9L、SBF2、SCAPER、SCN10A、SCN11A、SCN1A、SCN2A、SC N3A、SCN4A、SCN5A、SCN8A、SCN9A、SETBP1、SETD1A、SETD1B、SETD2、SETD5、S ETX、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, TCF 20, TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B, TNXB, TOGARAM1, TONSL, TRIO, TRIOBPIt may include one or more code regions of 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 one embodiment, the disclosed protein-coding gene may include one or more coding regions of CFTR, MDX, DYSF / TTN, DMPK, COL7A1, K14, MAPT, FVIII, HTT, RHO, DNA-PKcs, SMN2, or CD40L. In one embodiment, the disclosed protein-coding gene may include one or more coding regions of FXN, LMNA, or RYR2. In one embodiment, the disclosed protein-coding gene may include a portion of the disclosed protein-coding gene (e.g., exon 1 or exon 4).

[0102] In one embodiment, the 3' portion of the disclosed target endogenous pre-mRNA can be transspliced ​​with exogenous RNA. In one embodiment, the disclosed RNA targeting motif can bind to the target endogenous pre-mRNA. In one embodiment, the disclosed RNA targeting motif can bind to the 5' end of the target endogenous pre-mRNA. In one embodiment, the disclosed RNA targeting motif may be specific to endogenous pre-mRNA having one or more mutations. In one embodiment, the disclosed RNA targeting motif may be specific to endogenous pre-mRNA having one or more exon mutations. In one embodiment, the disclosed RNA targeting motif may be specific to endogenous pre-mRNA having one or more intron mutations.

[0103] In one embodiment, the disclosed RNA targeting motif may contain an antisense oligonucleotide. In one embodiment, the disclosed antisense oligonucleotide may contain approximately 15 to 50 nucleotides. In one embodiment, the disclosed antisense oligonucleotide may contain approximately 30 nucleotides. In one embodiment, the disclosed RNA targeting motif may be directed to an intron immediately 5' of the exon of the target endogenous pre-mRNA to be spliced.

[0104] In one embodiment, the disclosed 5' hemiintron may include a 5' splice site. In one embodiment, the disclosed 5' splice site may include a consensus 5' splice site. In one embodiment, the disclosed consensus 5' splice site may include MAG|GURAGU(SEQ ID NO: 13), where | represents an exon-intron junction, M represents A or C, and R represents A or G. In one embodiment, the disclosed 5' hemiintron may be recognized by nuclear splicing components in a host cell. In one embodiment, the disclosed 5' hemiintron may be recognized by a spliceosome in a host cell. In one embodiment, the disclosed 5' hemiintron may facilitate the trans-splicing of exogenous RNA to the exon immediately 3' to the target intron in endogenous pre-mRNA.

[0105] In one embodiment, the disclosed exogenous RNA to be transspliced ​​into the target endogenous pre-mRNA may contain one or more exons of a protein-coding gene. In one embodiment, the disclosed exogenous RNA to be transspliced ​​into the target endogenous pre-mRNA may contain the primary sequence of the coding sequence of one or more exons having one or more mutations. In one embodiment, the disclosed exogenous RNA may be transspliced ​​into the 3' end of the target endogenous pre-mRNA.

[0106] In one aspect of the disclosed exogenous RNA, the disclosed protein-coding genes are 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, ARHGEF 10, ARHGEF18, ARID1A, ARID1B, ARID2, ASH1L, ASPM, ASXL1, ASXL2, ASXL3, ATM, ATP7A, ATP7B, ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, BLM, BPTF, B RCA1, BRCA2, BRD4, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD, CAMTA1, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44, CFAP65, CFTR / ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1, CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1 A1, COL1A2, COL27A1, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1, CPAMD8, CPLANE1, CPS1, CPS F1, 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, ​​EY S, F5, F8, FANCA, FANCD2, FANCM, FAT1, FAT4, FBN1, FBN2, FLG, FLG2, FLNA, F LNB, FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2, FSIP2, FXN, FYCO1, GLI2 GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1, HERC 2. 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、KIAA0 586. KIAA1109, KIAA1549, KIDINS220, KIF14, KIF1A, KIF1B, KIF21A, KIF26 B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1, LAMA1, LAMA2, LAMA3, LA MA4, LAMA5, LAMB1, LAMB2, LAMC3, LCT, LMNA, LOXHD1, LPA, LRBA, LRP1, LRP2 LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD. MAGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5, MCM3AP, MED12, ME D12L, MED13, MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MY H11, MYH14, MYH2, MYH3, MYH6, MYH7, MYH7B, MYH8, MYH9, MYLK, MYO15A, MYO1 8B, MYO3A, MYO5A, MYO5B, MYO7A, MYO9A, NALCN, NBAS, NBEA, NBEAL2, NCAPD2NCAPD3、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、PL EC、PLEKHG2、PNPLA6、POGZ、POLA1、POLE、POLR1A、POLR2A、POLR3A、PRG4、PR KDC、PRPF8、PRR12、PRX、PTCH1、PTPN23、PTPRF、PTPRJ、PTPRQ、PXDN、QRICH2、 RAB3GAP2、RAI1、RALGAPA1、RANBP2、RB1CC1、RELN、RERE、REV3L、RIC1、RIMS 1、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、SMARC A4、SMCHD1、SNRNP200、SON、SPEF2、SPEG、SPG11、SPTA1、SPTAN1、SPTB、SPTBN 2、SPTBN4、SRCAP、STRC、SVIL、SYNE1、SYNGAP1、SYNJ1、SZT2、TAF1、TANC2、T CF20、TCOF1、TDRD9、TECPR2、TECTA、TENM3、TENM4、TET3、TEX14、TEX15、TG、T HOC2、TMEM94、TNC、TNIK、TNR、TNRC6B、TNXB、TOGARAM1、TONSL、TRIO、TRIOB P、TRIP11、TRIP12、TRPM1、TRPM6、TRPM7、TRRAP、TSC2、TTC37、TTN、TUBGCP6、In one embodiment, the disclosed protein-coding gene may include one or more coding regions of 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 one embodiment, the disclosed protein-coding gene may include one or more coding regions of CFTR, MDX, DYSF / TTN, DMPK, COL7A1, K14, MAPT, FVIII, HTT, RHO, DNA-PKcs, SMN2, or CD40L. In one embodiment, the disclosed protein-coding gene may include one or more coding regions of FXN, LMNA, or RYR2.

[0107] In one embodiment, the disclosed nucleic acid sequence to be transspliced ​​may encode LMNA / C (SEQ ID NO: 20) or a portion thereof. LMNA / C is known in the art (e.g., gene ID 4000), and this nucleotide sequence may include nucleotides 4974-62517 of accession number NG008692.2. The nuclear lamina consists of a two-dimensional matrix of proteins located adjacent to the inner nuclear membrane. Proteins of the lamin family constitute the matrix and are highly conserved in evolution. During mitosis, the lamina matrix is ​​reversibly degraded as lamin proteins are phosphorylated. Lamin proteins are involved in nuclear stability, chromatin structure, and gene expression. Vertebrate lamins consist of two types, A and B. Alternative splicing results in multiple transcriptional variants. Mutations in this gene cause several diseases, including Emery-Dreyfus muscular dystrophy, familial partial lipodystrophy, limb-girdle muscular dystrophy, dilated cardiomyopathy, Charcot-Marie-Tooth disease, and Hutchinson-Gilford progeria syndrome.

[0108] In one embodiment, the disclosed nucleic acid sequence to be transspliced ​​can encode DP71 or a portion thereof. DP71 is known in the art (e.g., gene ID 13405).

[0109] In one embodiment, the disclosed nucleic acid sequence to be transspliced ​​may encode CFTR (SEQ ID NO: 19) or a portion thereof. CFTR is known in the art (e.g., gene ID 1080), and this nucleotide sequence may include nucleotides 19180-207882 of accession number NG016465.4. This gene encodes a member of the ATP-binding cassette (ABC) transporter superfamily. The encoded protein functions as a chloride channel, unique among members of this protein family, and regulates the secretion and absorption of ions and water in epithelial tissues. Channel activation is mediated by a cycle of phosphorylation of the regulatory domain, ATP binding by the nucleotide-binding domain, and ATP hydrolysis. Mutations in this gene cause cystic fibrosis, which is the most common lethal genetic disorder in Nordic populations. The most frequently occurring mutation in cystic fibrosis, DeltaF508, results in impaired folding and transport of the encoded protein. Several pseudogenes have been identified in the human genome.

[0110] In one embodiment, the disclosed nucleic acid sequence to be transspliced ​​may encode DMPK (SEQ ID NO: 21) or a portion thereof. DMPK is known in the art (e.g., gene ID 1760), and its nucleotide sequence may include nucleotides 5068-17841 of accession number NG009784.1. DMPK is a serine-threonine kinase closely related to other kinases that interact with members of the Rho family of small GTPases. The substrates of this enzyme include myogenin, the β-subunit of the L-type calcium channel, and phosphoremane. 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, with increasing severity as the copy number of the repeat element increases. The repeat expansion is associated with local chromatin condensation that represses gene expression in this region. Several alternative splicing transcription variants of this gene have been reported, but the full length of some of these variants has not yet been determined.

[0111] In one embodiment, the disclosed gene may be DMD (dystrophin) (SEQ ID NO: 17). DMD is known in the art (e.g., gene ID 1756), and its nucleotide sequence may include nucleotides 5001-2225382 of accession number NG012232.1. DMD encodes a large protein containing an N-terminal actin-binding domain and multiple spectrin repeats over a genomic range of more than 2 Mb. 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 locus can cause Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or cardiomyopathy. The use of alternative promoters and alternative splicings results in numerous different transcriptional variants and protein isoforms for this gene.

[0112] In one embodiment, the disclosed nucleic acid sequence to be transspliced ​​may encode LRRK2 (SEQ ID NO: 18) or a portion thereof. LRRK2 is known in the art (e.g., gene ID 120892), and its nucleotide sequence may include nucleotides 5001-149275 of accession number NG011709.1. LRRK2 is a member of the leucine-rich repeat kinase family and encodes a protein having a repeat region, a leucine-rich repeat (LRR) domain, a kinase domain, a DFG-like motif, a RAS domain, a GTPase domain, an MLK-like domain, and a WD40 domain. The protein is mostly located in the cytoplasm but also associates with the outer mitochondrial membrane. Mutations in this gene are associated with Parkinson's disease.

[0113] In one embodiment, the disclosed exogenous RNA to be transspliced ​​may further include UTR.

[0114] In one embodiment, one or more disclosed RNA structures can bind to one or more RNA-binding proteins. In one embodiment, one or more disclosed RNA structures can bind to one or more double-stranded RNA-binding proteins (dsRBPs). In one embodiment, dsRBPs are known to those skilled in the art and include, but are not limited to, ADAR1, ADAR2, DICER, NFAR, PACT, PKR, RHA RNase III, Stauffen, TRBP, TSEN, or any combination thereof.

[0115] In one embodiment, one or more disclosed RNA structures may contain one of the sequences of SEQ ID NOs: 01 to 09. In one embodiment, one or more disclosed RNA structures may improve and / or enhance trans-splicing efficiency. In one embodiment, one or more disclosed RNA structures may stabilize pre-mRNA. In one embodiment, one or more disclosed RNA structures may localize RNA to the nucleus. In one embodiment, one or more disclosed RNA structures may stabilize the interaction between a targeted endogenous pre-mRNA molecule and the exogenous RNA to be trans-spliced. In one embodiment, the disclosed nucleic acid molecule may lack CRISPR-related proteins.

[0116] In one embodiment, the disclosed chimeric RNA transcript may comprise the 5' portion of the targeted endogenous pre-mRNA and the 3' portion of the exogenous RNA. In one embodiment, the disclosed chimeric RNA transcript may comprise the 3' portion of the targeted endogenous pre-mRNA and the 5' portion of the exogenous RNA.

[0117] In one embodiment, the disclosed target endogenous pre-mRNA and the disclosed exogenous RNA may encode the same protein-coding gene. In one embodiment, the disclosed target endogenous pre-mRNA and the disclosed exogenous RNA may contain one or more exons of the same protein-coding gene.

[0118] In one embodiment, the disclosed nucleic acid molecule may be packaged in a viral vector. In one embodiment, the disclosed viral vector may include an AAV vector. In one embodiment, the disclosed nucleic acid molecule may be packaged on a non-viral carrier. In one embodiment, the disclosed nucleic acid molecule may be incorporated into a plasmid. In one embodiment, the disclosed nucleic acid molecule may be incorporated into lipid nanoparticles.

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

[0120] In one embodiment, the disclosed nucleic acid molecule may further comprise one or more nuclear localization signals (NLSs). NLSs are known to those skilled in the art. In one embodiment, the disclosed NLS may comprise any NLS known in the art. As is known in the art (see, for example, Lu J et al. (2021) Cell Commun Signal. 19:60, which is incorporated herein by reference with respect to teachings on NLSs), nuclear localization signals (NLSs) are generally short peptides that act as signal fragments that mediate the transport of proteins from the cytoplasm to the nucleus.

[0121] In one embodiment, the disclosed nucleic acid molecule may further comprise one or more nucleo-retaining elements (NREs), NREs known to those skilled in the art. In one embodiment, the disclosed NRE may comprise SIRLOIN (SEQ ID NO: 15) or BORG (SEQ ID NO: 16).

[0122] In one embodiment, the disclosed nucleic acid molecule may further comprise one or more flavivirus genetic elements. In one embodiment, the flavivirus genetic elements may comprise one or more flavivirus 3' untranslated regions (3'UTRs), one or more subgenomic flavivirus RNA (sfRNA) elements, one or more flavivirus XRN1 resistance 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 International Publication 2022 / 182835 for a description of flavivirus genetic elements).

[0123] In one embodiment, the disclosed exogenous RNA may induce a splice event. In one embodiment, the disclosed 5' hemiintron may be recognized by nuclear splicing components within the host cell.

[0124] In one embodiment, the disclosed promoter for a 5' substitution construct may be tissue-specific or ubiquitous and may be constitutive or inductive depending on the desired expression pattern. The promoter may be native or exogenous and may be a natural or synthetic sequence. "Exogenous" means that the transcription start region is not found in the wild-type host into which it is introduced. In one embodiment, the disclosed promoter may be a promoter / enhancer. In one embodiment, the disclosed promoter for a disclosed nucleic acid molecule may be an endogenous promoter. In one embodiment, the disclosed endogenous promoter may be an endogenous promoter / enhancer. In one embodiment, the disclosed endogenous promoter or disclosed endogenous promoter / enhancer may generally be derived from a non-coding region located upstream of the transcription start site of the gene of interest. In one embodiment, the disclosed endogenous promoter or disclosed endogenous promoter / enhancer may be used for the constitutive and efficient expression of a disclosed protein-coding gene. In one embodiment, the disclosed promoter for one or more disclosed guide RNA sequences may be a CMV promoter or a CMV promoter / enhancer. CMV promoters and CMV promoter / enhancers are well known in the art. In one embodiment, the disclosed promoter for one or more disclosed guide RNA sequences may be any eukaryotic RNA polymerase II promoter.

[0125] Disclosed herein is an expression cassette comprising an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures. Disclosed herein is an expression cassette comprising an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures comprising one of the sequences of SEQ ID NOs. 01 to SEQ ID NOs. Disclosed herein is an expression cassette comprising an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures, wherein the nucleic acid molecule enables the 5' substitution of the target endogenous pre-mRNA. Disclosed herein is an expression cassette comprising an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures comprising one of the sequences of SEQ ID NOs. 01 to SEQ ID NOs. 09, wherein the nucleic acid molecule enables the 5' substitution of the target endogenous pre-mRNA.

[0126] In one embodiment, the expression of a disclosed protein-coding gene can be restored and / or reverted to wild-type, normal, or control expression levels. In one embodiment, a disclosed nucleic acid molecule can restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation. In one embodiment, a disclosed nucleic acid molecule can restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above). In one embodiment, restoring one or more features of cellular homeostasis and / or cellular functionality may include: (i) correcting cellular starvation in one or more cell types; (ii) normalizing aspects of autophagy pathways (e.g., correcting, preventing, reducing, and / or improving autophagy); (iii) improving, enhancing, restoring, and / or maintaining mitochondrial functionality and / or structural integrity; (iv) improving, enhancing, restoring, and / or maintaining organelle functionality and / or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the progression of multi-organ manifestations of a genetic disorder or impairment; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the progression of a genetic disorder or impairment; and (viii) one or more of any combination thereof. In one embodiment, restoring one or more features of cellular homeostasis may include improving, enhancing, restoring, and / or maintaining one or more features of the structural and / or functional integrity of the cell.

[0127] In one embodiment, restoring the activity and / or functionality of a deficient, incomplete, and / or mutant protein or enzyme may include restoration of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount compared to an existing level, such as a pre-treatment level. In one embodiment, the amount of restoration may be 10%–20%, 20%–30%, 30%–40%, 40%–50%, 50%–60%, 60%–70%, 70%–80%, 80%–90%, or 90%–100% greater than an existing level, such as a pre-treatment level. In one embodiment, the restoration may be measured against a control level or baseline level (determined, for example, using one or more subjects that do not have the deficient, incomplete, and / or mutant protein or enzyme). In one embodiment, the restoration may be partial or incomplete. In one embodiment, recovery may be complete or near-complete, such that the levels of expression, activity, and / or functionality are similar to those of the wild type or control level.

[0128] In one embodiment, the disclosed 5' substitution construct may be particularly useful in methods for treating subjects with autosomal dominant genetic disorders or disorders such as progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary ellipticosis, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, osteopetrosis, Marfan syndrome, neurofibromatosis, adult-onset polycystic kidney disease, protein C deficiency, osteogenesis imperfecta, Treacher Collins syndrome, tuberous sclerosis, and von Willebrand disease.

[0129] 3' Replacement construct Disclosed herein are nucleic acid molecules comprising one or more RNA structures, one or more RNA targeting motifs, a 3' hemiintron ligated to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​into a targeted endogenous pre-mRNA. Disclosed herein are nucleic acid molecules comprising one or more RNA structures containing one of the sequences of SEQ ID NOs. 01 to SEQ ID NOs. 09, one or more RNA targeting motifs, a 3' hemiintron ligated to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​into a targeted endogenous pre-mRNA. Disclosed herein are nucleic acid molecules comprising one or more RNA structures, one or more RNA targeting motifs, a 3' hemiintron ligated to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​into a targeted endogenous pre-mRNA, the nucleic acid molecule enabling the 3' substitution of the targeted endogenous pre-mRNA. Disclosed herein are nucleic acid molecules comprising one or more RNA structures containing one of the sequences of SEQ ID NOs: 01 to 09, one or more RNA targeting motifs, a 3' hemiintron ligated to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​to a target endogenous pre-mRNA, wherein the nucleic acid molecule enables a 3' substitution of the target endogenous pre-mRNA.

[0130] In one embodiment, the disclosed targeted endogenous pre-mRNA may contain one or more mutations in one or more exons. In one embodiment, one or more disclosed mutations may be located in the 3' portion of one or more exons of the pre-mRNA. In one embodiment, the disclosed targeted endogenous pre-mRNA may contain one or more mutations in one or more introns. In one embodiment, one or more disclosed mutations in one or more exons may contribute to the pathogenesis of one or more cells. In one embodiment, the disclosed cells may be present within the subject. In one embodiment, the subject may be a human patient and may be male or female. In one embodiment, the subject may have a genetic disorder or disability. In one embodiment, the subject may be treatment-naive. In one embodiment, one or more disclosed mutations may inhibit the translation of the encoded protein. In one embodiment, one or more disclosed mutations may modify the translation of the encoded protein. In one embodiment, during and / or after translation, one or more disclosed mutations may produce proteins with nonsense or missense mutations. In one embodiment, one or more disclosed exon mutations may contribute to the pathogenesis of one or more cells. In one embodiment, one or more disclosed intron mutations may contribute to the pathogenesis of one or more cells. In one embodiment, the disclosed targeted endogenous pre-mRNA may encode a protein-coding gene.

[0131] In one aspect of the disclosed target endogenous pre-mRNA, the disclosed protein-coding genes are 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, ARHG AP31, ARHGEF10, ARHGEF18, ARID1A, ARID1B, ARID2, ASH1L, ASPM, ASXL1, ASXL2, ASXL3, ATM, ATP7A, ATP7B, ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP 1, BLM, BPTF, BRCA1, BRCA2, BRD4, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD, CAM TA1, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CF AP43, 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, CPLA NE1, 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、DS P、DST、DUOX2、DYNC1H1、DYNC2H1、DYSF、EIF2AK4、EP300、EPG5、ERCC6、ERCC6 L2、EXPH5、EYS、F5、F8、FANCA、FANCD2、FANCM、FAT1、FAT4、FBN1、FBN2、FLG、 FLG2、FLNA、FLNB、FLNC、FLT4、FMN2、FN1、FRAS1、FREM1、FREM2、FSIP2、FXN、F YCO1、GLI2、GLI3、GPR179、GREB1L、GRIN2A、GRIN2B、GRIN2D、HCFC1、HECW2、 HERC1、HERC2、HFM1、HIVEP1、HIVEP2、HMCN1、HSPG2、HTT、HUWE1、HYDIN、IFT1 40, IFT172, IGF1R, IGF2R, IGF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A, KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B KDR、KIAA0586、KIAA1109、KIAA1549、KIDINS220、KIF14、KIF1A、KIF1B、KIF 21A、KIF26B、KIF7、KMT2A、KMT2B、KMT2C、KMT2D、KMT2E、KNL1、LAMA1、LAMA2、 LAMA3、LAMA4、LAMA5、LAMB1、LAMB2、LAMC3、LCT、LMNA、LOXHD1、LPA、LRBA、L RP1、LRP2、LRP4、LRP5、LRP6、LRPPRC、LRRK1、LRRK2、LTBP2、LTBP4、LYST、MAC F1、MADD、MAGI2、MAP1B、MAP3K1、MAPK8IP3、MAPKBP1、MAST1、MBD5、MCM3AP、 MED12、MED12L、MED13、MED13L、MED23、MEGF8、MET、MLH3、MPDZ、MSH6、MTOR、M YH10、MYH11、MYH14、MYH2、MYH3、MYH6、MYH7、MYH7B、MYH8、MYH9、MYLK、MYO1 5A、MYO18B、MYO3A、MYO5A、MYO5B、MYO7A、MYO9A、NALCN、NBAS、NBEA、NBEAL2、NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF, NF1, NFSC, NHS, NIN, NIPBL, NLRP1, NOTCH1, NOTCH2, NOTCH3, NPHP4, NRXN1, NRXN3, NSD1, NSD2, NUP155, NUP188 、NUP205、OBSCN、OBSL1、OTOF、OTOG、OTOGL、PARD3、PBRM1、PCDH15、PCLO、PC NT、PHIP、PI4KA、PIEZO1、PIEZO2、PIK3C2A、PIKFYVE、PKD1、PKD1L1、PKHD1、P LCE1、PLEC、PLEKHG2、PNPLA6、POGZ、POLA1、POLE、POLR1A、POLR2A、POLR3A、 PRG4、PRKDC、PRPF8、PRR12、PRX、PTCH1、PTPN23、PTPRF、PTPRJ、PTPRQ、PXDN、 QRICH2、RAB3GAP2、RAI1、RALGAPA1、RANBP2、RB1CC1、RELN、RERE、REV3L、RI C1、RIMS1、RIMS2、RNF213、ROBO1、ROBO2、ROBO3、ROS1、RP1、RP1L1、RTTN、RUS C2、RYR1、RYR2、SACS、SAMD9、SAMD9L、SBF2、SCAPER、SCN10A、SCN11A、SCN1A 、SCN2A、SCN3A、SCN4A、SCN5A、SCN8A、SCN9A、SETBP1、SETD1A、SETD1B、SETD 2、SETD5、SETX、SHANK2、SHANK3、SHROOM4、SI、SIPA1L3、SLIT2、SLX4、SMARC A2、SMARCA4、SMCHD1、SNRNP200、SON、SPEF2、SPEG、SPG11、SPTA1、SPTAN1、SP TB、SPTBN2、SPTBN4、SRCAP、STRC、SVIL、SYNE1、SYNGAP1、SYNJ1、SZT2、TAF1 、TANC2、TCF20、TCOF1、TDRD9、TECPR2、TECTA、TENM3、TENM4、TET3、TEX14、TE X15, TG, THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B, TNXB, TOGARAM1, TONSL, TR IO、TRIOBP、TRIP11、TRIP12、TRPM1、TRPM6、TRPM7、TRRAP、TSC2、TTC37、TTN、The disclosed protein-coding gene may include one or more coding regions of 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 one embodiment, the disclosed protein-coding gene may include one or more coding regions of CFTR, MDX, DYSF / TTN, DMPK, COL7A1, K14, MAPT, FVIII, HTT, RHO, DNA-PKcs, SMN2, or CD40L. In one embodiment, the disclosed protein-coding gene may include one or more coding regions of FXN, LMNA, or RYR2. In one embodiment, the disclosed protein-coding gene may include a portion of the disclosed protein-coding gene (e.g., exon 1 or exon 4).

[0132] In one embodiment, the 5' portion of the disclosed target endogenous pre-mRNA can be transspliced ​​with exogenous RNA.

[0133] In one embodiment, the disclosed RNA targeting motif can bind to a targeted endogenous pre-mRNA. In one embodiment, the disclosed RNA targeting motif can bind to the 3' end of a targeted endogenous pre-mRNA. In one embodiment, the disclosed RNA targeting motif may be specific to an endogenous pre-mRNA having one or more mutations. In one embodiment, the disclosed RNA targeting motif may be specific to an endogenous pre-mRNA having one or more exon mutations. In one embodiment, the disclosed RNA targeting motif may be specific to an endogenous pre-mRNA having one or more intron mutations. In one embodiment, the disclosed RNA targeting motif may contain an antisense oligonucleotide.

[0134] In one embodiment, the disclosed antisense oligonucleotide may contain approximately 15 to 50 nucleotides. In another embodiment, the disclosed antisense oligonucleotide may contain approximately 30 nucleotides. In another embodiment, the disclosed RNA targeting motif may be directed to an intron immediately 3' of the exon of the target endogenous pre-mRNA to be spliced.

[0135] In one embodiment, the disclosed 3' hemiintron may comprise (i) a 3' splice region including a branch point, (ii) a polypyrimidine tract, and (iii) a 3' splice acceptor site. In one embodiment, the disclosed branch point may comprise the sequence of SEQ ID NO: 10. In one embodiment, the disclosed 3' splice acceptor site may comprise the sequence YAG, where Y is a pyrimidine (SEQ ID NO: 11). In one embodiment, the disclosed 3' hemiintron may be recognized by nuclear splicing components in a host cell. In one embodiment, the disclosed 3' hemiintron may be recognized by spliceosomes in a host cell. In one embodiment, the disclosed 3' hemiintron may facilitate the transsplicing of exogenous RNA to the exon immediately 5' to the target intron in endogenous pre-mRNA.

[0136] In one embodiment, the disclosed exogenous RNA to be transspliced ​​into the target endogenous pre-mRNA may contain one or more exons of a protein-coding gene. In one embodiment, the disclosed exogenous RNA to be transspliced ​​into the target endogenous pre-mRNA may contain the primary sequence of the coding sequence of one or more exons having one or more mutations. In one embodiment, the disclosed exogenous RNA may be transspliced ​​into the 5' end of the target endogenous pre-mRNA.

[0137] In one aspect of the disclosed exogenous RNA, the disclosed protein-coding genes are 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, ARHGEF 10, ARHGEF18, ARID1A, ARID1B, ARID2, ASH1L, ASPM, ASXL1, ASXL2, ASXL3, ATM, ATP7A, ATP7B, ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, BLM, BPTF, B RCA1, BRCA2, BRD4, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD, CAMTA1, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44, CFAP65, CFTR / ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1, CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1 A1, COL1A2, COL27A1, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1, CPAMD8, CPLANE1, CPS1, CPS F1, 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, ​​EY S, F5, F8, FANCA, FANCD2, FANCM, FAT1, FAT4, FBN1, FBN2, FLG, FLG2, FLNA, F LNB, FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2, FSIP2, FXN, FYCO1, GLI2 GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1, HERC 2. 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、KIAA0 586. KIAA1109, KIAA1549, KIDINS220, KIF14, KIF1A, KIF1B, KIF21A, KIF26 B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1, LAMA1, LAMA2, LAMA3, LA MA4, LAMA5, LAMB1, LAMB2, LAMC3, LCT, LMNA, LOXHD1, LPA, LRBA, LRP1, LRP2 LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD. MAGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5, MCM3AP, MED12, ME D12L, MED13, MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MY H11, MYH14, MYH2, MYH3, MYH6, MYH7, MYH7B, MYH8, MYH9, MYLK, MYO15A, MYO1 8B, MYO3A, MYO5A, MYO5B, MYO7A, MYO9A, NALCN, NBAS, NBEA, NBEAL2, NCAPD2NCAPD3、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、PL EC、PLEKHG2、PNPLA6、POGZ、POLA1、POLE、POLR1A、POLR2A、POLR3A、PRG4、PR KDC、PRPF8、PRR12、PRX、PTCH1、PTPN23、PTPRF、PTPRJ、PTPRQ、PXDN、QRICH2、 RAB3GAP2、RAI1、RALGAPA1、RANBP2、RB1CC1、RELN、RERE、REV3L、RIC1、RIMS 1、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、SMARC A4、SMCHD1、SNRNP200、SON、SPEF2、SPEG、SPG11、SPTA1、SPTAN1、SPTB、SPTBN 2、SPTBN4、SRCAP、STRC、SVIL、SYNE1、SYNGAP1、SYNJ1、SZT2、TAF1、TANC2、T CF20、TCOF1、TDRD9、TECPR2、TECTA、TENM3、TENM4、TET3、TEX14、TEX15、TG、T HOC2、TMEM94、TNC、TNIK、TNR、TNRC6B、TNXB、TOGARAM1、TONSL、TRIO、TRIOB P、TRIP11、TRIP12、TRPM1、TRPM6、TRPM7、TRRAP、TSC2、TTC37、TTN、TUBGCP6、In one embodiment, the disclosed protein-coding gene may include one or more coding regions of 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 one embodiment, the disclosed protein-coding gene may include one or more coding regions of CFTR, MDX, DYSF / TTN, DMPK, COL7A1, K14, MAPT, FVIII, HTT, RHO, DNA-PKcs, SMN2, or CD40L. In one embodiment, the disclosed protein-coding gene may include one or more coding regions of FXN, LMNA, or RYR2.

[0138] In one embodiment, the disclosed nucleic acid sequence to be transspliced ​​may encode LMNA / C (SEQ ID NO: 20) or a portion thereof. LMNA / C is discussed above. In one embodiment, the disclosed nucleic acid sequence to be transspliced ​​may encode DP71 or a portion thereof. DP71 is discussed above. In one embodiment, the disclosed nucleic acid sequence to be transspliced ​​may encode CFTR (SEQ ID NO: 19) or a portion thereof. CFTR is discussed above. In one embodiment, the disclosed nucleic acid sequence to be transspliced ​​may encode DMPK (SEQ ID NO: 21) or a portion thereof. DMPK is discussed above. In one embodiment, the disclosed gene may be DMD (dystrophin) (SEQ ID NO: 17). DMD is discussed above.

[0139] In one embodiment, the disclosed exogenous RNA to be transspliced ​​may further include UTR.

[0140] In one embodiment, one or more disclosed RNA structures can bind to one or more RNA-binding proteins. In one embodiment, one or more disclosed RNA structures can bind to one or more double-stranded RNA-binding proteins (dsRBPs). In one embodiment, dsRBPs are known to those skilled in the art and include, but are not limited to, ADAR1, ADAR2, DICER, NFAR, PACT, PKR, RHA RNase III, Stauffen, TRBP, TSEN, or any combination thereof.

[0141] In one embodiment, one or more disclosed RNA structures can bind to one or more RNA-binding proteins. In one embodiment, one or more disclosed RNA structures can bind to one or more double-stranded RNA-binding proteins. In one embodiment, one or more disclosed RNA structures may contain any one sequence from SEQ ID NOs: 01 to SEQ ID NOs: 09. In one embodiment, one or more disclosed RNA structures may improve and / or enhance trans-splicing efficiency. In one embodiment, one or more disclosed RNA structures may stabilize pre-mRNA. In one embodiment, one or more disclosed RNA structures may localize RNA to the nucleus. In one embodiment, one or more disclosed RNA structures may stabilize the interaction between a targeted endogenous pre-mRNA molecule and the exogenous RNA to be trans-spliced.

[0142] In one embodiment, the disclosed nucleic acid molecule may lack a CRISPR-related protein.

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

[0144] In one embodiment, the disclosed nucleic acid molecule may be packaged in a viral vector. In one embodiment, the disclosed viral vector may include an AAV vector. In one embodiment, the disclosed nucleic acid molecule may be packaged on a non-viral carrier. In one embodiment, the disclosed nucleic acid molecule may be incorporated into a plasmid. In one embodiment, the disclosed nucleic acid molecule may be incorporated into lipid nanoparticles.

[0145] In one embodiment, the disclosed nucleic acid molecule may further include a polyadenylated sequence. In one embodiment, the disclosed nucleic acid molecule may further include a sequence for a promoter. In one embodiment, the disclosed 3' hemiintron may be recognized by a nuclear splicing component in a host cell. In one embodiment, the disclosed exogenous RNA may induce a splice event. In one embodiment, the disclosed nucleic acid molecule may further include a spacer region. In one embodiment, the disclosed spacer region may separate a 5' splice region from one or more RNA structures. In one embodiment, the disclosed spacer region may include any known spacer. In one embodiment, the disclosed spacer region may include a consensus splicing motif (e.g., U1 or U2). In one embodiment, the disclosed spacer region may include a limited number of consensus splicing motifs (e.g., U1 or U2). In one embodiment, the disclosed nucleic acid molecule may further include a nuclear localization signal (NLS). In one embodiment, the disclosed nucleic acid molecule may further comprise one or more nucleo-retaining elements (NREs), NREs known to those skilled in the art. In one embodiment, the disclosed NRE may comprise SIRLOIN (SEQ ID NO: 15) or BORG (SEQ ID NO: 16).

[0146] In one embodiment, the disclosed nucleic acid molecule may further comprise one or more flavivirus genetic elements. In one embodiment, the flavivirus genetic elements may comprise one or more flavivirus 3' untranslated regions (3'UTRs), one or more subgenomic flavivirus RNA (sfRNA) elements, one or more flavivirus XRN1 resistance 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 International Publication 2022 / 182835 for a description of flavivirus genetic elements).

[0147] In one embodiment, the disclosed nucleic acid molecule may comprise sequences of one or more regulatory elements (e.g., woodchuck hepatitis virus (WHV) post-transcriptional regulatory elements (WPRE), MALAT1-derived triple chains, hepatitis B virus (HPRE) PRE and iron response elements). For example, the disclosed regulatory elements may comprise a promoter operably linked to the disclosed nucleic acid molecule, the promoter driving the expression of a disclosed variant capsid protein, a disclosed encoded polypeptide, a disclosed encoded therapeutic agent, or both.

[0148] In one embodiment, the disclosed promoter for a 3' substitution construct may be tissue-specific or ubiquitous and may be constitutive or inductive depending on the desired expression pattern. The promoter may be native or exogenous and may be a natural or synthetic sequence. "Exogenous" means that the transcription start region is not found in the wild-type host into which it is introduced. In one embodiment, the disclosed promoter may be a promoter / enhancer. In one embodiment, the disclosed promoter for a disclosed nucleic acid molecule may be an endogenous promoter. In one embodiment, the disclosed endogenous promoter may be an endogenous promoter / enhancer. In one embodiment, the disclosed endogenous promoter or disclosed endogenous promoter / enhancer may generally be derived from a non-coding region located upstream of the transcription start site of the gene of interest. In one embodiment, the disclosed endogenous promoter or disclosed endogenous promoter / enhancer may be used for the constitutive and efficient expression of the disclosed gene. In one embodiment, the disclosed promoter for one or more disclosed guide RNA sequences may be a CMV promoter or a CMV promoter / enhancer. CMV promoters and CMV promoter / enhancers are well known in the art. In one embodiment, the disclosed promoter for one or more disclosed guide RNA sequences may 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' hemiintron ligated to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​into a targeted endogenous pre-mRNA. Disclosed herein is an expression cassette comprising one or more RNA structures comprising one of the sequences of SEQ ID NOs. 01 to SEQ ID NOs. 09, one or more RNA targeting motifs, a 3' hemiintron ligated to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​into a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3' substitution of the targeted endogenous pre-mRNA. Disclosed herein is an expression cassette comprising one or more RNA structures containing one of the sequences of SEQ ID NOs: 01 to 09, one or more RNA targeting motifs, a 3' hemiintron ligated to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables a 3' substitution of the targeted endogenous pre-mRNA.

[0150] In one embodiment, the expression of a disclosed protein-coding gene can be restored and / or reverted to wild-type, normal, or control expression levels. In one embodiment, a disclosed nucleic acid molecule can restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation. In one embodiment, a disclosed nucleic acid molecule can restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above). In one embodiment, restoring one or more features of cellular homeostasis and / or cellular functionality may include: (i) correcting cellular starvation in one or more cell types; (ii) normalizing aspects of autophagy pathways (e.g., correcting, preventing, reducing, and / or improving autophagy); (iii) improving, enhancing, restoring, and / or maintaining mitochondrial functionality and / or structural integrity; (iv) improving, enhancing, restoring, and / or maintaining organelle functionality and / or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the progression of multi-organ manifestations of a genetic disorder or impairment; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the progression of a genetic disorder or impairment; and (viii) one or more of any combination thereof. In one embodiment, restoring one or more features of cellular homeostasis may include improving, enhancing, restoring, and / or maintaining one or more features of the structural and / or functional integrity of a cell. In one embodiment, restoring the activity and / or functionality of a deficient, incomplete, and / or mutant protein or enzyme may include restoring 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount compared to an existing level, such as a pre-treatment level.In one embodiment, the amount of recovery may be 10%–20%, 20%–30%, 30%–40%, 40%–50%, 50%–60%, 60%–70%, 70%–80%, 80%–90%, or 90%–100% greater than the existing level, such as the pre-treatment level. In one embodiment, recovery may be measured against a control level or baseline level (determined, for example, using one or more subjects that do not have the deficient, incomplete, and / or mutant protein or enzyme). In one embodiment, recovery may be partial or incomplete. In one embodiment, recovery may be complete or near-complete, such that the levels of expression, activity, and / or functionality are similar to those of the wild-type or control level.

[0151] In one embodiment, the disclosed 3' substitution construct may be particularly useful in methods for treating subjects with autosomal dominant genetic disorders or disorders, such as progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary ellipticosis, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, osteopetrosis, Marfan syndrome, neurofibromatosis, adult-onset polycystic kidney disease, protein C deficiency, osteogenesis imperfecta, Treacher Collins syndrome, tuberous sclerosis, and von Willebrand disease.

[0152] 2. Transcriptome Engineering System Disclosed herein are transcriptome engineering systems comprising one or more disclosed 3' substitution constructs, one or more disclosed 5' substitution constructs, or any combination thereof. Disclosed herein are nucleic acid molecules comprising (i) exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures; (ii) nucleic acid molecules comprising exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures comprising any one sequence of SEQ ID NOs. 01 to SEQ ID NOs. 09; and (iii) exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, (iv) A transcriptome engineering system comprising one or more of the following: a nucleic acid molecule comprising a 5' hemiintron linked to an exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures, which enables the 5' substitution of a targeted endogenous pre-mRNA; and a nucleic acid molecule comprising an exogenous RNA to be transspliced ​​into a targeted endogenous pre-mRNA, a 5' hemiintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures containing one of sequence numbers 01 to 09, which enables the 5' substitution of a targeted endogenous pre-mRNA.

[0153] Disclosed herein are (i) nucleic acid molecules comprising one or more RNA structures, one or more RNA targeting motifs, a 3' hemiintron linked to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA; (ii) nucleic acid molecules comprising one or more RNA structures containing any one sequence of SEQ ID NOs. 01 to SEQ ID NOs. 09, one or more RNA targeting motifs, a 3' hemiintron linked to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA; and (iii) one or more RNA structures, one or more RNA targeting motifs, transspliced The nucleic acid molecule comprises a nucleic acid molecule that enables the 3' substitution of the target endogenous pre-mRNA, and (iv) one or more RNA structures containing one of sequence numbers 01 to 09, one or more RNA targeting motifs, a 3' hemiintron linked to the exogenous RNA that will be transspliced ​​into the target endogenous pre-mRNA, and one or more nucleic acid molecules that enable the 3' substitution of the target endogenous pre-mRNA.

[0154] Disclosed herein are (i) a nucleic acid molecule comprising an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures, and (ii) an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs (iii) a nucleic acid molecule comprising one or more RNA structures containing a line and one of the sequences of SEQ ID NOs: 01 to 09, and a nucleic acid molecule comprising an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures, enabling the 5' substitution of the target endogenous pre-mRNA, and (iv) a transspliced ​​into the target endogenous pre-mRNA A nucleic acid molecule comprising (v) one or more RNA structures comprising exogenous RNA to be spliced, a 5' hemiintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures comprising one of the sequences of SEQ ID NOs. 01 to SEQ ID NOs. 09, enabling the 5' substitution of the endogenous pre-mRNA to be targeted; (v) a nucleic acid molecule comprising one or more RNA structures, one or more RNA targeting motifs, a 3' hemiintron linked to the exogenous RNA to be transspliced, and exogenous RNA to be transspliced ​​into the endogenous pre-mRNA to be targeted; (vi) a nucleic acid molecule comprising one or more RNA structures comprising one of the sequences of SEQ ID NOs. 01 to SEQ ID NOs. 09, one or more RNA targeting motifs,The nucleic acid molecule comprises a nucleic acid molecule that enables the 3' substitution of the target endogenous pre-mRNA, and (viii) one or more RNA structures containing one of sequence numbers 01 to 09, one or more RNA targeting motifs, a 3' hemiintron linked to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​into the target endogenous pre-mRNA, and one or more nucleic acid molecules that enable the 3' substitution of the target endogenous pre-mRNA.

[0155] 3. Vector Disclosed herein are vectors comprising disclosed nucleic acid molecules. In one embodiment, the disclosed vector may be a nonviral vector or a viral vector. Disclosed herein are nonviral vectors comprising disclosed nucleic acid molecules. Disclosed herein are nonviral vectors comprising one or more disclosed nucleic acid molecules. Disclosed herein are viral vectors comprising disclosed nucleic acid molecules. Disclosed herein are viral vectors comprising one or more disclosed nucleic acid molecules. Disclosed herein are nonviral vectors or viral vectors comprising exogenous RNA to be transspliced ​​into a targeted endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures. Disclosed herein are non-viral or viral vectors containing a nucleic acid molecule comprising an exogenous RNA to be transspliced ​​into an endogenous pre-mRNA to be targeted, a 5' hemyintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures containing one of the sequences of SEQ ID NOs: 01 to 09.

[0156] Disclosed herein are non-viral or viral vectors containing a nucleic acid molecule comprising exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures, wherein the nucleic acid molecule enables the 5' substitution of the target endogenous pre-mRNA. Disclosed herein are non-viral or viral vectors containing a nucleic acid molecule comprising exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures containing one of the sequences of SEQ ID NOs. 01 to SEQ ID NOs. 09, wherein the nucleic acid molecule enables the 5' substitution of the target endogenous pre-mRNA. Disclosed herein are non-viral or viral vectors containing a nucleic acid molecule comprising one or more RNA structures, one or more RNA targeting motifs, a 3' hemiintron linked to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA. Disclosed herein are non-viral or viral vectors containing a nucleic acid molecule comprising one or more RNA structures containing one of the sequences of SEQ ID NOs: 01 to 09, one or more RNA targeting motifs, a 3' hemiintron linked to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA. Disclosed herein are non-viral or viral vectors containing a nucleic acid molecule comprising one or more RNA structures, one or more RNA targeting motifs, a 3' hemyintron ligated to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables a 3' substitution of the targeted endogenous pre-mRNA.Disclosed herein are non-viral or viral vectors comprising a nucleic acid molecule containing one or more RNA structures having one of the sequences of SEQ ID NOs: 01 to 09, one or more RNA targeting motifs, a 3' hemiintron ligated to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​to a target endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3' substitution of the target endogenous pre-mRNA.

[0157] Disclosed herein are nonviral vectors or viral vectors comprising one or more 5' substitution constructs. Disclosed herein are nonviral vectors or viral vectors comprising one or more 3' substitution constructs. Disclosed herein are nonviral vectors or viral vectors comprising one or more 5' substitution constructs and / or one or more 3' substitution constructs. In one embodiment, a disclosed 5' substitution construct to be used in combination with one or more other substitution constructs may include any disclosed 5' substitution construct. In one embodiment, the disclosed 5' substitution construct comprises (i) a nucleic acid molecule comprising an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures; (ii) a nucleic acid molecule comprising an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures comprising any one sequence of SEQ ID NOs: 01 to SEQ ID NOs: 09; and (iii) an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA A nucleic acid molecule comprising sex RNA, a 5' hemiintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures, which enables the 5' substitution of the targeted endogenous pre-mRNA; or (iv) a nucleic acid molecule comprising exogenous RNA to be transspliced ​​into the targeted endogenous pre-mRNA, a 5' hemiintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures comprising any one sequence of SEQ ID NOs. 01 to SEQ ID NOs. 09, which enables the 5' substitution of the targeted endogenous pre-mRNA; or (v) any combination thereof.

[0158] In one embodiment, a disclosed 3' substitution construct to be used in combination with one or more other substitution constructs may include any disclosed 3' substitution construct. In one embodiment, a disclosed 3' substitution construct is a nucleic acid molecule comprising (i) one or more RNA structures, one or more RNA targeting motifs, a 3' hemiintron linked to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA; (ii) a nucleic acid molecule comprising one or more RNA structures, one or more RNA targeting motifs, a 3' hemiintron linked to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA; and (iiii) one or more RNA structures, one or more RNA targeting motifs, The nucleic acid molecule may include (iv) a nucleic acid molecule that enables the 3' substitution of the target endogenous pre-mRNA, comprising a 3' hemiintron ligated to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​into the target endogenous pre-mRNA, or (v) any combination thereof.

[0159] In one aspect, the disclosed vectors can be formulated for administration via one or more routes. Such methods are well known to those skilled in the art and include the following routes: oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, intrauterine administration, intrahepatic administration, vaginal administration, ophthalmic administration, otologic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including but not limited to injectable forms such as intravenous administration, intrathecal administration, intraarterial administration, intramuscular administration, and subcutaneous administration. Administration can also include intrahepatic arterial administration or administration via the hepatic portal vein (HPV). Administration of the disclosed therapeutic agent, the disclosed pharmaceutical composition, or a combination thereof can include direct administration to the central nervous system (CNS) (e.g., within the parenchyma, intraventricularly, subarachnoid space within the cerebral cisterns, intrathecal (lumbar), deep gray matter delivery, convection-enhanced delivery to the deep gray matter) or the peripheral nervous system (PNS). Administration can be continuous or intermittent. Administration can include administering the viral vector and / or the generated optimized viral vector. Administration of the disclosed vectors can be continuous or intermittent.

[0160] In one aspect, a therapeutically effective amount of the disclosed AAV particles or the disclosed AAV vectors can include a range of from about 1×10 10 vg / kg to about 2×10 14 vg / kg. In one aspect, for example, the disclosed vectors can be administered at a dose of from about 1×10 11 to about 8×10 13 vg / kg or from about 1×10 12 to about 8×10 13 vg / kg. In one aspect, the disclosed vectors can be administered at a dose of from about 1×10 13 to about 6×10 13 vg / kg. In one aspect, the disclosed vectors are at least about 1×10 10 vg / kg, at least about 5×10 10 vg / kg, at least about 1×10 11 vg / kg, at least about 5×10 11 vg / kg, at least about 1×10 12 vg / kg, at least about 5×10 12vg / kg, at least about 1 × 10⁻⁶ 13 vg / kg, at least about 5 × 10⁻⁶ 13 vg / kg, or at least about 1 × 10⁻⁶ 14 It can be administered in doses of vg / kg. In one embodiment, the disclosed vector is approximately 1 × 10⁻⁶ 10 vg / kg or less, approximately 5×10 10 vg / kg or less, approximately 1×10 11 vg / kg or less, approximately 5×10 11 vg / kg or less, approximately 1×10 12 vg / kg or less, approximately 5×10 12 vg / kg or less, approximately 1×10 13 vg / kg or less, approximately 5×10 13 Less than vg / kg, or approximately 1 × 10⁻⁶ 14 It can be administered at doses of less than vg / kg. In one embodiment, the disclosed vector is administered at approximately 1 × 10⁻⁶ doses. 12 It can be administered in doses of vg / kg. In one embodiment, the disclosed vector is administered at approximately 1 × 10⁻⁶ doses. 11 It can be administered in doses of vg / kg. In one embodiment, the disclosed vector can be administered in single doses or multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses) as needed for the desired therapeutic outcome.

[0161] In one embodiment, a therapeutically effective amount of the disclosed AAV particles or disclosed AAV vector is approximately 1 × 10⁶ in total per subject. 12 vg~Approximately 1 x 10 per target 17 The range may include vg. In one embodiment, a therapeutically effective amount of the disclosed AAV particles or disclosed AAV vector totals approximately 1 × 10⁶ per subject. 12 vg, approximately 1 x 10 per target in total 13 vg, approximately 1 x 10 per target in total 14 vg, approximately 1 x 10 per target in total 15 vg, approximately 1 x 10 per target in total 16 vg, or approximately 1 x 10 per subject in total 17The range may include vg. In one embodiment, a therapeutically effective amount of the disclosed AAV particles or disclosed AAV vector may be delivered by retrograde ureteral infusion and / or renal artery administration, totaling approximately 1 × 10⁶ per subject. 12 vg~Approximately 1 x 10 per target 17 It can include the range of vg.

[0162] In one embodiment, the disclosed therapeutically effective amount of AAV particles is approximately 1 × 10⁻⁶ 6 DRP / mL ~ approximately 1 x 10 14 The range may include DRP / mL. In one embodiment, the disclosed pharmaceutical formulation is approximately 1 × 10⁻⁶ 6 DRP / mL, approximately 1 x 10 7 DRP / mL, approximately 1 x 10 8 DRP / mL, approximately 1 x 10 9 DRP / mL, approximately 1 x 10 10 DRP / mL, approximately 1 x 10 11 DRP / mL, approximately 1 x 10 12 DRP / mL, approximately 1 x 10 13 DRP / mL, or approximately 1 × 10⁻⁶ 14 It may contain DRP / mL. In one embodiment, the disclosed therapeutically effective amount of AAV particles or the disclosed vector may include a range determined by those skilled in the art.

[0163] In one embodiment, the disclosed nonviral vector may be a polymer-based vector, a peptide-based vector, a lipid nanoparticle, a solid lipid nanoparticle, or a cationic lipid-based vector. In one embodiment, the disclosed vector may include exosomes, extracellular vesicles, and virus-like particles.

[0164] In one embodiment, the disclosed viral vector may be an adenovirus vector, an AAV vector, a herpes simplex virus vector, a retrovirus vector, a lentivirus vector, an alphavirus vector, a flavivirus vector, a rhabdovirus vector, a measles virus vector, a Newcastle disease virus vector, a poxvirus vector, or a picornavirus vector. In one embodiment, the disclosed nucleic acid sequence may have a coding sequence of less than about 4.5 kilobases.

[0165] In one embodiment, the disclosed AAV vectors may include, but are not limited to, naturally isolated serotypes including AAV1, AAV2, AAV3 (including 3a and 3b), AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAVrh39, AAVrh43, AAVcy.7, as well as bovine AAV, goat AAV, canine AAV, equine AAV, sheep AAV, avian AAV, primate AAV, non-primate AAV, and any other viruses classified as AAV by the International Committee on Taxonomy of Viruses (ICTV). In one embodiment, AAV capsids may be chimeras created by capsid evolution or by rational capsid engineering from naturally isolated AAV variants to capture desirable serotype characteristics such as enhanced or specific tissue tropism and / or evasion of host immune responses. Naturally isolated AAV variants include, but are not limited to, AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV-1829, AAV2 Y / F, AAV2 T / V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A-String (e.g., AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep, AAV9.47-AS, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, and AAVcc.81. In one embodiment, the disclosed AAV vector may be AAV-Rh74 or a related variant (e.g., a capsid variant such as RHM4-1). In one embodiment, the disclosed AAV vector may be AAV.cc47. In one embodiment, the disclosed AAV vector may be AAV.cc81. In one embodiment, the disclosed AAV vector may be a self-complementary AAV.

[0166] In one embodiment, the disclosed vector may include one or more ITRs (e.g., ITRs derived from AAV2).

[0167] In one embodiment, the disclosed vector may further comprise one or more nuclear localization signals (NLSs). NLSs are known to those skilled in the art. In one embodiment, the disclosed NLS may comprise any NLS known in the art. As is known in the art (see, for example, Lu J et al. (2021) Cell Commun Signal. 19:60, which is incorporated herein by reference with respect to teaching of NLSs), nuclear localization signals (NLSs) are generally short peptides that act as signal fragments that mediate the transport of proteins from the cytoplasm to the nucleus.

[0168] In one embodiment, the disclosed vector may further comprise one or more nucleus-holding elements (NREs), NREs known to those skilled in the art. In one embodiment, the disclosed NRE may comprise SIRLOIN (SEQ ID NO: 15) or BORG (SEQ ID NO: 16).

[0169] In one embodiment, the disclosed vector may further comprise one or more flavivirus genetic elements. In one embodiment, the flavivirus genetic elements may comprise one or more flavivirus 3' untranslated regions (3'UTRs), one or more subgenomic flavivirus RNA (sfRNA) elements, one or more flavivirus XRN1 resistance 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 International Publication 2022 / 182835 for a description of flavivirus genetic elements).

[0170] In one embodiment, the disclosed vector may further comprise a nucleic acid sequence encoding a therapeutic protein, a therapeutic agent, and / or therapeutic RNA. In one embodiment, the disclosed therapeutic protein may include polypeptides and / or glycopeptides. In one embodiment, the disclosed therapeutic agent may include an oligonucleotide therapeutic agent. In one embodiment, the disclosed oligonucleotide therapeutic agent may be single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), antisense molecules, miRNA, morpholino, peptide nucleic acid (PNA), or analogs or conjugates thereof. In one embodiment, the disclosed therapeutic agent may be an ASO or RNAi. In one embodiment, the disclosed therapeutic agent may include a CRISPR-based endonuclease (e.g., Cas9). In one embodiment, the disclosed CRISPR-based endonuclease may be derived from a CRISPR / Cas type I, type II, or type III system. In one embodiment, the disclosed therapeutic RNA may include ribosomal RNA (rRNA), transfer RNA (tRNA), heteronuclear RNA (hnRNA), nuclear small RNA (snRNA), nucleolar small RNA (snoRNA), microRNA (miRNA), Piwi-interacting RNA (piRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), single guide RNA (sgRNA), non-coding RNA (ncRNA), long non-coding RNA (lncRNA), 7SL, Xist, short enhancer RNA (eRNA), circular RNA, intergenetic RNA, or any combination thereof. In one embodiment, the disclosed RNA may include lncRNA, siRNA, shRNA, sgRNA, circular RNA, snoRNA, miRNA, or any combination thereof. In one embodiment, the disclosed coding RNA may include functional non-coding RNA elements.

[0171] In one embodiment, the disclosed vector may include one or more promoters operably ligated to a disclosed nucleic acid molecule (e.g., a 5' substitution construct and / or a 3' substitution construct), a disclosed transgene, a disclosed sequence to be transspliced, and / or a disclosed nucleic acid sequence. In one embodiment of the disclosed vector, the disclosed nucleic acid molecule may be operably ligated to one or more transcriptional regulatory elements. In one embodiment, one or more transcriptional regulatory elements (e.g., the woodchuck hepatitis virus (WHV) post-transcriptional regulatory element (WPRE), the MALAT1-derived triple, the hepatitis B virus (HPRE) PRE and iron-responsive elements) may increase the transcription and / or expression of a disclosed nucleic acid molecule (e.g., a 5' substitution construct and / or a 3' substitution 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). In one embodiment, the disclosed promoter can be positioned 5' (upstream) or 3' (downstream) of a disclosed nucleic acid molecule (e.g., a 5' substitution construct and / or a 3' substitution 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) under its control. The distance between the disclosed promoter and the disclosed nucleic acid molecule (e.g., a 5' substitution construct and / or a 3' substitution 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) may be approximately the same as the distance between the promoter and the disclosed nucleic acid molecule under its control (e.g., a 5' substitution construct and / or a 3' substitution 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).As is known in the art, variations in this distance can be tolerated without loss of promoter function.

[0172] In one embodiment of the disclosed vector, the disclosed promoter may be tissue-specific or ubiquitous and may be constitutive or inductive depending on the desired expression pattern. The disclosed promoter may be native or exogenous and may be a natural or synthetic sequence. "Exogenous" means that the transcription start region is not found in the wild-type host into which it is introduced. In one embodiment, the disclosed promoter may be a promoter / enhancer. In one embodiment, the disclosed promoter for a disclosed nucleic acid molecule (e.g., a 5' substitution construct and / or a 3' substitution 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) may be an endogenous promoter. In one embodiment, the disclosed endogenous promoter may be an endogenous promoter / enhancer. In one embodiment, the disclosed endogenous promoter or disclosed endogenous promoter / enhancer may generally be derived from a non-coding region located upstream of the transcription start site of the gene of interest. In one embodiment, a disclosed endogenous promoter or a disclosed endogenous promoter / enhancer may be used for the constitutive and efficient expression of a disclosed protein-coding gene. In one embodiment, a disclosed promoter for a disclosed nucleic acid molecule (e.g., a 5' substitution construct and / or a 3' substitution 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) may be a CMV promoter or a CMV promoter / enhancer. CMV promoters and CMV promoters / enhancers are well known in the art.In one embodiment, the disclosed promoter for the disclosed nucleic acid molecule (e.g., a 5' substitution construct and / or a 3' substitution construct), the disclosed transgene, the disclosed sequence to be transspliced, and / or the disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and / or disclosed therapeutic RNA) may be any eukaryotic RNA polymerase II promoter. In one embodiment, AAV particles can be generated using the disclosed AAV vector. In one embodiment, AAV particles can be generated using the disclosed AAV vector, which under its control include the disclosed nucleic acid molecule (e.g., a 5' substitution construct and / or a 3' substitution construct), the disclosed transgene, the disclosed sequence to be transspliced, and / or the disclosed nucleic acid sequence (e.g., encoding the disclosed therapeutic protein and / or the disclosed therapeutic RNA).

[0173] Disclosed herein are AAV particles comprising, under their control, a disclosed nucleic acid molecule (e.g., a 5' substitution construct and / or a 3' substitution 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).

[0174] In one embodiment, the disclosed vector or disclosed AAV particles may be particularly useful in methods for treating subjects with autosomal dominant genetic disorders or disorders, such as progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary ellipticosis, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, osteopetrosis, Marfan syndrome, neurofibromatosis, adult-onset polycystic kidney disease, protein C deficiency, osteogenesis imperfecta, Treacher Collins syndrome, tuberous sclerosis, and von Willebrand disease.

[0175] 4. Pharmaceutical preparations Disclosed herein are pharmaceutical formulations comprising a disclosed nucleic acid molecule. Disclosed herein are pharmaceutical formulations comprising a disclosed nucleic acid molecule and a pharmaceutically acceptable carrier. Disclosed herein are pharmaceutical formulations comprising a disclosed vector. Disclosed herein are pharmaceutical formulations comprising a disclosed vector and a pharmaceutically acceptable carrier. Disclosed herein are pharmaceutical formulations comprising a disclosed AAV particle. Disclosed herein are pharmaceutical formulations comprising a disclosed AAV particle and a pharmaceutically acceptable carrier.

[0176] Disclosed herein are pharmaceutical formulations comprising a non-viral vector or viral vector containing a nucleic acid molecule comprising an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures. Disclosed herein are pharmaceutical formulations comprising a non-viral vector or viral vector containing an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures containing one of the sequences of SEQ ID NOs: 01 to 09. Disclosed herein are pharmaceutical formulations comprising a non-viral vector or viral vector containing a nucleic acid molecule comprising exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemi-intron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures, wherein the nucleic acid molecule enables the 5' substitution of the target endogenous pre-mRNA. Disclosed herein are pharmaceutical formulations comprising a non-viral vector or viral vector containing a nucleic acid molecule comprising exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemi-intron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures containing one of the sequences of SEQ ID NOs. 01 to SEQ ID NOs. The nucleic acid molecule enables the 5' substitution of the target endogenous pre-mRNA. Disclosed herein are pharmaceutical formulations comprising a non-viral vector or a viral vector containing a nucleic acid molecule comprising one or more RNA structures, one or more RNA targeting motifs, a 3' hemyintron ligated to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​to a target endogenous pre-mRNA.Disclosed herein are pharmaceutical formulations comprising a non-viral vector or viral vector containing a nucleic acid molecule comprising one or more RNA structures containing one of the sequences of SEQ ID NOs: 01 to 09, one or more RNA targeting motifs, a 3' hemiintron linked to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA. Disclosed herein are pharmaceutical formulations comprising a non-viral vector or viral vector containing a nucleic acid molecule comprising one or more RNA structures, one or more RNA targeting motifs, a 3' hemiintron linked to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3' substitution of the target endogenous pre-mRNA. Disclosed herein are pharmaceutical formulations comprising a non-viral vector or a viral vector containing a nucleic acid molecule comprising one or more RNA structures containing one of the sequences of SEQ ID NOs: 01 to 09, one or more RNA targeting motifs, a 3' hemiintron ligated to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​to a target endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3' substitution of the target endogenous pre-mRNA.

[0177] In one embodiment, the disclosed pharmaceutical formulation may include (i) one or more activators, (ii) a biological activator, (iii) one or more pharmaceutical activators, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved activators, or (vi) a combination thereof. In one embodiment, the disclosed composition may include one or more immunomodulators. In one embodiment, the disclosed composition may include one or more proteasome inhibitors. In one embodiment, the disclosed composition may include one or more immunosuppressants or immunosuppressive activators. In one embodiment, the immunosuppressive activator may be anti-thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate tomofetil (MMF), or a combination thereof. In one embodiment, the disclosed formulation may include an anaplerotic activator (e.g., a C7 compound such as triheptanoin or MCT).

[0178] In one embodiment, the disclosed formulation may include an RNA therapeutic agent. The RNA therapeutic agent may include RNA-mediated interference (RNAi) and / or antisense oligonucleotides (ASOs). In one embodiment, the disclosed RNA therapeutic agent may target any protein or enzyme that is overexpressed or overactive due to a deficient, incomplete, and / or mutant protein or enzyme. In one embodiment, the disclosed RNA therapeutic agent may include a therapeutic agent delivered via LNPs. In one embodiment, the disclosed formulation may include an enzyme or enzyme precursor for enzyme replacement therapy (ERT).

[0179] In one embodiment, the disclosed formulation may contain the disclosed small molecule. In one embodiment, the disclosed small molecule may help restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme.

[0180] In one embodiment, any disclosed pharmaceutical formulation may include one or more excipients and / or pharmaceutically acceptable carriers, which are known in the art and discussed above.

[0181] In one embodiment, a pharmaceutical formulation with a disclosed therapeutically effective dose is approximately 1 × 10⁻⁶ 10 vg / kg ~ approx. 2×10 14 The disclosed vector and / or disclosed AAV particles may be included in the range of vg / kg. In one embodiment, for example, the dose of the disclosed pharmaceutical formulation may be about 1 × 10⁻⁶ 11 ~Approx. 8×10 13 vg / kg or approximately 1 × 10⁻⁶ 12 ~Approx. 8×10 13 It may include vg / kg. In one embodiment, the dose of the disclosed pharmaceutical formulation is approximately 1 × 10⁻⁶ 13 ~Approx. 6×10 13 It may include vg / kg. In one embodiment, the dose of the disclosed pharmaceutical formulation is at least about 1 × 10⁻⁶ 10 , at least about 5 × 10 10 , at least about 1 × 10 11 , at least about 5 × 10 11 , at least about 1 × 10 12 , at least about 5 × 10 12 , at least about 1 × 10 13 , at least about 5 × 10 13 , or at least about 1 × 10 14 It may include vg / kg. In one embodiment, the dose of the disclosed pharmaceutical formulation is approximately 1 × 10⁻⁶ 10 Not exceeding vg / kg, approximately 5 x 10 10 Not exceeding vg / kg, approximately 1 × 10⁻⁶ 11 Not exceeding vg / kg, approximately 5 x 10 11 Not exceeding vg / kg, approximately 1 × 10⁻⁶ 12 Not exceeding vg / kg, approximately 5 x 10 12 Not exceeding vg / kg, approximately 1 × 10⁻⁶ 13 Not exceeding vg / kg, approximately 5 x 10 13 Do not exceed vg / kg or approximately 1 × 10⁻⁶ 14It may include a dosage not exceeding vg / kg. In one aspect, the dosage of the disclosed pharmaceutical preparation can be about 1×10 12 vg / kg. In one aspect, the dosage of the disclosed pharmaceutical preparation can be about 1×10 11 vg / kg. In one aspect, the dosage of the disclosed pharmaceutical preparation can include a single dose or multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 administrations) as required for the desired therapeutic result.

[0182] In one aspect, the therapeutically effective amount of the disclosed pharmaceutical preparation can include a range from a total of about 1×10 12 vg per subject to a total of about 1×10 17 vg per subject. In one aspect, the therapeutically effective amount of the disclosed pharmaceutical preparation can be a total of about 1×10 12 vg, a total of about 1×10 13 vg, a total of about 1×10 14 vg, a total of about 1×10 15 vg, a total of about 1×10 16 vg, or a total of about 1×10 17 vg per subject. In one aspect, the therapeutically effective amount of the disclosed pharmaceutical preparation can be about 1×10 6 DRP / mL to about 1×10 14 DRP / mL. In one aspect, the therapeutically effective amount of the disclosed pharmaceutical preparation can be about 1×10 6 DRP / mL, about 1×10 7 DRP / mL, about 1×10 8 DRP / mL, about 1×10 9 DRP / mL, about 1×10 10 DRP / mL, about 1×10 11 DRP / mL, about 1×10 12 DRP / mL, about 1×10 13 DRP / mL, or about 1×10 14 DRP / mL.

[0183] In one aspect, the therapeutically effective amount of the disclosed pharmaceutical preparation can include a range determined by those skilled in the art.

[0184] In one embodiment, the disclosed pharmaceutical formulation can be used to restore and / or reverse the expression of the disclosed protein-coding gene to wild-type, normal, or control expression levels. In one embodiment, the disclosed pharmaceutical formulation can restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation. In one embodiment, the disclosed nucleic acid molecule can restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above). In one embodiment, restoring one or more features of cellular homeostasis and / or cellular functionality may include: (i) correcting cellular starvation in one or more cell types; (ii) normalizing aspects of autophagy pathways (e.g., correcting, preventing, reducing, and / or improving autophagy); (iii) improving, enhancing, restoring, and / or maintaining mitochondrial functionality and / or structural integrity; (iv) improving, enhancing, restoring, and / or maintaining organelle functionality and / or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the progression of multi-organ manifestations of a genetic disorder or impairment; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the progression of a genetic disorder or impairment; and (viii) one or more of any combination thereof. In one embodiment, restoring one or more features of cellular homeostasis may include improving, enhancing, restoring, and / or maintaining one or more features of the structural and / or functional integrity of a cell. In one embodiment, restoring the activity and / or functionality of a deficient, incomplete, and / or mutant protein or enzyme may include restoring 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount compared to an existing level, such as a pre-treatment level.In one embodiment, the amount of recovery may be 10%–20%, 20%–30%, 30%–40%, 40%–50%, 50%–60%, 60%–70%, 70%–80%, 80%–90%, or 90%–100% greater than the existing level, such as the pre-treatment level. In one embodiment, recovery may be measured against a control level or baseline level (determined, for example, using one or more subjects that do not have the deficient, incomplete, and / or mutant protein or enzyme). In one embodiment, recovery may be partial or incomplete. In one embodiment, recovery may be complete or near-complete, such that the levels of expression, activity, and / or functionality are similar to those of the wild-type or control level.

[0185] In one embodiment, the disclosed pharmaceutical formulation may be particularly useful in treating subjects with autosomal dominant genetic disorders or disorders such as progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary ellipticosis, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, osteopetrosis, Marfan syndrome, neurofibromatosis, adult-onset polycystic kidney disease, protein C deficiency, osteogenesis imperfecta, Treacher Collins syndrome, tuberous sclerosis, and von Willebrand disease.

[0186] 5. Plasmid Disclosed herein are plasmids comprising one or more disclosed nucleic acid molecules. Disclosed herein are plasmids comprising one or more disclosed vectors. Disclosed herein are plasmids used for methods of producing disclosed compositions, such as disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations. Plasmids and the use of plasmids are known in the art. Disclosed herein are plasmids comprising a sequence or fragment thereof described in any one of SEQ ID NOs. 25 to SEQ ID NOs. 33. Disclosed herein are plasmids comprising a sequence or fragment thereof having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence or fragment thereof described in any one of SEQ ID NOs. 25 to SEQ ID NOs. 33. Disclosed herein are plasmids comprising a sequence or fragment thereof having at least 40% to 60%, at least 60% to 80%, at least 80% to 90%, or at least 90% to 100% identity with any one of the sequences described in SEQ ID NOs. 25 to 33.

[0187] 6.Cells Disclosed herein are cells comprising disclosed nucleic acid molecules, disclosed vectors, and / or disclosed plasmids. Disclosed herein are cells transduced with one or more disclosed viral vectors. Disclosed herein are cells transfected with one or more disclosed nucleic acid molecules. Techniques for achieving transfection and transduction are known in the art, and the use of transfected or transduced cells is known in the art. In one embodiment, disclosed herein are human cell lines transduced with one or more disclosed viral vectors, or human cell lines transfected with one or more disclosed nucleic acids, human cell lines transfected with one or more disclosed nonviral vectors, or human cell lines transfected with one or more disclosed plasmids. In one embodiment, disclosed herein are human cell lines having one or more genetic disorders or genetic defects that have come into contact with one or more nucleic acid molecules, one or more disclosed vectors, and / or one or more disclosed pharmaceutical formulations. Disclosed herein are one or more disclosed nucleic acid molecules, one or more disclosed vectors, one or more disclosed plasmids, and / or cells obtained from subjects treated with one or more disclosed pharmaceutical formulations.

[0188] 7.Animals Disclosed herein are one or more disclosed nucleic acid molecules, one or more disclosed substitution constructs, one or more disclosed vectors, one or more disclosed AAV particles, one or more disclosed pharmaceutical formulations, and / or animals treated with one or more disclosed plasmids. Transgenic animals, as well as techniques for producing transgenic animals, are known in the art.

[0189] 8. Library Disclosed herein are libraries of one or more disclosed nucleic acid molecules. Disclosed herein are libraries of one or more disclosed 5' substitution constructs. Disclosed herein are libraries of one or more disclosed 3' substitution constructs. Disclosed herein are libraries of one or more disclosed 5' substitution constructs and / or disclosed 3' substitution constructs. Disclosed herein are libraries of one or more disclosed vectors. Disclosed herein are libraries of one or more disclosed vectors comprising one or more disclosed 5' substitution constructs, one or more disclosed 3' constructs, or any combination thereof. Disclosed herein are libraries of one or more disclosed AAV particles comprising one or more disclosed 5' substitution constructs, one or more disclosed 3' constructs, or any combination thereof. Disclosed herein are libraries of one or more disclosed plasmids.

[0190] 9. Kit Disclosed herein are kits comprising one or more disclosed nucleic acid molecules, disclosed vectors or disclosed AAV particles, disclosed pharmaceutical formulations, or any combination thereof. Disclosed herein are kits comprising one or more disclosed nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof. In one embodiment, the kit may include a disclosed nucleic acid molecule, a disclosed vector or disclosed AAV particles, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, or a combination thereof, and one or more activators. "Activators" and "therapeutic agents" are known in the art and are described above.

[0191] In one embodiment, one or more activators can treat, prevent, inhibit, and / or mitigate one or more comorbidities of a subject. In one embodiment, one or more activators can treat, inhibit, prevent, and / or mitigate cellular and / or metabolic complications associated with deficient, incomplete, and / or mutant proteins or enzymes.

[0192] In one embodiment, the disclosed kit may include at least two components that constitute the kit. These components together constitute a functional unit for a predetermined purpose (e.g., treating a subject diagnosed with or suspected of having a genetic disorder or genetic defect). The individual component parts may be packaged physically together or separately. For example, a kit including instructions for using the kit may or may not physically include the instructions together with the other individual component parts. Alternatively, the instructions may be supplied as a separate component part, either in paper or electronic form, supplied on a computer-readable memory device, downloaded from an internet website, or provided as a recorded presentation. In one embodiment, the kit for use in the disclosed manner may include one or more containers that hold the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, disclosed RNA therapeutics, or combinations thereof, and a label or accompanying document including instructions for use. In one embodiment, suitable containers may include, for example, bottles, vials, syringes, blister packs, etc. Containers may be formed from a variety of materials, such as glass or plastic. The container may hold the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or combinations thereof, and may have a sterile access port (for example, the container may be a vial or intravenous solution bag with a stopper that can be punctured with a subcutaneous needle). The label or accompanying information may indicate that the disclosed nucleic acid molecules, disclosed vectors, disclosed AAV particles, disclosed pharmaceutical formulations, disclosed RNA therapeutic agents, or combinations thereof may be used to treat, prevent, inhibit, and / or alleviate a disease or disorder, or complications and / or symptoms associated with the disease or disorder. The disclosed kit may include additional components necessary for administration, such as other buffers, diluents, filters, needles, and syringes. In one embodiment, the disclosed kit may be used in any disclosed manner.In one embodiment, the disclosed kit can be used to generate one or more chimeric RNA molecules. In one embodiment, the disclosed kit can be used to treat a genetic disorder or genetic defect. In one embodiment, the disclosed kit can be used to inhibit and / or minimize disease progression.

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

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

[0195] Disclosed herein is a method for generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule, the nucleic acid molecule comprising an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures, wherein the resulting chimeric RNA transcript comprises the 3' portion of the target endogenous pre-mRNA and the 5' portion of the exogenous RNA.

[0196] Disclosed herein is a method for generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule, the nucleic acid molecule comprising one or more RNA structures comprising an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron ligated to the transspliced ​​exogenous RNA, one or more RNA targeting motifs, and one of the sequences of SEQ ID NOs. 01 to SEQ ID NOs. 09, wherein the resulting chimeric RNA transcript comprises the 3' portion of the target endogenous pre-mRNA and the 5' portion of the exogenous RNA.

[0197] Disclosed herein is a method for generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule, the nucleic acid molecule comprising an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures, the nucleic acid molecule enabling a 5' substitution of the target endogenous pre-mRNA, the resulting chimeric RNA transcript comprising a 3' portion of the target endogenous pre-mRNA and a 5' portion of the exogenous RNA.

[0198] Disclosed herein is a method for generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule, the nucleic acid molecule comprising one or more RNA structures comprising an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one of the sequences of SEQ ID NOs. 01 to SEQ ID NOs. 09, wherein the nucleic acid molecule enables a 5' substitution of the target endogenous pre-mRNA, and the resulting chimeric RNA transcript comprises the 3' portion of the target endogenous pre-mRNA and the 5' portion of the exogenous RNA.

[0199] Disclosed herein is a method for generating a chimeric RNA molecule in a cell, the method comprising contacting an endogenous pre-mRNA in the cell with a disclosed 3' substitution construct, the resulting chimeric RNA transcript comprising a 5' portion of the targeted endogenous pre-mRNA and a 3' portion of the exogenous RNA.

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

[0201] Disclosed herein is a method for generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule, the nucleic acid molecule comprising one or more RNA structures, one or more RNA targeting motifs, a 3' hemiintron linked to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, the resulting chimeric RNA transcript may comprise a 5' portion of the target endogenous pre-mRNA and a 3' portion of the exogenous RNA.

[0202] Disclosed herein is a method for generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule, the nucleic acid molecule comprising one or more RNA structures containing one of the sequences of SEQ ID NOs. 01 to SEQ ID NOs. 09, one or more RNA targeting motifs, a 3' hemiintron ligated to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​to a target endogenous pre-mRNA, wherein the resulting chimeric RNA transcript may comprise a 5' portion of the target endogenous pre-mRNA and a 3' portion of the exogenous RNA.

[0203] Disclosed herein is a method for generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule, the nucleic acid molecule comprising one or more RNA structures, one or more RNA targeting motifs, a 3' hemiintron linked to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, the nucleic acid molecule enabling a 3' substitution of the target endogenous pre-mRNA, and the resulting chimeric RNA transcript may comprise a 5' portion of the target endogenous pre-mRNA and a 3' portion of the exogenous RNA.

[0204] Disclosed herein is a method for generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule, the nucleic acid molecule comprising one or more RNA structures containing one of the sequences of SEQ ID NOs: 01 to 09, one or more RNA targeting motifs, a 3' hemiintron ligated to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​to a target endogenous pre-mRNA, the nucleic acid molecule enabling a 3' substitution of the target endogenous pre-mRNA, and the resulting chimeric RNA transcript may contain a 5' portion of the target endogenous pre-mRNA and a 3' portion of the exogenous RNA.

[0205] In one embodiment, one or more cells may be present within the subject. In one embodiment, the subject may be diagnosed with or suspected of having a genetic disorder or defect. In one embodiment, the disorder or defect may include any disorder or defect caused by a disclosed gene, or a missing, deficient, and / or mutant gene. In one embodiment, the subject may be a subject requiring treatment for a disclosed disorder or defect (e.g., a genetic disorder or defect). Genetic disorders and defects are discussed extensively herein.

[0206] In one embodiment, the disclosed method for generating a chimeric RNA molecule may further include identifying a target that requires the generation of the chimeric RNA molecule.

[0207] In one aspect of a disclosed method for generating a chimeric RNA molecule, the disclosed vector or 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 the following routes: oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, intrauterine administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraotoral administration, otological administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including, but not limited to, injectable forms such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration may also include intra-arterial administration or administration via the hepatic portal vein (HPV). The administration of the disclosed therapeutic agents, disclosed pharmaceutical compositions, or combinations thereof may include direct administration to the central nervous system (CNS) (e.g., intraparenchymal, intraventricular, intracisional subarachnoid space, intra-subarachnoid space (lumbar spine), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or the peripheral nervous system (PNS). Administration may be continuous or intermittent.

[0208] In one embodiment, administration may be carried out by one or more ex vivo methods, such as an ex vivo perfusion protocol. In one embodiment, the ex vivo perfusion protocol may be used on one or more cells, tissues and / or organs affected by a genetic disease or disorder obtained from a subject. In one embodiment, one or more cells and / or one or more tissues and / or one or more organs may be obtained from a subject in need and subjected to an ex vivo perfusion and / or treatment and / or contact protocol, and returned to the subject in need, and one or more cells may produce chimeric RNA transcripts.

[0209] In one embodiment, a disclosed method for generating a chimeric RNA molecule in a cell may include verifying a transsplicing event and / or the generation of a chimeric RNA molecule. Verification of a transsplicing event and / or the generation of a chimeric RNA molecule can be achieved using methods and techniques known in the art (e.g., sequencing, Northern blotting, FISH, PCR, RNA-Seq, 3'RACE, 5'RACE, etc.).

[0210] In one embodiment, a disclosed method for generating a chimeric RNA molecule may include preparing a disclosed 5' substitution construct, a disclosed 3' substitution construct, a disclosed nonviral vector or a disclosed viral vector, a disclosed nucleic acid molecule, a disclosed pharmaceutical formulation, or any combination thereof.

[0211] In one embodiment, the disclosed method may be carried out ex vivo. In one embodiment, administration may be carried out by one or more ex vivo methods, such as an ex vivo perfusion protocol. In one embodiment, the ex vivo perfusion protocol may be used on one or more cells, tissues and / or organs affected by a genetic disease or disorder obtained from a subject. In one embodiment, one or more cells and / or one or more tissues and / or one or more organs may be obtained from a subject in need and subjected to an ex vivo perfusion and / or treatment and / or contact protocol, and returned to the subject in need, and one or more cells may produce chimeric RNA transcripts.

[0212] In one aspect, the disclosed method can restore the activity and / or functionality of a missing, incomplete, and / or mutant protein or enzyme, and can include a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to an existing level such as a pre-treatment level. In one 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 an existing level such as a pre-treatment level. In one aspect, the restoration can be measured against a control level or a reference level (e.g., determined using one or more subjects without the missing, incomplete, and / or mutant protein or enzyme). In one aspect, the restoration can be partial or incomplete. In one aspect, the restoration can be complete or nearly complete such that the levels of expression, activity, and / or functionality are similar to those of the wild type or control level, and / or the expression level of one or more protein-coding genes can be restored to the wild type, normal, or control expression level.

[0213] In one aspect, the disclosed method can restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation. In one aspect, the disclosed method can restore the functionality and / or structural integrity of a missing, incomplete, and / or mutant protein or enzyme (e.g., such as that encoded by one of the genes presented above).

[0214] In one aspect, restoring one or more characteristics of cellular homeostasis and / or cellular functionality includes the following: (i) correcting cellular starvation in one or more cell types, (ii) normalizing aspects of the autophagy pathway (e.g., correcting, preventing, reducing, and / or improving autophagy, etc.), (iii) improving, enhancing, restoring, and / or maintaining mitochondrial functionality and / or structural integrity, (iv) improving, enhancing, restoring, and / or maintaining organelle functionality and / or structural integrity, (v) correcting enzyme dysregulation, (vi) reversing, inhibiting, preventing, stabilizing, and / or delaying the progression of the multi-organ system manifestations of a genetic disease or disorder, (vii) reversing, inhibiting, preventing, stabilizing, and / or delaying the progression of a genetic disease or disorder, (viii) and may include one or more of any combination thereof. In one aspect, restoring one or more characteristics of cellular homeostasis may include improving, enhancing, restoring, and / or maintaining one or more characteristics of the structural and / or functional integrity of the cell.

[0215] In one aspect, the disclosed chimeric RNAs can be particularly useful in methods of treating subjects having autosomal dominant genetic diseases or disorders such as, for example, progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary elliptocytosis, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, marble bone disease, Marfan syndrome, neurofibromatosis, adult polycystic kidney, protein C deficiency, osteogenesis imperfecta, Treacher Collins syndrome, tuberous sclerosis, von Willebrand disease, and the like.

[0216] D. Methods of Treating a Genetic Disease or Genetic Disorder Disclosed herein are methods for treating genetic disorders or disorders, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a disclosed non-viral vector or a disclosed viral vector or a pharmaceutical formulation thereof to a subject in need thereof, the resulting chimeric RNA molecule being able to restore one or more characteristics of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation.

[0217] Disclosed herein is a method for treating a genetic disorder or impairment, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of the disclosed non-viral vector or the disclosed viral vector or a pharmaceutical formulation thereof to a subject in need thereof, the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes provided above).

[0218] Disclosed herein are methods for treating genetic disorders or disorders, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule comprising an exogenous RNA to be transspliced ​​into an endogenous pre-mRNA targeted to a target subject in need thereof, wherein the nucleic acid molecule comprises a 5' hemyintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures, the resulting chimeric RNA molecule may restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or the resulting chimeric RNA molecule may restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above).

[0219] Disclosed herein are methods for treating genetic disorders or disorders, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule containing an exogenous RNA to be transspliced ​​into an endogenous pre-mRNA targeted to a target subject in need thereof, the nucleic acid molecule comprising one or more RNA structures comprising a 5' hemyintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one of the sequences of SEQ ID NOs. 01 to SEQ ID NOs. 09, the resulting chimeric RNA molecule may restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or the resulting chimeric RNA molecule may restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above).

[0220] Disclosed herein are methods for treating genetic disorders or disorders, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule containing an exogenous RNA to be transspliced ​​into a targeted endogenous pre-mRNA, the nucleic acid molecule comprising a 5' hemyintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures, the nucleic acid molecule enabling a 5' substitution of the targeted endogenous pre-mRNA, the resulting chimeric RNA molecule may restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or the resulting chimeric RNA molecule may restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above).

[0221] Disclosed herein are methods for treating genetic disorders or disorders, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule containing an exogenous RNA to be transspliced ​​into a targeted endogenous pre-mRNA, the nucleic acid molecule comprising one or more RNA structures comprising a 5' hemyintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one of the sequences of SEQ ID NOs. 01 to SEQ ID NOs. 09, the nucleic acid molecule enabling a 5' substitution of the targeted endogenous pre-mRNA, the resulting chimeric RNA molecule may restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or the resulting chimeric RNA molecule may restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above).

[0222] Disclosed herein are methods for treating genetic disorders or disorders, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing one or more RNA structures to a subject in need thereof, wherein the nucleic acid molecule comprises one or more RNA targeting motifs, a 3' hemyintron linked to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​to a targeted endogenous pre-mRNA, the resulting chimeric RNA molecule may restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or the resulting chimeric RNA molecule may restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above).

[0223] Disclosed herein are methods for treating a genetic disorder or impairment, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector to a subject in need thereof, the nucleic acid molecule comprising one or more RNA structures containing one of the sequences of SEQ ID NOs: 01 to 09, the nucleic acid molecule comprising one or more RNA targeting motifs, a 3' hemiintron linked to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​to a targeted endogenous pre-mRNA, the resulting chimeric RNA molecule may restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or the resulting chimeric RNA molecule may restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above).

[0224] Disclosed herein are methods for treating a genetic disorder or impairment, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing one or more RNA structures to a subject in need thereof, wherein the nucleic acid molecule comprises one or more RNA targeting motifs, a 3' hemyintron linked to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​to a targeted endogenous pre-mRNA, the nucleic acid molecule enabling a 3' substitution of the targeted endogenous pre-mRNA, the resulting chimeric RNA molecule may restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or the resulting chimeric RNA molecule may restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above).

[0225] Disclosed herein is a method for treating a genetic disorder or impairment, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector to a subject in need thereof, the nucleic acid molecule comprising one or more RNA structures containing one of the sequences of SEQ ID NOs: 01 to 09, the nucleic acid molecule comprising one or more RNA targeting motifs, a 3' hemiintron linked to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​to a targeted endogenous pre-mRNA, the nucleic acid molecule enabling a 3' substitution of the targeted endogenous pre-mRNA. The resulting chimeric RNA molecule may restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or the resulting chimeric RNA molecule may restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above).

[0226] Disclosed herein are methods for treating genetic disorders or disorders, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral vector, viral vector, AAV particles, or a pharmaceutical formulation thereof to a subject in need thereof, comprising (i) one or more 5' substitution constructs, (ii) one or more 3' substitution constructs, or (iii) one or more 5' substitution constructs and / or one or more 3' substitution constructs, the resulting chimeric RNA molecule can restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above).

[0227] In one embodiment of the disclosed treatment method, the disclosed 5' substitution construct to be used in combination with one or more other substitution constructs may include any disclosed 5' substitution construct. In one embodiment, the disclosed 5' substitution construct comprises (i) a nucleic acid molecule comprising an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures; (ii) a nucleic acid molecule comprising an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures comprising any one sequence of SEQ ID NOs: 01 to SEQ ID NOs: 09; and (iii) an exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA A nucleic acid molecule comprising sex RNA, a 5' hemiintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures, which enables the 5' substitution of the targeted endogenous pre-mRNA; or (iv) a nucleic acid molecule comprising exogenous RNA to be transspliced ​​into the targeted endogenous pre-mRNA, a 5' hemiintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures comprising any one sequence of SEQ ID NOs. 01 to SEQ ID NOs. 09, which enables the 5' substitution of the targeted endogenous pre-mRNA; or (v) any combination thereof.

[0228] In one embodiment of the disclosed treatment method, the disclosed 3' substitution construct to be used in combination with one or more other substitution constructs may include any disclosed 3' substitution construct. In one embodiment, the disclosed 3' substitution construct comprises (i) a nucleic acid molecule comprising one or more RNA structures, one or more RNA targeting motifs, a 3' hemiintron linked to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA; (ii) a nucleic acid molecule comprising one or more RNA structures, one or more RNA targeting motifs, a 3' hemiintron linked to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​into a target endogenous pre-mRNA; and (iiii) one or more RNA structures, one or more RNA targeting motifs, The nucleic acid molecule may include (iv) a nucleic acid molecule that enables the 3' substitution of the target endogenous pre-mRNA, comprising a 3' hemiintron ligated to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​into the target endogenous pre-mRNA, or (v) any combination thereof.

[0229] In one aspect of the disclosed treatment method, the subject may have a genetic disorder or disability related to and / or caused by one or more disclosed genes, including the genes disclosed in Section VII(B)(1).

[0230] In one aspect of the disclosed treatment method, the expression of the disclosed protein-coding gene can be restored and / or returned to wild-type, normal, or control expression levels. In one aspect, the disclosed method for treating a genetic disorder or impairment can restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme. In one aspect, the disclosed treatment method may include restoring one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation. In one aspect, restoring one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation includes restoring the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme. In one embodiment, restoring one or more features of cellular homeostasis and / or cellular functionality may include: (i) correcting cellular starvation in one or more cell types; (ii) normalizing aspects of autophagy pathways (e.g., correcting, preventing, reducing, and / or improving autophagy); (iii) improving, enhancing, restoring, and / or maintaining mitochondrial functionality and / or structural integrity; (iv) improving, enhancing, restoring, and / or maintaining organelle functionality and / or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the progression of multi-organ manifestations of a genetic disorder or impairment; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the progression of a genetic disorder or impairment; and (viii) one or more of any combination thereof. In one embodiment, restoring one or more features of cellular homeostasis may include improving, enhancing, restoring, and / or maintaining one or more features of the structural and / or functional integrity of the cell.In one embodiment, restoring the activity and / or functionality of a deficient, incomplete, and / or mutant protein or enzyme may include restoration of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount compared to an existing level, such as a pre-treatment level. In one embodiment, the amount of restoration may be 10%–20%, 20%–30%, 30%–40%, 40%–50%, 50%–60%, 60%–70%, 70%–80%, 80%–90%, or 90%–100% greater than an existing level, such as a pre-treatment level. In one embodiment, the restoration may be measured against a control level or baseline level (determined, for example, using one or more subjects that do not have the deficient, incomplete, and / or mutant protein or enzyme). In one embodiment, the restoration may be partial or incomplete. In one embodiment, recovery may be complete or near-complete, such that the levels of expression, activity, and / or functionality are similar to those of the wild type or control level.

[0231] In one embodiment, the disclosed treatment method may further include monitoring metabolic and / or physiological improvements in the subject after an administration step and / or multiple administration steps. In one embodiment, a clinician may determine and / or decide on the metabolic and / or physiological state of the subject over time to identify one or more improvements and / or one or more declines. In one embodiment of the disclosed method, a clinician may use the metabolic and / or physiological state of the subject, and / or trends in the metabolic and / or physiological state of the subject, and / or trends, to make treatment decisions and / or modify one embodiment of the disclosed method and / or continue treatment for the subject and / or continue administering the disclosed AAV particles, disclosed vectors, disclosed nucleic acid molecules, disclosed pharmaceutical formulations, disclosed therapeutic agents and / or disclosed immunomodulators, or any combination thereof. In one embodiment, metabolic and / or physiological data may inform the clinician and the treatment plan.

[0232] In one aspect of the disclosed treatment method, techniques for monitoring, measuring, and / or evaluating the restoration of one or more characteristics of cellular homeostasis and / or cellular functionality may include both qualitative (or subjective) means and quantitative (or objective) means. These means are known to those skilled in the art. For example, representative regulatory variables and sensors related to systemic homeostasis are shown below. [Table 6]

[0233] In one aspect, the disclosed treatment method may further include subjecting the subject to one or more invasive or non-invasive diagnostic evaluations. Diagnostic evaluations are known in the art. In one aspect, the disclosed non-invasive diagnostic evaluations can include X-rays, computed tomography (CT) scans, magnetic resonance imaging (MRI) scans, ultrasounds, positron emission tomography (PET) scans, or any combination thereof. In one aspect, the disclosed invasive diagnostic evaluations can include tissue biopsies or exploratory surgeries.

[0234] In one aspect, the disclosed treatment method can be used to repair diseased cell types and / or dysfunctional cell types within cells, tissues, and / or organs having one or more diseases and / or disorders.

[0235] In one aspect, the disclosed treatment method can be used to improve and / or enhance the quality of life of the subject when compared to the pre-treatment level. In one aspect, using the disclosed treatment method, the quality of life of the subject can be improved by at least 50% when compared to the pre-treatment quality of life of the subject.

[0236] In one embodiment, the disclosed treatment method can be used to improve and / or enhance the quality of life of a subject compared to a pre-treatment level. In one embodiment, the disclosed treatment method can be used to improve the quality of life of a subject by at least 50% compared to a pre-treatment level.

[0237] In one embodiment, the disclosed treatment method can be used to alleviate and / or reduce one or more symptoms associated with and / or related to a genetic disorder and / or genetic defect in a subject. In one embodiment, the disclosed treatment method can be used to prevent the development of undesirable physiological changes, diseases, pathological symptoms or disorders in a subject. In one embodiment, the disclosed treatment method can be used to inhibit physiological changes, diseases, pathological symptoms or disorders in a subject, i.e., to halt their progression. In one embodiment, the disclosed treatment method can be used to alleviate physiological changes, diseases, pathological symptoms or disorders in a subject, i.e., to induce disease regression.

[0238] In one embodiment of the disclosed treatment method, administration to a subject may include 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 one aspect of the disclosed treatment method, administering the disclosed nucleic acid molecule, the disclosed vector, the disclosed AAV particle, the disclosed pharmaceutical preparation, or any combination thereof can include one or more routes. Such methods are well known to those skilled in the art and include the following routes: oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, intrauterine administration, intrahepatic administration, vaginal administration, ophthalmic administration, otological administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including but not limited to those by injectable forms such as intravenous administration, intrathecal administration, intraarterial administration, intramuscular administration, and subcutaneous administration. Administration can also include intrahepatic arterial administration or administration via the hepatic portal vein (HPV). Administration of the disclosed nucleic acid molecule, the disclosed vector, the disclosed AAV particle, the disclosed pharmaceutical preparation, or any combination thereof can include direct administration to the central nervous system (CNS) (e.g., within the parenchyma, intraventricularly, subarachnoid space within the cerebral cistern, subarachnoid space (lumbar), deep gray matter delivery, convection-enhanced delivery to the deep gray matter) or the peripheral nervous system (PNS). Administration can be continuous or intermittent.

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

[0241] In one aspect of the disclosed method for treating a genetic disease or disorder, a therapeutically effective amount of the disclosed vector or the disclosed pharmaceutical preparation is about 1×10 10 vg / kg to about 2×10 14 vg / kg can be included. In one aspect, for example, a therapeutically effective amount of the disclosed vector or the disclosed pharmaceutical preparation is about 1×10 11 vg / kg to about 8×10 13 vg / kg or about 1×10 12 vg / kg to about 8×10 13 vg / kg can be included. In one aspect, a therapeutically effective amount of the disclosed vector or the disclosed pharmaceutical preparation is about 1×10 13 vg / kg to about 6×1013 The dosage may include vg / kg. In one embodiment, the disclosed vector or disclosed pharmaceutical formulation in a therapeutically effective amount contains at least about 1 × 10⁶ 10 vg / kg, at least about 5 × 10⁻⁶ 10 vg / kg, at least about 1 × 10⁻⁶ 11 vg / kg, at least about 5 × 10⁻⁶ 11 vg / kg, at least about 1 × 10⁻⁶ 12 vg / kg, at least about 5 × 10⁻⁶ 12 vg / kg, at least about 1 × 10⁻⁶ 13 vg / kg, at least about 5 × 10⁻⁶ 13 vg / kg, or at least about 1 × 10⁻⁶ 14 The dosage may include vg / kg. In one embodiment, the disclosed vector or disclosed pharmaceutical formulation in a therapeutically effective amount is approximately 1 × 10⁻⁶ 10 vg / kg or less, approximately 5 × 10 10 vg / kg or less, approximately 1 × 10⁻⁶ 11 vg / kg or less, approximately 5 × 10 11 vg / kg or less, approximately 1 × 10⁻⁶ 12 vg / kg or less, approximately 5 × 10 12 vg / kg or less, approximately 1 × 10⁻⁶ 13 vg / kg or less, approximately 5 × 10 13 vg / kg or less, or about 1 × 10⁻⁶ 14 It may include doses of vg / kg or less. In one embodiment, the disclosed vector or disclosed pharmaceutical formulation in a therapeutically effective amount is approximately 1 × 10⁻⁶ 12 vg / kg or approximately 1 × 10⁻⁶ 11 Doses may include vg / kg. In one embodiment, a disclosed therapeutically effective vector or disclosed pharmaceutical formulation may be administered in single doses or multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses), depending on the need for the desired therapeutic outcome.

[0242] In one embodiment, the disclosed vector or disclosed pharmaceutical formulation in a therapeutically effective amount totals approximately 1 × 10⁶ per subject. 12 vg~Approximately 1 x 10 per target17 The range may include vg. In one embodiment, the disclosed vector or disclosed pharmaceutical formulation in a therapeutically effective amount totals approximately 1 × 10 per subject. 12 vg, approximately 1 x 10 per target in total 13 vg, approximately 1 x 10 per target in total 14 vg, approximately 1 x 10 per target in total 15 vg, approximately 1 x 10 per target in total 16 vg, or approximately 1 x 10 per subject in total 17 It can include the range of vg.

[0243] In one embodiment, the disclosed vector or disclosed pharmaceutical formulation in a therapeutically effective amount is approximately 1 × 10⁻⁶ 6 DRP / mL ~ approximately 1 x 10 14 It may contain DRP / mL. In one embodiment, the disclosed vector or disclosed pharmaceutical formulation in a therapeutically effective amount is approximately 1 × 10⁻⁶ 6 DRP / mL, approximately 1 x 10 7 DRP / mL, approximately 1 x 10 8 DRP / mL, approximately 1 x 10 9 DRP / mL, approximately 1 x 10 10 DRP / mL, approximately 1 x 10 11 DRP / mL, approximately 1 x 10 12 DRP / mL, approximately 1 x 10 13 DRP / mL, or approximately 1 × 10⁻⁶ 14 It may include DRP / mL. In one embodiment, the disclosed therapeutically effective amount of the vector or disclosed pharmaceutical formulation may include a range determined by those skilled in the art.

[0244] In one embodiment, a disclosed method for treating a genetic disorder or impairment may further include administering a therapeutically effective dose of a therapeutic agent to a target. The therapeutic agent may be any disclosed agonist that produces a desired clinical outcome.

[0245] In one embodiment, a disclosed method for treating a genetic disorder or impairment may further include monitoring the subject for adverse effects. In one embodiment, if no adverse effects occur, the method may further include continuing treatment of the subject. In one embodiment, if adverse effects occur, the method may further include modifying the treatment process. Methods for monitoring the health of a subject may include both subjective and objective criteria (as discussed above). Such methods are known to those skilled in the art.

[0246] In one embodiment, the disclosed treatment method may further include administering a therapeutically effective amount of an agonist capable of correcting one or more characteristics of a dysregulated metabolic or enzymatic pathway to a target. In one embodiment, such an agonist may include an enzyme for enzyme replacement therapy. In one embodiment, the disclosed enzyme may substitute any enzyme in a dysregulated or dysfunctional metabolic or enzymatic pathway. In one embodiment, the disclosed treatment method may include substituting one or more enzymes in a dysregulated or dysfunctional metabolic pathway.

[0247] In one embodiment, a disclosed method for treating a genetic disorder or impairment may further include administering one or more immunomodulators. In one embodiment, the disclosed immunomodulator may be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or a combination thereof. In one embodiment, the disclosed immunomodulator may be bortezomib or SVP-rapamycin. In one embodiment, the disclosed immunomodulator may be tacrolimus. In one embodiment, the disclosed immunomodulator (e.g., methotrexate) may be administered transiently in low to high doses. In one embodiment, the disclosed immunomodulator may be administered in doses of approximately 0.1 mg / kg body weight to approximately 0.6 mg / kg body weight. In one embodiment, the disclosed immunomodulator may be administered in doses of approximately 0.4 mg / kg body weight. In one embodiment, the disclosed immunomodulator may be administered at a daily dose of approximately 0.4 mg / kg body weight for 3 to 5 cycles or more, for a maximum of 3 days per cycle. In one embodiment, the disclosed immunomodulator can be administered at a daily dose of approximately 0.4 mg / kg body weight for at least 3 cycles, with each cycle lasting 3 days. In one embodiment, a person skilled in the art can determine an appropriate number of cycles. In one embodiment, the disclosed immunomodulator can be administered as many times as necessary to achieve the desired clinical effect.

[0248] In one embodiment, the disclosed immunomodulator may be administered orally about one hour before the disclosed therapeutic agent. In one embodiment, the disclosed immunomodulator may be administered subcutaneously about 15 minutes before the disclosed therapeutic agent. In one embodiment, the disclosed immunomodulator may be administered together with the disclosed therapeutic agent. In one embodiment, the disclosed immunomodulator may be administered orally about one hour or several days before the disclosed nucleic acid molecule, the disclosed vector, the disclosed pharmaceutical formulation, or a combination thereof. In one embodiment, the disclosed immunomodulator may be administered subcutaneously about 15 minutes or several days before the disclosed nucleic acid molecule, the disclosed vector, the disclosed pharmaceutical formulation, or a combination thereof. In one embodiment, the disclosed immunomodulator may be administered together with the disclosed nucleic acid molecule, the disclosed vector, the disclosed pharmaceutical formulation, or a combination thereof.

[0249] In one embodiment, a disclosed method for treating a genetic disorder or impairment may further include administering one or more proteasome inhibitors (e.g., bortezomib, carfilzomib, marizomib, ixazomib, and oprozomib). In one embodiment, the proteasome inhibitor may be an agent that acts on plasma cells (e.g., daratumumab). In one embodiment, the agonist acting on plasma cells may 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, prelixafor, ixazomib citrate, pamidronate disodium, panobinostat, prelixafor, pomalidomide, pomalidomide, lenalidomide, selinexol, thalidomide, thalidomide, bortezomib, selinexol, zoledronic acid, or zoledronic acid.

[0250] In one embodiment, the disclosed method for addressing stability may further include administering one or more proteasome inhibitors or plasma cell-acting agents before administering the disclosed nucleic acid molecule, the disclosed vector, the disclosed AAV particles, the disclosed pharmaceutical formulation, or any combination thereof. In one embodiment, the disclosed treatment method may include administering one or more proteasome inhibitors or plasma cell-acting agents along with administering the disclosed nucleic acid molecule, the disclosed vector, the disclosed AAV particles, the disclosed pharmaceutical formulation, or any combination thereof. In one embodiment, the disclosed treatment method may include administering one or more proteasome inhibitors or plasma cell-acting agents after administering the disclosed nucleic acid molecule, the disclosed vector, the disclosed AAV particles, the disclosed pharmaceutical formulation, or any combination thereof. In one embodiment, the disclosed treatment method may further include administering one or more proteasome inhibitors more than once. In one embodiment, the disclosed treatment method may include repeated administration of one or more proteasome inhibitors over time.

[0251] In one embodiment, a disclosed method for treating a genetic disorder or impairment may further include administering one or more immunosuppressants. In one embodiment, the immunosuppressants may be, but are not limited to, azathioprine, methotrexate, sirolimus, anti-thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate-tomofetil (MMF), steroids, or combinations thereof. In one embodiment, a disclosed treatment method may include administering one or more immunosuppressants in more than one dose. In one embodiment, a disclosed method may include administering one or more immunosuppressants repeatedly over time. In one embodiment, a disclosed treatment method may include administering compounds that target or alter antigen presentation, humoral immune responses, cell-mediated immune responses, or innate immune responses.

[0252] In one embodiment, a disclosed method for treating a genetic disorder or impairment may further include administering a compound that exerts a therapeutic effect on B cells, and / or a compound that targets or alters antigen presentation, humoral immune response, or cell-mediated immune response. In one embodiment, the disclosed compound may be rituximab, methotrexate, intravenous gamma globulin, anti-CD4 antibody, anti-CD2 antibody, anti-FcRN antibody, BTK inhibitor, anti-IGF1R antibody, CD19 antibody (e.g., inebilizumab), anti-IL6 antibody (e.g., tocilizumab), anti-CD40 antibody, IL2 mutein, or a combination thereof. Also disclosed herein are Treg infusions (e.g., antigen-specific Treg cells against AAV) that can be administered as a means of aiding immune tolerance.

[0253] In one embodiment, the disclosed treatment method may further include administering lipid nanoparticles (LNPs). In one embodiment, LNPs may target organs. In one embodiment, LNPs may target the liver or the testes. For example, in one embodiment, LNP-encapsulated mRNA therapy for systemic delivery to a subject may have the potential to restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme.

[0254] In one embodiment, a disclosed method for treating a genetic disorder or impairment may further include treating a subject that has acquired or may acquire neutralizing antibodies (AB) to a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof. In one embodiment, treating a subject that has acquired or may acquire neutralizing antibodies may include plasma exchange and immunosuppression. In one embodiment, a disclosed method may include using immunosuppression to reduce T cell, B cell and / or plasma cell populations and to reduce the innate immune response, inflammatory response, and antibody levels overall. In one embodiment, a disclosed method may include administering an IgG degrading agent that depletes existing neutralizing antibodies. In one embodiment, a disclosed method may include administering IdeS or IdeZ, rapamycin, and / or SVP-rapamycin to a subject. In one embodiment, a disclosed treatment method may include administering tacrolimus. In one embodiment, the disclosed IgG degrading agent is a bacterial IdeS or IdeZ.

[0255] In one embodiment, the disclosed treatment method may include repeating the disclosed administration step, for example, repeated administration of a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immunomodulator, a disclosed proteasome inhibitor, a disclosed immunosuppressant, a disclosed compound that exerts a therapeutic effect on B cells, and / or a disclosed compound that targets or alters antigen presentation, humoral immune response, or cell-mediated immune response.

[0256] In one embodiment, the disclosed treatment method may include modifying one or more of the disclosed steps. For example, modifying one or more steps of the disclosed method may include modifying or changing one or more features or aspects of one or more steps in the disclosed method. For example, in one embodiment, the method may be modified by changing the amount of one or more 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 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 duration of administration of one or more 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.

[0257] In one embodiment, the disclosed treatment method may be modified by changing the amount of one or more disclosed therapeutic agents, disclosed immunomodulators, disclosed proteasome inhibitors, disclosed immunosuppressants, disclosed compounds that exert a therapeutic effect on B cells, and / or disclosed compounds that target or alter antigen presentation, humoral immune responses, or cell-mediated immune responses administered to a subject; or by changing the frequency of administration of one or more disclosed therapeutic agents, disclosed immunomodulators, disclosed proteasome inhibitors, disclosed immunosuppressants, disclosed compounds that exert a therapeutic effect on B cells, and / or disclosed compounds that target or alter antigen presentation, humoral immune responses, or cell-mediated immune responses administered to a subject.

[0258] In one embodiment, the disclosed treatment method may include the simultaneous administration of one or more 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 immunomodulators, one or more disclosed proteasome inhibitors, one or more disclosed immunosuppressants, one or more disclosed compounds that exert a therapeutic effect on B cells, one or more disclosed compounds that target or alter antigen presentation, humoral immune responses or cell-mediated immune responses, or any combination thereof. In one embodiment, the disclosed immunomodulators may be administered before or after the administration of the disclosed therapeutic agents.

[0259] In one embodiment, a disclosed method for treating a genetic disorder or impairment may further include generating a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof.

[0260] In one embodiment, the disclosed treatment method may further include gene editing of one or more related genes (e.g., a missing, incomplete, and / or mutant protein or enzyme), and editing includes, but is not limited to, single gene knockout, loss of function screening of multiple genes in one, gene knock-in, or a combination thereof.

[0261] In one embodiment, a disclosed method for treating a genetic disorder or impairment may further include administering an oligonucleotide therapeutic agent to a subject. The disclosed oligonucleotide therapeutic agent may include single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), antisense molecules, miRNA, morpholino, peptide nucleic acid (PNA), or analogs or conjugates thereof. In one embodiment, the disclosed oligonucleotide therapeutic agent may be an ASO or RNAi. In one embodiment, the disclosed oligonucleotide therapeutic agent may include one or more modifications at any applicable position. In one embodiment, the disclosed oligonucleotide therapeutic agent may include a CRISPR-based endonuclease. In one embodiment, the disclosed endonuclease may be Cas9. In one embodiment, the disclosed Cas9 may be derived from Staphylococcus aureus or Streptococcus pyogenes. In one embodiment, the disclosed treatment method may include administering the disclosed RNA therapeutic agent to a subject.

[0262] In one embodiment, a disclosed method for treating a genetic disorder or impairment may further include generating and / or validating one or more 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 one embodiment, a disclosed treatment method may inhibit and / or minimize one or more features of disease progression in a subject (e.g., the genetic disorder or impairment described above).

[0263] In one embodiment, the disclosed treatment method can delay and / or reduce one or more characteristics of disease progression in a subject (e.g., a genetic disorder or genetic defect as described above).

[0264] In one embodiment, the disclosed method may be particularly useful in treating subjects with autosomal dominant genetic disorders or disorders such as progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary ellipticosis, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, osteopetrosis, Marfan syndrome, neurofibromatosis, adult-onset polycystic kidney disease, protein C deficiency, osteogenesis imperfecta, Treacher Collins syndrome, tuberous sclerosis, and von Willebrand disease.

[0265] E. Methods to inhibit and / or minimize disease progression Disclosed herein are methods for inhibiting and / or minimizing disease progression, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a disclosed non-viral vector or a disclosed viral vector or a pharmaceutical formulation thereof to a subject in need thereof, the resulting chimeric RNA molecule being able to restore one or more characteristics of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation.

[0266] Disclosed herein are methods for inhibiting and / or minimizing disease progression, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of the disclosed non-viral vector or the disclosed viral vector or its pharmaceutical formulation to a subject in need thereof, the resulting chimeric RNA molecule being able to restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes provided above).

[0267] Disclosed herein are methods for inhibiting and / or minimizing disease progression, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule comprising an exogenous RNA to be transspliced ​​into an endogenous pre-mRNA targeted to a target subject, wherein the nucleic acid molecule comprises a 5' hemyintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures, the resulting chimeric RNA molecule may restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or the resulting chimeric RNA molecule may restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above).

[0268] Disclosed herein are methods for inhibiting and / or minimizing disease progression, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule comprising an exogenous RNA to be transspliced ​​into an endogenous pre-mRNA targeted to a target subject in need thereof, the nucleic acid molecule comprising one or more RNA structures comprising a 5' hemyintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one of the sequences of SEQ ID NOs. 01 to SEQ ID NOs. 09, the resulting chimeric RNA molecule may restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or the resulting chimeric RNA molecule may restore the functionality and / or structural integrity of a missing, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above).

[0269] Disclosed herein are methods for inhibiting and / or minimizing disease progression, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule containing an exogenous RNA to be transspliced ​​into a targeted endogenous pre-mRNA, the nucleic acid molecule comprising a 5' hemyintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures, the nucleic acid molecule enabling a 5' substitution of the targeted endogenous pre-mRNA, the resulting chimeric RNA molecule may restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or the resulting chimeric RNA molecule may restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above).

[0270] Disclosed herein are methods for inhibiting and / or minimizing disease progression, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule containing an exogenous RNA to be transspliced ​​into a targeted endogenous pre-mRNA, the nucleic acid molecule comprising one or more RNA structures comprising a 5' hemyintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one of the sequences of SEQ ID NOs. 01 to SEQ ID NOs. 09, the nucleic acid molecule enabling a 5' substitution of the targeted endogenous pre-mRNA, the resulting chimeric RNA molecule may restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or the resulting chimeric RNA molecule may restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above).

[0271] Disclosed herein are methods for inhibiting and / or minimizing disease progression, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing one or more RNA structures to a subject requiring such action, the nucleic acid molecule comprising one or more RNA targeting motifs, a 3' hemyintron linked to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​to a targeted endogenous pre-mRNA, the resulting chimeric RNA molecule may restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or the resulting chimeric RNA molecule may restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above).

[0272] Disclosed herein are methods for inhibiting and / or minimizing disease progression, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector to a subject requiring such inhibition and / or minimization, the nucleic acid molecule comprising one or more RNA targeting motifs, a 3' hemi-intron linked to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​to a targeted endogenous pre-mRNA, the resulting chimeric RNA molecule may restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or the resulting chimeric RNA molecule may restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above).

[0273] Disclosed herein are methods for inhibiting and / or minimizing disease progression, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing one or more RNA structures to a subject requiring such action, the nucleic acid molecule comprising one or more RNA targeting motifs, a 3' hemyintron linked to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​to a targeted endogenous pre-mRNA, the nucleic acid molecule enabling a 3' substitution of the targeted endogenous pre-mRNA, the resulting chimeric RNA molecule may restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or the resulting chimeric RNA molecule may restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above).

[0274] Disclosed herein is a method for inhibiting and / or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector to a subject requiring such inhibition and / or minimization, the nucleic acid molecule comprising one or more RNA structures containing one or more sequences of SEQ ID NOs: 01 to 09, the nucleic acid molecule comprising one or more RNA targeting motifs, a 3' hemiintron linked to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​to a targeted endogenous pre-mRNA, the nucleic acid molecule enabling a 3' substitution of the targeted endogenous pre-mRNA, the resulting chimeric RNA molecule may restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or the resulting chimeric RNA molecule may restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above).

[0275] Disclosed herein are methods for inhibiting and / or minimizing disease progression, the methods comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral vector, viral vector, AAV particles, or a pharmaceutical formulation thereof to a subject requiring such action, comprising (i) one or more 5' substitution constructs, (ii) one or more 3' substitution constructs, or (iii) one or more 5' substitution constructs and / or one or more 3' substitution constructs, the resulting chimeric RNA molecule can restore one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme (e.g., one encoded by one of the genes presented above).

[0276] In one embodiment of a disclosed method for inhibiting and / or minimizing disease progression, the disclosed 5' substitution construct to be used in combination with one or more other substitution constructs may include any disclosed 5' substitution construct. In one embodiment, the disclosed 5' substitution construct comprises (i) a nucleic acid molecule comprising an exogenous RNA to be transspliced ​​into a targeted endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures; (ii) a nucleic acid molecule comprising an exogenous RNA to be transspliced ​​into a targeted endogenous pre-mRNA, a 5' hemiintron ligated to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures comprising any one sequence of SEQ ID NOs: 01 to SEQ ID NOs: 09; and (iii) an exogenous RNA to be transspliced ​​into a targeted endogenous pre-mRNA A nucleic acid molecule comprising sex RNA, a 5' hemiintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures, which enables the 5' substitution of the targeted endogenous pre-mRNA; or (iv) a nucleic acid molecule comprising exogenous RNA to be transspliced ​​into the targeted endogenous pre-mRNA, a 5' hemiintron linked to the exogenous RNA to be transspliced, one or more RNA targeting motifs, and one or more RNA structures comprising any one sequence of SEQ ID NOs. 01 to SEQ ID NOs. 09, which enables the 5' substitution of the targeted endogenous pre-mRNA; or (v) any combination thereof.

[0277] In one embodiment of a disclosed method for inhibiting and / or minimizing disease progression, a disclosed 3' substitution construct to be used in combination with one or more other substitution constructs may include any disclosed 3' substitution construct. In one embodiment, a disclosed 3' substitution construct is a nucleic acid molecule comprising (i) one or more RNA structures, one or more RNA targeting motifs, a 3' hemiintron linked to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​into a targeted endogenous pre-mRNA; (ii) a nucleic acid molecule comprising one or more RNA structures, one or more RNA targeting motifs, a 3' hemiintron linked to an exogenous RNA to be transspliced, and an exogenous RNA to be transspliced ​​into a targeted endogenous pre-mRNA; and (iiii) one or more RNA structures, one or more RNA targeting motifs, The nucleic acid molecule may include (iv) a nucleic acid molecule that enables the 3' substitution of the target endogenous pre-mRNA, comprising a 3' hemiintron ligated to the exogenous RNA to be transspliced, and the exogenous RNA to be transspliced ​​into the target endogenous pre-mRNA, or (v) any combination thereof.

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

[0279] In one aspect of a disclosed method for inhibiting and / or minimizing disease progression, the expression of a disclosed protein-coding gene can be restored and / or returned to wild-type, normal, or control expression levels. In one aspect of a method for inhibiting and / or minimizing the progression of a genetic disease or disorder, a disclosed method for treating a genetic disease or disorder can restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme. In one aspect, a disclosed method for inhibiting and / or minimizing disease progression may include restoring one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation. In one aspect, restoring one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation includes restoring the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme. In one embodiment, restoring one or more features of cellular homeostasis and / or cellular functionality may include: (i) correcting cellular starvation in one or more cell types; (ii) normalizing aspects of autophagy pathways (e.g., correcting, preventing, reducing, and / or improving autophagy); (iii) improving, enhancing, restoring, and / or maintaining mitochondrial functionality and / or structural integrity; (iv) improving, enhancing, restoring, and / or maintaining organelle functionality and / or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the progression of multi-organ manifestations of a genetic disorder or impairment; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the progression of a genetic disorder or impairment; and (viii) one or more of any combination thereof. In one embodiment, restoring one or more features of cellular homeostasis may include improving, enhancing, restoring, and / or maintaining one or more features of the structural and / or functional integrity of the cell.In one embodiment, restoring the activity and / or functionality of a deficient, incomplete, and / or mutant protein or enzyme may include restoration of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount compared to an existing level, such as a pre-treatment level. In one embodiment, the amount of restoration may be 10%–20%, 20%–30%, 30%–40%, 40%–50%, 50%–60%, 60%–70%, 70%–80%, 80%–90%, or 90%–100% greater than an existing level, such as a pre-treatment level. In one embodiment, the restoration may be measured against a control level or baseline level (determined, for example, using one or more subjects that do not have the deficient, incomplete, and / or mutant protein or enzyme). In one embodiment, the restoration may be partial or incomplete. In one embodiment, recovery may be complete or near-complete, such that the levels of expression, activity, and / or functionality are similar to those of the wild type or control level.

[0280] In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression may further include monitoring metabolic and / or physiological improvements in the subject after a dose step and / or multiple dose steps. In one embodiment, a clinician may determine and / or decide on the metabolic and / or physiological state of the subject over time to identify one or more improvements and / or one or more declines. In one embodiment of the disclosed method, a clinician may use the metabolic and / or physiological state of the subject, and / or trends in the metabolic and / or physiological state of the subject, and / or trends, to make treatment decisions and / or modify one embodiment of the disclosed method and / or continue treatment for the subject and / or continue administering the disclosed AAV particles, disclosed vectors, disclosed nucleic acid molecules, disclosed pharmaceutical formulations, disclosed therapeutic agents and / or disclosed immunomodulators, or any combination thereof. In one embodiment, metabolic and / or physiological data may inform the clinician and the treatment plan.

[0281] In one embodiment of a disclosed method for inhibiting and / or minimizing disease progression, techniques for monitoring, measuring, and / or evaluating the restoration of one or more features of cellular homeostasis and / or cellular functionality may include both qualitative (or subjective) and quantitative (or objective) means. These means are known to those skilled in the art. For example, representative regulators and sensors related to systemic homeostasis are shown below.

[0282] In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression may further include subjecting a subject to one or more invasive or non-invasive diagnostic evaluations. Diagnostic evaluations are known in the art. In one embodiment, the disclosed non-invasive diagnostic evaluation may include radiography, computed tomography (CT) scans, magnetic resonance imaging (MRI) scans, ultrasound, positron emission tomography (PET) scans, or any combination thereof. In one embodiment, the disclosed invasive diagnostic evaluation may include tissue biopsy or exploratory surgery.

[0283] In one embodiment, diseased cell types and / or dysfunctional cell types in cells, tissues, and / or organs having one or more diseases and / or disorders can be repaired using a disclosed method that inhibits and / or minimizes disease progression.

[0284] In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression can be used to improve and / or enhance the quality of life of a subject compared to a pre-treatment level. In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression can be used to improve and / or enhance the quality of life of a subject compared to a pre-treatment level. In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression can be used to improve and / or enhance the quality of life of a subject compared to a pre-treatment level. In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression can be used to improve the quality of life of a subject compared to a pre-treatment level.

[0285] In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression can be used to mitigate and / or reduce one or more symptoms associated with and / or related to a genetic disorder and / or genetic defect in a subject. In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression can be used to prevent the occurrence of undesirable physiological changes, diseases, pathological symptoms or disorders in a subject. In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression can be used to inhibit physiological changes, diseases, pathological symptoms or disorders in a subject, i.e., to halt their progression. In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression can be used to mitigate physiological changes, diseases, pathological symptoms or disorders in a subject, i.e., to induce disease regression.

[0286] In one embodiment of a disclosed method for inhibiting and / or minimizing disease progression, administration to a subject may include 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 one embodiment of a disclosed method for inhibiting and / or minimizing disease progression, administration of a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof may include one or more routes. Such methods are well known to those skilled in the art and include the following routes: oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, intrauterine administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraotoral administration, otological administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including, but not limited to, injectable forms such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration may also include intra-arterial administration or administration via the hepatic portal vein (HPV). Administration of the disclosed nucleic acid molecules, disclosed vectors, disclosed AAV particles, disclosed pharmaceutical formulations, or any combination thereof may include direct administration to the central nervous system (CNS) (e.g., intraparenchymal, intraventricular, intracisional subarachnoid space, intra-subarachnoid space (lumbar), deep gray matter delivery, convective-enhanced delivery to deep gray matter) or the peripheral nervous system (PNS). Administration may be continuous or intermittent.

[0288] In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression may employ multiple routes of administration to a subject. In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression may employ a first route of administration which may be the same as or different from a second and / or subsequent route of administration.

[0289] In one aspect of a disclosed method for inhibiting and / or minimizing the progression of a genetic disorder or disability, a therapeutically effective amount of the disclosed vector or disclosed pharmaceutical formulation is approximately 1 × 10⁻⁶ 10 vg / kg ~ approx. 2×10 14 The range may include vg / kg. In one embodiment, for example, the disclosed vector or disclosed pharmaceutical formulation in a therapeutically effective amount is approximately 1 × 10⁻⁶ 11 vg / kg ~ approx. 8×10 13 vg / kg or approximately 1 × 10⁻⁶ 12vg / kg ~ approx. 8×10 13 The dosage may include vg / kg. In one embodiment, the disclosed vector or disclosed pharmaceutical formulation in a therapeutically effective amount is approximately 1 × 10⁻⁶ 13 vg / kg ~ approx. 6×10 13 The dosage may include vg / kg. In one embodiment, the disclosed vector or disclosed pharmaceutical formulation in a therapeutically effective amount contains at least about 1 × 10⁶ 10 vg / kg, at least about 5 × 10⁻⁶ 10 vg / kg, at least about 1 × 10⁻⁶ 11 vg / kg, at least about 5 × 10⁻⁶ 11 vg / kg, at least about 1 × 10⁻⁶ 12 vg / kg, at least about 5 × 10⁻⁶ 12 vg / kg, at least about 1 × 10⁻⁶ 13 vg / kg, at least about 5 × 10⁻⁶ 13 vg / kg, or at least about 1 × 10⁻⁶ 14 The dosage may include vg / kg. In one embodiment, the disclosed vector or disclosed pharmaceutical formulation in a therapeutically effective amount is approximately 1 × 10⁻⁶ 10 vg / kg or less, approximately 5 × 10 10 vg / kg or less, approximately 1 × 10⁻⁶ 11 vg / kg or less, approximately 5 × 10 11 vg / kg or less, approximately 1 × 10⁻⁶ 12 vg / kg or less, approximately 5 × 10 12 vg / kg or less, approximately 1 × 10⁻⁶ 13 vg / kg or less, approximately 5 × 10 13 vg / kg or less, or about 1 × 10⁻⁶ 14 It may include doses of vg / kg or less. In one embodiment, the disclosed vector or disclosed pharmaceutical formulation in a therapeutically effective amount is approximately 1 × 10⁻⁶ 12 vg / kg or approximately 1 × 10⁻⁶ 11 Doses may include vg / kg. In one embodiment, the therapeutically effective amount of the disclosed vector or disclosed pharmaceutical formulation may be administered in single doses or multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses), depending on the need for the desired therapeutic outcome.

[0290] In one embodiment, the therapeutically effective amount of the disclosed vector or disclosed pharmaceutical formulation is approximately 1 × 10⁻¹⁶ per subject in total. 12 vg~Approximately 1 x 10 per target 17 The range may include vg. In one embodiment, the therapeutically effective amount of the disclosed vector or disclosed pharmaceutical formulation is approximately 1 × 10⁶ in total per subject. 12 vg, approximately 1 x 10 per target in total 13. vg, approximately 1 x 10 per target in total 14 vg, approximately 1 x 10 per target in total 15. vg, approximately 1 x 10 per target in total 16 vg, or approximately 1 x 10 per subject in total 17. It can include the range of vg.

[0291] In one embodiment, the therapeutically effective amount of the disclosed vector or disclosed pharmaceutical formulation is approximately 1 × 10⁻⁶ 6 DRP / mL ~ approximately 1 x 10 14 It may contain DRP / mL. In one embodiment, the therapeutically effective dose of the disclosed vector or disclosed pharmaceutical formulation is approximately 1 × 10⁻⁶ 6 DRP / mL, approximately 1 x 10 7 DRP / mL, approximately 1 x 10 8 DRP / mL, approximately 1 x 10 9 DRP / mL, approximately 1 x 10 10 DRP / mL, approximately 1 x 10 11 DRP / mL, approximately 1 x 10 12 DRP / mL, approximately 1 x 10 13 DRP / mL, or approximately 1 × 10⁻⁶ 14 It may contain DRP / mL. In one embodiment, the therapeutically effective dose of the disclosed vector or disclosed pharmaceutical formulation may include a range determined by those skilled in the art.

[0292] In one embodiment, a disclosed method for inhibiting and / or minimizing the progression of a genetic disorder or disability may further include administering a therapeutically effective dose of a therapeutic agent to a target. The therapeutic agent may be any disclosed agonist that produces a desired clinical outcome.

[0293] In one embodiment, a disclosed method for inhibiting and / or minimizing the progression of a genetic disorder or disability may further include monitoring the subject for adverse effects. In one embodiment, if no adverse effects are observed, the method may further include continuing the treatment of the subject. In one embodiment, if adverse effects are observed, the method may further include modifying the treatment step. Methods for monitoring the health of a subject may include both subjective and objective criteria (as discussed above). Such methods are known to those skilled in the art.

[0294] In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression may further include administering a therapeutically effective amount of an agonist capable of correcting one or more characteristics of a dysregulated metabolic or enzymatic pathway to a target. In one embodiment, such an agonist may include an enzyme for enzyme replacement therapy. In one embodiment, the disclosed enzyme may substitute for any enzyme in a dysregulated or dysfunctional metabolic or enzymatic pathway. In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression may include substituting one or more enzymes in a dysregulated or dysfunctional metabolic pathway.

[0295] In one embodiment, a disclosed method for inhibiting and / or minimizing the progression of a genetic disorder or impairment may further include administering one or more immunomodulators. In one embodiment, the disclosed immunomodulator may be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or a combination thereof. In one embodiment, the disclosed immunomodulator may be bortezomib or SVP-rapamycin. In one embodiment, the disclosed immunomodulator may be tacrolimus. In one embodiment, the disclosed immunomodulator (e.g., methotrexate) may be administered transiently in low to high doses. In one embodiment, the disclosed immunomodulator may be administered in doses of approximately 0.1 mg / kg body weight to approximately 0.6 mg / kg body weight. In one embodiment, the disclosed immunomodulator may be administered in doses of approximately 0.4 mg / kg body weight. In one embodiment, the disclosed immunomodulator may be administered at a daily dose of approximately 0.4 mg / kg body weight for 3 to 5 cycles or more, for a maximum of 3 days per cycle. In one embodiment, the disclosed immunomodulator may be administered at a daily dose of approximately 0.4 mg / kg body weight for at least 3 cycles, for 3 days per cycle. In one embodiment, a person skilled in the art can determine an appropriate number of cycles. In one embodiment, the disclosed immunomodulator may be administered as many times as necessary to achieve the desired clinical effect.

[0296] In one embodiment, the disclosed immunomodulator may be administered orally about one hour before the disclosed therapeutic agent. In one embodiment, the disclosed immunomodulator may be administered subcutaneously about 15 minutes before the disclosed therapeutic agent. In one embodiment, the disclosed immunomodulator may be administered together with the disclosed therapeutic agent. In one embodiment, the disclosed immunomodulator may be administered orally about one hour or several days before the disclosed nucleic acid molecule, the disclosed vector, the disclosed pharmaceutical formulation, or a combination thereof. In one embodiment, the disclosed immunomodulator may be administered subcutaneously about 15 minutes or several days before the disclosed nucleic acid molecule, the disclosed vector, the disclosed pharmaceutical formulation, or a combination thereof. In one embodiment, the disclosed immunomodulator may be administered together with the disclosed nucleic acid molecule, the disclosed vector, the disclosed pharmaceutical formulation, or a combination thereof.

[0297] In one embodiment, a disclosed method for inhibiting and / or minimizing the progression of a genetic disorder or impairment may further include administering one or more proteasome inhibitors (e.g., bortezomib, carfilzomib, marizomib, ixazomib, and oprozomib). In one embodiment, the proteasome inhibitor may be an agent that acts on plasma cells (e.g., daratumumab). In one embodiment, the agonist acting on plasma cells may 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, prelixafor, ixazomib citrate, pamidronate disodium, panobinostat, prelixafor, pomalidomide, pomalidomide, lenalidomide, selinexol, thalidomide, thalidomide, bortezomib, selinexol, zoledronic acid, or zoledronic acid.

[0298] In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression stability may further include administering one or more proteasome inhibitors or plasma cell-acting agents before administering a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof. In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression may include administering one or more proteasome inhibitors or plasma cell-acting agents along with administering a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof. In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression may include administering one or more proteasome inhibitors or plasma cell-acting agents after administering a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof. In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression may further include administering one or more proteasome inhibitors in more than one dose. In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression may include administering one or more proteasome inhibitors repeatedly over time.

[0299] In one embodiment, a disclosed method for inhibiting and / or minimizing the progression of a genetic disorder or impairment may further include administering one or more immunosuppressants. In one embodiment, the immunosuppressants may be, but are not limited to, azathioprine, methotrexate, sirolimus, anti-thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate-tomofetil (MMF), steroids, or combinations thereof. In one embodiment, a disclosed method for inhibiting and / or minimizing the progression of a disease may include administering one or more immunosuppressants in more than one dose. In one embodiment, a disclosed method may include administering one or more immunosuppressants repeatedly over time. In one embodiment, a disclosed method for inhibiting and / or minimizing the progression of a disease may include administering compounds that target or alter antigen presentation, humoral immune responses, cell-mediated immune responses, or innate immune responses.

[0300] In one embodiment, a disclosed method for inhibiting and / or minimizing the progression of a genetic disorder or impairment may further include administering a compound that exerts a therapeutic effect on B cells and / or a compound that targets or alters antigen presentation, humoral immune response, or cell-mediated immune response. In one embodiment, the disclosed compound may be rituximab, methotrexate, intravenous gamma globulin, anti-CD4 antibody, anti-CD2 antibody, anti-FcRN antibody, BTK inhibitor, anti-IGF1R antibody, CD19 antibody (e.g., inebilizumab), anti-IL6 antibody (e.g., tocilizumab), anti-CD40 antibody, IL2 mutein, or a combination thereof. Also disclosed herein are Treg infusions (e.g., antigen-specific Treg cells against AAV) that can be administered as a means of aiding immune tolerance.

[0301] In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression may further include administering lipid nanoparticles (LNPs). In one embodiment, LNPs may target organs. In one embodiment, LNPs may target the liver or testes. For example, in one embodiment, LNP-encapsulated mRNA therapy for systemic delivery to a subject may have the potential to restore the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme.

[0302] In one embodiment, a disclosed method for inhibiting and / or minimizing the progression of a genetic disorder or impairment may further include treating a subject that has acquired or may acquire neutralizing antibodies (AB) to a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof. In one embodiment, treating a subject that has acquired or may acquire neutralizing antibodies may include plasmapheresis and immunosuppression. In one embodiment, a disclosed method may include using immunosuppression to reduce T cell, B cell and / or plasma cell populations and to reduce the innate immune response, inflammatory response, and antibody levels overall. In one embodiment, a disclosed method for inhibiting and / or minimizing the progression of a disease may include administering an IgG degrader that depletes existing neutralizing antibodies. In one embodiment, a disclosed method may include administering IdeS or IdeZ, rapamycin, and / or SVP-rapamycin to a subject. In one embodiment, a disclosed method for inhibiting and / or minimizing the progression of a disease may include administering tacrolimus. In one embodiment, the disclosed IgG degrading agent is a bacterial IdeS or IdeZ.

[0303] In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression may include repeating a disclosed administration step, for example, administering a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immunomodulator, a disclosed proteasome inhibitor, a disclosed immunosuppressant, a disclosed compound that exerts a therapeutic effect on B cells, and / or a disclosed compound that targets or alters antigen presentation, humoral immune response, or cell-mediated immune response.

[0304] In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression may include modifying one or more of the disclosed steps. For example, modifying one or more steps of the disclosed method may include modifying or changing one or more features or aspects of one or more steps in the disclosed method. For example, in one embodiment, the method may be modified by changing the amount of one or more 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 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 duration of administration of one or more 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.

[0305] In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression may be altered by varying the amount of one or more disclosed therapeutic agents, disclosed immunomodulators, disclosed proteasome inhibitors, disclosed immunosuppressants, disclosed compounds that exert a therapeutic effect on B cells, and / or disclosed compounds that target or alter antigen presentation, humoral immune responses, or cell-mediated immune responses administered to a subject, or by varying the frequency of administration of one or more disclosed therapeutic agents, disclosed immunomodulators, disclosed proteasome inhibitors, disclosed immunosuppressants, disclosed compounds that exert a therapeutic effect on B cells, and / or disclosed compounds that target or alter antigen presentation, humoral immune responses, or cell-mediated immune responses administered to a subject.

[0306] In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression may include the simultaneous administration of one or more 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 immunomodulators, one or more disclosed proteasome inhibitors, one or more disclosed immunosuppressants, one or more disclosed compounds that exert a therapeutic effect on B cells, one or more disclosed compounds that target or alter antigen presentation, humoral immune responses or cell-mediated immune responses, or any combination thereof. In one embodiment, a disclosed immunomodulator may be administered before or after administration of a disclosed therapeutic agent. In one embodiment, a disclosed method for inhibiting and / or minimizing disease progression of a genetic disorder or impairment may further include generating a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof.

[0307] In one embodiment, a disclosed treatment method for inhibiting and / or minimizing disease progression may further include gene editing of one or more relevant genes (e.g., those listed in Table 1 below). [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0308] In one embodiment, the disclosed method may be particularly useful in treating subjects with autosomal dominant genetic disorders or disorders such as progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary ellipticosis, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, osteopetrosis, Marfan syndrome, neurofibromatosis, adult-onset polycystic kidney disease, protein C deficiency, osteogenesis imperfecta, Treacher Collins syndrome, tuberous sclerosis, and von Willebrand disease. [Examples]

[0309] VIII. Examples Because pathogenic mutations exist in over 500 genes that exceed the packaging capacity of AAV, several attempts have been made to circumvent this barrier to large gene expression. While these techniques differ, the general approach remains broadly similar across strategies. In short, a dual AAV vector approach is employed, where the DNA sequence encoding the target protein is split and packaged into separate vectors. When target cells are simultaneously infected with the two vectors, the genomes of the two vectors recombine with each other via reverse repeat sequences or overlapping complementary sequences to form a single genome with a reconfigured DNA sequence expressing the complete protein of the target. While this strategy is viable, the efficiency of inter-genomic recombination limits its feasibility for widespread adoption.

[0310] Separately, other efforts to correct the phenotypic effects of genes ineligible for classical AAV-mediated gene therapy have led to the conceivable approach of manipulating endogenous messenger RNA, the conduit between DNA and protein. An RNA editing strategy known as spliceosome-mediated RNA trans-splicing (SMART) was developed as a strategy to introduce large, precise modifications to the primary structure of RNA transcripts, regardless of the length of the target transcript. The goal of this approach is to hijack the cell's RNA processing mechanism to incorporate the desired sequence into the endogenous transcript. In this strategy, a recombinant RNA molecule containing three essential components—an RNA targeting motif, a hemiintron sequence, and the primary sequence of the desired RNA to be joined to the endogenous RNA transcript—is introduced into the cell. The RNA targeting motif consists of an antisense oligonucleotide stretch against the intron of the pre-mRNA to be endogenously targeted. The hemiintron is recognized by the spliceosome during Watson-Crick base pairing between the RNA targeting motif and the endogenous intron. Depending on the desired splicing reaction, either the 5' hemiintron facilitates the splicing of the trans-splicing molecule to the exon immediately 3' of the targeted intron, or the 3' hemiintron ligated to the exogenous RNA to be trans-spliced ​​facilitates the splicing of the trans-splicing molecule to the exon immediately 5' of the targeted intron. This results in the production of a mature chimeric RNA transcript, each consisting of either the recombinant 5' start of the transcript bound to the endogenous 3' sequence, or the endogenous 5' start of the transcript bound to the recombinant 3' sequence. The utility of this strategy is that a single AAV vector only needs to package a genome capable of producing a trans-splicing RNA molecule containing a portion of the gene sequence, eliminating the need to deliver the full-length protein-coding sequence to the cell. Since the entire recombinant RNA region of the chimeric RNA product can be specified by the user, the trans-splicing RNA can contain the wild-type sequence of the 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 antisense RNA sequences hinder the widespread use of this technique in research and clinical settings. While promising, the indefinite expression of CRISPR proteins as therapeutic agents (e.g., long-term expression) presents challenges arising from immunogenicity and off-target effects. Furthermore, CRISPR-based approaches often require two constructs to be delivered (i.e., a CRISPR effector protein and an RNA trans-splicing construct), resulting in high-dose requirements and low correction efficiency. Disclosed herein is a system for transcriptome engineering that does not require a CRISPR system. Many endogenous RNA-binding ribonucleoproteins exist in human cells, and the system disclosed herein utilizes several RNA structures that interact with known human ribonucleoproteins to enable trans-splicing. As detailed herein, by incorporating these RNA structures into trans-splicing RNA, effective trans-splicing of many targeted endogenous pre-mRNAs has been achieved. This made it possible to rewrite large stretches of mRNA.

[0311] Example 1 Verification of trans-splicing efficiency To assay trans-splicing efficiency, a green fluorescence-based screening system was designed and constructed. The first step was to construct a reporter by splitting an open reading frame of green fluorescent protein (EGFP) into two halves. An intron was inserted between the two halves. Thus, if this construct is spliced ​​in cis, it produces mature RNA encoding EGFP and expresses the green fluorescent protein at translation.

[0312] Fluorescence expression was abolished by introducing an early stop codon into the first half of the EGFP open reading frame's "exons" (Figure 1 (left)). When this construct is cis-spliced, the early stop codon present in the first half of the EGFP open reading frame stops translation of the full-length protein, thus preventing the expression of green fluorescence.

[0313] Green fluorescence expression could be restored by encoding the first half of the correct open reading frame of EGFP, followed by delivery of a trans-splicing RNA containing hemiintrons and guide RNA sequences. Therefore, green fluorescence was used as an indirect readout of trans-splicing efficiency (Figure 1). This reporter system was central to our downstream assays.

[0314] The initial trans-splicing RNA delivered to these cells (hereinafter referred to as SMaRT or spliceosome-mediated RNA trans-splicing) contained only the first half of EGFP, a hemi-intron, and a 30 bp antisense targeting domain. The sequence was then cloned into a trans-splicing RNA vector to determine if it could enhance trans-splicing efficiency. These new constructs were transfected into HEK293 cells with a split GFP reporter, and after 48 hours, green fluorescence intensity (percent GFP% and mean fluorescence intensity) was assayed by flow cytometry as a surrogate for trans-splicing efficiency (Figures 2A and 2B). Through this process, three RNA structures were obtained that significantly enhanced trans-splicing efficiency (RNA structures 4, 5, and 7). In Figures 2A and 2B, the RNA targeting motif of RYR2 is shown as Sequence ID No. 23.

[0315] Next, we investigated whether any member of this panel of RNA sequences enhanced the 3' trans-splicing approach. To do this, we used the same split GFP reporter and made a slight modification, moving the stop codon from the first half to the second half of the open reading frame (Figure 1 (right)). This construct alone does not express green fluorescence when delivered to HEK293 cells because cis-splicing retains the early stop codon. To restore EGFP expression, we used trans-splicing RNA that completed the open reading frame of EGFP during proper splicing. Similar to the 5' editing approach, we used trans-splicing RNA containing a 30 bp antisense targeting motif, followed by a hemi-intron and the second half of the EGFP open reading frame (this construct was SMaRT). Then, to improve trans-splicing efficiency, we introduced the same panel of RNA constructs into this RNA molecule.

[0316] The novel constructs were co-transfected into HEK293 cells with a split GFP reporter. After 48 hours, green fluorescence intensity (percent GFP% and mean fluorescence intensity) was assayed by flow cytometry as a surrogate for trans-splicing efficiency (Figures 3A-3B). Through this process, four RNA structures were identified that yielded significantly enhanced trans-splicing efficiency (RNA structures 1, 2, 10, and 11). This panel was repeated with a second intron-targeted intron (Figures 3C-3D), and a similar trend was observed in the multiplier of change in editing efficiency. In Figures 3A-3B, the RNA targeting motif for RYR2 is at SEQ ID NO: 23, and in Figures 3C-3D, the RNA targeting motif for LMNA is at SEQ ID NO: 22.

[0317] Following these results, the trans-splicing assay was repeated for the top hits (RNA structures 1 and 2). A second control RNA structure (i.e., a direct repeat derived from Ruminococcus flavefaciens XPD3002 (RfxCas13d)) was also used. The rationale for this control was to confirm that the enhancement of RNA editing was inherent to these RNA structures and not an artifact due to the inclusion of additional sequence length / complexity in the trans-splicing RNA. Indeed, only RNA structures 1 and 2 appeared to enhance editing efficiency, while the Cas13 direct repeat had a slightly detrimental effect on trans-splicing efficiency (Figures 4A-4B).

[0318] Finally, the novel target intron was cloned into a split GFP reporter and assayed for editing efficiency with RNA structure 2 and several guide RNA candidates. Editing was observed in over 50% of GFP-positive cells with this novel target (Figures 5A-5B). In Figures 5A-5B, the RNA targeting motif of FXN is shown as Sequence ID No. 24. In Table 2, structures 4 and 5 contain cloning sites inside that correspond to the insertion sites of the guide RNAs, which are shown in bold. [Table 2] [Table 3]

[0319] Example 2 Transsplicing of mutations in DMD DMD (dystrophin) is known in the art (e.g., gene ID 1756), and its nucleotide sequence may include nucleotides 5001-2225382 of accession number NG012232.1. DMD encodes a large protein containing an N-terminal actin-binding domain and multiple spectrin repeats over a genomic range of more than 2 Mb. The encoded protein (SEQ ID NO: 17) 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 locus can cause Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or cardiomyopathy. Currently, approximately 1750 pathogenic variants of DMD have been reported. The following details constructs to address more than 1000 of these pathogenic variants. Here, a plasmid is used to generate a 5' substitution construct for DMD based on the schematic diagram shown in Figure 7A. The 5' substitution construct targets exons 1–23 of the DMD (SEQ ID NO: 33). The 5' substitution construct repairs one or more of the 618 mutations identified in Table 4 below. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11] [Table 4-12] [Table 4-13] [Table 4-14] [Table 4-15]

[0320] AAV vectors based on plasmid constructs shown in Figure 7A are delivered to subjects with one or more 5' mutations in DMD (e.g., Table 4). A therapeutically effective dose of AAV vector (e.g., approximately 1 × 10⁻¹⁶) is delivered. 10 vg / kg ~ approx. 2×10 14 Following administration of vg / kg, one or more target cells, tissues, and / or organs generate chimeric RNA molecules encoding corrected and / or restored DMD. Confirmation of chimeric RNA molecule generation is performed by taking a sample from the target and checking the expression level of corrected and / or restored DMD (by comparing the post-treatment level of activating / functional DMD to the pre-treatment level of activating / functional DMD in the target). The target experiences inhibition and / or minimization of DMD disease progression. The target's quality of life improves. Here, a plasmid is used to generate a 3' substitution construct for DMD based on the schematic diagram shown in Figure 7B. The 3' substitution construct targets exons 53-79 of DMD (SEQ ID NO: 32). The 3' substitution construct repairs one or more of the 446 mutations identified in Table 5 below. [Table 5-1] Table 5-2 Table 5-3 Table 5-4 Table 5-5 Table 5-6 Table 5-7 Table 5-8 Table 5-9 Table 5-10 Table 5-11 Table 5-12 Table 5-13 Table 5-14 Table 5-15 Table 5-16 Table 5-17 Table 5-18

[0321] AAV vectors based on plasmid constructs shown in Figure 7B are delivered to subjects with one or more 3' mutations (e.g., Table 5). A therapeutically effective dose of AAV vector (e.g., approximately 1 × 10⁻¹⁶) is delivered. 10 vg / kg ~ approx. 2×10 14 Following administration of vg / kg, one or more target cells, tissues, and / or organs generate chimeric RNA molecules encoding corrected and / or restored DMD. Confirmation of chimeric RNA molecule generation is performed by taking samples from the target and checking the expression levels of corrected and / or restored DMD (by comparing the post-treatment level of activating / functional DMD to the pre-treatment level of activating / functional DMD in the target). The target experiences inhibition and / or minimization of DMD disease progression. The target's quality of life improves.

[0322] Summary of Examples The compositions and methods disclosed herein are superior to previously disclosed compositions and methods such as SMART. The disclosed systems offer numerous advantages. These surprising and unexpected advantages include: (i) No additional effector protein is required (e.g., a CRISPR-based one that enables trans-splicing); (ii) The effector RNA structure can be modified to bind to the endogenous splicing mechanism, enabling substitution of the 3' or 5' end of the trans-splicing mRNA; (iii) The construct can be delivered using a non-viral delivery system (e.g., LNP) within a single AAV vector or as a trans-splicing RNA fragment; (iv) The ability to edit large stretches of mRNA composed of multiple exons; (v) The ability to correct mRNA by a "knockdown and substitution" approach using a single trans-splicing construct without the need to provide two different constructs for knockdown and substitution, which is particularly useful in autosomal dominant genetic disorders; (vi) The ability to prevent overexpression of any gene, with correction dependent on the level of the endogenous mutant transcript based on the provided trans-splicing fragment (therefore, the corrected mRNA will not be expressed at levels higher than the endogenous disease transcript). While comprehensive, this list of benefits is not exhaustive.

[0323] Overall, the platform described herein is for efficient RNA editing that is independent of CRISPR-based targeting and superior to simple antisense base pairing. This enables efficient rewriting of large stretches of RNA, which impacts human health and basic biology. Therapeutically, it allows for gene rewriting and correction of dominant-negative mutations that exceed the packaging capacity of AAV while maintaining the expression of the target transcript at endogenous levels.

Claims

1. One or more RNA structures containing one of the sequences from SEQ ID NO: 01 to SEQ ID NO: 09, One or more RNA targeting motifs, 3' hemiintron, and Exogenous RNA that will be transspliced ​​into the target endogenous pre-mRNA Nucleic acid molecules, including those mentioned above.

2. The exogenous RNA that will be transspliced ​​into the target endogenous pre-mRNA, 5' Hemi-Intron, One or more RNA targeting motifs, One or more RNA structures containing one of the sequences from SEQ ID NO: 01 to SEQ ID NO: 09 Nucleic acid molecules, including those mentioned above.

3. The nucleic acid molecule according to claim 1 or claim 2, wherein the target endogenous pre-mRNA contains one or more mutations in one or more exons.

4. The nucleic acid molecule according to claim 3, wherein the one or more mutations are located in the 3' portion of the target endogenous pre-mRNA.

5. The nucleic acid molecule according to claim 3, wherein the one or more mutations are located in the 5' portion of the target endogenous pre-mRNA.

6. The nucleic acid molecule according to claim 1, wherein the target endogenous pre-mRNA encodes a protein-coding gene.

7. The protein-coding gene is 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, CAMTA1, CARMIL2, CC2D2A, CCDDC88A, CCDDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44, CFAP65, CFTR / ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1, CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1, CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1, DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11, DNAH17, DNAH2, DNAH5, DNAH7, DNAH8, DNAH9, DNMBP, DNMT1, DOCK2, DOCK3DASH、DASH7、DASH8、DASH、DASH、S 、DY2、DYS101、DYS201、DYSY TWO4、SH300、SHA5、SHAS6、SHASHAS2、S DAY5、DYS、55、D8、DYSHY、DYSH2、DYS W、HY1、HYS4、HY11、HY22、HYH、HYH2 CHEEKS、DREYTH、DREY4、DRE22、DRE1、S JOK1、JOK1、JOK2、JOK2、JOK1 、LOVE2、LOVE3、LOVE179、LOVE14、LOVE20、 ROKE20、ROKE20、ROSE1、ROSE2、ROSE1 、HAR2、HAR1、HARH1、HARH2、HAR1 、SIC2、SIC、SIC1、SIC10、SIC140、1 C172、CH1S、SCH10、SH10、1 11、SYS2、SYS4、SYS1、SYS2、2 H1K、HARSH、HAR1、HARSHY、HARSHY、HAR 30、CH050、HAR56、HARSH、HARSH、HARSH、 ROSE586、ROSE1100、ROSE1549、ROSE1 220、CH14 J26、CHY7、CHY2Y、CHY2H、HYH T2D、KY20、HY1、CHY1、HYH2、CHY 3、SPIRITS、SPIRITS5、SPIRITS1、SPIRITS2、SPIRITS3 4. LOVE、LOVE1、LOVE1、LOVE、LOVE1 LOVE2、LOVE4、LOVE5、LOVE6、LOVE LOVE、LOVE1 、LOVE2、LOVE2、LOVE4、LOVE、ROSE1、N CHRONIC 、SPIRITS13、SPIRITS31、SPIRITS3 CHRISTI1、CHR1、CHR55、CHRISTE、CHR1 、DY123、DY13、DY133、DY23 8. CHRIST、CHR3、CHROSX、CHRON 、FN11、FYN14、FYN2、FYN3、DYN6 、DYS3、DYN8、DYN9、DYS 180、DYS30、DYS53、DYSYS、9 ROCK、ROSHY、ROSHY、ROSHY2、ROSHY 22、SYSCH3、SYSY、SYSHYS、SYSH10、NFASC、NHS、NIN、NIPBL、NLRP1、NOTCH 1、NOTCH2、NOTCH3、NPHP4、NRXN1、NRX N3、NSD1、NSD2、NUP155、NUP188、NUP205、OBSCN、OBSL1、OTOF、OTOG、OTOGLぁPARD3、PBRM1、PCDH15、PCLO、PCNT、PH IP、PI4KA、PIEZO1、PIEZO2、PIK3C2A、 PIKFYVE、PKD1、PKD1L1、PKHD1、PLCE1 、PLEC、PLEKHG2、PNPLA6、POGZ、POLA1、 POLE、POLR1A、POLR2A、POLR3A、PRG4、 PRKDC、PRPF8、PRR12、PRX、PTCH1、PTP N23、PTPRF、PTPRJ、PTPRQ、PXDN、QRIC H2、RAB3GAP2、RAI1、RALGAPA1、RANBP2 、RB1CC1、RELN、RERE、REV3L、RIC1、RI MS1、RIMS2、RNF213、ROBO1、ROBO2、RO BO3、ROS1、RP1、RP1L1、RTTN、RUSC2、R YR1、RYR2、SACS、SAMD9、SAMD9L、SBF2、 SCAPER、SCN10A、SCN11A、SCN1A、SCN2 A、SCN3A、SCN4A、SCN5A、SCN8A、SCN9A 、SETBP1、SETD1A、SETD1B、SETD2、SET D5、SETX、SHANK2、SHANK3、SHROOM4、S I、SIPA1L3、SLIT2、SLX4、SMARCA2、SM ARCA4、SMCHD1、SNRNP200、SON、SPEF2 、SPEG、SPG11、SPTA1、SPTAN1、SPTB、S PTBN2、SPTBN4、SRCAP、STRC、SVIL、SYN E1、SYNGAP1、SYNJ1、SZT2、TAF1、TANC 2、TCF20、TCOF1、TDRD9、TECPR2、TECT A、TENM3、TENM4、TET3、TEX14、TEX15、TG、THOC2、TMEM94、TNC、TNIK、TNR、TNR C6B、TNXB、TOGARAM1、TONSL、TRIO、TR IOBP、TRIP11、TRIP12、TRPM1、TRPM6、 TRPM7、TRRAP、TSC2、TTC37、TTN、TUBG CP6、UBR1、UNC80、USH2A、USP9X、VCAN、A nucleic acid molecule according to claim 6, comprising one or more coding regions from among VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62, WDR81, WNK1, WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292, ZNF335, ZNF407, ZNF462, or ZNF469.

8. A nucleic acid molecule according to any one of claims 3 to 7, wherein the one or more mutations in one or more exons contribute to the pathogenesis in one or more cells.

9. The nucleic acid molecule according to claim 8, wherein one or more cells are included in the target.

10. The nucleic acid molecule according to claim 1, wherein the 5' portion of the target endogenous pre-mRNA is transspliced ​​with the exogenous RNA.

11. The nucleic acid molecule according to claim 2, wherein the 3' portion of the target endogenous pre-mRNA is transspliced ​​with the exogenous RNA.

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

13. The nucleic acid molecule according to claim 12, wherein the RNA targeting motif includes an antisense oligonucleotide.

14. The nucleic acid molecule according to claim 13, wherein the antisense oligonucleotide comprises about 15 to about 50 nucleotides.

15. The nucleic acid molecule according to claim 1, wherein the RNA targeting motif binds to the 3' end of the target endogenous pre-mRNA.

16. The nucleic acid molecule according to claim 2, wherein the RNA targeting motif binds to the 5' end of the target endogenous pre-mRNA.

17. The nucleic acid molecule according to claim 3, wherein the RNA targeting motif is specific to endogenous pre-mRNA having one or more mutations.

18. The nucleic acid molecule according to claim 1, wherein the RNA targeting motif is directed to an intron immediately 3' of the exon of the target endogenous pre-mRNA with which it will be spliced.

19. The nucleic acid molecule according to claim 2, wherein the RNA targeting motif is directed to an intron immediately 5' of the exon of the target endogenous pre-mRNA with which it will be spliced.

20. The nucleic acid molecule according to claim 1, wherein the 3' hemiintron is linked to the exogenous RNA which will be transspliced ​​together with the endogenous mRNA.

21. The nucleic acid molecule according to claim 1, wherein the 3' hemiintron is recognized by nuclear splicing components in the host cell.

22. The nucleic acid molecule according to claim 1, wherein the 3' hemiintron comprises (i) a 3' splice region including a branching point, (ii) a polypyrimidine tract, and (iii) a 3' splice acceptor site.

23. The nucleic acid molecule according to claim 2, wherein the 5' hemiintron is linked to the exogenous RNA which will be transspliced ​​together with the endogenous mRNA.

24. The nucleic acid molecule according to claim 2, wherein the 5' hemiintron is recognized by nuclear splicing components in the host cell.

25. The nucleic acid molecule according to claim 2, wherein the 5' hemiintron includes a 5' splice site.

26. The nucleic acid molecule according to any one of the preceding claims, wherein the one or more RNA structures are bound to one or more RNA-binding proteins.

27. The nucleic acid molecule according to any of the preceding claims, 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 RNA and the exogenous RNA which will be trans-spliced ​​together with the target endogenous pre-mRNA molecule, or (v) any combination thereof.

28. A viral vector comprising a nucleic acid molecule according to any one of claims 1 to 27.

29. The vector according to claim 28, A pharmaceutical formulation comprising one or more pharmaceutically acceptable carriers.

30. A method for generating a chimeric RNA molecule, wherein the method is Contacting one or more endogenous pre-mRNAs targeted within cells with a nucleic acid molecule according to any one of claims 1 to 27 or a vector according to claim 28. Includes, A method wherein the resulting chimeric RNA molecule contains a transspliced ​​nucleic acid sequence.

31. The method according to claim 30, wherein one or more of the aforementioned cells are present within the target.

32. The method according to claim 31, wherein the subject has been diagnosed with or is suspected of having a genetic disorder or genetic defect.

33. A method for treating a genetic disorder or genetic defect, wherein the method is To generate a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of the vector described in claim 28 or the pharmaceutical formulation described in claim 29 to a target that requires it. Includes, The resulting chimeric RNA molecule contains a transspliced ​​nucleic acid sequence. A method for restoring one or more characteristics of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation using the resulting chimeric RNA molecule.

34. The method according to claim 33, wherein restoring one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation includes restoring the functionality and / or structural integrity of a deficient, incomplete, and / or mutant protein or enzyme.

35. The therapeutically effective amount of the vector is approximately 1 × 10 １０ vg ~ approx. 2×10 １４ The method according to claim 33, including vg.

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

37. The method according to claim 33, further comprising administering a therapeutically effective amount of one or more immunomodulators to the subject.

38. The method according to any one of claims 33 to 37, further comprising repeating the administration step.

39. The method according to any one of claims 33 to 38, further comprising monitoring the subject for adverse effects.

40. The method according to any one of claims 33 to 39, wherein restoring one or more features of cellular homeostasis and / or cellular functionality and / or metabolic dysfunction includes (i) correcting cellular starvation in one or more cells, (ii) correcting, preventing, reducing, and / or improving autophagy, (iii) improving, enhancing, restoring, and / or maintaining mitochondrial functionality and / or structural integrity, (iv) improving, enhancing, restoring, and / or maintaining organelle functionality and / or structural integrity, (v) correcting enzyme dysregulation, (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the progression of multi-organ manifestations of a genetic disorder or impairment, (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the progression of a genetic disorder or impairment, or (viiii) any combination thereof.

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