Compositions and methods for engineering transcriptomes

Abstract

Disclosed herein are CRISPR-free compositions for the production of chimeric RNA molecules via trans-splicing and methods of producing the chimeric RNA molecules, and methods of using chimeric RNA molecules produced via trans-splicing to treat and / or prevent genetic diseases or conditions.

Description

I. Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 493,696, filed March 31, 2023, the entire contents of which are incorporated herein by reference.

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

[0003] III. References to Sequence Lists The sequence list submitted on March 29, 2024, in an XML file named “23-2078-WO_Sequence Listing”, created on March 29, 2024, is 95 kilobytes in size and is incorporated herein by reference in accordance with 37 CFR §1.52(e)(5).

[0004] IV. Background Technology In eukaryotes, chromosomal DNA is transcribed into precursor RNA information (pre-mRNA), which contains protein-coding regions (exons) and intercalated non-protein-coding regions (introns). Prior to processing, these pre-mRNA molecules lack sequences for ribosome-initiated translation due to the retention of non-coding intron sequences. Therefore, before nuclear export, the exons of the pre-mRNA transcript are joined by a cellular mechanism called splicing. This mechanism is characterized by a double transesterification mediated by a large multi-ribonucleoprotein structure called the spliceosome. In the first transesterification, the branching point sequence of the intercalated intron attacks the 5' splice site, forming a lasso structure. This reaction releases the 5' splice site to attack the 3' splice site, thereby removing the intercalated intron and joining the adjacent exon. After all intron sequences are removed, the precursor information matures into a mature RNA transcript capable of translation, which is transported to the ribosome, where it is decoded to make cellular proteins.

[0005] In mammalian cells, mutations in transcriptionally active regions of chromosomal DNA produce pre-mRNA with the same mutation. If the mutation is located in a non-coding region, it can alter or eliminate the processing of the pre-mRNA. If the mutation is located in an exon region of the pre-mRNA, the mutation will be passed on to the mature mRNA sequence. These mutations can help suppress the complete translation of the encoded protein (speechless mutation) or counterproductively modify the primary structure of the encoded protein (missense mutation). In summary, these genetically encoded mutations can play a role in promoting pathogenesis in eukaryotes.

[0006] The goal of gene therapy is to correct genetic abnormalities through adoptive gene transfer of recombinant nucleic acids carrying sequences that produce the protein products of mutated genes. This strategy, often referred to as "classical gene therapy," has proven to be a safe and effective phenotypic correction strategy for genetic disorders, and several gene therapy products are available on the market.

[0007] However, current gene therapy approaches are limited by the following reasons: toxicity caused by transcript overexpression, inability to deliver large transcripts exceeding the packaging capacity of AAV vectors (i.e., the standard vectors for gene therapy with a packaging capacity of ~4.7 KB), inability to correct and replace large segments of pathogenic mRNAs, and inability to correct and replace autosomal dominant inheritance with a single transcript and / or method.

[0008] Therefore, there is an urgent need for a minimally invasive, definitive therapy to address the root causes and sequelae of symptoms associated with these various genetic diseases and conditions. Accordingly, this disclosure provides compositions and methods for generating chimeric RNA molecules via trans-splicing (and without CRISPR) and for treating and / or preventing genetic diseases and / or conditions, which can be used alone or in combination with other treatments.

[0009] V. Brief Description of the Attached Drawings Figure 1 (Left side) shows the mechanism of 5' reverse splicing, while Figure 1(Right side) illustrates the mechanism of 3' trans-splicing. The left side diagram first shows a construct transfected to confirm trans-splicing for 5' substitution. If the RNA species is cis-spliced, the stop codon in the first half of the open reading frame blocks translation. If the trans-spliced ​​RNA successfully edits the RNA, the EGFP open reading frame is restored and fluorescent expression is restored. The right side diagram first shows a construct transfected to confirm trans-splicing for 3' substitution. If the RNA species is cis-spliced, the stop codon in the second half of the open reading frame blocks translation. If the trans-spliced ​​RNA successfully edits the RNA, the EGFP open reading frame is restored and fluorescent expression is restored. GRAFT is for guide RNA helper fragment trans-splicing.

[0010] Figure 2A – Figure 2B A set of 5'-splicing motifs is shown. These are flow cytometry data from co-transfection experiments of 5'-trans-splicing candidate molecules. Figure 2A In this study, the percentage of cells expressing green fluorescence was measured when a trans-spliced ​​RNA candidate was delivered to cells. RNA structures 1-11 are plotted on the x-axis, and the percentage of GFP-positive cells is plotted on the y-axis. Figure 2B In this study, the average fluorescence intensity of cells was measured when trans-spliced ​​RNA candidates were delivered. RNA structures 1-11 were plotted on the x-axis, and the average fluorescence intensity was plotted on the y-axis.

[0011] Figure 3A – Figure 3D A set of 3' trans-splicing motifs is shown. Flow cytometry data from co-transfection experiments of 3' trans-splicing candidate molecules are presented. Figure 3A In this study, the percentage of cells expressing green fluorescence was measured when a trans-spliced ​​RNA candidate was delivered to the cells. RNA structures 1-11 were plotted on the x-axis, and the percentage of GFP-positive cells was plotted on the y-axis. Figure 3B In this study, the average fluorescence intensity of cells was measured when trans-spliced ​​RNA candidates were delivered. RNA structures 1-11 were plotted on the x-axis, and the average fluorescence intensity was plotted on the y-axis. Measurements were performed in the same manner for different target introns. Figure 3C and Figure 3D The data in the middle.

[0012] Figure 4A – Figure 4B This section shows replication validation of the top-ranked 3' trans-splicing RNA candidates. Flow cytometry data from co-transfection experiments using 3' trans-splicing candidate molecules are also presented. Figure 4AIn this study, the percentage of cells expressing green fluorescence was measured when a trans-spliced ​​RNA candidate was delivered to cells. The RNA structure was plotted on the x-axis, and the percentage of GFP-positive cells was plotted on the y-axis. Figure 4B In this study, the average fluorescence intensity of cells was measured when trans-spliced ​​RNA candidates were delivered. The RNA structure was plotted on the x-axis, and the average fluorescence intensity was plotted on the y-axis.

[0013] Figure 5A – Figure 5B This demonstrates the replication validation of top-ranked 3' trans-splicing RNA candidates targeting novel targets. Flow cytometry data from co-transfection experiments using 3' trans-splicing candidate molecules were measured. Figure 5A In this study, the percentage of cells expressing green fluorescence was measured when a trans-spliced ​​RNA candidate was delivered to the cells. RNA structures 1-11 were plotted on the x-axis, and the percentage of GFP-positive cells was plotted on the y-axis. Figure 5B In this study, the average fluorescence intensity of cells was measured when trans-spliced ​​RNA candidates were delivered. RNA structures 1-11 were plotted on the x-axis, and the average fluorescence intensity was plotted on the y-axis.

[0014] Figure 6A – Figure 6G Plasmid maps of the constructs used in Example 1 and / or disclosed herein are provided. Figure 6A An example 3' replacement builder (empty) is shown. Figure 6B The 3' replacement construct for RYR2 (3-GRAFT-RYR2), and Figure 6C The 5' replacement builder for RYR2 (5-GRAFT-RYR2) is shown. Figure 6D The 3' alternative construct for LMNA (3-GRAFT-LMNA) is shown, while Figure 6E The 3' alternative builder for FXN is shown (3-GRAFT-FXN). Figure 6F Showing the Split GFP reporter construct for LMNA, while Figure 6G Showing the split GFP reporter construct for RYR2.

[0015] Figure 7A The 5' replacement construct for DMD in Example 2 is shown. Figure 7B The 3' alternative construct for DMD in Example 2 is shown.

[0016] VI. Brief Description of the Invention This article discloses a nucleic acid molecule comprising exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' hemiintron; one or more RNA targeting motifs; and one or more RNA structures.

[0017] This article discloses a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA.

[0018] This article discloses a non-viral or viral vector containing a nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' hemiintron; one or more RNA targeting motifs; and one or more RNA structures.

[0019] This article discloses a non-viral or viral vector containing a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA.

[0020] This article discloses a pharmaceutical formulation comprising a non-viral vector or a viral vector, wherein the non-viral vector or viral vector comprises a nucleic acid molecule, wherein the nucleic acid molecule comprises an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' hemiintron; one or more RNA targeting motifs; and one or more RNA structures.

[0021] This article discloses a pharmaceutical formulation comprising a non-viral vector or a viral vector, wherein the non-viral vector or viral vector comprises a nucleic acid molecule, wherein the nucleic acid molecule comprises one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA.

[0022] This article discloses a method for treating a genetic disease or condition, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a publicly disclosed nonviral vector or a publicly disclosed viral vector or a pharmaceutical preparation thereof to a subject in need, wherein the resulting chimeric RNA molecule can restore cellular homeostasis and / or one or more aspects of cellular functional and / or metabolic disorders.

[0023] This article discloses a method for treating genetic diseases or conditions, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a publicly disclosed nonviral vector or a publicly disclosed viral vector or a pharmaceutical preparation thereof to a subject in need, wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of a missing, defective, and / or mutated protein or enzyme.

[0024] This article discloses a method for treating a genetic disease or condition, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a nonviral vector, a viral vector, an AAV particle, or a pharmaceutical preparation thereof to a subject in need, wherein the nonviral vector, viral vector, AAV particle, or pharmaceutical preparation thereof comprises (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, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic disorders, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes.

[0025] This article discloses 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 publicly disclosed nonviral vector or a publicly disclosed viral vector or a pharmaceutical preparation thereof to a subject in need of the molecule, wherein the resulting chimeric RNA molecule can restore cellular homeostasis and / or one or more aspects of cellular functional and / or metabolic dysregulation.

[0026] This article discloses 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 publicly disclosed nonviral vector or a publicly disclosed viral vector or a pharmaceutical preparation thereof to a subject in need thereof, wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective and / or mutated proteins or enzymes.

[0027] This article discloses 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 nonviral vector, viral vector, AAV particle, or pharmaceutical preparation thereof to a subject in need of the molecule, said nonviral vector, viral vector, AAV particle, or pharmaceutical preparation 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, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes.

[0028] VII. Detailed Explanation This disclosure describes formulations, complex compositions, kits, capsules, containers, and / or methods thereof. It should be understood that various aspects of the invention are not limited to specific synthetic methods unless otherwise stated, or to specific reagents unless otherwise stated, and therefore variations are naturally possible. It should also be understood that the terminology used herein is for descriptive purposes only and is not intended to be limiting. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the invention, exemplary methods and materials are now described.

[0029] All publications mentioned herein are incorporated herein by reference to disclose and describe methods and / or materials relating to the cited publications. The publications discussed herein are provided only because their disclosures predate the filing date of this application. Nothing herein should be construed as an admission that the invention is not entitled to precedence over such publications by virtue of prior invention.

[0030] A. Definition Before disclosing and describing the compounds, compositions, articles, systems, devices, and / or methods of the present invention, it should be understood that they are not limited to specific synthetic methods unless otherwise stated, or to specific reagents unless otherwise stated, and therefore variations are naturally permissible. It should also be understood that the terminology used herein is for descriptive purposes only and is not intended to be limiting. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the present invention, exemplary methods and materials are described hereafter.

[0031] This disclosure describes the inventive concept with reference to specific embodiments. However, it is intended to cover all modifications, equivalents, and alternatives to the inventive concept consistent with this disclosure.

[0032] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly indicates otherwise.

[0033] The phrase "consistently composed of..." limits the scope of the claim to the components described in the composition or the steps described in the method, as well as those that do not materially affect the essential and novel features of the claimed composition or the claimed method. The phrase "consisting of..." excludes any components, steps, or elements not recited in the claim. The phrase "comprising" is synonymous with "including," "containing," or "characterized as," and is inclusive or open-ended. "Comprising" does not exclude additional, unlisted components or steps.

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

[0035] A range herein may be expressed as "about" a particular value and / or "about" another particular value. When expressing such a range, a further aspect includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation using the antecedent "about," it should be understood that the particular value forms a further aspect. It should be further understood that the endpoints of each range are significant relative to and independent of the other endpoint. It should also be understood that many values ​​are disclosed herein, and each value is also disclosed herein as "about" that particular value, in addition to the value itself. For example, if the value "10" is disclosed, "about 10" is also disclosed. It should 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] The reference to parts by weight of a particular element or component in the specification and the final claims indicates the weight relationship between that element or component and any other elements or components in the composition or article expressed in parts by weight. Therefore, in a composite containing 2 parts by weight of component X and 5 parts by weight of component Y, X and Y are present in a 2:5 weight ratio, and are present in this ratio regardless of whether the composite contains any other components.

[0037] As used herein, the terms "optional" or "optionally" mean that the events or conditions described below may or may not occur, and the specification includes both scenarios in which the events or conditions occur and scenarios in which they do not occur. In one aspect, the disclosed method may optionally include one or more additional steps, such as repeating the application step or changing the application step.

[0038] As used herein, "isolated" refers to nucleic acid molecules or sequences that have been substantially separated from, produced separately from, or purified from other biological components in the cells or tissues of an organism in which the component is present. These other biological components include, for example, other cells, chromosomes, and extrachromosomal DNA and RNA, and proteins. "Isolated" nucleic acids and proteins include those purified using standard purification methods. The term also covers nucleic acids and proteins prepared through 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 the treatment, such as a human. The term "subject" also includes domestic 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.). Therefore, the subject of the methods disclosed herein can be a vertebrate, such as a mammal, fish, bird, reptile, or amphibian. Alternatively, the subject of the methods disclosed herein can be a human, a non-human primate, a horse, pig, rabbit, dog, sheep, goat, cattle, cat, guinea pig, or rodent. This term does not indicate a particular age or sex and is therefore intended to cover adult and child subjects, as well as fetuses, whether male or female. In one aspect, the subject can be a human patient. In another aspect, the subject can have a disease or condition, be suspected of having a disease or condition, or be at risk of developing a disease or condition (e.g., a genetic disease or condition). In another aspect, the subject can be untreated.

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

[0041] As used herein, the term "diagnostic" indicates that an examination has been conducted by a technician, such as a physician, and that the individual has been found to have a condition that can be diagnosed or treated using one or more publicly available nucleic acid molecules, publicly available vectors, publicly available pharmaceutical preparations, or combinations thereof, or using one or more publicly available methods. For example, "diagnosed with a disease or condition" indicates that an examination has been conducted by a technician, such as a physician, and that the individual has been found to have a condition (e.g., a genetic disease or condition) that can be treated using one or more publicly available nucleic acid molecules, publicly available vectors, publicly available pharmaceutical preparations, or combinations thereof, or using one or more publicly available methods. Similarly, "suspected to have a disease or condition" may indicate that an examination has been conducted by a technician, such as a physician, and that the individual has been found to have a condition that may be treated using one or more publicly available nucleic acid molecules, publicly available vectors, publicly available pharmaceutical preparations, or combinations thereof, or using one or more publicly available methods (e.g., a genetic disease or condition). In one aspect, the examination can be physical and may involve various tests (e.g., blood tests, genotyping, biopsies, etc.) and assays (e.g., enzyme assays), or combinations thereof.

[0042] "Patient" refers to a subject suffering from a disease or condition (such as a genetic disease or condition). In one aspect, a patient can refer to a subject who has been diagnosed with or is suspected of having a disease or condition. In another aspect, a patient can refer to a subject who has been diagnosed with or is suspected of having a disease or condition and is seeking or receiving treatment for a disease or condition.

[0043] As used herein, phrases such as "identified as requiring treatment of a disease or condition" refer to selecting a subject based on the need for treatment of a disease or condition. For example, based on an early diagnosis by someone skilled in the art, a subject may be identified as requiring treatment of a disease or condition (e.g., a genetic disease or condition), and then treatment for the genetic disease or condition may be administered. In one aspect, the identification may be performed by someone different from the person making the diagnosis. In another aspect, the administration may be performed by the person making the diagnosis.

[0044] As used herein, “inhibition,” “inhibition,” and “prevention” refer to a reduction or decrease in activity, level, response, condition, severity, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of activity, level, response, condition, severity, disease, or other biological parameter. It can also include, for example, a 10% inhibition or reduction of activity, level, response, condition, severity, disease, or other biological parameter compared to a natural or control level (e.g., a subject without a disease or condition, such as a genetic disease or condition). Therefore, in one aspect, inhibition or reduction compared to a natural or control level can be any reduction of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount between these values. In another aspect, inhibition or reduction compared to a natural or control level can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%. In one respect, the inhibition or reduction compared to the natural or control level can be 0-25%, 25-50%, 50-75%, or 75-100%. In another respect, the natural or control level can be the pre-disease or pre-symptom level.

[0045] The terms “treatment” or “managing” or “treating” include palliative care, which is treatment designed to eliminate symptoms rather than cure a disease, pathological condition, or symptom; preventative care, which aims to minimize or partially or completely suppress the development of a related disease, pathological condition, or symptom; and supportive care, which is treatment used to complement another specific therapy aimed at improving a related disease, pathological condition, or symptom. In one aspect, the term covers any treatment of a subject (including mammals, such as humans) and includes: (i) preventing the occurrence of undesirable physiological changes, diseases, pathological conditions, or symptoms in subjects who may be susceptible to the disease but have not yet been diagnosed with it; (ii) suppressing physiological changes, diseases, pathological conditions, or symptoms, i.e., halting their development; or (iii) eliminating physiological changes, diseases, pathological conditions, or symptoms, i.e., causing the disease to regress. For example, in one aspect, treating a disease or symptom can reduce the severity of an identified disease or symptom in a subject by 1%–100% compared to a control (e.g., an individual without a genetic disease or symptom). In one aspect, treatment can refer to a reduction in the severity of a disease or condition (e.g., a genetic disease or condition) by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. For example, treatment of a disease or condition can reduce one or more symptoms of the disease or condition in a subject by 1%–100% compared to a control (e.g., an individual without a genetic disease or condition). In another aspect, treatment can refer to a reduction in one or more symptoms of a identified disease or condition by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. It should be understood that treatment does not necessarily mean a cure or complete elimination or eradication of a disease or condition. However, in one aspect, treatment can mean a cure or complete elimination or eradication of a disease or condition.

[0046] As used herein, the terms “prevention,” “taking preventative measures,” or “avoidance” refer to stopping, avoiding, eliminating, preemptively preventing, terminating, or hindering the occurrence of something, particularly by taking early action. It should be understood that the use of “reduction,” “inhibition,” or “prevention” herein also explicitly discloses the use of the other two terms unless otherwise specifically stated. In one aspect, it aims to prevent a disease or condition with chromatin dysregulation and / or abnormalities in chromatin regulation. The terms “prevention,” “taking preventative measures,” and “avoidance” also refer to preventative or preventative measures used to protect or prevent a subject (e.g., an individual) who does not have a given disease or condition (e.g., a genetic disease or condition) or related complications from progressing to that complication.

[0047] As used herein, the terms “administer” and “application” refer to any method of providing a subject with one or more disclosed nucleic acid molecules, disclosed carriers, disclosed pharmaceutical formulations, or combinations thereof. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, intrauterine administration, intrahepatic administration, intravaginal administration, ocular administration, intraauricular administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable methods such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration may also include intrahepatic artery administration or administration via the portal vein (HPV). Administration of disclosed nucleic acid molecules, disclosed carriers, 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 aspect, a person skilled in the art can determine an effective dose, effective regimen, and effective route of administration for one or more of the disclosed nucleic acid molecule, disclosed carrier, disclosed pharmaceutical preparation, or combination thereof, to treat or prevent a disease or condition (e.g., a genetic disease or condition). In another aspect, a person skilled in the art can also modify, alter, or modify one aspect of the administration procedure to improve the efficacy of one or more of the disclosed nucleic acid molecule, disclosed carrier, disclosed pharmaceutical preparation, or combination thereof.

[0049] "Measurement" refers to the absolute quantification of a particular analyte (e.g., an mRNA sequence containing a specific 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 direct or indirect measurements of abundance (e.g., the amount of a single mRNA transcript that can be quantified, or the amount of an mRNA sequence amplified under certain conditions for a certain period of time that can be used as an alternative to quantifying a single transcript) or both.

[0050] As used herein, "modifying the method" can include modifying or altering one or more features or aspects of one or more steps of the disclosed method. For example, in one aspect, the method can be modified by changing the amount of one or more of the disclosed nucleic acid molecule, disclosed carrier, disclosed pharmaceutical preparation, or combination thereof administered to a subject, or by changing the frequency of administration of one or more of the disclosed nucleic acid molecule, disclosed carrier, disclosed pharmaceutical preparation, or combination thereof to a subject, by changing the duration of administration of one or more of the disclosed nucleic acid molecule, disclosed carrier, disclosed pharmaceutical preparation, or combination thereof to a subject, or by replacing one or more of the disclosed components and / or reagents with similar or equivalent components and / or reagents. This also applies to all disclosed therapeutic agents, immunomodulators, immunosuppressants, proteasome inhibitors, etc.

[0051] As used herein, the term "pharmaceutical-grade carrier" refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders intended for recombination into sterile injectable solutions or dispersions prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or media include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethyl cellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. In one aspect, the drug carrier employed can be solid, liquid, or gaseous. In one aspect, examples of solid carriers may include lactose, kaolin, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, and stearic acid. In one aspect, examples of liquid carriers may include syrup, peanut oil, olive oil, and water. In one aspect, examples of gaseous carriers may include carbon dioxide and nitrogen. Any convenient drug medium may be used in preparing the disclosed compositions for oral dosage forms. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, etc., can be used to form oral liquid preparations, such as suspensions, elixirs, and solutions; while carriers, such as starch, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrants, etc., can be used to form oral solid preparations, such as powders, capsules, and tablets. Tablets and capsules are preferred oral dosage units where solid drug carriers are used due to their ease of administration. Optionally, tablets can be coated using standard aqueous or non-aqueous techniques. For example, by using coating materials such as lecithin, suitable flowability can be maintained in the case of dispersions by maintaining the desired particle size and by using surfactants. These compositions may also contain excipients such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of microbial action can be ensured by including various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. Isotonic agents, such as sugars and sodium chloride, may also be desired. Extended absorption of injectable drug formulations can be achieved by incorporating agents that delay absorption, such as aluminum monostearate and gelatin. Injectable reservoir forms are prepared by forming microcapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolic acid, 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. Reservoir injectable formulations are also prepared by encapsulating the drug in tissue-compatible liposomes or microemulsions. Injectable formulations can be sterilized, for example, by filtration through a bacterial trap or by incorporation of a sterilizing agent that can be dissolved or dispersed in sterile water or other sterile injectable media as a sterile solid composition prior to use. Suitable inert carriers may include sugars, such as lactose. Ideally, 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 an inert substance commonly used as a diluent, medium, preservative, binder, or stabilizer, and includes, but is not limited to, proteins (e.g., serum albumin), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylates, etc.), surfactants (e.g., SDS, polysorbates, nonionic surfactants, etc.), sugars (e.g., sucrose, maltose, trehalose, etc.), and polyols (e.g., mannitol, sorbitol, etc.). For reference, see also Remington's Pharmaceutical Sciences, (1990) Mack Publishing Co., Easton, Pa., the entire contents of which are incorporated herein by reference.

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

[0054] As used herein, the term "contact" refers to the act of bringing together one or more disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical preparations, or combinations thereof with a target region or intended target region in such a manner that one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical preparations, or combinations thereof directly or indirectly act on the intended target or target region. A target region may include one or more cells, and in one aspect, the one or more cells may be in a subject. A 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 aspect, a target region or intended target region may be any cell or any organ infected by a disease or condition (e.g., a genetic disease or condition). In one aspect, a target region or intended target region may be any organ, tissue, or cell affected by a disease or condition (e.g., a genetic disease or condition).

[0055] As used herein, “determine” can refer to measuring or clarifying the presence and severity of a disease or condition, such as a genetic disease or condition. Methods and techniques used to determine the presence and / or severity of a disease or condition are generally known in the medical field. For example, ways of identifying and / or diagnosing the presence, severity, or both of a disease or condition (e.g., a genetic disease or condition) are well-known in the art.

[0056] As used herein, “effective amount” and “effective quantity” can refer to an amount sufficient to achieve a desired outcome, such as treatment and / or prevention of a disease or condition (e.g., a genetic disease or condition) or a suspected disease or condition. As used herein, the terms “effective amount” and “effective quantity” can also refer to an amount sufficient to achieve a desired effect on an undesirable condition (e.g., a disease or condition). For example, a “therapeutic effective amount” refers to an amount sufficient to achieve a desired therapeutic outcome or to have an effect on an undesirable symptom, but generally insufficient to cause adverse side effects. In one aspect, a “therapeutic effective amount” represents an amount of a disclosed nucleic acid molecule, a disclosed carrier, or a disclosed pharmaceutical preparation that (i) treats a particular disease, condition, or condition (e.g., a genetic disease or condition), (ii) reduces, alleviates, or eliminates one or more symptoms of a particular disease, condition, or condition (e.g., a genetic disease or condition), or (iii) delays the onset of one or more symptoms of a particular disease, condition, or condition (e.g., a genetic disease or condition). The specific effective therapeutic dose level for any particular patient will depend on a variety of factors, including the condition being treated and its severity; the disclosed nucleic acid molecule, disclosed carrier, disclosed pharmaceutical preparation used; the disclosed method used; the patient's age, weight, general health condition, sex, and diet; the time of administration; the route of administration; the excretion rate of the disclosed nucleic acid molecule, disclosed carrier, or disclosed pharmaceutical preparation used; the duration of treatment; the drugs used in combination with or concurrently with the disclosed nucleic acid molecule, disclosed carrier, or disclosed pharmaceutical preparation; and other similar factors well known in the medical field. For example, starting with a dose of the disclosed nucleic acid molecule, disclosed carrier, or disclosed pharmaceutical preparation at a level below what is required to achieve the desired therapeutic effect and gradually increasing the dose until the desired effect is achieved is within the scope of the art. If necessary, the effective daily dose can be divided into multiple doses for the purpose of administration. Therefore, a single dose of the disclosed nucleic acid molecule, disclosed carrier, or disclosed pharmaceutical preparation may contain such an amount or an approximation thereof to constitute the daily dose. If there are any contraindications, the dose may be adjusted by an individual physician. The dose can be varied and may be administered daily in one or more doses for one or more days. For the appropriate dosage of a given class of pharmaceutical products, guidance can be found in the literature. In further aspects, the preparation can be administered in a “preventatively effective amount”; that is, an amount that effectively prevents disease or condition, such as disease or ailment caused by a missing, defective, and / or mutated protein or enzyme.

[0057] As used herein, "RNA therapeutic agent" can refer to the use of oligonucleotides to target RNA. RNA therapeutic agents offer the prospect of uniquely targeting precise nucleic acids involved in a specific disease with greater specificity, improved efficacy, and reduced toxicity. This may be particularly effective for genetic diseases, where targeting RNA rather than proteins is most advantageous. In one aspect, therapeutic RNA may comprise one or more expression sequences. As known in the art, expression sequences may include RNAi, shRNA, mRNA, non-coding RNA (ncRNA), antisense such as antisense RNA, miRNA, morpholino oligonucleotides, peptide-nucleotides (PNA), or ssDNA (containing natural and modified nucleotides, including but not limited to LNA, BNA, 2'-O-Me-RNA, 2'-MEO-RNA, 2'-F-RNA), or analogs or conjugates thereof. In one aspect, the disclosed therapeutic RNA may include 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), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), pseudogenes, rRNAs, or tRNAs. In one aspect, the ncRNA may be a piwi-interacting RNA (piRNA), a primary miRNA (pri-miRNA), or a pre-mature miRNA (pre-miRNA). In one aspect, the disclosed therapeutic RNA or RNA therapeutic agent may include antisense oligonucleotides (ASOs) that inhibit mRNA translation, oligonucleotides that act via the RNA interference (RNAi) pathway, RNA molecules that behave like enzymes (ribosomal enzymes), RNA oligonucleotides that bind to proteins and other cellular molecules, and ASOs that bind to mRNA and form structures recognized by RNase H, thereby causing mRNA target cleavage. In one aspect, the RNA therapeutic agent may include both ASOs that inhibit mRNA translation and RNAi. Generally, as is known in the art, RNAi specifically and posttranscribedly manipulates sequences by activating ribonucleases that, together with other enzymes and complexes, synergistically degrade RNA after the original RNA target has been cleaved into smaller fragments, while antisense oligonucleotides bind to their target nucleic acids via Watson-Crick base pairing and inhibit or alter gene expression via steric hindrance, splicing alteration, initiation of target degradation, or other events.

[0058] As used herein, “small molecule” can refer to any organic or inorganic material that is not a polymer. Small molecules do not include large macromolecules, such as large proteins (e.g., proteins with a molecular weight exceeding 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 a molecular weight exceeding 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000), or large polysaccharides (e.g., polysaccharides with a molecular weight exceeding 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000). In one aspect, “small molecule” can, for example, be a drug that can readily enter cells due to its low molecular weight. In one respect, small molecules can be used in combination with disclosed compositions in disclosed methods.

[0059] In one aspect, the term "ex vivo" can generally refer to activities occurring outside an organism or subject, such as experiments, modifications, differentiations, manipulations, and / or measurements performed in or on living tissue in an artificial environment outside an organism. In one aspect, ex vivo experiments, modifications, differentiations, manipulations, and / or measurements can occur with minimal alteration to natural conditions. In one aspect, "ex vivo" can include living cells, tissues, or organs (e.g., cells requiring trans-splicing against one or more protein-coding genes) taken from a subject or donor subject and cultured and / or maintained and / or perfused in laboratory equipment, typically under sterile conditions and typically for a limited duration (e.g., a few hours or up to about 24 hours, up to about 48 hours, up to about 72 hours, up to about 96 hours, up to about 120 hours, up to about 144 hours, up to about 168 hours or longer, depending on the environment and / or desired characteristics). In one aspect, tissues, cells, or organs can be collected, frozen, and subsequently thawed for ex vivo processing.

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

[0061] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. Proteins 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 a short chain of amino acids, including, for example, native peptides, recombinant peptides, synthetic peptides, or any combination thereof. Proteins and peptides can include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins.

[0062] As used herein, “nucleic acid,” “oligonucleotide,” or “polynucleotide” refers to at least two nucleotides covalently linked together. A single-stranded description can also define the sequence of the complementary strand. Therefore, nucleic acid can encompass the complementary strand of the single-stranded nucleic acid described. Many variants of nucleic acids can be used for the same purpose as a given nucleic acid. Therefore, nucleic acid can encompass substantially the same nucleic acid and its complement. A single strand can provide a probe that can hybridize with a target sequence under strict hybridization conditions. Therefore, nucleic acid can encompass probes that hybridize under strict hybridization conditions. Nucleic acids can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA, genomic DNA, and cDNA, RNA, or a hybrid, wherein the nucleic acid can contain combinations of deoxyribonucleotides and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or by recombinant methods. Similarly, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid construct,” “nucleotide sequence,” and “polynucleotide” as used herein can refer to linear or branched, single-stranded or double-stranded RNA or DNA, or hybrids thereof. The term can encompass RNA / DNA hybrids. When dsRNA is synthesized, less common bases such as inosine, 5-methylcytosine, 6-methyladenine, and hypoxanthine can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides containing C-5 propyne analogs of uridine and cytidine have been shown to bind RNA with high affinity and are effective antisense inhibitors of gene expression. Other modifications can also be made, such as modifications to the 2'-hydroxyl group in the phosphodiester backbone or the ribose group of RNA. “Synthetic” nucleic acids or polynucleotides as used herein refer to nucleic acids or polynucleotides that are not found in nature but are artificially constructed and are therefore not products of nature.

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

[0064] A “fragment” or “part” of a nucleotide sequence can be understood as a nucleotide sequence that is reduced in length (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides) relative to a reference nucleic acid or nucleotide sequence, and comprises, is substantially composed of, or is composed of the following nucleotide sequences: consecutive nucleotides that are 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%) to the reference nucleic acid or nucleotide sequence. Such a nucleic acid fragment or part according to this disclosure may (where appropriate) be included in a larger polynucleotide in which the nucleic acid fragment or part is a component. In one aspect, a fragment or portion of a nucleotide or nucleic acid sequence may include a sequence encoding an exon with one or more mutations.

[0065] A “fragment” or “part” of an amino acid sequence can be understood as representing an amino acid sequence that is shorter than a reference amino acid sequence (e.g., shorter 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 contains, is substantially composed of or is composed of the following amino acid sequences: identical or nearly identical to the reference amino acid 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). According to this disclosure, such amino acid fragments or portions may be included in the larger amino acid sequence in which they constitute when appropriate.

[0066] The terms “heterologous” or “recombinant” nucleotide or amino acid sequence are used interchangeably in this document and may refer to a nucleotide or amino acid sequence that is not naturally associated with the host cell to which it is introduced, including multiple non-natural copies of naturally occurring nucleotide or amino acid sequences.

[0067] As used herein, the term "endogenous" can refer to genes, proteins, compounds, or activities (e.g., pre-mRNA) that are normally present in host cells. As used herein, the term "exogenous" nucleic acid molecule, construct, or sequence (e.g., RNA sequence to be trans-spliced) can refer to a nucleic acid molecule or portion thereof that is not native to the host cell but may be homologous to a nucleic acid molecule or portion thereof derived from the host cell.

[0068] Different nucleic acids or proteins that share homology can be referred to as "homologous sequences." The term homologous sequences includes homologous sequences from the same and other species, and orthologous sequences from the same and other species. "Homology" refers to the level of similarity between two or more nucleic acid and / or amino acid sequences, expressed as 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. Therefore, the disclosed compositions and methods may include homologous sequences of disclosed nucleotide sequences and / or disclosed polypeptide sequences.

[0069] As used herein, “orthologs” can refer to homologous nucleotide and / or amino acid sequences in different species arising from a common ancestral gene during speciation. Homologs of disclosed nucleotide sequences or disclosed polypeptides may have substantial sequence identity with the disclosed nucleotide sequence or disclosed polypeptide (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” or “complementary” means that nucleic acids can be paired in Watson-Crick (e.g., AT / U and CG) or Hoogsteen manner between nucleotides or nucleotide analogs of a nucleic acid molecule. “Complementarity” refers to a property shared between two nucleic acid sequences such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.

[0071] As used herein, "promoter" or "multiple promoters" are known in the art. A variety of 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 gene expression pattern. Promoters can be natural (endogenous) or exogenous (exogenous), and can be natural or synthetic sequences. Exogenous or exogenous means that the transcription start region is absent in the wild-type host in which the transcription start region is introduced.

[0072] "Tissue-specific promoters" are known in the art, including but 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 those known in the art, including but not limited to thyroxine-binding globulin (TBG) promoters, α1-microglobulin / bikuin enhancers / thyroxine-binding globulin promoters, human albumin (hALB) promoters, thyroxine-binding globulin promoters, α-1-antitrypsin promoters, bovine albumin (bAlb) promoters, mouse albumin (mAlb) promoters, human α1-antitrypsin (hAAT) promoters, ApoEhAAT promoters containing ApoE enhancers and hAAT promoters, transthyretin (TTR) promoters, liver fatty acid-binding protein promoters, hepatitis B virus (HBV) promoters, DC172 promoters containing hAAT promoters and α1-microglobulin enhancers, DC190 promoters containing human albumin promoters and prothrombin enhancers, or any other natural or synthetic liver-specific promoters. In one aspect, a liver-specific promoter may include approximately 845 bp and includes a thyroid hormone-binding globulin promoter sequence (2382 to 13), two copies of an α1-microglobulin / bicubin enhancer sequence (22,804 to 22,704), and a 71 bp leader sequence, as described by Ill CR et al. (1997).

[0074] Ubiquitous / constitutive promoters are known in the art and include, but are not limited to, the CMV major immediate early enhancer / chicken β-actin promoter, the cytomegalovirus (CMV) major immediate early promoter, the elongation factor 1-a (EFl-a) promoter, the simian vacuolating virus 40 (SV40) promoter, the AmpR promoter, the PyK 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 Rous sarcoma virus promoter, or any other natural or synthetic ubiquitous / constitutive promoter.

[0075] As used in this article, "inducible promoter" refers to a promoter that can be modulated by positive or negative control. Factors that can modulate inducible promoters 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" refers to an AAV that has a capsid that is serologically distinct from other AAV serotypes. Serological uniqueness can be determined by the lack of cross-reactivity between antibodies against an AAV compared to another AAV. This difference in cross-reactivity is typically due to differences in capsid protein sequence / antigenic determinants (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of the AAV serotype).

[0077] As used herein, "tropism" refers to the specificity of AAV capsid proteins present in AAV viral particles for infecting a specific type of cell or tissue. The tropism of the AAV capsid for a specific 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 specific type of cell or tissue using standard assays well known in the art (e.g., those disclosed in the embodiments of this application). As used herein, the terms "liver tropism" or "liver tropism" refer to tropism for the liver or liver tissue and cells (including hepatocytes).

[0078] "Sequence identity" and "sequence similarity" can be determined by aligning two peptide or two nucleotide sequences using global or local alignment algorithms. When a sequence is optimally aligned, it can then be described as "substantially identical" or "substantially similar." For example, sequence similarity or identity can be determined by searching databases such as FASTA, BLAST, etc., but hits should be retrieved and compared side-by-side to determine sequence identity. Two proteins, two protein domains, or two nucleic acid sequences can be considered to have "substantially identical sequence identity" if the percentage of sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 98%, or higher, preferably 90%, 95%, 98%, 99%, or higher. Such sequences are also referred to herein as "variants," for example, other variants of deleted, defective, and / or mutated proteins or enzymes. It should be understood that sequences with substantial sequence identity do not necessarily have the same length and can differ in length. For example, sequences with the same nucleotide sequence but one having additional nucleotides on the 3'- and / or 5'- sides are 100% identical.

[0079] As used herein, "codon optimization" can 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 natural sequence with codons that are more frequently or most frequently used in the gene in the host cell, while preserving the natural amino acid sequence. Various species exhibit a particular preference for certain codons of specific amino acids. As considered herein, genes can be tailored based on codon optimization for optimal gene expression in a given organism. Codon usage tables are readily available, for example, in codon usage databases. Numerous methods and software tools for codon optimization have been previously reported (see, for example, genomes.urv.es / OPTIMIZER / ).

[0080] In one respect, “RNA editing” can be post-transcriptional modification, where the nucleotide sequence of precursor mRNA (pre-mRNA) is altered through base insertion, deletion, or modification. The extent of RNA editing varies, ranging from hundreds of bases in trypanosome mitochondrial DNA to a single base in mammalian nuclear genes. Even a single base change in pre-mRNA can convert a codon of one amino acid to a codon of another or a stop codon. This type of recoding can significantly affect the structure and function of proteins and can lead to multiple variants of proteins derived from a single gene.

[0081] In one respect, insertional and deletional RNA editing can involve adding and deleting specific nucleotides or sequences of nucleotides from pre-mRNA. In another respect, substitutional RNA editing via base modification has been observed in higher eukaryotes, where bases are modified without altering the length of the pre-mRNA.

[0082] As used herein, “immune tolerance,” “immunologic tolerance,” and “immune tolerance” refer to the state of the immune system’s unresponsive or blunted response to substances capable of evoking an immune response in a subject (e.g., publicly available nucleic acid molecules, publicly available vectors, publicly available transgenic products, publicly available pharmaceutical preparations, publicly available therapeutic agents, etc.). Immune tolerance is induced by prior exposure to a specific antigen. It can be determined in subjects by measuring antibodies against a specific antigen or by liver-restricted transgenic expression using a viral vector (e.g., AAV). Low antibody titers or the absence of antibody titers over time are indicators of immune tolerance. For example, in some implementations, immune tolerance can be determined by having an IgG antibody titer less than or equal to about 12,000, 11,500, 11,000, 10,500, 10,000, 9,500, 9,000, 8,500, 8,000, 7,500, 7,000, 6,500, or 6,000 after gene therapy (e.g., administration of a transgenic protein or enzyme that is missing, defective, or mutated).

[0083] As is known in the art, antibodies (Abs) can mitigate AAV infection through a variety of mechanisms, mediating their rapid clearance from circulation by binding to the AAV capsid and blocking key steps in transduction, such as cell surface attachment and uptake, endosome escape, productive transport to the nucleus, or uncoating, as well as promoting AAV opsonization by phagocytes. For example, in humans, serological studies have revealed a high prevalence of NAbs in the global population, with approximately 67% of individuals possessing antibodies against AAV1, 72% against AAV2, and approximately 40% against AAV serotypes 5 through 9. Vector immunogenicity represents a major challenge in the reapplication of AAV vectors.

[0084] In one aspect, this document also discloses partially self-complementary parvovirus (e.g., disclosed AAV) genomes, plasmid vectors encoding said parvovirus genomes, and parvovirus (e.g., disclosed AAV) particles comprising these genomes. In another aspect, this document provides a plasmid vector containing a nucleotide sequence encoding a disclosed parvovirus genome (e.g., disclosed AAV). In yet another aspect, this document provides a partially self-complementary parvovirus genome comprising a payload construct, a parvovirus ITR flanking the payload construct, and a self-complementary region flanking one ITR. The self-complementary region may contain nucleotide sequences complementary to the payload construct. A disclosed self-complementary region may have a length less than the entire length of the payload construct.

[0085] In one aspect, the publicly disclosed self-complementary region of the publicly disclosed parvovirus genome may contain a minimum length while still having a length less than the entire length of the payload construct. In another aspect, the publicly disclosed self-complementary region may contain a length of at least 50 bases, at least 100 bases, at least 200 bases, at least 300 bases, at least 400 bases, at least 500 bases, at least 600 bases, at least 700 bases, at least 800 bases, at least 900 bases, or at least 1,000 bases.

[0086] In one aspect, a “self-complementary parvovirus genome” can be a single-stranded polynucleotide having a first parvovirus ITR sequence, a heterologous sequence (e.g., a payload construct containing, for example, the desired gene), a second parvovirus ITR sequence, a second heterologous sequence, and a third parvovirus ITR sequence in the 5' to 3' direction, wherein the second heterologous sequence is complementary to the first heterologous 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 has a length less than the entire length of the first heterologous sequence (e.g., the 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 complementary to a portion of the heterologous sequence and having a length less than the entire length of the heterologous sequence in the 5' to 3' or 3' to 5' direction.

[0087] As used herein, "immunomodulatory" refers to the ability of publicly disclosed nucleic acid molecules, publicly disclosed vectors, publicly disclosed pharmaceutical preparations, or publicly disclosed reagents to alter (modulate) one or more aspects of the immune system. The immune system protects an organism from infection and foreign antigens through cellular and humoral mechanisms involving lymphocytes, macrophages, and other antigen-presenting cells. These cellular and humoral mechanisms are mutually regulated through a variety of cell-cell interactions and through the elaboration of soluble factors (including lymphokines and antibodies) that have autocrine, paracrine, and endocrine effects on immune cells.

[0088] As used herein, "immunomodulator" refers to an agent capable of modulating a given immune response to a desired level (e.g., in immune enhancement, immunosuppression, or induction of immune tolerance). Examples of immunomodulators include, but are not limited to, disclosed immunomodulators that may include aspirin, azathioprine, belimumab, betamethasone dipropionate, betamethasone valerate, bortezomib, brebrutinib, cyazathioprine, cyclophosphamide, cyclosporine, deoxyguanidine, didemnin B, fluocinolone acetonide, leucovorin, ibuprofen, IL-6 inhibitors (e.g., sarilumab), indomethacin, inebilizumab, intravenous gamma globulin (IVIG), methotrexate, methylprednisolone, and mycophenolate mofetil. (mofetil), naproxen, prednisolone, prednisolone, prednisolone, indomethacin, rapamycin, rituximab, sirolimus, sulindac, synthetic vaccine particles containing rapamycin (SVP-rapamycin or ImmTOR), thalidomide, tocilizumab, tometidine, 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-IGFIR antibody, IL2 mutant protein, BTK inhibitor, or combinations thereof. In one aspect, the disclosed immunomodulatory agent may include one or more Treg (regulatory T cell) infusions (e.g., antigen-specific Treg cells against AAV). In one aspect, the disclosed immunomodulatory agent may be bortezomib or SVP-rapamycin. In one aspect, immunomodulatory agents can be administered via any suitable route of administration, including but not limited to intrauterine, intra-CSF, intrathecal, intravenous, subcutaneous, transdermal, intradermal, intramuscular, oral, percutaneous, intraperitoneal (IP), or vaginal administration. In another aspect, a combination of routes can be used to administer a disclosed immunomodulatory agent. Administration may also include intrahepatic artery administration or administration via the portal vein (HPV). Administration of immunomodulatory agents can be continuous or intermittent, and may include a combination of one or more routes.

[0089] As used herein, the term "immunotolerant" refers to a lack of response to antigens (e.g., vectors, therapeutic proteins, transgenic products, etc.). Immunotolerant promoters can reduce, mitigate, or prevent transgene-induced immune responses that may be associated with gene therapy. Assays known in the art for measuring immune responses, such as immunohistochemical assays of cytotoxic T-cell responses, can be used to determine whether one or more promoters confer immunotolerant properties.

[0090] As used herein, the term "packaging insert" refers to the instruction leaflet typically included in the commercial packaging of a therapeutic product, which contains information about indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic products.

[0091] As used herein in the context of administering other therapies (e.g., other agents), the term "combination" includes the use of more than one therapy (e.g., pharmaceutical therapy). Administration in combination with one or more other therapeutic agents includes simultaneous (e.g., concurrent) and sequential administration in any order. The use of the term "combination" does not limit the order in which the therapies are administered to the subject. As a non-limiting example, a first therapy (e.g., a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical preparation, or a combination thereof) may be administered to a subject who has or is diagnosed with a disease or condition (e.g., a hereditary disease or condition) before (e.g., at 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 hour ... It can be administered in 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks), in parallel or after (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks or longer).

[0092] Components for preparing the disclosed nucleic acid molecules, disclosed carriers, or disclosed pharmaceutical formulations, as well as the disclosed nucleic acid molecules, disclosed carriers, or disclosed pharmaceutical formulations used in the methods disclosed herein, are disclosed herein. These and other materials are disclosed herein, and it should be understood that when combinations, subsets, interactions, groups, etc., of these materials are disclosed, while specific mentions of every different individual and collective combination and arrangement of these compounds are not explicitly disclosed, each is specifically considered and described herein. For example, if a particular compound is disclosed and discussed, and many modifications that can be made to a number of molecules including the compound are discussed, then the compound and possible modifications, as well as every combination and arrangement thereof, are specifically considered, unless otherwise expressly indicated to the contrary. Thus, if a class of molecules A, B, and C and a class of molecules D, E, and F are disclosed, and an example of the combination molecule AD is disclosed, then each is considered individually and collectively, even if each is not individually enumerated, implying that combinations AE, AF, BD, BE, BF, CD, CE, and CF are disclosed. Similarly, any subsets or combinations of these are also disclosed. Thus, for example, subgroups of AE, BF, and CE should be considered disclosed. This concept applies to all aspects of this application, including but not limited to steps in methods of preparing and using the compositions of the invention. Therefore, if various additional steps are available, it should be understood that each of these additional steps can be performed using any particular embodiment or combination of embodiments of the method of the invention.

[0093] B. Compositions for transcriptome engineering 1. Nucleic acid molecules 5' Replacement builder This article discloses a nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures.

[0094] This article discloses a nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising the sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09.

[0095] This article discloses a nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures, wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA.

[0096] This article discloses a nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising the sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA.

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

[0098] In one respect, the exposed cells can be in the subject. In another respect, the subject can be a human patient and can be male or female. In another respect, the subject can have a genetic disease or condition. In another respect, the subject can be untreated.

[0099] In one aspect, one or more publicly disclosed mutations can inhibit the translation of the encoded protein. In another aspect, one or more publicly disclosed mutations can modify the translation of the encoded protein. In another aspect, one or more publicly disclosed mutations can produce the encoded protein with nonsense or missense mutations.

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

[0101] In one aspect, the disclosed targeted endogenous pre-mRNA may be a primary transcript of a protein-coding gene. In another aspect, the disclosed protein-coding gene may contain one or more coding regions of the following: 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, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44, CFAP65, CFTR / ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1,CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1, CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1, DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11, DNAH17, DNAH2, DNAH5, DNAH7, DNAH8, DNAH9, and DNMBP. DNMT1, DOCK2, DOCK3, DOCK6, DOCK7, DOCK8, DSCAM, DSP, DST, DUOX2, DYNC1H1, DYNC2H1, DYSF. EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, ​​EYS, F5, F8, FANCA, FANCD2, FANCM, FAT1 FAT4, FBN1, FBN2, FLG, FLG2, FLNA, FLNB, FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2, FSIP2. FXN, FYCO1, GLI2, GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1 HERC2, HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R. IGF2R, IGSF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A. KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220. KIF14, KIF1A, KIF1B, KIF21A, KIF26B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1 LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMC3, LCT, LMNA, LOXHD1, LPA, LRBA. LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD. MAGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5, MCM3AP, MED12, MED12L, MED13. MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MYH11, MYH14, MYH2, MYH3, MYH6. MYH7, MYH7B, MYH8, MYH9, MYLK, MYO15A, MYO18B, MYO3A, MYO5A, MYO5B, MYO7A, MYO9A, NALCN NBAS, NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF, NF1, NFASC, NHS, NIN, NIPBL NLRP1、NOTCH1、NOTCH2、NOTCH3、NPHP4、NRXN1、NRXN3、NSD1、NSD2、NUP155、NUP188、NUP205、 OBSCN、OBSL1、OTOF、OTOG、OTOGL、PARD3、PBRM1、PCDH15、PCLO、PCNT、PHIP、PI4KA、PIEZO1、 PIEZO2、PIK3C2A、PIKFYVE、PKD1、PKD1L1、PKHD1、PLCE1、PLEC、PLEKHG2、PNPLA6、POGZ、 POLA1、POLE、POLR1A、POLR2A、POLR3A、PRG4、PRKDC、PRPF8、PRR12、PRX、PTCH1、PTPN23、 PTPRF、PTPRJ、PTPRQ、PXDN、QRICH2、RAB3GAP2、RAI1、RALGAPA1、RANBP2、RB1CC1、RELN、RERE、 REV3L、RIC1、RIMS1、RIMS2、RNF213、ROBO1、ROBO2、ROBO3、ROS1、RP1、RP1L1、RTTN、RUSC2、 RYR1、RYR2、SACS、SAMD9、SAMD9L、SBF2、SCAPER、SCN10A、SCN11A、SCN1A、SCN2A、SCN3A、 SCN4A、SCN5A、SCN8A、SCN9A、SETBP1、SETD1A、SETD1B、SETD2、SETD5、SETX、SHANK2、SHANK3、 SHROOM4、SI、SIPA1L3、SLIT2、SLX4、SMARCA2、SMARCA4、SMCHD1、SNRNP200、SON、SPEF2、SPEG、 SPG11、SPTA1、SPTAN1、SPTB、SPTBN2、SPTBN4、SRCAP、STRC、SVIL、SYNE1、SYNGAP1、SYNJ1、 SZT2、TAF1、TANC2、TCF20、TCOF1、TDRD9、TECPR2、TECTA、TENM3、TENM4、TET3、TEX14、TEX15、 TG, THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B, TNXB, TOGARAM1, TONSL, TRIO, TRIOBP, TRIP11, TRIP12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, 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 NF469. In one respect, publicly available protein-coding genes can contain CFTR, MDX, DYSF / TTN, DMPK, COL7A1, K14, MAPT, FVIII, HTT, RHO, DNA-PKcs, SMN2 or CD40L One or more coding regions. In one respect, a publicly disclosed protein-coding gene may contain... FXN , LMNA or RYR2 One or more coding regions. In one respect, a publicly disclosed protein-coding gene may contain a portion of a publicly disclosed protein-coding gene (e.g., exon 1 or exon 4, etc.).

[0102] In one aspect, the 3' portion of the disclosed targeted endogenous pre-mRNA can be trans-spliced ​​using exogenous RNA. In one aspect, the disclosed RNA targeting motif can bind to the targeted endogenous pre-mRNA. In one aspect, the disclosed RNA targeting motif can bind to the 5' end of the targeted endogenous pre-mRNA. In one aspect, the disclosed RNA targeting motif can be specific to endogenous pre-mRNAs with one or more mutations. In one aspect, the disclosed RNA targeting motif can be specific to endogenous pre-mRNAs with one or more exon mutations. In one aspect, the disclosed RNA targeting motif can be specific to endogenous pre-mRNAs with one or more intron mutations.

[0103] In one aspect, the disclosed RNA targeting motif may contain an antisense oligonucleotide. In another aspect, the disclosed antisense oligonucleotide may contain about 15 to about 50 nucleotides. In another aspect, the disclosed antisense oligonucleotide may contain about 30 nucleotides. In another aspect, the disclosed RNA targeting motif may be directed to an intron adjacent to the 5' of the exon of the targeted endogenous pre-mRNA to be spliced.

[0104] In one aspect, the disclosed 5' hemiintron may include a 5' splice site. In one aspect, the disclosed 5' splice site may include a shared 5' splice site. In one aspect, the disclosed resulting 5' splice site may include MAG|GURAGU (SEQ ID NO: 13), where | denotes exon-intron linkage, where M=A or C, and where R=A or G. In one aspect, the disclosed 5' hemiintron can be recognized by nuclear splicing components in host cells. In one aspect, the disclosed 5' hemiintron can be recognized by spliceosomes in host cells. In one aspect, the disclosed 5' hemiintron can facilitate trans-splicing of exogenous RNA to the 3' exon of an intron immediately adjacent to the target intron in endogenous pre-mRNA.

[0105] In one aspect, the disclosed exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA may contain one or more exons of a protein-coding gene. In another aspect, the disclosed exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA may contain a primary sequence of a coding sequence of one or more exons having one or more mutations. In another aspect, the disclosed exogenous RNA may be trans-spliced ​​to the 3' end of the targeted endogenous pre-mRNA.

[0106] In one aspect of publicly disclosed exogenous RNA, a publicly disclosed protein-coding gene may contain one or more of the following coding regions: 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, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44, CFAP65, CFTR / ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1; CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, COL2A1 COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1 CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1. DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11, DNAH17, DNAH2, DNAH5, DNAH7, DNAH8, DNAH9, and DNMBP. DNMT1, DOCK2, DOCK3, DOCK6, DOCK7, DOCK8, DSCAM, DSP, DST, DUOX2, DYNC1H1, DYNC2H1, DYSF. EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, ​​EYS, F5, F8, FANCA, FANCD2, FANCM, FAT1 FAT4, FBN1, FBN2, FLG, FLG2, FLNA, FLNB, FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2, FSIP2. FXN, FYCO1, GLI2, GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1 HERC2, HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R. IGF2R, IGSF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A. KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220. KIF14, KIF1A, KIF1B, KIF21A, KIF26B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1 LAMA1、LAMA2、LAMA3、LAMA4、LAMA5、LAMB1、LAMB2、LAMC3、LCT、LMNA、LOXHD1、LPA、LRBA、 LRP1、LRP2、LRP4、LRP5、LRP6、LRPPRC、LRRK1、LRRK2、LTBP2、LTBP4、LYST、MACF1、MADD、 MAGI2、MAP1B、MAP3K1、MAPK8IP3、MAPKBP1、MAST1、MBD5、MCM3AP、MED12、MED12L、MED13、 MED13L、MED23、MEGF8、MET、MLH3、MPDZ、MSH6、MTOR、MYH10、MYH11、MYH14、MYH2、MYH3、MYH6、 MYH7、MYH7B、MYH8、MYH9、MYLK、MYO15A、MYO18B、MYO3A、MYO5A、MYO5B、MYO7A、MYO9A、NALCN、 NBAS、NBEA、NBEAL2、NCAPD2、NCAPD3、NEB、NEXMIF、NEXMIF、NF1、NFASC、NHS、NIN、NIPBL、 NLRP1、NOTCH1、NOTCH2、NOTCH3、NPHP4、NRXN1、NRXN3、NSD1、NSD2、NUP155、NUP188、NUP205、 OBSCN、OBSL1、OTOF、OTOG、OTOGL、PARD3、PBRM1、PCDH15、PCLO、PCNT、PHIP、PI4KA、PIEZO1、 PIEZO2、PIK3C2A、PIKFYVE、PKD1、PKD1L1、PKHD1、PLCE1、PLEC、PLEKHG2、PNPLA6、POGZ、 POLA1、POLE、POLR1A、POLR2A、POLR3A、PRG4、PRKDC、PRPF8、PRR12、PRX、PTCH1、PTPN23、 PTPRF、PTPRJ、PTPRQ、PXDN、QRICH2、RAB3GAP2、RAI1、RALGAPA1、RANBP2、RB1CC1、RELN、RERE、 REV3L、RIC1、RIMS1、RIMS2、RNF213、ROBO1、ROBO2、ROBO3、ROS1、RP1、RP1L1、RTTN、RUSC2、 RYR1、RYR2、SACS、SAMD9、SAMD9L、SBF2、SCAPER、SCN10A、SCN11A、SCN1A、SCN2A、SCN3A、 SCN4A, SCN5A, SCN8A, SCN9A, SETBP1, SETD1A, SETD1B, SETD2, SETD5, SETX, SHANK2, SHANK3, SHROOM4, SI, SIPA1L3, SLIT2, SLX4, SMARCA2, SMARCA4, SMCHD1, SNRNP200, SON, SPEF2, SPEG, SPG11, SPTA1, SPTAN1, SPTB, SPTBN2, SPTBN4, SRCAP, STRC, SVIL, SYNE1, SYNGAP1, SYNJ1, SZT2, TAF1, TANC2, TCF20, TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B, TNXB, TOGARAM1, TONSL, TRIO, TRIOBP, TRIP11, TRIP12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, 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 respect, publicly available protein-coding genes can contain CFTR, MDX, DYSF / TTN, DMPK, COL7A1, K14, MAPT, FVIII, HTT, RHO, DNA-PKcs, SMN2 or CD40L One or more coding regions. In one respect, a publicly disclosed protein-coding gene may contain... FXN, LMNA or RYR2 One or more encoding regions.

[0107] In one aspect, the disclosed nucleic acid sequence to be trans-spliced ​​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 the nucleotide sequence may contain nucleotides 4974-62517 of accession number NG008692.2. The nuclear layer consists of a two-dimensional matrix of proteins located adjacent to the nuclear membrane. Proteins of the lamin family constitute the matrix and are highly conserved in evolution. During mitosis, the nuclear matrix is ​​reversibly degraded as lamins are phosphorylated. Lamins are involved in nuclear stability, chromatin structure, and gene expression. Vertebrate lamins consist of two types, A and B. Alternative splicing produces a variety of transcript variants. Mutations in this gene can lead to several diseases: Emery-Dreifuss muscular dystrophy, familial partial lipodystrophy, limb-girdle muscular dystrophy, dilated cardiomyopathy, Charcot-Marie-Tooth disease, and Hutchinson-Gilford progeria.

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

[0109] In one aspect, the disclosed nucleic acid sequence to be trans-spliced ​​may encode CFTR (SEQ ID NO: 19) or a portion thereof. CFTR is known in the art (e.g., Gene ID 1080), and the nucleotide sequence may contain nucleotides 19180-207882 in accession number NG016465.4. This gene encodes a member of the ATP-binding cassette (ABC) transporter superfamily. The encoded protein acts as a chloride ion channel, making it unique among members of this protein family, and controls the secretion and uptake of ions and water in epithelial tissues. Channel activation is mediated by a cycle of phosphorylation of the regulatory domain, ATP binding of the nucleotide-binding domain, and ATP hydrolysis. Mutations in this gene lead to cystic fibrosis, the most common fatal genetic disease in populations of Nordic descent. The most common 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 aspect, the disclosed nucleic acid sequence to be trans-spliced ​​may encode DMPK (SEQ ID NO: 21) or a portion thereof. DMPK is known in the art (e.g., Gene ID 1760), and the nucleotide sequence may contain nucleotides 5068-17841 of accession number NG009784.1. DMPK is a serine-threonine kinase, closely associated with other kinases that interact with the Rho family members of small GTPases. The substrates of this enzyme include myopoietin (the β-subunit of an L-type calcium channel) and phosphatases. The 3' untranslated region of this gene contains 5-38 copies of a CTG trinucleotide repeat sequence. Amplification of this unstable motif to 50-5,000 copies causes type I myotonic dystrophy, the severity of which increases with the copy number of the repeat element. Repeat amplification is associated with condensation that disrupts local chromatin structure and gene expression in this region. Several alternative splicing transcript variants of this gene have been described, but the full-length nature of some of these variants has not been determined.

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

[0112] In one aspect, the disclosed nucleic acid sequence to be trans-spliced ​​may encode LRRK2 (SEQ ID NO: 18) or a portion thereof. LRRK2 is known in the art (e.g., Gene ID 120892), and the nucleotide sequence may contain 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. This protein is primarily located in the cytoplasm but is also associated with the outer mitochondrial membrane. Mutations in this gene are associated with Parkinson's disease.

[0113] In one respect, the exogenous RNA to be trans-spliced ​​may further include a UTR.

[0114] In one aspect, one or more disclosed RNA structures may bind to one or more RNA-binding proteins. In another aspect, one or more disclosed RNA structures may bind to one or more double-stranded RNA-binding proteins (dsRBPs). In another aspect, 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 aspect, one or more disclosed RNA structures may comprise sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09. In one aspect, one or more disclosed RNA structures may improve and / or enhance trans-splicing efficiency. In one aspect, one or more disclosed RNA structures may stabilize pre-mRNA. In one aspect, one or more disclosed RNA structures may localize RNA to the cell nucleus. In one aspect, one or more disclosed RNA structures may stabilize the interaction between the targeted endogenous pre-mRNA molecule and the exogenous RNA to be trans-spliced. In one aspect, the disclosed nucleic acid molecule may lack CRISPR-related proteins.

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

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

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

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

[0120] In one aspect, the disclosed nucleic acid molecule may further include one or more nuclear localization signals. NLS are known to those skilled in the art. In another aspect, the disclosed NLS may include 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, whose teachings on NLS are incorporated herein by reference), nuclear localization signals (NLS) are typically short peptides that act as signal fragments mediating the transport of proteins from the cytoplasm to the nucleus.

[0121] In one aspect, the disclosed nucleic acid molecule may further include one or more nuclear retention elements (NREs). NREs are known to those skilled in the art. In one aspect, the disclosed NRE may include SIRLOIN (SEQ ID NO:15) or BORG (SEQ ID NO:16).

[0122] In one aspect, the disclosed nucleic acid molecule may further comprise one or more flavivirus genetic elements. In another aspect, a flavivirus genetic element may comprise one or more flavivirus 3' untranslated region (3'UTR), 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. (For a description of flavivirus genetic elements, see WO 2022 / 182835).

[0123] In one respect, exposed exogenous RNA can induce splicing events. In another respect, exposed 5' half-introns can be recognized by nuclear splicing components within the host cell.

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

[0125] This document discloses an expression cassette comprising: exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09. This document discloses an expression cassette comprising: exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures, wherein the nucleic acid molecules enable 5' substitution of the targeted endogenous pre-mRNA. This article discloses an expression cassette comprising: an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising the sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the nucleic acid molecules enable 5' substitution of the targeted endogenous pre-mRNA.

[0126] In one respect, the expression of the disclosed protein-coding gene can be restored and / or returned to wild-type, normal, or control expression levels. In one respect, the disclosed nucleic acid molecule can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic dysregulation. In one respect, the disclosed nucleic acid molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above). In one aspect, one or more aspects of restoring cellular homeostasis and / or cellular function may include one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (e.g., correcting, preventing, reducing, and / or alleviating autophagy); (iii) improving, enhancing, restoring, and / or preserving mitochondrial function and / or structural integrity; (iv) improving, enhancing, restoring, and / or preserving organelle function and / or structural integrity; (v) correcting enzymatic dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a multisystemic manifestation of a genetic disease or condition; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a genetic disease or condition, or (viii) any combination thereof. In one aspect, one or more aspects of restoring cellular homeostasis may include one or more aspects of improving, enhancing, restoring, and / or preserving cellular structural and / or functional integrity.

[0127] In one aspect, restoration of the activity and / or function of a missing, defective, and / or mutated protein or enzyme may include restoration of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount compared to a pre-existing level, such as a pre-treatment level. In one aspect, 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% above the pre-existing level (e.g., pre-treatment level). In one aspect, restoration may be measured against a control or reference level (e.g., determined using one or more subjects who do not have the missing, defective, and / or mutated protein or enzyme). In one aspect, restoration may be partial or incomplete. In one aspect, restoration may be complete or near-complete, such that the levels of expression, activity, and / or function are similar to wild-type or control levels.

[0128] In one aspect, the disclosed 5' substitution construct can be used specifically in methods for treating subjects with autosomal dominant genetic diseases or conditions, such as progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary elliptosis, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, marble bone disease, Marfan syndrome, neurofibromatosis, polycystic kidney disease (adults), protein C deficiency, osteogenesis imperfecta, Treacher Collins syndrome, tuberous sclerosis, Von Willebrand's disease, etc.

[0129] 3' Replacement builder This document discloses a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA. This document discloses a nucleic acid molecule comprising one or more RNA structures comprising sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA. This document discloses a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA. This article discloses a nucleic acid molecule comprising one or more RNA structures, said RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA.

[0130] In one aspect, the disclosed targeted endogenous pre-mRNA may contain one or more mutations in one or more exons. In one aspect, one or more disclosed mutations may be in the 3' portion of one or more exons of the pre-mRNA. In one aspect, the disclosed targeted endogenous pre-mRNA may contain one or more mutations in one or more introns. In one aspect, one or more disclosed mutations in one or more exons may contribute to the pathogenesis of one or more cells. In one aspect, the disclosed cells may be in a subject. In one aspect, the subject may be a human patient and may be male or female. In one aspect, the subject may have a genetic disease or condition. In one aspect, the subject may be untreated. In one aspect, one or more disclosed mutations may inhibit the translation of the encoded protein. In one aspect, one or more disclosed mutations may modify the translation of the encoded protein. In one aspect, during and / or after translation, one or more disclosed mutations may produce a protein with nonsense or missense mutations. In one aspect, one or more disclosed exon mutations may contribute to the pathogenesis of one or more cells. In one aspect, one or more disclosed intron mutations may contribute to the pathogenesis of one or more cells. In one respect, the publicly disclosed targeted endogenous pre-mRNA can encode protein-coding genes.

[0131] In one aspect of the publicly disclosed targeted endogenous pre-mRNA, the disclosed protein-coding gene may contain one or more of the following coding regions: ABCA ​ ​ ​ 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, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2 CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44 CFAP65, CFTR / ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1 CNTNAP1、COL11A1、COL11A2、COL12A1、COL17A1、COL18A1、COL1A1、COL1A2、COL27A1、COL2A1、 COL3A1、COL4A1、COL4A2、COL4A3、COL4A4、COL4A5、COL4A6、COL5A1、COL5A2、COL6A3、COL7A1、 CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1 DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11,DNAH17,DNAH2,DNAH5,DNAH7,DNAH8,DNAH9,DNAMBP.” DNMT1、DOCK2、DOCK3、DOCK6、DOCK7、DOCK8、DSCAM、DSP、DST、DUOX2、DYNC1H1、DYNC2H1、DYSF、 EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, ​​EYS, F5, F8, FANCA, FANCD2, FANCM, FAT1 FAT4、FBN1、FBN2、FLG、FLG2、FLNA、FLNB、FLNC、FLT4、FMN2、FN1、FRAS1、FREM1、FREM2、FSIP2、 FXN、FYCO1、GLI2、GLI3、GPR179、GREB1L、GRIN2A、GRIN2B、GRIN2D、HCFC1、HECW2、HERC1、 HERC2、HFM1、HIVEP1、HIVEP2、HMCN1、HSPG2、HTT、HUWE1、HYDIN、IFT140、IFT172、IGF1R、 IGF2R、IGSF1、INSR、INTS1、IQSEC2、ITGB4、ITPR1、ITPR2、JMJD1C、KALRN、KANK1、KAT6A、 KAT6B、KDM3B、KDM5B、KDM5C、KDM6A、KDM6B、KDR、KIAA0586、KIAA1109、KIAA1549、KIDINS220、 KIF14、KIF1A、KIF1B、KIF21A、KIF26B、KIF7、KMT2A、KMT2B、KMT2C、KMT2D、KMT2E、KNL1、 LAMA1、LAMA2、LAMA3、LAMA4、LAMA5、LAMB1、LAMB2、LAMC3、LCT、LMNA、LOXHD1、LPA、LRBA、 LRP1、LRP2、LRP4、LRP5、LRP6、LRPPRC、LRRK1、LRRK2、LTBP2、LTBP4、LYST、MACF1、MADD、 MAGI2、MAP1B、MAP3K1、MAPK8IP3、MAPKBP1、MAST1、MBD5、MCM3AP、MED12、MED12L、MED13、 MED13L、MED23、MEGF8、MET、MLH3、MPDZ、MSH6、MTOR、MYH10、MYH11、MYH14、MYH2、MYH3、MYH6、 MYH7, MYH7B, MYH8, MYH9, MYLK, MYO15A, MYO18B, MYO3A, MYO5A, MYO5B, MYO7A, MYO9A, NALCN NBAS, NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF, NF1, NFASC, NHS, NIN, NIPBL NLRP1、NOTCH1、NOTCH2、NOTCH3、NPHP4、NRXN1、NRXN3、NSD1、NSD2、NUP155、NUP188、NUP205、 OBSCN、OBSL1、OTOF、OTOG、OTOGL、PARD3、PBRM1、PCDH15、PCLO、PCNT、PHIP、PI4KA、PIEZO1、 PIEZO2、PIK3C2A、PIKFYVE、PKD1、PKD1L1、PKHD1、PLCE1、PLEC、PLEKHG2、PNPLA6、POGZ、 POLA1, POLE, POLR1A, POLR2A, POLR3A, PRG4, PRKDC, PRPF8, PRR12, PRX, PTCH1, PTPN23, PTPRF, PTPRJ, PTPRQ, PXDN, QRICH2, RAB3GAP2, RAI1, RALGAPA1, RANBP2, RB1CC1, RELN, RERE, REV3L, RIC1, RIMS1, RIMS2, RNF213, ROBO1, ROBO2, ROBO3, ROS1, RP1, RP1L1, RTTN, RUSC2, RYR1, RYR2, SACS, SAMD9, SAMD9L, SBF2, SCAPER, SCN10A, SCN11A, SCN1A, SCN2A, SCN3A,SCN4A, SCN5A, SCN8A, SCN9A, SETBP1, SETD1A, SETD1B, SETD2, SETD5, SETX, SHANK2, SHANK3, SHROOM4, SI, SIPA1L3, SLIT2, SLX4, SMARCA2, SMARCA4, SMCHD1, SNRNP200, SON, SPEF2, SPEG, SPG11, SPTA1, SPTAN1, SPTB, SPTBN2, SPTBN4, SRCAP, STRC, SVIL, SYNE1, SYNGAP1, SYNJ1, SZT2, TAF1, TANC2, TCF20, TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B, TNXB, TOGARAM1, TONSL, TRIO, TRIOBP, TRIP11, TRIP12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, 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 respect, publicly available protein-coding genes can contain CFTR, MDX, DYSF / TTN, DMPK, COL7A1, KI4, MAPT, FVIII HTT, RHO, DNA-PKcs, SMN2 or CD40L One or more coding regions. In one respect, a publicly disclosed protein-coding gene may contain... FXN, LMNA or RYR2 One or more coding regions. In one respect, a publicly disclosed protein-coding gene may contain a portion of a publicly disclosed protein-coding gene (e.g., exon 1 or exon 4, etc.).

[0132] In one respect, the 5' portion of the publicly disclosed targeted endogenous pre-mRNA can be trans-spliced ​​with exogenous RNA.

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

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

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

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

[0137] In one aspect of publicly disclosed exogenous RNA, a publicly disclosed protein-coding gene may contain one or more of the following coding regions: ABCA1, ABCA12, ABC13, ABCA2, ABCA3, ABCA4, ABCA5, ABCC1, ABC22, ABC6, ABC8. 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, 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, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2 CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44. CFAP65, CFTR / ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1; CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, COL2A1 COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1 CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1. DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11, DNAH17, DNAH2, DNAH5, DNAH7, DNAH8, DNAH9, and DNMBP. DNMT1, DOCK2, DOCK3, DOCK6, DOCK7, DOCK8, DSCAM, DSP, DST, DUOX2, DYNC1H1, DYNC2H1, DYSF. EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, ​​EYS, F5, F8, FANCA, FANCD2, FANCM, FAT1 FAT4, FBN1, FBN2, FLG, FLG2, FLNA, FLNB, FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2, FSIP2. FXN, FYCO1, GLI2, GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1 HERC2, HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R. IGF2R, IGSF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A. KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220. KIF14, KIF1A, KIF1B, KIF21A, KIF26B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1 LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMC3, LCT, LMNA, LOXHD1, LPA, LRBA. LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD. MAGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5, MCM3AP, MED12, MED12L, MED13. MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MYH11, MYH14, MYH2, MYH3, MYH6. MYH7, MYH7B, MYH8, MYH9, MYLK, MYO15A, MYO18B, MYO3A, MYO5A, MYO5B, MYO7A, MYO9A, NALCN. NBAS, NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF, NF1, NFASC, NHS, NIN, NIPBL NLRP1、NOTCH1、NOTCH2、NOTCH3、NPHP4、NRXN1、NRXN3、NSD1、NSD2、NUP155、NUP188、NUP205、 OBSCN、OBSL1、OTOF、OTOG、OTOGL、PARD3、PBRM1、PCDH15、PCLO、PCNT、PHIP、PI4KA、PIEZO1、 PIEZO2、PIK3C2A、PIKFYVE、PKD1、PKD1L1、PKHD1、PLCE1、PLEC、PLEKHG2、PNPLA6、POGZ、 POLA1、POLE、POLR1A、POLR2A、POLR3A、PRG4、PRKDC、PRPF8、PRR12、PRX、PTCH1、PTPN23、 PTPRF、PTPRJ、PTPRQ、PXDN、QRICH2、RAB3GAP2、RAI1、RALGAPA1、RANBP2、RB1CC1、RELN、RERE、 REV3L、RIC1、RIMS1、RIMS2、RNF213、ROBO1、ROBO2、ROBO3、ROS1、RP1、RP1L1、RTTN、RUSC2、 RYR1、RYR2、SACS、SAMD9、SAMD9L、SBF2、SCAPER、SCN10A、SCN11A、SCN1A、SCN2A、SCN3A、 SCN4A、SCN5A、SCN8A、SCN9A、SETBP1、SETD1A、SETD1B、SETD2、SETD5、SETX、SHANK2、SHANK3、 SHROOM4、SI、SIPA1L3、SLIT2、SLX4、SMARCA2、SMARCA4、SMCHD1、SNRNP200、SON、SPEF2、SPEG、 SPG11、SPTA1、SPTAN1、SPTB、SPTBN2、SPTBN4、SRCAP、STRC、SVIL、SYNE1、SYNGAP1、SYNJ1、 SZT2、TAF1、TANC2、TCF20、TCOF1、TDRD9、TECPR2、TECTA、TENM3、TENM4、TET3、TEX14、TEX15、 TG、THOC2、TMEM94、TNC、TNIK、TNR、TNRC6B、TNXB、TOGARAM1、TONSL、TRIO、TRIOBP、TRIP11、 TRIP12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, 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 respect, publicly available protein-coding genes can contain CFTR, MDX, DYSF / TTN, DMPK, COL7A1, KI4, MAPT, FVIII HTT, RHO, DNA-PKcs, SMN2 or CD40L One or more coding regions. In one respect, a publicly disclosed protein-coding gene may contain... FXN, LMNA or RYR2 One or more encoding regions.

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

[0139] In one respect, the exogenous RNA to be trans-spliced ​​may further include a UTR.

[0140] In one aspect, one or more disclosed RNA structures may bind to one or more RNA-binding proteins. In another aspect, one or more disclosed RNA structures may bind to one or more double-stranded RNA-binding proteins (dsRBPs). In another aspect, 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 aspect, one or more disclosed RNA structures can bind to one or more RNA-binding proteins. In one aspect, one or more disclosed RNA structures can bind to one or more double-stranded RNA-binding proteins. In one aspect, one or more disclosed RNA structures can comprise a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09. In one aspect, one or more disclosed RNA structures can improve and / or enhance trans-splicing efficiency. In one aspect, one or more disclosed RNA structures can stabilize pre-mRNA. In one aspect, one or more disclosed RNA structures can localize RNA to the cell nucleus. In one aspect, one or more disclosed RNA structures can stabilize the interaction between the targeted internal pre-mRNA molecule and the exogenous RNA to be trans-spliced.

[0142] In one respect, publicly available nucleic acid molecules may lack CRISPR-related proteins.

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

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

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

[0146] On the one hand, the disclosed nucleic acid molecule may further include one or more flavivirus genetic elements. On the other hand, a flavivirus genetic element may include one or more flavivirus 3' untranslated region (3'UTR), 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. (For a description of flavivirus genetic elements, see WO 2022 / 182835).

[0147] In one aspect, the disclosed nucleic acid molecule may contain the sequence of one or more regulatory elements, such as a post-transcriptional regulatory element (WPRE) of marmot hepatitis virus (WHV), a triple helix from MALAT1, a PRE (HPRE) of hepatitis B virus, and an iron response element. For example, the disclosed regulatory element may contain a promoter operatively linked to the disclosed nucleic acid molecule, wherein the promoter drives the expression of a disclosed variant capsid protein, a disclosed encoded polypeptide, a disclosed encoded therapeutic agent, or both.

[0148] In one aspect, the disclosed promoter for the 3' substitution construct may be tissue-specific or ubiquitous, and may be constitutive or inducible, depending on the desired expression pattern. The promoter may be natural or exogenous, and may be a natural or synthetic sequence. Exogenous means that the transcription start region is absent in the wild-type host in which the transcription start region is introduced. In one aspect, the disclosed promoter may be a promoter / enhancer. In one aspect, the disclosed promoter for the disclosed nucleic acid molecule may be an endogenous promoter. In one aspect, the disclosed endogenous promoter may be an endogenous promoter / enhancer. In one aspect, the disclosed endogenous promoter or disclosed endogenous promoter / enhancer is generally obtained from a non-coding region upstream of the transcription start site of the target gene. In one aspect, the disclosed endogenous promoter or disclosed endogenous promoter / enhancer can be used for the constitutive and efficient expression of the disclosed gene. In one aspect, 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 promoters / enhancers are well known in the art. In one respect, the public promoter for one or more public guide RNA sequences can be any eukaryotic RNA polymerase II promoter.

[0149] This document discloses an expression cassette comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA. This document discloses an expression cassette comprising one or more RNA structures comprising sequences of any one of SEQ ID NO:01-SEQ ID NO:09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA. This document discloses an expression cassette comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA, wherein the nucleic acid molecules enable 3' substitution of the targeted endogenous pre-mRNA. This document discloses an expression cassette comprising one or more RNA structures, said RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA, wherein the nucleic acid molecules enable 3' substitution of the targeted endogenous pre-mRNA.

[0150] In one respect, the expression of a disclosed protein-coding gene can be restored and / or returned to wild-type, normal, or control expression levels. In one respect, a disclosed nucleic acid molecule can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic dysregulation. In one respect, a disclosed nucleic acid molecule can restore the functional and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above). In one aspect, one or more aspects of restoring cellular homeostasis and / or cellular function may include one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (e.g., correcting, preventing, reducing, and / or alleviating autophagy); (iii) improving, enhancing, restoring, and / or preserving mitochondrial function and / or structural integrity; (iv) improving, enhancing, restoring, and / or preserving organelle function and / or structural integrity; (v) correcting enzymatic dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a multisystemic manifestation of a genetic disease or condition; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a genetic disease or condition, or (viii) any combination thereof. In one aspect, one or more aspects of restoring cellular homeostasis may include one or more aspects of improving, enhancing, restoring, and / or preserving cellular structural and / or functional integrity. In one aspect, restoration of the activity and / or function of a missing, defective, and / or mutated protein or enzyme may include restoration of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount compared to a pre-existing level, such as a pre-treatment level. In one aspect, 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% higher than a pre-existing level, such as a pre-treatment level. In one aspect, restoration may be measured against a control or reference level (e.g., determined using one or more subjects without the missing, defective, and / or mutated protein or enzyme). In one aspect, restoration may be partial or incomplete. In one aspect, restoration may be complete or near-complete, such that the levels of expression, activity, and / or function are similar to wild-type or control levels.

[0151] In one aspect, the disclosed 3' substitution construct can be used specifically in methods for treating subjects with autosomal dominant genetic diseases or conditions (e.g., progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary elliptic polycythemia, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, marble bone disease, Marfan syndrome, neurofibromatosis, polycystic kidney disease (adults), protein C deficiency, osteogenesis imperfecta, TracyCollins syndrome, tuberous sclerosis, Villebrand disease, etc.).

[0152] 2. Transcriptome Engineering System This document discloses a transcriptome engineering system comprising one or more disclosed 3' substitution constructs, one or more disclosed 5' substitution constructs, or any combination thereof. The document discloses a transcriptome engineering system comprising one or more of the following: (i) a nucleic acid molecule comprising exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures; (ii) a nucleic acid molecule comprising exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising SEQ ID NO: 01-SEQ ID NO: (iii) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA; and (iv) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising a sequence comprising any one of SEQ ID NO: 01-SEQ ID NO: 09 wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA.

[0153] This document discloses a transcriptome engineering system comprising one or more of the following: (i) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; (ii) a nucleic acid molecule comprising one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; (iii) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA; and (iv) a nucleic acid molecule comprising one or more RNA structures comprising a sequence of SEQ ID NO: 01-Sequence of any one of SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to the exogenous RNA to be trans-spliced; and the exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3' substitution of the targeted endogenous pre-mRNA.

[0154] This paper discloses a transcriptome engineering system comprising one or more of the following: (i) a nucleic acid molecule containing exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures; (ii) a nucleic acid molecule containing exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising SEQ ID NO: 01-SEQ ID NO: The sequence of any one of SEQ ID NO: 09; (iii) a nucleic acid molecule comprising a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' half-intron linked to the foreign RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA; and (iv) a nucleic acid molecule comprising a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' half-intron linked to the foreign RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising SEQ ID NO: 01-SEQ ID NO: (v) A nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; (vi) A nucleic acid molecule comprising one or more RNA structures, wherein the RNA structures comprise SEQ ID NO: 01-SEQ ID NO: (vii) A nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; and (viii) A nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA; and (viii) A nucleic acid molecule comprising one or more RNA structures, said RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; and a 3' half-intron linked to a foreign RNA to be trans-spliced.And the exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3' substitution of the targeted endogenous pre-mRNA.

[0155] 3. Carrier This document discloses a vector comprising a disclosed nucleic acid molecule. In one aspect, the disclosed vector may be a non-viral vector or a viral vector. This document discloses a non-viral vector comprising a disclosed nucleic acid molecule. This document discloses a non-viral vector comprising one or more disclosed nucleic acid molecules. This document discloses a viral vector comprising a disclosed nucleic acid molecule. This document discloses a viral vector comprising one or more disclosed nucleic acid molecules. This document discloses a non-viral or viral vector comprising a nucleic acid molecule comprising a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the foreign RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures. This document discloses a non-viral or viral vector comprising a nucleic acid molecule comprising a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the foreign RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09.

[0156] This document discloses a non-viral or viral vector containing a nucleic acid molecule, comprising a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the foreign RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures, wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA. This document also discloses a non-viral or viral vector containing a nucleic acid molecule, comprising a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the foreign RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising the sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA. Finally, this document discloses a non-viral or viral vector containing a nucleic acid molecule, wherein the nucleic acid molecule comprises one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to the foreign RNA to be trans-spliced; and the foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA. This document discloses a non-viral or viral vector comprising a nucleic acid molecule containing one or more RNA structures, the RNA structures comprising sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA. The nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA. This document discloses a non-viral or viral vector comprising a nucleic acid molecule, said nucleic acid molecule comprising one or more RNA structures, said RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' hemiintron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA. This document discloses a nonviral or viral vector comprising one or more 5' substitution constructs. This document discloses a nonviral or viral vector comprising one or more 3' substitution constructs. This document discloses a nonviral or viral vector comprising one or more 5' substitution constructs and / or one or more 3' substitution constructs. In one aspect, the disclosed 5' substitution construct to be used in combination with one or more other substitution constructs may comprise any disclosed 5' substitution construct. In one aspect, the disclosed 5' substitution construct may comprise (i) a nucleic acid molecule comprising exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures; (ii) a nucleic acid molecule comprising exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising SEQ ID NO: 01-SEQ ID NO: The sequence of any one of SEQ ID NO: 09, (iii) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA, or (iv) a nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09 wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA, or (v) any combination thereof.

[0157] In one aspect, a publicly disclosed 3' alternative builder to be used in combination with one or more other alternative builder may contain any publicly disclosed 3' alternative builder. In one aspect, the disclosed 3' substitution construct may comprise (i) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; (ii) a nucleic acid molecule comprising one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; (iii) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA; (iv) a nucleic acid molecule comprising one or more RNA structures comprising a sequence of SEQ ID NO: 01-The sequence of any one of SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to the exogenous RNA to be trans-spliced; and the exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA, or (v) any combination thereof.

[0158] In one aspect, the disclosed carriers can be formulated for administration via one or more routes. These methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, intrauterine administration, intrahepatic administration, intravaginal administration, ocular administration, intraauricular administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable methods such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration may also include intra-hepatic artery administration or administration via the portal vein (HPV). Administration of the disclosed therapeutic agents, disclosed pharmaceutical compositions, or combinations thereof may include direct administration to the CNS (e.g., intraparenchymal, intravenous, intrasheathic cisterns, intrasheath (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or PNS. Administration may be continuous or intermittent. Administration may include administration of viral vectors and / or optimized viral vectors produced. Administration of the disclosed carriers may be continuous or intermittent.

[0159] In one aspect, the therapeutically effective amount of the disclosed AAV particles or the disclosed AAV carrier may include approximately 1 × 10⁻⁶. 10 vg / kg to approximately 2×10 14 The range is vg / kg. In one aspect, for example, the disclosed carrier can be in the range of about 1×10 11 Approximately 8×10 13 vg / kg or approximately 1×10 12 Approximately 8×10 13 Administered at a dose of vg / kg. In one respect, the disclosed carrier can be administered at approximately 1×10 13 Approximately 6×10 13 The dosage is administered at a rate of vg / kg. In one aspect, the disclosed carrier can be administered at a dosage of at least about 1 × 10⁻⁶. 10 At least approximately 5×10 10 At least approximately 1×10 11 At least approximately 5×10 11 At least approximately 1×10 12 At least approximately 5×10 12 At least approximately 1×10 13 At least approximately 5×10 13 Or at least about 1×10 14 vg / kg. In one aspect, the disclosed carrier can be administered at a dose not exceeding about 1 × 10 10 No more than approximately 5×10 10 No more than approximately 1×10 11 No more than approximately 5×10 11 No more than approximately 1×10 12 No more than approximately 5×10 12 No more than approximately 1×10 13 No more than approximately 5×10 13 Or no more than about 1×10 14 vg / kg. In one respect, the disclosed vector can be 1×10 12 Administered at a dose of vg / kg. In one aspect, the disclosed carrier can be administered at a dose of 1×10... 11 The dosage is administered in vg / kg. In one aspect, the disclosed carrier can be administered in single or multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed to achieve the desired therapeutic outcome.

[0160] In one respect, the therapeutically effective amount of the disclosed AAV particles or the disclosed AAV carrier may include approximately 1 × 10⁻⁶ per subject in total. 12 vg to each subject total approximately 1×10 17.The range. In one aspect, the therapeutically effective amount of a disclosed AAV particle or a disclosed AAV carrier may include approximately 1 × 10⁻⁶ per subject in total. 12 vg, approximately 1×10 per subject 13. vg, approximately 1×10 per subject 14 vg, approximately 1×10 per subject 15. vg, approximately 1×10 per subject 16 vg or approximately 1×10 per subject in total 17. The range of vg. In one aspect, a therapeutically effective dose of the disclosed AAV particles or the disclosed AAV carrier can be delivered by retrograde ureteral infusion and / or renal artery administration, and can include approximately 1 × 10⁻⁶ per subject in total. 12 vg to each subject total approximately 1×10 17. The range of vg.

[0161] In one respect, the therapeutically effective amount of the disclosed AAV particles may include approximately 1 × 10⁻⁶. 6 DRP / mL to approximately 1×10 14 DRP / mL. In one aspect, the disclosed pharmaceutical formulation may include about 1 × 10⁻⁶ DRP / mL. 6 DRP / mL, 1×10 7 DRP / mL, 1×10 8 DRP / mL, 1×10 9 DRP / mL, 1×10 10 DRP / mL, 1×10 11 DRP / mL, 1×10 12 DRP / mL, 1×10 13 DRP / mL or 1×10 14 DRP / mL. In one aspect, the therapeutically effective amount of a disclosed AAV particle or a disclosed AAV carrier may include a range determined by a technician.

[0162] In one aspect, the disclosed non-viral vector can be a polymer-based vector, a peptide-based vector, a lipid nanoparticle, a solid lipid nanoparticle, or a cationic lipid-based vector. In another aspect, the disclosed vector can include exosomes, extracellular vesicles, and virus-like particles.

[0163] In one respect, the disclosed viral vector may be an adenovirus vector, AAV vector, herpes simplex virus vector, retrovirus vector, lentivirus vector, alphavirus vector, flavivirus vector, rhabdovirus vector, measles virus vector, Newcastle disease virus vector, poxvirus vector, or piconemavirus vector. In another respect, the disclosed nucleic acid sequence may have a coding sequence of less than approximately 4.5 kilobases.

[0164] In one aspect, the disclosed AAV vector may include naturally isolated serotypes, including but not limited to 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, horse AAV, sheep AAV, bird AAV, primate AAV, non-primate AAV, and any other virus classified as AAV by the International Committee on Taxonomy of Viruses (ICTV). In another aspect, the AAV capsid may be a chimera generated through capsid evolution or through rational capsid engineering from naturally isolated AAV variants to capture desired serotype characteristics, such as enhanced or specific tissue tropism and / or host immune response evasion. 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, and AAV9.47-AS, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, and AAVcc.81. In one aspect, the disclosed AAV vector may be AAV-Rh74 or a related variant (e.g., a capsid variant such as RHM4-1). In another aspect, the disclosed AAV vector may be AAV.cc47. In one respect, the publicly available AAV carrier may be AAV.cc81. In another respect, the publicly available AAV carrier may be a self-complementary AAV.

[0165] In one respect, the publicly disclosed carrier may contain one or more ITRs (e.g., an ITR from AAV2).

[0166] In one aspect, the disclosed vector may further comprise one or more nuclear localization signals (NLS). NLS are known to those skilled in the art. In one aspect, the disclosed NLS may comprise any NLS known in the art. As known in the art (see, for example, Lu J et al. (2021) Cell Commun Signal. 19:60, whose teachings on NLS are incorporated herein by reference), nuclear localization signals (NLS) are typically short peptides that act as signal fragments mediating the transport of proteins from the cytoplasm to the nucleus.

[0167] In one aspect, the disclosed vector may further comprise one or more nuclear retention elements (NREs). NREs are known to those skilled in the art. In one aspect, the disclosed NRE may comprise SIRLOIN (SEQ ID NO:15) or BORG (SEQ ID NO:16).

[0168] On the one hand, the disclosed vector may further include one or more flavivirus genetic elements. In one aspect, flavivirus genetic elements may include one or more flavivirus 3' untranslated regions (3'UTR), 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. (For a description of flavivirus genetic elements, see WO 2022 / 182835).

[0169] In one aspect, the disclosed vector may further comprise a nucleic acid sequence encoding a therapeutic protein, therapeutic agent, and / or therapeutic RNA. In one aspect, the disclosed therapeutic protein may include a polypeptide and / or glycopeptide. In one aspect, the disclosed therapeutic agent may include an oligonucleotide therapeutic agent. In one aspect, the disclosed oligonucleotide therapeutic agent may be single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), antisense molecule, miRNA, morpholinonucleotide, peptide nucleic acid (PNA), or analogs or conjugates thereof. In one aspect, the disclosed therapeutic agent may be ASO or RNAi. In one aspect, the disclosed therapeutic agent may comprise a CRISPR-based endonuclease (e.g., Cas9). In one aspect, the disclosed CRISPR-based endonuclease may be derived from a CRISPR / Cas Type I, II, or Type III system.

[0170] In one aspect, the disclosed therapeutic RNA may comprise ribosomal RNA (rRNA), transfer RNA (tRNA), heterogeneous nuclear RNA (hnRNA), small nuclear RNA (snRNA), small nucleolar 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, intergenic RNA, or any combination thereof. In one aspect, the disclosed RNA may include lncRNA, siRNA, shRNA, sgRNA, circular RNA, snoRNA, miRNA, or any combination thereof. In one aspect, the disclosed coding RNA may include functional non-coding RNA elements.

[0171] In one aspect, the disclosed vector may contain one or more promoters operatively linked 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 trans-spliced, and / or a disclosed nucleic acid sequence. In one aspect, the disclosed nucleic acid molecule may be operatively linked to one or more transcriptional regulatory elements. In one aspect, one or more regulatory elements (e.g., a post-transcriptional regulatory element (WPRE) of marmot hepatitis virus (WHV), a triple helix from MALAT1, a PRE (HPRE) of hepatitis B virus, and an iron response element) may increase the transcription and / or expression of 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 trans-spliced, and / or the disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and / or a disclosed therapeutic RNA). In one aspect, the disclosed promoter may be located at the 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 trans-spliced, and / or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and / or a disclosed therapeutic RNA) under its control. The distance between the disclosed promoter and 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 trans-spliced, and / or the disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and / or a disclosed therapeutic RNA) may be substantially the same as the distance between the promoter and 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 trans-spliced, and / or the disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and / or a disclosed therapeutic RNA) under its control. As is known in the art, variations in this distance can be modulated without loss of promoter function.

[0172] In one aspect of the disclosed vector, the disclosed promoter can be tissue-specific or ubiquitous, and can be constitutive or inducible, depending on the desired expression pattern. The disclosed promoter can be natural or exogenous, and can be a natural or synthetic sequence. Exogenous means that the transcription start region is not present in the wild-type host in which the transcription start region is introduced. In one aspect, the disclosed promoter can be a promoter / enhancer. In one aspect, the disclosed promoter for the disclosed nucleic acid molecule (e.g., 5' substitution constructs and / or 3' substitution constructs), the disclosed transgene, the disclosed sequence to be trans-spliced, and / or the disclosed nucleic acid sequence (e.g., encoding the disclosed therapeutic protein and / or the disclosed therapeutic RNA) can be an endogenous promoter. In one aspect, the disclosed endogenous promoter can be an endogenous promoter / enhancer. In one aspect, the disclosed endogenous promoter or disclosed endogenous promoter / enhancer can generally be obtained from a non-coding region upstream of the transcription start site of the target gene. In one aspect, a disclosed endogenous promoter or a disclosed endogenous promoter / enhancer can be used for the constitutive and efficient expression of a disclosed protein-coding gene. In one aspect, 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 trans-spliced, and / or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and / or a disclosed therapeutic RNA) can be a CMV promoter or a CMV promoter / enhancer. CMV promoters and CMV promoters / enhancers are well known in the art. In one aspect, 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 trans-spliced, and / or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and / or a disclosed therapeutic RNA) can be any eukaryotic RNA polymerase II promoter. In one aspect, a disclosed AAV vector can be used to generate AAV particles. In one aspect, a disclosed AAV vector can be used to generate AAV particles containing disclosed nucleic acid molecules (e.g., 5' substitution constructs and / or 3' substitution constructs) under its control, disclosed transgenes, disclosed sequences to be trans-spliced, and / or disclosed nucleic acid sequences (e.g., encoding disclosed therapeutic proteins and / or disclosed therapeutic RNAs).

[0173] This article discloses AAV particles containing publicly disclosed nucleic acid molecules (e.g., 5' substitution constructs and / or 3' substitution constructs) under its control, publicly disclosed transgenes, publicly disclosed sequences to be trans-spliced, and / or publicly disclosed nucleic acid sequences (e.g., encoding publicly disclosed therapeutic proteins and / or publicly disclosed therapeutic RNAs).

[0174] In one respect, the disclosed vector or disclosed AAV particles can be used specifically in methods for treating subjects with autosomal dominant genetic diseases or conditions (e.g., progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary elliptic polycythemia, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, marble bone disease, Marfan syndrome, neurofibromatosis, polycystic kidney disease (adults), protein C deficiency, osteogenesis imperfecta, TracyCollins syndrome, tuberous sclerosis, von Willebrand disease, etc.).

[0175] 4. Pharmaceutical preparations This article discloses a pharmaceutical formulation comprising a disclosed nucleic acid molecule. This article discloses a pharmaceutical formulation comprising a disclosed nucleic acid molecule and a pharmaceutically acceptable carrier. This article discloses a pharmaceutical formulation comprising a disclosed carrier. This article discloses a pharmaceutical formulation comprising a disclosed carrier and a pharmaceutically acceptable carrier. This article discloses a pharmaceutical formulation comprising disclosed AAV particles. This article discloses a pharmaceutical formulation comprising disclosed AAV particles and a pharmaceutically acceptable carrier.

[0176] This article discloses a pharmaceutical formulation comprising a non-viral or viral vector containing nucleic acid molecules, comprising exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures. This article discloses a pharmaceutical formulation comprising a non-viral or viral vector containing nucleic acid molecules, comprising exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures, wherein the nucleic acid molecules enable 5' substitution of the targeted endogenous pre-mRNA. This article discloses a pharmaceutical formulation comprising a non-viral or viral vector containing a nucleic acid molecule, comprising a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the foreign RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA. This article also discloses a pharmaceutical formulation comprising a non-viral or viral vector containing a nucleic acid molecule, wherein the nucleic acid molecule comprises one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to the foreign RNA to be trans-spliced; and the foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA. This document discloses a pharmaceutical formulation comprising a non-viral or viral vector containing a nucleic acid molecule, said nucleic acid molecule comprising one or more RNA structures, said RNA structures comprising sequences of any one of SEQ ID NO:01-SEQ ID NO:09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA. The nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA.This article discloses a pharmaceutical formulation comprising a non-viral or viral vector containing a nucleic acid molecule, said nucleic acid molecule comprising one or more RNA structures, said RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA.

[0177] In one aspect, the disclosed pharmaceutical formulation may comprise (i) one or more active agents, (ii) bioactive agents, (iii) one or more pharmaceutically active agents, (iv) one or more immunotherapeutic agents, (v) one or more clinically approved pharmaceutical agents, or (vi) combinations thereof. In one aspect, the disclosed composition may comprise one or more immunomodulators. In one aspect, the disclosed composition may comprise one or more proteasome inhibitors. In one aspect, the disclosed composition may comprise one or more immunosuppressive agents or immunosuppressants. In one aspect, the immunosuppressant may be anti-thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), or a combination thereof. In one aspect, the disclosed formulation may comprise an anaplegic agent (e.g., a C7 compound, such as triheptanylglycerol or MCT).

[0178] In one aspect, the disclosed formulation may comprise an RNA therapeutic agent. The RNA therapeutic agent may comprise RNA-mediated interference (RNAi) and / or antisense oligonucleotides (ASO). In one aspect, the disclosed RNA therapeutic agent may target any protein or enzyme that is overexpressed or overactive due to deletion, defective, and / or mutation. In one aspect, the disclosed RNA therapeutic agent may comprise a therapy delivered via LNP. In one aspect, the disclosed formulation may comprise an enzyme or enzyme precursor for enzyme replacement therapy (ERT).

[0179] In one respect, a disclosed formulation may contain a disclosed small molecule. In another respect, a disclosed small molecule may help restore the functionality and / or structural integrity of a missing, defective, and / or mutated protein or enzyme.

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

[0181] In one aspect, the therapeutically effective amount of the disclosed pharmaceutical formulation may include approximately 1 × 10⁻⁶ of the disclosed carrier and / or disclosed AAV particles. 10vg / kg to approximately 2×10 14. The range of vg / kg. In one aspect, for example, the dosage of a disclosed pharmaceutical preparation may include about 1 × 10⁻⁶. 11 Approximately 8×10 13 vg / kg or approximately 1×10 12 Approximately 8×10 13 vg / kg. In one aspect, the dosage of the disclosed pharmaceutical formulation may include about 1 × 10 13 Approximately 6×10 13 vg / kg. In one aspect, the dosage of the disclosed pharmaceutical formulation may include at least about 1 × 10 10 At least approximately 5×10 10 At least approximately 1×10 11 At least approximately 5×10 11 At least approximately 1×10 12 At least approximately 5×10 12 At least approximately 1×10 13 At least approximately 5×10 13 Or at least about 1×10 14 vg / kg. In one aspect, the dosage of the disclosed pharmaceutical formulation may include no more than about 1 × 10 10 No more than approximately 5×10 10 No more than approximately 1×10 11 No more than approximately 5×10 11 No more than approximately 1×10 12 No more than approximately 5×10 12 No more than approximately 1×10 13 No more than approximately 5×10 13 Or no more than about 1×10 14 vg / kg. In one aspect, the dosage of the disclosed pharmaceutical formulation may include about 1 × 10 12 vg / kg. In one aspect, the dosage of the disclosed pharmaceutical formulation may include about 1 × 10 11 vg / kg. In one aspect, the dosage of the disclosed pharmaceutical formulation may include single or multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed according to the desired therapeutic outcome.

[0182] In one respect, the therapeutically effective amount of the disclosed pharmaceutical formulation may include approximately 1 × 10⁻⁶ per subject in total. 12 vg to each subject total approximately 1×10 17. The range of vg. In one aspect, the therapeutically effective dose of the disclosed drug formulation may include approximately 1 × 10⁻⁶ per subject in total. 12 vg, approximately 1×10 per subject 13.vg, approximately 1×10 per subject 14 vg, approximately 1×10 per subject 15. vg, approximately 1×10 per subject 16 vg or approximately 1×10 per subject in total 17. The range of vg. In one aspect, the therapeutically effective amount of a disclosed pharmaceutical preparation may include about 1 × 10 6 DRP / mL to approximately 1×10 14 DRP / mL. In one aspect, the therapeutically effective amount of a disclosed pharmaceutical formulation may include approximately 1 × 10⁻⁶. 6 DRP / mL, 1×10 7 DRP / mL, 1×10 8 DRP / mL, 1×10 9 DRP / mL, 1×10 10 DRP / mL, 1×10 11 DRP / mL, 1×10 12 DRP / mL, 1×10 13 DRP / mL or 1×10 14 DRP / mL.

[0183] In one respect, the therapeutically effective amount of a disclosed pharmaceutical preparation may include a range determined by a technician.

[0184] In one aspect, the disclosed pharmaceutical formulation can be used to restore and / or return a disclosed protein-coding gene to wild-type, normal, or control expression levels. In one aspect, the disclosed pharmaceutical formulation can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic disorders. In one aspect, the disclosed nucleic acid molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above). In one aspect, one or more aspects of restoring cellular homeostasis and / or cellular function may include one or more of 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 mitigating autophagy); (iii) improving, enhancing, restoring, and / or preserving mitochondrial function and / or structural integrity; (iv) improving, enhancing, restoring, and / or preserving organelle function and / or structural integrity; (v) correcting enzymatic dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a multisystemic manifestation of a genetic disease or condition; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a genetic disease or condition, or (viii) any combination thereof. In one aspect, one or more aspects of restoring cellular homeostasis may include one or more aspects of improving, enhancing, restoring, and / or preserving cellular structural and / or functional integrity. In one aspect, restoration of the activity and / or function of a missing, defective, and / or mutated protein or enzyme may include restoration of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount compared to a pre-existing level, such as a pre-treatment level. In one aspect, 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% higher than a pre-existing level, such as a pre-treatment level. In one aspect, restoration may be measured against a control or reference level (e.g., determined using one or more subjects without the missing, defective, and / or mutated protein or enzyme). In one aspect, restoration may be partial or incomplete. In one aspect, restoration may be complete or near-complete, such that the levels of expression, activity, and / or function are similar to wild-type or control levels.

[0185] In one respect, the disclosed pharmaceutical formulations may be used specifically in the treatment of subjects with autosomal dominant genetic diseases or conditions (e.g., progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary elliptic polycythemia, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, marble bone disease, Marfan syndrome, neurofibromatosis, polycystic kidney disease (adults), protein C deficiency, osteogenesis imperfecta, TracyCollins syndrome, tuberous sclerosis, von Willebrand disease, etc.).

[0186] 5. Plasmids This document discloses plasmids comprising one or more disclosed nucleic acid molecules. This document discloses plasmids comprising one or more disclosed vectors. This document discloses plasmids for use in methods of preparing 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. This document discloses a plasmid comprising a sequence or fragment thereof shown in any one of SEQ ID NO: 25-SEQ ID NO: 33. This document discloses a plasmid 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 with the sequence shown in any one of SEQ ID NO: 25-SEQ ID NO: 33. This article discloses a plasmid comprising a sequence or fragment thereof having at least 40%-60%, at least 60%-80%, at least 80%-90%, or at least 90%-100% identity with the sequence shown in any one of SEQ ID NO: 25-SEQ ID NO: 33.

[0187] 6. Cells This document discloses a cell comprising a disclosed nucleic acid molecule, a disclosed vector, and / or a disclosed plasmid. This document discloses cells transduced with one or more disclosed viral vectors. This document discloses 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 aspect, this document discloses human cell lines transduced with one or more disclosed viral vectors or transfected with one or more disclosed nucleic acids, one or more disclosed non-viral vectors, or one or more disclosed plasmids. In one aspect, this document discloses human cell lines having one or more genetic diseases or conditions, contacted with one or more nucleic acid molecules, one or more disclosed vectors, and / or one or more disclosed pharmaceutical preparations. This document discloses treatment of cells obtained from a subject with one or more disclosed nucleic acid molecules, one or more disclosed vectors, one or more disclosed plasmids, and / or one or more disclosed pharmaceutical preparations.

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

[0189] 8. Document Library This document discloses libraries of one or more publicly disclosed nucleic acid molecules. This document discloses libraries of one or more publicly disclosed 5' substitution constructs. This document discloses libraries of one or more publicly disclosed 3' substitution constructs. This document discloses libraries of one or more publicly disclosed 5' substitution constructs and / or publicly disclosed 3' substitution constructs. This document discloses libraries of one or more publicly disclosed vectors. This document discloses libraries of one or more publicly disclosed vectors containing one or more publicly disclosed 5' substitution constructs, one or more publicly disclosed 3' constructs, or any combination thereof. This document discloses libraries of one or more publicly disclosed AAV particles containing one or more publicly disclosed 5' substitution constructs, one or more publicly disclosed 3' constructs, or any combination thereof. This document discloses libraries of one or more publicly disclosed plasmids.

[0190] 9. Reagent kit This document discloses a kit comprising one or more disclosed nucleic acid molecules, one or more disclosed vectors or disclosed AAV particles, disclosed pharmaceutical preparations, or any combination thereof. In one aspect, the kit may comprise disclosed nucleic acid molecules, disclosed vectors or disclosed AAV particles, disclosed pharmaceutical preparations, disclosed therapeutic agents, or combinations thereof, and one or more reagents. "Reagents" and "therapeutic agents" are known in the art and as described above.

[0191] In one aspect, one or more agents can treat, prevent, inhibit, and / or alleviate one or more comorbidities in a subject. In another aspect, one or more active agents can treat, inhibit, prevent, and / or alleviate cellular and / or metabolic complications associated with missing, defective, and / or mutated proteins or enzymes.

[0192] In one aspect, a disclosed kit may contain at least two components constituting the kit. These components together constitute a functional unit for a given purpose (e.g., treating a subject diagnosed with or suspected of having a genetic disease or condition). The individual components may be physically packaged together or separately. For example, a kit containing instructions for use may or may not physically contain instructions with other individual components. Instead, the instructions may be provided as a separate component, in paper or electronic form, available on a computer-readable storage device or downloaded from an internet website, or presented as a recording. In one aspect, a kit for a disclosed method may contain one or more containers holding the disclosed nucleic acid molecule, disclosed vector, disclosed pharmaceutical preparation, disclosed RNA therapeutic agent, or a combination thereof, and a label or packaging insert with instructions for use. In one aspect, suitable containers include, for example, bottles, vials, syringes, blister packs, etc. Containers may be formed from various materials such as glass or plastic. Containers may hold the disclosed nucleic acid molecule, disclosed vector, disclosed pharmaceutical preparation, or a combination thereof, and may have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). Labels or packaging inserts may indicate that the disclosed nucleic acid molecules, disclosed vectors, disclosed AAV particles, disclosed pharmaceutical preparations, disclosed RNA therapeutics, or combinations thereof, can be used to treat, prevent, inhibit, and / or alleviate diseases or conditions or complications and / or symptoms associated with diseases or conditions. The disclosed kits may contain additional components required for administration, such as additional buffers, diluents, filters, needles, and syringes. In one aspect, the disclosed kits can be used in any of the disclosed methods. In one aspect, the disclosed kits can be used to generate one or more chimeric RNA molecules. In one aspect, the disclosed kits can be used to treat genetic diseases or conditions. In one aspect, the disclosed kits can be used to inhibit and / or minimize disease progression.

[0193] C. Methods for producing chimeric RNA molecules This article discloses a method for generating chimeric RNA molecules in cells, the method comprising contacting endogenous pre-mRNA in the cell with a disclosed 5' substitution construct, wherein the resulting chimeric RNA transcript comprises the 3' portion of the targeted endogenous pre-mRNA and the 5' portion of the exogenous RNA.

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

[0195] This article discloses a method for generating chimeric RNA molecules, the method comprising contacting one or more cells with a nucleic acid molecule containing an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures, wherein the resulting chimeric RNA transcript contains the 3' portion of the targeted endogenous pre-mRNA and the 5' portion of the exogenous RNA.

[0196] This article discloses a method for generating chimeric RNA molecules, the method comprising contacting one or more cells with a nucleic acid molecule containing an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures containing sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the resulting chimeric RNA transcript contains the 3' portion of the targeted endogenous pre-mRNA and the 5' portion of the exogenous RNA.

[0197] This article discloses a method for generating chimeric RNA molecules, the method comprising contacting one or more cells with a nucleic acid molecule containing an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA transcript contains a 3' portion of the targeted endogenous pre-mRNA and a 5' portion of the exogenous RNA.

[0198] This article discloses a method for generating chimeric RNA molecules, the method comprising contacting one or more cells with a nucleic acid molecule containing an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures containing sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA, and wherein the resulting chimeric RNA transcript contains a 3' portion of the targeted endogenous pre-mRNA and a 5' portion of the exogenous RNA.

[0199] This article discloses a method for generating chimeric RNA molecules in cells, the method comprising contacting endogenous pre-mRNA in the cell with a disclosed 3' substitution construct, wherein the resulting chimeric RNA transcript comprises the 5' portion of the targeted endogenous pre-mRNA and the 3' portion of the exogenous RNA.

[0200] This article discloses a method for generating chimeric RNA molecules, the method comprising contacting one or more cells with a disclosed 3' substitution construct, wherein the resulting chimeric RNA transcript comprises the 5' portion of a targeted endogenous pre-mRNA and the 3' portion of an exogenous RNA.

[0201] This article discloses a method for generating chimeric RNA molecules, the method comprising contacting one or more cells with a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the resulting chimeric RNA transcript may comprise a 5' portion of the targeted endogenous pre-mRNA and a 3' portion of the foreign RNA.

[0202] This article discloses a method for generating chimeric RNA molecules, the method comprising contacting one or more cells with a nucleic acid molecule comprising one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the resulting chimeric RNA transcript may comprise a 5' portion of the targeted endogenous pre-mRNA and a 3' portion of the foreign RNA.

[0203] This article discloses a method for generating chimeric RNA molecules, the method comprising contacting one or more cells with a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA, and wherein the resulting chimeric RNA transcript may comprise a 5' portion of the targeted endogenous pre-mRNA and a 3' portion of the foreign RNA.

[0204] This article discloses a method for generating chimeric RNA molecules, the method comprising contacting one or more cells with a nucleic acid molecule comprising one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA, and wherein the resulting chimeric RNA transcript may comprise a 5' portion of the targeted endogenous pre-mRNA and a 3' portion of the foreign RNA.

[0205] In one aspect, one or more cells may be present in the subject. In one aspect, the subject may be diagnosed with or may be suspected of having a genetic disease or condition. In one aspect, a disease or condition may include any disease or condition caused by a known gene or a missing, defective, and / or mutated gene. In one aspect, the subject may be a subject who requires treatment for a known disease or condition (e.g., a genetic disease or condition). Genetic diseases and conditions are discussed extensively herein.

[0206] In one respect, the disclosed methods for generating chimeric RNA molecules may further include identifying subjects for whom chimeric RNA molecules need to be generated.

[0207] In one aspect of the disclosed method for generating chimeric RNA molecules, 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, but are not limited to, oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, intrauterine administration, intrahepatic administration, intravaginal administration, ocular administration, intraauricular administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable methods such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration may also include intrahepatic artery administration or administration via the portal vein (HPV). Administration of the disclosed therapeutic agent, disclosed pharmaceutical composition, or combination thereof may include direct administration to the CNS (e.g., intraparenchymal, intravenous, intrasheathic cisterns, intrasheath (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or PNS. Administration may be continuous or intermittent.

[0208] In one aspect, administration can be performed via one or more ex vivo methods, such as an ex vivo perfusion protocol. In another aspect, an ex vivo perfusion protocol can be used in conjunction with one or more cells, tissues, and / or organs obtained from a subject affected by a genetic disease or condition. In another aspect, one or more cells and / or one or more tissues and / or one or more organs can be obtained from a subject in need, can be used for ex vivo perfusion and / or treatment and / or contact protocols, and can be returned to the subject in need, wherein said one or more cells produce chimeric RNA transcripts.

[0209] In one aspect, the disclosed methods for generating chimeric RNA molecules in cells may include verifying trans-splicing events and / or the generation of chimeric RNA molecules. Verification of trans-splicing events and / or the generation of chimeric RNA molecules may be performed using methods and techniques known in the art, such as sequencing, northern blotting, FISH, PCR, RNA-Seq, 3'RACE, 5'RACE, etc.

[0210] In one aspect, disclosed methods for generating chimeric RNA molecules may include preparing disclosed 5' substitution constructs, disclosed 3' substitution constructs, disclosed nonviral vectors or disclosed viral vectors, disclosed nucleic acid molecules, disclosed pharmaceutical preparations, or any combination thereof.

[0211] In one aspect, the disclosed method can be performed in vitro. In another aspect, administration can be performed via one or more in vitro methods, such as an in vitro perfusion protocol. In one aspect, the in vitro perfusion protocol can be used in conjunction with one or more cells, tissues, and / or organs obtained from a subject affected by a genetic disease or condition. In one aspect, one or more cells and / or one or more tissues and / or one or more organs can be obtained from a subject in need, an in vitro perfusion and / or treatment and / or contact protocol can be performed, and they can be returned to the subject in need, wherein one or more cells produce chimeric RNA transcripts.

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

[0213] In one aspect, the disclosed methods can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic disorders. In another aspect, the disclosed methods can restore the functional and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0214] In one aspect, one or more aspects of restoring cellular homeostasis and / or cellular function may include one or more of 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 mitigating autophagy); (iii) improving, enhancing, restoring, and / or preserving mitochondrial function and / or structural integrity; (iv) improving, enhancing, restoring, and / or preserving organelle function and / or structural integrity; (v) correcting enzymatic dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a multisystemic manifestation of a genetic disease or condition; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a genetic disease or condition, or (viii) any combination thereof. In one aspect, one or more aspects of restoring cellular homeostasis may include one or more aspects of improving, enhancing, restoring, and / or preserving cellular structural and / or functional integrity.

[0215] In one respect, the disclosed chimeric RNA can be used specifically in methods for treating subjects with autosomal dominant genetic diseases or conditions (e.g., progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary elliptic polycythemia, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, marble bone disease, Marfan syndrome, neurofibromatosis, polycystic kidney disease (adults), protein C deficiency, osteogenesis imperfecta, TracyCollins syndrome, tuberous sclerosis, Villebrand disease, etc.).

[0216] D. Methods for treating genetic diseases or hereditary conditions This article discloses a method for treating a genetic disease or condition, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a publicly disclosed nonviral vector or a publicly disclosed viral vector or a pharmaceutical preparation thereof to a subject in need, wherein the resulting chimeric RNA molecule can restore cellular homeostasis and / or one or more aspects of cellular functional and / or metabolic disorders.

[0217] This article discloses a method for treating a genetic disease or condition, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a disclosed nonviral vector or a disclosed viral vector or a pharmaceutical preparation thereof to a subject in need of the molecule, wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0218] This article discloses a method for treating a genetic disease or condition, 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 containing a nucleic acid molecule to a subject in need, the nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA-targeting motifs; one or more RNA structures, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic disorders, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0219] This article discloses a method for treating a genetic disease or condition, 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 containing a nucleic acid molecule to a subject in need, the nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic disorders, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0220] This article discloses a method for treating a genetic disease or condition, 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 containing a nucleic acid molecule to a subject in need of the molecule; the nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA-targeting motifs; and one or more RNA structures wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic disorders, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0221] This article discloses a method for treating a genetic disease or condition, 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 containing a nucleic acid molecule to a subject in need, the nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic disorders, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0222] This article discloses a method for treating a genetic disease or condition, 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 containing a nucleic acid molecule to a subject in need of the molecule, the nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' hemiintron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic disorders, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0223] This article discloses a method for treating a genetic disease or condition, 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 containing a nucleic acid molecule to a subject in need, the nucleic acid molecule comprising one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic disorders, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0224] This article discloses a method for treating a genetic disease or condition, 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 containing a nucleic acid molecule to a subject in need of the molecule, the nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic disorders, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0225] This document discloses a method for treating a genetic disease or condition, 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 containing a nucleic acid molecule to a subject in need, the nucleic acid molecule comprising one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic disorders, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0226] This article discloses a method for treating a genetic disease or condition, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a nonviral vector, a viral vector, an AAV particle, or a pharmaceutical preparation thereof to a subject in need of the molecule, said nonviral vector, viral vector, AAV particle, or pharmaceutical preparation 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, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic disorders, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

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

[0228] In one aspect of the disclosed treatment method, a disclosed 3' alternative construct to be used in combination with one or more other alternative constructs may contain any disclosed 3' alternative construct. In one aspect, the disclosed 3' substitution construct may comprise (i) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; (ii) a nucleic acid molecule comprising one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; (iii) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA; (iv) a nucleic acid molecule comprising one or more RNA structures comprising a sequence of SEQ ID NO: 01-The sequence of any one of SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to the exogenous RNA to be trans-spliced; and the exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA, or (v) any combination thereof.

[0229] In one aspect of the disclosed treatment method, the subject may suffer from a genetic disease or condition that is associated with and / or caused by one or more disclosed genes, including those 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 disease or condition can restore the functionality and / or structural integrity of a missing, defective, and / or mutated protein or enzyme. In one aspect, the disclosed treatment method may include one or more aspects of restoring cellular homeostasis and / or cellular function and / or metabolic disorders. In one aspect, one or more aspects of restoring cellular homeostasis and / or cellular function and / or metabolic disorders include restoring the functionality and / or structural integrity of a missing, defective, and / or mutated protein or enzyme. In one aspect, one or more aspects of restoring cellular homeostasis and / or cellular function may include one or more of 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 mitigating autophagy); (iii) improving, enhancing, restoring, and / or preserving mitochondrial function and / or structural integrity; (iv) improving, enhancing, restoring, and / or preserving organelle function and / or structural integrity; (v) correcting enzymatic dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a multisystemic manifestation of a genetic disease or condition; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a genetic disease or condition, or (viii) any combination thereof. In one aspect, one or more aspects of restoring cellular homeostasis may include one or more aspects of improving, enhancing, restoring, and / or preserving cellular structural and / or functional integrity. In one aspect, the restoration of the activity and / or function of a missing, defective, and / or mutated protein or enzyme (e.g., encoded by a protein-coding gene as described above) may include a restoration of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount compared to a pre-existing level, such as a pre-treatment level. In one aspect, 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% higher than a pre-existing level, such as a pre-treatment level. In one aspect, restoration may be measured against a control or reference level (e.g., determined using one or more subjects without the missing, defective, and / or mutated protein or enzyme). In one aspect, restoration may be partial or incomplete. In one aspect, restoration may be complete or near-complete, such that the levels of expression, activity, and / or function are similar to wild-type or control levels.

[0231] In one aspect, the disclosed treatment method may further include monitoring the subject's metabolic and / or physiological improvements after one administration step and / or after multiple administration steps. In one aspect, clinicians may measure and / or determine the subject's metabolic and / or physiological status over time to identify one or more improvements and / or one or more attenuations. In one aspect of the disclosed method, clinicians may use the subject's metabolic and / or physiological status and / or trends and / or tendencies in the subject's metabolic and / or physiological status to make treatment decisions and / or modify one aspect of the disclosed method and / or continue treating the subject and / or continue administering the disclosed AAV particles, the disclosed carrier, the disclosed nucleic acid molecule, the disclosed pharmaceutical preparation, the disclosed therapeutic agent, and / or the disclosed immunomodulator, or any combination thereof. In one aspect, metabolic and / or physiological data may be communicated to clinicians and the treatment plan.

[0232] In one aspect of the disclosed treatment methods, techniques for monitoring, measuring, and / or assessing the restoration of one or more aspects of cellular homeostasis and / or cellular function may include qualitative (or subjective) methods as well as quantitative (or objective) methods. These methods are known to those skilled in the art. For example, representative regulatory variables and sensors related to systemic homeostasis are provided below.

[0233] In one aspect, the disclosed treatment method may further include performing one or more invasive or non-invasive diagnostic assessments on the subject. The diagnostic assessments are known in the art. In one aspect, the disclosed non-invasive diagnostic assessments may include x-ray, computed tomography (CT) scan, magnetic resonance imaging (MRI) scan, ultrasound, positron emission tomography (PET)... PET Scanning or any combination thereof. In one aspect, open invasive diagnostic evaluation may include tissue biopsy or exploratory surgery.

[0234] In one respect, the disclosed treatment methods can be used to repair diseased and / or dysfunctional cell types in one or more diseased and / or disordered cells, tissues and / or organs.

[0235] In one respect, the disclosed treatment method can be used to improve and / or enhance the subject's quality of life compared to pre-treatment levels. In another respect, the disclosed treatment method can improve the subject's quality of life by at least 50% when compared to the subject's pre-treatment quality of life.

[0236] In one respect, the disclosed treatment method can be used to improve and / or enhance the subject's quality of life compared to pre-treatment levels. In another respect, the disclosed treatment method can improve the subject's quality of life by at least 50% when compared to the subject's pre-treatment quality of life.

[0237] In one aspect, the disclosed treatment method can be used to diminish and / or reduce one or more symptoms associated with and / or related to a subject's genetic disease and / or genetic condition. In one aspect, the disclosed treatment method can be used to prevent the occurrence of undesirable physiological changes, diseases, pathological conditions, or symptoms in a subject. In one aspect, the disclosed treatment method can be used to inhibit physiological changes, diseases, pathological conditions, or symptoms in a subject, i.e., to halt their development. In one aspect, the disclosed treatment method can be used to eliminate physiological changes, diseases, pathological conditions, or symptoms in a subject, i.e., to induce disease regression.

[0238] In one aspect of a publicly disclosed treatment method, administration to a subject may include contacting one or more cells with one or more publicly disclosed nucleic acid molecules, a publicly disclosed carrier or publicly disclosed AAV particles, a publicly disclosed pharmaceutical preparation, or any combination thereof.

[0239] In one aspect of the disclosed treatment method, administration of the disclosed nucleic acid molecule, disclosed carrier, disclosed AAV particles, 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, but are not limited to, oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, intrauterine administration, intrahepatic administration, intravaginal administration, ocular administration, intraauricular administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable methods such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration may also include intrahepatic artery administration or administration via the portal vein (HPV). Administration of the disclosed nucleic acid molecule, disclosed carrier, disclosed AAV particles, disclosed pharmaceutical formulation, or any combination thereof may include direct administration to the CNS (e.g., intraparenchymal, intraventricular, intrasheathic cisterns, intrasheath (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or PNS. Administration may be continuous or intermittent.

[0240] In one respect, the disclosed treatment method may be administered to the subject via multiple routes. In another respect, the disclosed treatment method may be administered via a first route, which may be the same as or different from a second and / or subsequent route.

[0241] In one aspect of a publicly disclosed method for treating a genetic disease or condition, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical preparation may include approximately 1 × 10⁻⁶. 10 vg / kg to approximately 2×10 14. The range of vg / kg. In one aspect, for example, the therapeutically effective amount of a disclosed carrier or a disclosed pharmaceutical formulation may include about 1 × 10⁻⁶. 11 vg / kg to approximately 8×1013 vg / kg or approximately 1×10 12 vg / kg to approximately 8×10 13 A dose of vg / kg. In one aspect, for example, the therapeutically effective amount of a disclosed carrier or a disclosed pharmaceutical formulation may include about 1 × 10⁻⁶. 13 vg / kg to approximately 6×10 13 A dose of vg / kg. In one aspect, the therapeutically effective amount of a disclosed carrier or a disclosed pharmaceutical preparation may include at least about 1 × 10⁻⁶. 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 12 vg / kg, at least about 1×10 13 vg / kg, at least about 5×10 13 vg / kg or at least about 1×10 14 A dose of vg / kg. In one aspect, the therapeutically effective amount of a disclosed carrier or a disclosed pharmaceutical preparation may include no more than about 1 × 10⁻⁶. 10 vg / kg, not exceeding approximately 5×10 10 vg / kg, not exceeding approximately 1×10 11 vg / kg, not exceeding approximately 5×10 11 vg / kg, not exceeding approximately 1×10 12 vg / kg, not exceeding approximately 5×10 12 vg / kg, not exceeding approximately 1×10 13 vg / kg, not exceeding approximately 5×10 13 Or no more than about 1×10 14 A dose of vg / kg. In one aspect, the therapeutically effective amount of a disclosed carrier or a disclosed pharmaceutical preparation may include about 1 × 10⁻⁶. 12 vg / kg or approximately 1×10 11 A dose of vg / kg. In one aspect, the therapeutically effective amount of a disclosed carrier or a disclosed pharmaceutical preparation may include administration in single or multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses) as needed to achieve the desired therapeutic outcome.

[0242] In one respect, the therapeutically effective amount of a disclosed carrier or a disclosed pharmaceutical preparation may include approximately 1 × 10⁻⁶ per subject in total. 12 vg to each subject total approximately 1×10 17.In terms of the scope of vg, in one aspect, the therapeutically effective amount of a disclosed carrier or disclosed pharmaceutical preparation may include approximately 1 × 10⁻⁶ per subject in total. 12 vg, approximately 1×10 per subject 13. vg, approximately 1×10 per subject 14 vg, approximately 1×10 per subject 15. vg, approximately 1×10 per subject 16 vg or approximately 1×10 per subject in total 17. The range of vg.

[0243] In one aspect, the therapeutically effective amount of a disclosed carrier or a disclosed pharmaceutical preparation may include approximately 1 × 10⁻⁶. 6 DRP / mL to approximately 1×10 14 DRP / mL. In one aspect, the therapeutically effective amount of a disclosed carrier or a disclosed pharmaceutical formulation may include about 1 × 10⁻⁶. 6 DRP / mL, 1×10 7 DRP / mL, 1×10 8 DRP / mL, 1×10 9 DRP / mL, 1×10 10 DRP / mL, 1×10 11 DRP / mL, 1×10 12 DRP / mL, 1×10 13 DRP / mL or 1×10 14 DRP / mL. In one aspect, the therapeutically effective amount of a disclosed carrier or a disclosed pharmaceutical preparation may include a range determined by a person skilled in the art.

[0244] In one aspect, publicly disclosed methods for treating genetic diseases or conditions may further include administering a therapeutically effective amount of a therapeutic agent to a subject. The therapeutic agent can be any publicly disclosed agent that achieves the desired clinical outcome.

[0245] In one aspect, the disclosed method for treating a genetic disease or condition may further include monitoring the subject for adverse reactions. In one aspect, in the absence of adverse reactions, the method may further include continuing treatment of the subject. In one aspect, in the presence of adverse reactions, the method may further include modifying the treatment steps. Methods for monitoring the health of the subject may include subjective and objective criteria (and as described above). Such methods are known to those skilled in the art.

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

[0247] In one aspect, the disclosed method of treating a genetic disease or condition may further include administering one or more immunomodulatory agents. In one aspect, the disclosed immunomodulatory agent may be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or a combination thereof. In one aspect, the disclosed immunomodulatory agent may be bortezomib or SVP-rapamycin. In one aspect, the disclosed immunomodulatory agent may be tacrolimus. In one aspect, the disclosed immunomodulatory agent, such as methotrexate, may be administered in an instantaneous low to high dose. In one aspect, the disclosed immunomodulatory agent may be administered at a dose of about 0.1 mg / kg body weight to about 0.6 mg / kg body weight. In one aspect, the disclosed immunomodulatory agent may be administered at a dose of about 0.4 mg / kg body weight. In one aspect, the disclosed immunomodulatory agent may be administered at a daily dose of about 0.4 mg / kg body weight for 3 to 5 or more cycles, each cycle lasting up to three days. In one aspect, the disclosed immunomodulatory agent may be administered at a daily dose of about 0.4 mg / kg body weight for at least 3 cycles, each cycle lasting three days. In one aspect, those skilled in the art can determine the appropriate number of cycles. In one respect, publicly available immunomodulators can be administered multiple times as needed to achieve the desired clinical effect.

[0248] In one aspect, the disclosed immunomodulatory agent may be administered orally approximately 1 hour before the disclosed therapeutic agent. In another aspect, the disclosed immunomodulatory agent may be administered subcutaneously approximately 15 minutes before the disclosed therapeutic agent. In one aspect, the disclosed immunomodulatory agent may be administered concurrently with the disclosed therapeutic agent. In one aspect, the disclosed immunomodulatory agent may be administered orally approximately 1 hour or several days before the disclosed nucleic acid molecule, disclosed carrier, disclosed pharmaceutical preparation, or a combination thereof. In one aspect, the disclosed immunomodulatory agent may be administered subcutaneously approximately 15 minutes or several days before the disclosed nucleic acid molecule, disclosed carrier, disclosed pharmaceutical preparation, or a combination thereof. In one aspect, the disclosed immunomodulatory agent may be administered concurrently with the disclosed nucleic acid molecule, disclosed carrier, disclosed pharmaceutical preparation, or a combination thereof.

[0249] In one aspect, the disclosed methods of treating genetic diseases or conditions may further include the administration of one or more proteasome inhibitors (e.g., bortezomib, carfilzomib, marizomib, ixazomib, and oprozomib). In another aspect, the proteasome inhibitor may be an agent that acts on plasma cells (e.g., daratumumab). In one respect, the reagents acting on plasma cells can be melphalan hydrochloride, melphalan, disodium pamidronate, carmustine, carfilzomib, cyclophosphamide, daratumumab, liposome doxorubicin hydrochloride, liposome doxorubicin hydrochloride, elotuzumab, melphalan hydrochloride, pambistat, ixazomib citrate, carfilzomib, lenalidomide, melphalan hydrochloride, prasaf, ixazomib citrate, disodium pamidronate, pambistat, prasaf, pomalidomide, lenalidomide, celiniso, thalidomide, thalidomide, bortezomib, celiniso, zoledronic acid, or zoledronic acid.

[0250] In one aspect, the disclosed method for achieving therapeutic stability may further include administering one or more proteasome inhibitors or plasma cell-acting agents prior to the administration of the disclosed nucleic acid molecule, the disclosed carrier, the disclosed AAV particles, the disclosed pharmaceutical formulation, or any combination thereof. In one aspect, the disclosed treatment method may include administering one or more proteasome inhibitors or plasma cell-acting agents concurrently with the administration of the disclosed nucleic acid molecule, the disclosed carrier, the disclosed AAV particles, the disclosed pharmaceutical formulation, or any combination thereof. In one aspect, the disclosed treatment method may include administering one or more proteasome inhibitors or one or more plasma cell-acting agents after the administration of the disclosed nucleic acid molecule, the disclosed carrier, the disclosed AAV particles, the disclosed pharmaceutical formulation, or any combination thereof. In one aspect, the disclosed treatment method may further include administering one or more proteasome inhibitors more than once. In one aspect, the disclosed treatment method may include repeated administration of one or more proteasome inhibitors over time.

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

[0252] In one aspect, the disclosed methods for treating genetic diseases or conditions may further include administering compounds that exert a therapeutic effect against B cells and / or compounds that target or alter antigen presentation or humoral or cell-mediated immune responses. In one aspect, the disclosed compounds may be rituximab, methotrexate, intravenous gamma globulin, anti-CD4 antibody, anti-CD2, anti-FcRN antibody, BTK inhibitor, anti-IGFIR antibody, CD19 antibody (e.g., inebilizumab), anti-IL6 antibody (e.g., tocilizumab), antibody against CD40, IL2 mutant protein, or combinations thereof. This document also discloses Treg infusions (e.g., antigen-specific Treg cells against AAV) that can be administered as a means of aiding immune tolerance.

[0253] In one aspect, the disclosed treatment method may further include the administration of lipid nanoparticles (LNPs). In one aspect, the LNPs may be organ-targeted. In one aspect, the LNPs may be liver-targeted or testis-targeted. For example, in one aspect, mRNA therapies encapsulated with LNPs for systemic delivery to a subject have the potential to restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes.

[0254] In one aspect, the disclosed methods for treating a genetic disease or condition may further include treating a subject who has developed or may develop neutralizing antibodies (ABs) to the disclosed nucleic acid molecule, the disclosed vector, the disclosed AAV particles, the disclosed pharmaceutical preparation, or any combination thereof. In one aspect, treating a subject who has developed or may develop neutralizing antibodies may include plasmapheresis and immunosuppression. In one aspect, the disclosed methods may include using immunosuppression to reduce overall T-cell, B-cell, and / or plasma cell populations, thereby reducing innate immune responses, inflammatory responses, and antibody levels. In one aspect, the disclosed methods may include administering an IgG degrading agent that depletes pre-existing neutralizing antibodies. In one aspect, the disclosed methods may include administering IdeS or IdeZ, rapamycin, and / or SVP-rapamycin to a subject. In one aspect, the disclosed treatment may include administering tacrolimus. In one aspect, the disclosed IgG degrading agent is bacterial IdeS or IdeZ.

[0255] In one aspect, disclosed treatment methods may include repeated disclosed administration steps, such as repeated administration of disclosed nucleic acid molecules, disclosed carriers, disclosed AAV particles, disclosed pharmaceutical formulations, disclosed therapeutic agents, disclosed immunomodulators, disclosed proteasome inhibitors, disclosed immunosuppressants, disclosed compounds that exert therapeutic effects against B cells, and / or disclosed compounds that target or alter antigen presentation or humoral or cell-mediated immune responses.

[0256] In one aspect, a disclosed treatment method may include modifications to one or more disclosed steps. For example, modifying one or more steps of a disclosed method may include modifying or altering one or more features or aspects of one or more steps of the disclosed method. For example, in one aspect, 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 carriers, one or more disclosed pharmaceutical preparations, 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 carriers, one or more disclosed pharmaceutical preparations, or any combination thereof 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 carriers, one or more disclosed pharmaceutical preparations, or any combination thereof to a subject.

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

[0258] In one aspect, the disclosed treatment methods may include the concurrent administration of one or more disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed carriers, one or more disclosed pharmaceutical preparations, 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 therapeutic effects on B cells, one or more disclosed compounds that target or modify antigen presentation or humoral or cell-mediated immune responses, or any combination thereof. In one aspect, the disclosed immunomodulators may be administered before or after the administration of the disclosed therapeutic agents.

[0259] In one aspect, disclosed methods for treating genetic diseases or conditions may further include producing disclosed nucleic acid molecules, disclosed vectors, disclosed AAV particles, disclosed pharmaceutical preparations, or any combination thereof.

[0260] In one aspect, the disclosed treatment methods may further include gene editing of one or more related genes (e.g., deleted, defective, and / or mutated proteins or enzymes), wherein the editing includes, but is not limited to, single gene knockout, screening multiple genes for loss of function at one time, gene knock-in, or combinations thereof.

[0261] In one aspect, the disclosed method of treating a genetic disease or condition 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 aspect, the disclosed oligonucleotide therapeutic agent may be ASO or RNAi. In one aspect, the disclosed oligonucleotide therapeutic agent may contain one or more modifications at any applicable position. In one aspect, the disclosed oligonucleotide therapeutic agent may include a CRISPR-based endonuclease. In one aspect, the disclosed endonuclease may be Cas9. In one aspect, the disclosed Cas9 may be derived from Staphylococcus aureus or Streptococcus pyogenes. In one aspect, the disclosed treatment method may include administering the disclosed RNA therapeutic agent to a subject.

[0262] In one aspect, the disclosed methods for treating genetic diseases or conditions 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 preparations, or any combination thereof. In another aspect, the disclosed treatment methods may inhibit and / or minimize one or more aspects of disease progression in a subject (e.g., the genetic diseases or conditions described above).

[0263] In one respect, the disclosed treatment methods can slow down and / or reduce one or more aspects of disease progression in the subject (e.g., genetic diseases or genetic conditions as described above).

[0264] In one respect, the disclosed method can be specifically used to treat subjects with autosomal dominant genetic diseases or conditions (e.g., progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary elliptic polycythemia, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, marble bone disease, Marfan syndrome, neurofibromatosis, polycystic kidney disease (adults), protein C deficiency, osteogenesis imperfecta, TracyCollins syndrome, tuberous sclerosis, Villebrand disease, etc.).

[0265] E. Methods to inhibit and / or minimize disease progression This article discloses 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 publicly disclosed nonviral vector or a publicly disclosed viral vector or a pharmaceutical preparation thereof to a subject in need of the molecule, wherein the resulting chimeric RNA molecule can restore cellular homeostasis and / or one or more aspects of cellular functional and / or metabolic dysregulation.

[0266] This article discloses 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 publicly disclosed nonviral vector or a publicly disclosed viral vector or a pharmaceutical preparation thereof to a subject in need thereof, wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0267] This article discloses 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 nonviral or viral vector containing a nucleic acid molecule to a subject in need of the molecule, the nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA-targeting motifs; and one or more RNA structures, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0268] This article discloses 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 containing a nucleic acid molecule to a subject in need of the molecule; the nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0269] This article discloses 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 containing a nucleic acid molecule to a subject in need of the molecule; the nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA-targeting motifs; and one or more RNA structures wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0270] This article discloses 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 nonviral or viral vector containing a nucleic acid molecule to a subject in need of the molecule; the nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0271] This article discloses 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 containing a nucleic acid molecule to a subject in need of the molecule, the nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0272] This article discloses 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 containing a nucleic acid molecule to a subject in need of the molecule, the nucleic acid molecule comprising one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0273] This article discloses 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 containing a nucleic acid molecule to a subject in need of the molecule, the nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0274] This article discloses 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 containing a nucleic acid molecule to a subject in need of the molecule, the nucleic acid molecule comprising one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0275] This article discloses 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 nonviral vector, viral vector, AAV particle, or pharmaceutical preparation thereof to a subject in need thereof, said nonviral vector, viral vector, AAV particle, or pharmaceutical preparation 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, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0276] In one aspect of the disclosed methods for inhibiting and / or minimizing disease progression, the disclosed 5' alternative construct to be used in combination with one or more other alternative constructs may comprise any disclosed 5' alternative construct. In one aspect, the disclosed 5' alternative construct may comprise (i) a nucleic acid molecule comprising exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures; (ii) a nucleic acid molecule comprising exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising SEQ ID NO: 01-SEQ ID NO: The sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09, (iii) a nucleic acid molecule comprising a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' half-intron linked to the foreign RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA, or (iv) a nucleic acid molecule comprising a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' half-intron linked to the foreign RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09 wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA, or (v) any combination thereof.

[0277] In one aspect of a publicly disclosed method for inhibiting and / or minimizing disease progression, a publicly disclosed 3' alternative construct to be used in combination with one or more other alternative constructs may contain any publicly disclosed 3' alternative construct. In one aspect, the disclosed 3' substitution construct may comprise (i) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; (ii) a nucleic acid molecule comprising one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; (iii) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA; (iv) a nucleic acid molecule comprising one or more RNA structures comprising a sequence of SEQ ID NO: 01-The sequence of any one of SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to the exogenous RNA to be trans-spliced; and the exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted 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 suffer from a genetic disease or condition associated with and / or caused by one or more disclosed genes, including those disclosed in Section VII(B)(1).

[0279] In one aspect of the disclosed methods for inhibiting and / or minimizing disease progression, the expression of the disclosed protein-coding genes can be restored and / or returned to wild-type, normal, or control expression levels. In one aspect, the disclosed methods for inhibiting and / or minimizing the progression of a genetic disease or condition can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression may include one or more aspects of restoring cellular homeostasis and / or cellular function and / or metabolic disorders. In one aspect, one or more aspects of restoring cellular homeostasis and / or cellular function and / or metabolic disorders include restoring the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes. In one aspect, one or more aspects of restoring cellular homeostasis and / or cellular function may include one or more of 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 alleviating autophagy); (iii) improving, enhancing, restoring, and / or preserving mitochondrial function and / or structural integrity; (iv) improving, enhancing, restoring, and / or preserving organelle function and / or structural integrity; (v) correcting enzymatic dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a multisystemic manifestation of a genetic disease or condition; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a genetic disease or condition, or (viii) any combination thereof. In one aspect, one or more aspects of restoring cellular homeostasis may include one or more aspects of improving, enhancing, restoring, and / or preserving cellular structural and / or functional integrity. In one aspect, the restoration of the activity and / or functionality of a missing, defective, and / or mutated protein or enzyme (e.g., encoded by a protein-coding gene as described above) may include a restoration of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount compared to a pre-existing level, such as a pre-treatment level. In one aspect, 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% higher than a pre-existing level, such as a pre-treatment level. In one aspect, restoration may be measured against a control or reference level (e.g., determined using one or more subjects without the missing, defective, and / or mutated protein or enzyme). In one aspect, restoration may be partial or incomplete. In one aspect, restoration may be complete or near-complete, such that the levels of expression, activity, and / or functionality are similar to wild-type or control levels.

[0280] In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression may further include monitoring the subject's metabolic and / or physiological improvements after one administration step and / or after multiple administration steps. In one aspect, clinicians may measure and / or determine the subject's metabolic and / or physiological status over time to identify one or more improvements and / or one or more attenuations. In one aspect of the disclosed methods, clinicians may use the subject's metabolic and / or physiological status and / or trends and / or tendencies in the subject's metabolic and / or physiological status to make treatment decisions and / or modify one aspect of the disclosed methods and / or continue treatment of the subject and / or continue administration of the disclosed AAV particles, disclosed carriers, disclosed nucleic acid molecules, disclosed pharmaceutical formulations, disclosed therapeutic agents, and / or disclosed immunomodulators, or any combination thereof. In one aspect, metabolic and / or physiological data may be communicated to clinicians and to the treatment plan.

[0281] In one aspect of the disclosed methods for inhibiting and / or minimizing disease progression, techniques for monitoring, measuring, and / or assessing the restoration of one or more aspects of cellular homeostasis and / or cellular function may include qualitative (or subjective) and quantitative (or objective) methods. These methods are known to those skilled in the art. For example, representative regulatory variables and sensors related to systemic homeostasis are provided above.

[0282] In one aspect, disclosed methods for inhibiting and / or minimizing disease progression may further include performing one or more invasive or non-invasive diagnostic assessments on the subject. Diagnostic assessments are known in the art. In one aspect, disclosed non-invasive diagnostic assessments may include x-ray, computed tomography (CT) scan, magnetic resonance imaging (MRI) scan, ultrasound, positron emission tomography (PET)... PET Scanning or any combination thereof. In one aspect, open invasive diagnostic evaluation may include tissue biopsy or exploratory surgery.

[0283] In one respect, disclosed methods for inhibiting and / or minimizing disease progression can be used to repair diseased and / or dysfunctional cell types in one or more diseased and / or dysfunctional cells, tissues and / or organs.

[0284] In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression, compared to pre-treatment levels, can be used to improve and / or enhance the subject's quality of life. In another aspect, when compared to the subject's pre-treatment quality of life, the disclosed methods for inhibiting and / or minimizing disease progression can improve the subject's quality of life by at least 50%. In another aspect, when compared to pre-treatment levels, the disclosed methods for inhibiting and / or minimizing disease progression can be used to improve and / or enhance the subject's quality of life. In another aspect, when compared to the subject's pre-treatment quality of life, the disclosed methods for inhibiting and / or minimizing disease progression can improve the subject's quality of life by at least 50%.

[0285] In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression can be used to reduce and / or alleviate one or more symptoms related to and / or associated with a subject's genetic disease and / or genetic condition. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression can be used to prevent the occurrence of undesirable physiological changes, diseases, pathological conditions, or symptoms in a subject. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression can be used to inhibit physiological changes, diseases, pathological conditions, or symptoms in a subject, i.e., to halt their development. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression can be used to eliminate physiological changes, diseases, pathological conditions, or symptoms in a subject, i.e., to induce disease regression.

[0286] In one aspect of publicly disclosed methods for inhibiting and / or minimizing disease progression, administration to a subject may include contacting one or more cells with one or more publicly disclosed nucleic acid molecules, publicly disclosed carriers or publicly disclosed AAV particles, publicly disclosed pharmaceutical formulations, or any combination thereof.

[0287] In one aspect of the disclosed methods for inhibiting and / or minimizing disease progression, administration of the disclosed nucleic acid molecule, disclosed carrier, disclosed AAV particles, 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, but are not limited to, oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, intrauterine administration, intrahepatic administration, intravaginal administration, ocular administration, intraauricular administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable methods such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration may also include intra-hepatic artery administration or administration via the portal vein (HPV). Administration of the disclosed nucleic acid molecule, disclosed carrier, disclosed AAV particles, disclosed pharmaceutical formulation, or any combination thereof may include direct administration to the CNS (e.g., intraparenchymal, intraventricular, intrathecal cisterns, intrathecal (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or PNS. Application can be continuous or intermittent.

[0288] In one aspect, the disclosed methods for inhibiting and / or minimizing the progression of a genetic disease or condition may be administered to the subject via multiple routes of administration. In another aspect, the disclosed methods for inhibiting and / or minimizing disease progression may be administered via 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 disease or condition, the therapeutically effective amount of the disclosed carrier or disclosed pharmaceutical preparation may include about 1 × 10⁻⁶. 10 vg / kg to approximately 2×10 14. The range of vg / kg. In one aspect, for example, the therapeutically effective amount of a disclosed carrier or a disclosed pharmaceutical formulation may include about 1 × 10⁻⁶. 11 vg / kg to approximately 8×10 13 vg / kg or approximately 1×10 12 vg / kg to approximately 8×10 13 A dose of vg / kg. In one aspect, the therapeutically effective amount of a disclosed carrier or a disclosed pharmaceutical preparation may include about 1 × 10⁻⁶. 13 vg / kg to approximately 6×10 13 A dose of vg / kg. In one aspect, the therapeutically effective amount of a disclosed carrier or a disclosed pharmaceutical preparation may include at least about 1 × 10⁻⁶. 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 1×1012 vg / kg, at least about 5×10 12 vg / kg, at least about 1×10 13 vg / kg, at least about 5×10 13 vg / kg or at least about 1×10 14 A dose of vg / kg. In one aspect, the therapeutically effective amount of a disclosed carrier or a disclosed pharmaceutical preparation may include no more than about 1 × 10⁻⁶. 10 vg / kg, not exceeding approximately 5×10 10 vg / kg, not exceeding approximately 1×10 11 vg / kg, not exceeding approximately 5×10 11 vg / kg, not exceeding approximately 1×10 12 vg / kg, not exceeding approximately 5×10 12 vg / kg, not exceeding approximately 1×10 13 vg / kg, not exceeding approximately 5×10 13 Or no more than about 1×10 14 A dose of vg / kg. In one aspect, the therapeutically effective amount of a disclosed carrier or a disclosed pharmaceutical preparation may include about 1 × 10⁻⁶. 12 vg / kg or approximately 1×10 11 A dose of vg / kg. In one respect, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical preparation may be administered in single or multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic outcome.

[0290] In one respect, the therapeutically effective amount of a disclosed carrier or a disclosed pharmaceutical preparation may include approximately 1 × 10⁻⁶ per subject in total. 12 vg to each subject total approximately 1×10 17. The range of vg. In one aspect, the therapeutically effective amount of a disclosed carrier or a disclosed pharmaceutical preparation may include approximately 1 × 10⁻⁶ per subject in total. 12 Each subject totals approximately 1×10 13. vg, approximately 1×10 per subject 14 vg, approximately 1×10 per subject 15. vg, approximately 1×10 per subject 16 vg or approximately 1×10 per subject in total 17. The range of vg.

[0291] In one aspect, the therapeutically effective amount of a disclosed carrier or a disclosed pharmaceutical preparation may include approximately 1 × 10⁻⁶. 6 DRP / mL to approximately 1×10 14DRP / mL. In one aspect, the therapeutically effective amount of a disclosed carrier or a disclosed pharmaceutical formulation may include about 1 × 10⁻⁶. 6 DRP / mL, 1×10 7 DRP / mL, 1×10 8 DRP / mL, 1×10 9 DRP / mL, 1×10 10 DRP / mL, 1×10 11 DRP / mL, 1×10 12 DRP / mL, 1×10 13 DRP / mL or 1×10 14 DRP / mL. In one aspect, the therapeutically effective amount of a disclosed carrier or a disclosed pharmaceutical preparation may include a range determined by a person skilled in the art.

[0292] In one aspect, publicly disclosed methods for inhibiting and / or minimizing the progression of a genetic disease or condition may further include administering a therapeutically effective amount of a therapeutic agent to the subject. The therapeutic agent may be any publicly disclosed agent that achieves the desired clinical outcome.

[0293] In one aspect, methods for inhibiting and / or minimizing the progression of a genetic disease or condition may further include monitoring the subject for adverse reactions. In one aspect, in the absence of adverse reactions, the method may further include continuing treatment of the subject. In one aspect, in the presence of adverse reactions, the method may further include modifying the treatment steps. Methods for monitoring the health of the subject may include both subjective and objective criteria (and as described above). Such methods are known to those skilled in the art.

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

[0295] In one aspect, the disclosed method for inhibiting and / or minimizing the progression of a genetic disease or condition may further include administration of one or more immunomodulatory agents. In one aspect, the disclosed immunomodulatory agent may be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or combinations thereof. In one aspect, the disclosed immunomodulatory agent may be bortezomib or SVP-rapamycin. In one aspect, the disclosed immunomodulatory agent may be tacrolimus. In one aspect, the disclosed immunomodulatory agent, such as methotrexate, may be administered in an instantaneous low to high dose. In one aspect, the disclosed immunomodulatory agent may be administered at a dose of about 0.1 mg / kg body weight to about 0.6 mg / kg body weight. In one aspect, the disclosed immunomodulatory agent may be administered at a dose of about 0.4 mg / kg body weight. In one aspect, the disclosed immunomodulatory agent may be administered at a daily dose of about 0.4 mg / kg body weight for 3 to 5 or more cycles, each cycle lasting up to three days. In one aspect, the disclosed immunomodulatory agent may be administered at a daily dose of about 0.4 mg / kg body weight for at least 3 cycles, each cycle lasting three days. In one respect, those skilled in the art can determine the appropriate number of cycles. In another respect, the disclosed immunomodulators can be administered multiple times as needed to achieve the desired clinical effect.

[0296] In one aspect, the disclosed immunomodulatory agent may be administered orally approximately 1 hour before the disclosed therapeutic agent. In another aspect, the disclosed immunomodulatory agent may be administered subcutaneously approximately 15 minutes before the disclosed therapeutic agent. In one aspect, the disclosed immunomodulatory agent may be administered concurrently with the disclosed therapeutic agent. In one aspect, the disclosed immunomodulatory agent may be administered orally approximately 1 hour or several days before the disclosed nucleic acid molecule, disclosed carrier, disclosed pharmaceutical preparation, or a combination thereof. In one aspect, the disclosed immunomodulatory agent may be administered subcutaneously approximately 15 minutes or several days before the disclosed nucleic acid molecule, disclosed carrier, disclosed pharmaceutical preparation, or a combination thereof. In one aspect, the disclosed immunomodulatory agent may be administered concurrently with the disclosed nucleic acid molecule, disclosed carrier, disclosed pharmaceutical preparation, or a combination thereof.

[0297] In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression may further include the administration of one or more proteasome inhibitors (e.g., bortezomib, carfilzomib, marizomib, ixazomib, and opzomib). In another aspect, the proteasome inhibitor may be an agent that acts on plasma cells (e.g., daratumumab). In one respect, the reagents acting on plasma cells can be melphalan hydrochloride, melphalan, disodium pamidronate, carmustine, carfilzomib, cyclophosphamide, daratumumab, liposome doxorubicin hydrochloride, liposome doxorubicin hydrochloride, erlotuzumab, melphalan hydrochloride, pamidronate, ixazomib citrate, carfilzomib, lenalidomide, melphalan hydrochloride, prasaf, ixazomib citrate, disodium pamidronate, pamidronate, prasaf, pomalidomide, lenalidomide, celinido, thalidomide, thalidomide, bortezomib, celinido, zoledronic acid, or zoledronic acid.

[0298] In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression stability may further include administering one or more proteasome inhibitors or plasma cell-acting agents prior to the administration of the disclosed nucleic acid molecule, the disclosed carrier, the disclosed AAV particles, the disclosed pharmaceutical formulation, or any combination thereof. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression may include administering one or more proteasome inhibitors or one or more plasma cell-acting agents in parallel with the administration of the disclosed nucleic acid molecule, the disclosed carrier, the disclosed AAV particles, the disclosed pharmaceutical formulation, or any combination thereof. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression may include administering one or more proteasome inhibitors or one or more plasma cell-acting agents after the administration of the disclosed nucleic acid molecule, the disclosed carrier, the disclosed AAV particles, the disclosed pharmaceutical formulation, or any combination thereof. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression may further include administering one or more proteasome inhibitors more than once. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression may include repeatedly administering one or more proteasome inhibitors over time.

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

[0300] In one aspect, the disclosed methods for inhibiting and / or minimizing the progression of a genetic disease or condition may further include administering compounds that exert a therapeutic effect against B cells and / or compounds that target or alter antigen presentation or humoral or cell-mediated immune responses. In one aspect, the disclosed compounds may be rituximab, methotrexate, intravenous gamma globulin, anti-CD4 antibody, anti-CD2, anti-FcRN antibody, BTK inhibitor, anti-IGF1R antibody, CD19 antibody (e.g., inelolizumab), anti-IL6 antibody (e.g., tocilizumab), antibody against CD40, IL2 mutant protein, or combinations thereof. This document also discloses Treg infusions (e.g., antigen-specific Treg cells against AAV) that can be administered as a means of aiding immune tolerance.

[0301] In one aspect, disclosed methods for inhibiting and / or minimizing disease progression may further include the administration of lipid nanoparticles (LNPs). In one aspect, LNPs may be organ-targeted. In one aspect, LNPs may be liver-targeted or testis-targeted. For example, in one aspect, mRNA therapies encapsulated with LNPs for systemic delivery to a subject have the potential to restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes.

[0302] In one aspect, the disclosed methods for inhibiting and / or minimizing the progression of a genetic disease or condition may further include treating a subject who has developed or may develop neutralizing antibodies (ABs) to the disclosed nucleic acid molecule, the disclosed vector, the disclosed AAV particles, the disclosed pharmaceutical formulation, or any combination thereof. In one aspect, treating a subject who has developed or may develop neutralizing antibodies may include plasmapheresis and immunosuppression. In one aspect, the disclosed methods may include using immunosuppression to reduce T-cell, B-cell, and / or plasma cell populations in general, thereby reducing innate immune responses, inflammatory responses, and antibody levels. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression may include administering an IgG degrading agent that depletes pre-existing neutralizing antibodies. In one aspect, the disclosed methods may include administering IdeS or IdeZ, rapamycin, and / or SVP-rapamycin to a subject. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression may include administering tacrolimus. In one aspect, the disclosed IgG degrading agent is a bacterially derived IdeS or IdeZ.

[0303] In one aspect, disclosed methods for inhibiting and / or minimizing disease progression may include repeated disclosed administration steps, such as repeated administration of disclosed nucleic acid molecules, disclosed carriers, disclosed AAV particles, disclosed pharmaceutical formulations, disclosed therapeutic agents, disclosed immunomodulators, disclosed proteasome inhibitors, disclosed immunosuppressants, disclosed compounds that exert therapeutic effects against B cells, and / or disclosed compounds that target or alter antigen presentation or humoral or cell-mediated immune responses.

[0304] In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression may include modifications to one or more disclosed steps. For example, modifying one or more steps of the disclosed method may include modifying or altering one or more features or aspects of one or more steps of the disclosed method. For example, in one aspect, 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 carriers, one or more disclosed pharmaceutical preparations, 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 carriers, one or more disclosed pharmaceutical preparations, or any combination thereof 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 carriers, one or more disclosed pharmaceutical preparations, or any combination thereof to a subject.

[0305] In one aspect, the disclosed method of inhibiting and / or minimizing disease progression can be modified by changing the amount administered to a subject of one or more disclosed therapeutic agents, disclosed immunomodulators, disclosed proteasome inhibitors, disclosed immunosuppressants, disclosed compounds that act against B cells and / or disclosed compounds that target or alter antigen presentation or humoral or cell-mediated immune responses, or by changing the frequency of administration of one or more disclosed therapeutic agents, disclosed immunomodulators, disclosed proteasome inhibitors, disclosed immunosuppressants, disclosed compounds that act against B cells and / or disclosed compounds that target or alter antigen presentation or humoral or cell-mediated immune responses.

[0306] In one aspect, disclosed methods for inhibiting and / or minimizing disease progression may include the concurrent 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 act against B cells, one or more disclosed compounds that target or modify antigen presentation or humoral or cell-mediated immune responses, or any combination thereof. In one aspect, the disclosed immunomodulators may be administered before or after the administration of the disclosed therapeutic agents. In one aspect, disclosed methods for inhibiting and / or minimizing disease progression of a genetic disease or condition may further include generating disclosed nucleic acid molecules, disclosed vectors, disclosed AAV particles, disclosed pharmaceutical formulations, or any combination thereof.

[0307] In one aspect, publicly disclosed methods 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).

[0308] Table 1 – Large Genes and Identification of Affected Chromosomes and Mutations#

[0309] In one respect, the disclosed method can be specifically used to treat subjects with autosomal dominant genetic diseases or conditions (e.g., progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary elliptic polycythemia, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, marble bone disease, Marfan syndrome, neurofibromatosis, polycystic kidney disease (adults), protein C deficiency, osteogenesis imperfecta, TracyCollins syndrome, tuberous sclerosis, Villebrand disease, etc.).

[0310] VIII. Examples Because pathogenic mutations exceeding AAV packaging capacity exist in over 500 genes, efforts have been made to circumvent this barrier to large gene expression. While these techniques differ, general approaches remain broadly similar in strategy. In short, a dual AAV vector approach is employed; the DNA sequence encoding the target protein is split and packaged into separate vectors. After co-infection of target cells with both vectors, the genomes of the two vectors recombine with each other via inverted repeat sequences or overlapping complementary sequences, forming a single genome carrying a reconstructed DNA sequence expressing the complete target protein. While this strategy is feasible, the efficiency of recombination between genomes limits its widespread adoption.

[0311] Furthermore, other efforts to phenotypically correct genes unsuitable for classical AAV-mediated gene therapy have inspired a method involving the manipulation of endogenous messenger RNA (the conduit between DNA and protein). An RNA editing strategy called spliceosome-mediated RNA transsplicing (SMART) has been developed as a strategy to introduce large, precise modifications into the primary structure of RNA transcripts, independent of target transcript length. The aim of this method is to hijack cellular RNA processing mechanisms to incorporate the desired sequence into endogenous transcripts. In this strategy, a recombinant RNA molecule is introduced into the cell, containing three essential components: an RNA-targeting motif, a hemiintron sequence, and the primary sequence of the desired RNA to be ligated into the endogenous RNA transcript. The RNA-targeting motif consists of an oligonucleotide segment antisense to the intron of the endogenous target pre-mRNA. After the RNA-targeting motif pairs with the Watson-Crick bases of the endogenous intron, the hemiintron is recognized by the spliceosome. Depending on the desired splicing response, a 5' hemiintron promotes the splicing of the trans-splicing molecule to the 3' exon immediately adjacent to the targeted intron, or a 3' hemiintron linked to the exogenous RNA to be trans-spliced ​​promotes the splicing of the trans-splicing molecule to the 5' exon immediately adjacent to the targeted intron. This produces a mature chimeric RNA transcript consisting of either a recombinant 5' initiator linked to an endogenous 3' sequence or an endogenous 5' initiator linked to a recombinant 3' sequence. The advantage of this strategy is that a single AAV vector only needs to package the genome capable of producing trans-spliced ​​RNA molecules containing a portion of the sequence for the gene, avoiding the need to deliver the full-length protein-coding sequence to the cell. Because the entire recombinant RNA region of the chimeric RNA product can be specified by the user, the trans-spliced ​​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 new protein. However, similar to the split AAV vector approach, the low specificity and efficiency of RNA targeting via antisense RNA sequences hinder the widespread use of this technology in research and clinical settings. Despite its promise, unrestricted expression of CRISPR proteins (e.g., long-term expression) as a therapy presents challenges due to immunogenicity and off-target effects. Furthermore, CRISPR-based methods typically require the delivery of two constructs (i.e., a CRISPR effector protein and an RNA trans-splicing construct), leading to high dose requirements and low correction efficiency. This paper discloses a system for transcriptome engineering that does not require a CRISPR system. Many endogenous RNAs bind to ribonucleoproteins 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, efficient trans-splicing of many targeted endogenous pre-mRNAs is achieved by incorporating these RNA structures into the trans-splicing RNA. This allows for the rewriting of large segments of mRNA.

[0312] Example 1 Verification of the efficiency of reverse splicing To determine trans-splicing efficiency, a green fluorescence-based screening system was designed and constructed. The first step was to construct a reporter construct in which the green fluorescent protein (EGFP) open reading frame was split in two. Introns were then inserted between the two halves. Therefore, if this construct is cis-spliced, it will produce mature RNA encoding EGFP, which will express a green fluorophore upon translation.

[0313] Next, we eliminated fluorescent expression by introducing an early stop codon into the first "exon" of the EGFP open reading frame. Figure 1 (Left side) If the construct is cis-splicing, there will be no green fluorescence expression because the premature stop codon in the first half of the EGFP open reading frame terminates the translation of the full-length protein.

[0314] Green fluorescence expression can be restored by delivering trans-splicing RNA encoding the correct open reading frame for the first half of EGFP, followed by a half-intron and guide RNA sequence. Therefore, green fluorescence is used as an indirect reading of trans-splicing efficiency. Figure 1 This reporting subsystem is the mainstay of our downstream measurements.

[0315] The first 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 half-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 splitting GFP reporter, and after 48 hours, the green fluorescence intensity (percentage GFP% and mean fluorescence intensity) was measured by flow cytometry as a representative of trans-splicing efficiency. Figure 2A and Figure 2B Through this process, three (3) RNA structures produced significantly enhanced trans-splicing efficiency (RNA structures 4, 5, and 7). Figures 2A-2B The RNA targeting base sequence for RYR2 is listed in SEQ ID NO: 23.

[0316] Next, we examined whether any member of this RNA sequence group enhanced the 3' trans-splicing method. For this, we used the same splitting GFP reporter, but with a slight modification: the stop codon was moved from the first half of the open reading frame to the second half. Figure 1 (Right side) When delivered to HEK293 cells, this construct itself did not express green fluorescence because cis-splicing preserved the premature stop codon. To restore EGFP expression, trans-splicing RNA was used, which completed the EGFP open reading frame after proper splicing. Similar to the 5' editing method, trans-splicing RNA containing a 30 bp antisense targeting motif, followed by a half-intron and the latter half of the EGFP open reading frame (this construct was SMaRT). The same set of RNA structures was then introduced into this RNA molecule to improve trans-splicing efficiency.

[0317] The novel construct was co-transfected with a splitting GFP reporter into HEK 293 cells. Then, 48 hours later, the green fluorescence intensity (GFP % percentage and mean fluorescence intensity) was measured by flow cytometry (representing trans-splicing efficiency). Figures 3A-3B Using this method, four RNA structures (RNA structures 1, 2, 10, and 11) were identified that produced significantly enhanced trans-splicing efficiency. This group was repeated on the target intron of the second intron. Figures 3C-3D Furthermore, a similar trend was observed in the multiple-fold change in editing efficiency. Figures 3A-3B In the sequence, the RNA targeting motif for RYR2 is in SEQ ID NO: 23, while Figures 3C-3D The RNA targeting motif for LMNA is shown in SEQ ID NO: 22.

[0318] Following these results, trans-splicing assays were repeated for the highest hits (RNA structures 1 and 2). A second control RNA structure (i.e., a direct repeat from Ruminococcus flavefaciens XPD3002 (RfxCas13d)) was also used. The rationale for this control was to ensure that the enhancement of RNA editing was unique to these RNA structures, rather than an artificial phenomenon involving additional sequence length / complexity in the trans-splicing RNA. In fact, only RNA structures 1 and 2 showed enhanced editing efficiency, while the Cast13 direct repeat sequence had a slightly adverse effect on trans-splicing efficiency. Figures 4A-4B ).

[0319] Finally, the new target intron was cloned into the splitting GFP reporter, and editing efficiency was determined using RNA structure 2 and several guide RNA candidates. Editing was observed at this new target in over 50% of GFP-positive cells. Figures 5A-5B ).exist Figures 5A-5B In Table 2, the RNA targeting motif for FXN is listed in SEQ ID NO: 24. Structures 4 and 5 contain cloning sites in which guide RNA is inserted; these are the sequences in bold.

[0320] Table 2 – List of RNA structures

[0321] Table 3 - List of plasmids used in the examples

[0322] Example 2 Trans-splicing mutations in DMD DMD (dystrophin) is known in the art (e.g., Gene ID 1756), and its nucleotide sequence may contain nucleotides 5001-2225382 in accession number NG 012232.1. DMD spans a genome greater than 2 Mb and encodes a large protein containing an N-terminal actin-binding domain and multiple spectrin repeats. The encoded protein (SEQ ID NO: 17) forms a component of the dystrophin-glycoprotein complex (DGC), which bridges the internal cytoskeleton and extracellular matrix. Deletions, duplications, and point mutations at this gene locus can lead to Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or cardiomyopathy. Currently, approximately 1750 pathogenic mutations of DMD have been reported. The following details constructs that address over 1000 of these pathogenic mutations. Here, constructs based on… Figure 7A The plasmid illustrated in the diagram generates a 5' substitution construct for DMD. The 5' substitution construct targets exons 1-23 of DMD (SEQ ID NO:33). The 5' substitution construct corrects one or more of the 618 mutations identified in Table 4 below.

[0323] Table 4 - List of mutations in exons 1-23 of DMD

[0324] Based on Figure 7A The AAV vector of the plasmid construct shown is delivered to subjects with one or more 5' mutations in DMD (e.g., Table 4). A therapeutically effective amount of AAV vector (e.g., approximately 1 × 10⁻⁶) is administered. 10 vg / kg to approximately 2×10 14 Following administration of .vg / kg, cells, tissues, and / or organs of one or more subjects produced chimeric RNA molecules encoding corrected and / or restored DMD. The production of chimeric RNA molecules was confirmed by obtaining samples from the subjects and verifying the expression levels of corrected and / or restored DMD (by comparing post-treatment levels of surgical / functional DMD with pre-treatment levels of the subjects' surgical / functional DMD). Subjects experienced suppression and / or minimization of DMD disease progression. Subjects experienced improvement in quality of life. Here, a method based on... Figure 7B The plasmid illustrated in the diagram generates a 3' substitution construct for DMD. The 3' substitution construct targets exons 53-79 of DMD (SEQ ID NO: 32). The 3' substitution construct corrects one or more of the 446 mutations identified in Table 5 below.

[0325] Table 5 - List of mutations in exons 53-79 of DMD

[0326] Based on Figure 7B The AAV vectors of the plasmid constructs shown are delivered to subjects with one or more 3' mutations (e.g., Table 5). A therapeutically effective amount of the AAV vector (e.g., approximately 1 × 10⁻⁶) is administered. 10 vg / kg to approximately 2×10 14 Following treatment (vg / kg), cells, tissues, and / or organs of one or more subjects produced chimeric RNA molecules encoding corrected and / or restored DMD. The production of chimeric RNA molecules was confirmed by obtaining samples from the subjects and verifying the expression levels of corrected and / or restored DMD (by comparing post-treatment levels of surgical / functional DMD with pre-treatment levels of the subjects' surgical / functional DMD). Subjects experienced suppression and / or minimization of DMD disease progression. Subjects experienced improvement in quality of life.

[0327] Implementation Examples Overview The compositions and methods disclosed herein are superior to those previously disclosed, such as SMART. The advantages conferred by the disclosed system are numerous. These surprising and unexpected advantages include: (i) the elimination of the need for additional effector proteins (e.g., enabling trans-splicing based on CRISPR); (ii) the ability to modify effector RNA structures to bind to endogenous splicing mechanisms and enable 3' or 5' end substitution of pre-splicing mRNA; (iii) the ability to deliver constructs in a single AAV vector or as a trans-splicing RNA fragment using non-viral delivery systems (e.g., LNP); (iv) the ability to edit large segments of mRNA constituting multiple exons; (v) the ability to correct mRNA in a single "knockdown and replacement" method, particularly useful in autosomal dominant genetic disorders, using a single trans-splicing construct without having to provide two different constructs for knockdown and replacement; and (vi) the ability to prevent any gene overexpression, with correction based on the provided trans-splicing fragment, which depends on the level of the endogenous mutant transcript (therefore, the corrected mRNA will never be expressed at a level higher than that of the endogenous disease transcript). While comprehensive, this list of advantages is not exhaustive.

[0328] In summary, this article describes an efficient RNA editing platform that does not rely on CRISPR-based targeting and outperforms simple antisense base pairing. This enables the efficient rewriting of large segments of RNA, with implications for human health and basic biology. As a therapeutic agent, this will allow for the rewriting of genes beyond AAV packaging capabilities, correcting dominant-negative mutations while maintaining the expression of target transcripts at endogenous levels.

Claims

1. Nucleic acid molecules, which include: One or more RNA structures comprising the sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; One or more RNA targeting motifs; 3' half intron; and The exogenous RNA is to be trans-spliced ​​to the targeted endogenous pre-mRNA.

2. Nucleic acid molecules, which include: The exogenous RNA is to be trans-spliced ​​to the targeted endogenous pre-mRNA; 5' hemi-intron; One or more RNA targeting motifs; One or more RNA structures comprising the sequence of any one of SEQ ID NO: 01-SEQ ID NO:

09.

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

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

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

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

7. The nucleic acid molecule of claim 6, wherein the protein-coding gene comprises one or more coding regions of: ABCA1 , ABCA12 , ABCA13 , ABCA2 , ABCA3 , ABCA4 , ABCA5 , ABCC1 , ABCC2 , ABCC6 , ABCC8 , ABCC9 , ACAN , ADAMTS13 , ADCY10 , ADGRV1 , AGL , AGRN , AHDC1 , ALK , ALMS1 , 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 、 CCDC88A 、 CCDC88C 、 CCNB3 、 CDH23 、 CDK13 、 CDK5RAP2 、 CELSR1 、 CEMIP2 、 CENPE 、 CENPF 、 CENPJ 、 CEP152 、 CEP164 、 CEP250 、 CEP290 、 CFAP43 、 CFAP44 、 CFAP65 、 CFTR / ABCC7 、 CHD1 、 CHD2 、 CHD3 、 CHD4 、 CHD7 、 CHD8 、 CIC 、 CIT 、 CLIP1 、 CLTC 、 CNOT1 、 CNTNAP1 、 COL11A1 、 COL11A2 、 COL12A1 、 COL17A1 、 COL18A1 、 COL1A1 、 COL1A2 、 COL27A1 、 COL2A1 、 COL3A1 、 COL4A1 、 COL4A2 、 COL4A3 、 COL4A4 、 COL4A5 、 COL4A6 、 COL5A1 、 COL5A2 、 COL6A3 、 COL7A1 、 CPAMD8 、 CPLANE1 、 CPS1 、 CPSF1 、 CRB1 、 CREBBP 、 CUBN 、 CUL7 、 CUX1 、 DCC 、 DCHS1 、 DEPDC5 、 DICER1 、 DIP2B 、 DLC1 、 DMD 、 DMXL2 、 DNAH1 、 DNAH1 、 DNAH17 、 DNAH2 、 DNAH5 、 DNAH7 、 DNAH8 、 DNAH9 、 DNMBP 、 DNMT1 、 DOCK2 、 DOCK3 、 DOCK6 、 DOCK7 、 DOCK8 、 DSCAM 、 DSP 、 DST 、 DUOX2 、 DYNC1H1 、 DYNC2H1 、 DYSF 、 EIF2AK4 、 EP300 、 EPG5 、 ERCC6 、 ERCC6L2 、 EXPH5 、 EYS 、 F5 、 F8 、 FANCA 、 FANCD2 、 FANCM 、 FAT1 、 FAT4 、 FBN1 、 FBN2 、 FLG 、 FLG2 、 FLNA 、 FLNB 、 FLNC 、 FLT4 、 FMN2 、 FN1 、 FRAS1 、 FREM1 、 FREM2 、 FSIP2 、 FXN 、 FYCO1 、 GLI2 、 GLI3 、 GPR179 、 GREB1L 、 GRIN2A 、 GRIN2B 、 GRIN2D 、 HCFC1 、 HECW2 、 HERC1 、 HERC2 、 HFM1 、 HIVEP1 、 HIVEP2 、 HMCN1 、 HSPG2 、 HTT 、 HUWE1 、 HYDIN 、 IFT140 、 IFT172 、 IGF1R 、 IGF2R 、 IGSF1 、 INSR 、 INTS1 、 IQSEC2 、 ITGB4 、 ITPR1 、 ITPR2 、 JMJD1C 、 KALRN 、 KANK1 、 KAT6A 、 KAT6B 、 KDM3B 、 KDM5B 、 KDM5C 、 KDM6A 、 KDM6B 、 KDR 、 KIAA0586 、 KIAA1109 、 KIAA1549 、 KIDINS2 、 KIF1 、 KIF1A 、 KIF1B 、 KIF21A 、 KIF26B 、 KIF7 、 KMT2A 、 KMT2B 、 KMT2C 、 KMT2D 、 KMT2E 、 KNL1 、 LAMA1 、 LAMA2 、 LAMA3 、 LAMA4 、 LAMA5 、 LAMB1 、 LAMB2 、 LAMC3 、 LCT 、 LMNA 、 LOXHD1 、 LPA 、 LRBA 、 LRP1 、 LRP2 、 LRP4 、 LRP5 、 LRP6 、 LRPPRC 、 LRRK1 、 LRRK2 、 LTBP2 、 LTBP4 、 LYST 、 MACF1 、 MADD 、 MAGIC2 、 MAP1B 、 MAP3K1 、 MAPK8IP3 、 MAPKBP1 、 MAST1 、 MBD5 、 MCM3AP 、 MED12 、 MED12L 、 MED1 、 MED13L 、 MED23 、 MEGF8 、 ALSO 、 MLH3 、 MPDZ 、 MSH6 、 MTOR 、 MYH1 、 MYH1 、 MYH1 、 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 、 NOTCH2 、 NOTCH3 、 NPHP4 、 NRXN1 、 NRXN3 、 NSD1 、 NSD2 、 NUP155 、 NUP188 、 NUP205 、 OBSCN 、 OBSL1 、 OTOF 、 OTOG 、 OTOGL 、 PARD3 、 PBRM1 、 PCDH15 、 PCLO 、 PCNT 、 PHIP 、 PI4KA 、 PIEZO1 、 PIEZO2 、 PIK3C2A 、 PIKFYVE 、 PKD1 、 PKD1L1 、 PKHD1 、 PLCE1 、 PLEC 、 PLEKHG2 、 PNPLA6 、 POGZ 、 POLA1 、 POLE 、 POLR1A 、 POLR2A 、 POLR3A 、 PRG4 、 PRKDC 、 PRPF8 、 PRR12 、 PRX 、 PTCH1 、 PTPN23 、 PTPRF 、 PTPRJ 、 PTPRQ 、 PXDN 、 QRICH2 、 RAB3GAP2 、 RAI1 、 RALGAPA1 、 RANBP2 、 RB1CC1 、 RELN 、 RERE 、 REV3L 、 RIC1 、 RIMS1 、 RIMS 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 8. The nucleic acid molecule of any one of claims 3-7, wherein one or more mutations in one or more exons contribute to the pathogenesis in one or more cells.

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

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

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

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

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

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

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

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

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

18. The nucleic acid molecule of claim 1, wherein the RNA targeting motif is directed to an intron immediately adjacent to the 3' of an exon of the targeted endogenous pre-mRNA to be spliced.

19. The nucleic acid molecule of claim 2, wherein the RNA targeting motif is directed to an intron immediately adjacent to the 5' of the exon of the targeted endogenous pre-mRNA to be spliced.

20. The nucleic acid molecule of claim 1, wherein the 3' half-intron is linked to the exogenous RNA to be trans-spliced ​​with the endogenous mRNA.

21. The nucleic acid molecule of claim 1, wherein the 3' half-intron is recognized by nuclear splicing components in the host cell.

22. The nucleic acid molecule of claim 1, wherein the 3' half-intron comprises (i) a 3' splice region containing a branch point, (ii) a polypyrimidine string, and (iii) a 3' splice acceptor site.

23. The nucleic acid molecule of claim 2, wherein the 5' half-intron is linked to the exogenous RNA to be trans-spliced ​​with the endogenous mRNA.

24. The nucleic acid molecule of claim 2, wherein the 5' half-intron is recognized by the nuclear splicing component in the host cell.

25. The nucleic acid molecule of claim 2, wherein the 5' half-intron contains a 5' splicing site.

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

27. A nucleic acid molecule according to any one of the preceding claims, wherein 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 cell nucleus, (iv) stabilize the interaction between the targeted endogenous pre-mRNA molecule and the exogenous RNA to be trans-spliced, or (v) any combination thereof.

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

29. Pharmaceutical preparations comprising: The carrier of claim 28; and One or more pharmaceutically acceptable carriers.

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

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

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

33. A method for treating a genetic disease or hereditary condition, the method comprising: By administering a therapeutically effective amount of the carrier of claim 28 or the pharmaceutical preparation of claim 29 to a subject in need, a chimeric RNA molecule is generated in one or more cells; The resulting chimeric RNA molecule contains a trans-spliced ​​nucleic acid sequence; and The resulting chimeric RNA molecules restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic disorders.

34. The method of claim 33, wherein restoring one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic disorders includes restoring the functional and / or structural integrity of missing, defective and / or mutated proteins or enzymes.

35. The method of claim 33, wherein the therapeutically effective amount of the carrier comprises about 1 × 10⁻⁶. 10 vg to approximately 2×10 14 vg.

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

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

38. The method of any one of claims 33-37, further comprising the step of repeated application.

39. The method of any one of claims 33-38, further comprising monitoring subjects for adverse effects.

40. The method of any one of claims 33-39, wherein one or more aspects of restoring cellular homeostasis and / or cellular functional and / or metabolic dysfunction include (i) correcting cellular starvation in one or more cells; (ii) correcting, preventing, reducing and / or alleviating autophagy; (iii) improving, enhancing, restoring and / or preserving mitochondrial functional and / or structural integrity; (iv) improving, enhancing, restoring and / or preserving organelle functional and / or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing and / or slowing the rate of progression of a multisystemic manifestation of a genetic disease or condition; (vii) reversing, inhibiting, preventing, stabilizing and / or slowing the rate of progression of a genetic disease or condition, or (viii) any combination thereof.

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