Compositions comprising xbp1 fragments and methods of use in gene therapy

Abstract

Disclosed herein are compositions comprising endoplasmic reticulum (ER) stress sensor variants. Disclosed herein are methods of using the disclosed compositions to maintain and / or establish ER homeostasis following transgene expression.

Description

COMPOSITIONS COMPRISING XBP1 FRAGMENTS AND METHODS OF USE IN GENE THERAPYI. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 515,957 filed 27 July 2023 and to U.S. Provisional Application No. 63 / 665,118 filed 27 June 2024, each of which is incorporated herein in its entirety.II. REFERENCE TO THE SEQUENCE LISTING

[0002] The Sequence Listing submitted 26 July 2024 as an XML file named “23-2092- WO_Sequence Listing”, created on 26 July 2024 and having a size of 451 kilobytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).III. BACKGROUND

[0003] Excess protein expression in the endoplasmic reticulum (ER) exceeding its folding capacity sets off ER stress responses, also known as the unfolded protein response (UPR). UPR senses when the ER has accumulated an excess of unfolded plasma membrane and secretory proteins, which constitute approximately 30% of the total proteome in most eukaryotic cells. Upon activation of UPR, transcription of genes that serve to reinforce the ER’s protein folding capacity is triggered as necessary. However, if ER homeostasis is not established in a timely manner, then UPR pathway eventually induces cell death by apoptosis.

[0004] Gene therapy using adeno-associated virus (AAV) as the delivery agent has emerged as an efficacious therapeutic modality for monogenic diseases bolstered by more than 3000 patients treated over more than 20 years. Despite the overall success of AAV-based gene delivery, several major challenges remain unresolved. One of these challenges is the inability to regulate the transgene copy number per cell, which generates significant heterogeneity in per cell protein expression levels. In cells that have high protein burden after AAV administration, UPR activation may engender apoptosis, leading to lowered long-term expression of the transgene. For example, following AAV -mediated liver delivery of Factor VIII (FVIII) for treatment of hemophilia A, transient UPR activation was observed in response to B -domain deleted single chain FVIII (BDD-FVIII) transduction in mice.

[0005] As AAV delivery systems improve and transduction efficiencies increase, high protein load per cell may result in a more intense UPR activation and sub-optimal long-term prognosis, which calls for an effective strategy to re-establish ER homeostasis upon UPR activation in cells with high protein load after AAV administration. Similar challenges exist for mRN A therapeutics, where overexpression is an issue. The compositions and methods described herein overcome these challenges.IV. BRIEF DESCRIPTION OF THE FIGURES

[0006] FIG. 1 shows that IREla RNase splices XBP1 mRNA, which encodes a potent transcription factor that activates expression of UPR target genes involved in ER proteostasis and cell pathophysiology. IREla RNase can also cleave ER- associated mRNAs or non-coding functional RNAs, leading to their degradation through regulated IRE 1 -dependent decay (RIDD), which modulates the protein folding load, cell metabolism, inflammation and inflammasome signaling pathways. The IREla cytosolic domain may also serve as a scaffold to recruit adaptor proteins, for example tumor necrosis factor receptor- associated factor (TRAF) family members, thereby activating inflammatory responses under non- canonical ER stress conditions. Here, IRElais Inositol-Requiring Enzyme la; CHOPis CCAAT / enhancer-binding protein homologous protein; and RIDD is Regulated IRE 1 -depend ent decay.

[0007] FIG. 2 shows the proapoptotic UPR schematic. Under ER stress, the PERK-eIF2aUPR branch induces translation of ATF4, which can activate expression of the proapoptotic factor CCAAT / enhancer- binding protein homologous protein (CHOP) and GADD34. GADD34 targets protein phosphatase 1 (PPI) to dephosphorylate eIF2a and thereby restore mRNA translation. Constitutive repressor of eIF2a phosphorylation (CReP) also serves as a cofactor to provide PPI specificity for phosphorylated eIF2a under ER stress. CHOP promotes ER stress- induced apoptosis by modulating GADD34, death receptor 5 (DR5) and the members of the BCL-2 or BH3- only family, including NOXA, BIM and PUMA, to stimulate protein synthesis and exacerbating protein folding defect. Furthermore, the IRE la UPR branch is involved in caspase 2- dependent, caspase 8- dependent or BAX / BAK- dependent apoptosis through RIDD or activation of TRAF2-JUN N-terminal kinase (JNK) signaling. The IRE la-mediated RIDD also regulates thioredoxin- interacting protein (TXNIP) to activate inflammasome- dependent and caspase 1— IL-1 P- dependent sterile inflammation, leading to apoptosis. In addition, Ca2+ release from the ER via inositol 1,4,5- trisphosphate receptor (IP3R), which interacts with the ER- located antiapoptotic proteins BAX inhibitor 1 (BI-1) and GRINA, contributes to mitochondrial reactive oxygen species release and the activation of the BAX / BAK-dependent apoptosome. miRNA means microRNA.

[0008] FIG. 3 shows that IREla splices out a 26-nucleotide region from XBPlu mRNA when UPR is activated.

[0009] FIG. 4A - FIG. 4B shows IREla endonuclease spliced XBP1 mRNA under ER stress. FIG. 4A shows AAV delivered transgene mRNA underwent splicing by IREla on the ER membrane. When protein load in the ER lumen was high, UPR was triggered. IREla spliced out a 26-nucleotide region in XBP1 fragment (XBP IF) tagged therapeutic transcripts, which resulted in a shift in the reading frame. Consequently, protein expression decreased, and ER stress wasmitigated. FIG. 4B shows endogenous XBP1 mRNA splicing by IREla on the ER membrane. When UPR was activated in a cell, IREla spliced out a 26-nucleotide region in endogenous full- length XBP 1 (XBP lu) mRNA on the ER membrane. This shorter XBP 1 (XBP 1 s) mRNA encoded a transcription factor which activated downstream UPR pathway genes. Here, XBP IF is engineered XBP1 ER sensor fragment.

[0010] FIG. 5A - FIG. 5B show B-domain deleted recombinant human factor VIII (BDD FVHI) triggers ER stress and XBP1F2 tagged BDD FVIII is spliced by IREla. HEK293 cells were transfected with CBA-XBP1F2-BDD FVIII and CBA-BDD FVIII in a 24-well plate. 24 hours later, RNA was collected and converted to cDNA to assess ER stress and splicing of the engineered therapeutic construct. FIG. 5A shows PCR for endogenous XBP 1 splicing confirmed that BDD FVIII expression triggers ER stress since both CBA-BDD FVIII transfected wells (lanes 2-4) and CBA-XBP1F2-FVIII transfected wells (lanes 5-7) have the double banding pattern seen in ER stress conditionsunlike the non-transfected control (lane 1). FIG. 5B shows 26-nucleotide region in the ER sensor domain of the therapeutic RNA spliced out with triggering of ER stress and IREla activation.

[0011] FIG. 6 shows that tagging BDD FVIII with XBP1F2 / 3 / 4 / 5 ER sensor fragments significantly reduced UPR activation in HEK293 cells. HEK293 cells were transfected with constructs above. Cellular RNA was extracted 24 hours later and was converted to cDNA. QPCR reaction was performed for GRP78 / BiP, a marker of ER stress. Delta-delta Ct method was applied to normalize values to RPL19, a housekeeping gene, and to non-transfected control cDNA. Compared to CBA-BDD FVIII condition, ER stress was significantly mitigated for constructs with the ER stress sensor domain. Ordinary one-way ANOVA test was applied to the results.

[0012] FIG. 7 shows that endogenous XBP1 spliced when HEK293 cells express engineered leronlimab constructs, indicating that these constructs trigger ER stress. HEK293 cells were transfected with a CMV promoter driven leronlimab monoclonal antibody with different selfcleaving peptides linking the heavy and the light chains. All leronlimab constructs trigger ER stress in cells, as indicated by the double banding pattern, absent in the non-transfected cells only control. This result corroborates that our engineered ER stress domains can be utilized for multiple therapeutic genes.

[0013] FIG. 8A - FIG. 8C show that the XBP lu-eGFP transcript gets spliced by IREla under chemically induced ER stress. FIG. 8 A shows a plasmid that was designed with a Cbh promoter driving XBPlu mRNA incorporated upstream of enhanced green fluorescent protein (eGFP) without a start codon. Fusion mRNA was designed in a way that when the 26 nucleotide XBPlu intron is removed, a frameshift in eGFP protein-coding ORF halted protein production. FIG. 8B shows that a plasmid was transfected into HEK293 cells in a 24-well plate. Then, 48 hours aftertransfection, either ER stress inducing thapsigargin or DMSO was added. 1 hour and 3 hours after incubation, RNA was collected and converted to cDNA. Full length XBPlu-eGFP expression was reduced by >50% after 1 hour and 3 hours incubation with 1 pM thapsigargin. BiP / GRP78 (ER stress marker) expression increased by > 2x and > 4 x at 1 hour and 3 hours with 1 pM thapsigargin incubation, respectively. Two-tailed unpaired t-test was applied to results. FIG. 8C provides the appearance of the splicing product with added thapsigargin in RT-PCR gel confirming XBPlu-eGFP splicing.

[0014] FIG. 9A - FIG. 9C show that fragments of XBPlu mRNA (XBP1F) decreased percent change in eGFPd2 expression under induced ER stress. FIG. 9A shows the regions of XBPlu mRNA included in the ER stress sensor. P2A, a self -cleaving peptide, separates ER stress sensor from eGFP degron (eGFPd2) which is a variant of eGFP with a shorter half-life. Here, HR2 is Hydrophobic Region 2; AP is Arrest Peptide; and CP is Calreticulin Peptide. FIG. 9B shows that HEK293 cells were transfected with a plasmid that has a CB A promoter driving XBP IF 1 construct in FIG. 9A. 48 hours after transfection, 1 pM thapsigargin was added to wells. Cells were collected and flow-analyzed at the given timepoints. Mean Fluorescence Intensity (MFI) at each timepoint was calculated as percent increase from expression at Time 0. A CBA-eGFPd2 construct with no ER stress sensor was used as control. FIG. 9C shows that 12 hours after incubation with thapsigargin, cell media was changed. EGFP expression was quantified by flow cytometry and percent change in MFI relative to Time 0 was determined. FIG. 9D shows the experiment in FIG. 9B was repeated for XBPlF2-eGFPd2 construct. Two-way ANOVA was applied to results.

[0015] FIG. 10A - FIG. 10C show that IREla splices out intron in in vitro transcribed XBPlu- eGFP mRNA under chemically induced ER stress. FIG. 10A shows that XBPlu-eGFP was cloned into a T7 plasmid and in vitro transcribed to generate capped XBPlu-eGFP mRNA. After column clean-up, 1000 ng IVT RNA was transfected into HEK293 cells. FIG. 10B shows that 1 pM thapsigargin or DMSO was added to cells 48 hours after transfection. RNA was extracted 3 hours after incubation and converted to cDNA. cDNAwas PCR amplified, and resulting amplicon was Sanger sequenced to confirm existence of multiple RNA species after the intron. FIG. 10C shows RT-qPCR was performed to determine the abundance of full-length XBPlu-eGFP transcripts. As the forward primer was on the intron, reduction in signal for thapsigargin treated samples indicating XBPlu-eGFP mRNA splicing under chemically induced ER stress.

[0016] FIG. HA - FIG. 11C shows that incorporating XBP1F in front of B-domain deleted Factor VIII (BDD FVIII) reduced ER stress marker expression in HEK293 and Huh7 cells. FIG. HA shows that HEK293 (top) and Huh7 (bottom) cells were transfected with CB A-XBP1F-BDD FVIII plasmids. 48 hours after transfection, RNA was collected and converted to cDNA. RT-qPCR was performed to calculate BDD FVIII mRNA abundance for all constructs. FIG. 11B shows that HEK293 cells were transfected with CBA-XBP1F1-BDD FVIII, CBA-XBP1F2-BDD FVIII, and CBA-BDD FVIII. At 12 hours, 24 hours, and 48 hours after transfection, cells were lysed to collect protein. Equal amount of protein was run on an SDS-PAGE gel that was probed for FVIII (top) and vinculin (bottom). Protein expression for XBP1F1 and XBP1F2 at different timepoints was confirmed. FIG. 11C shows that BiP / GRP78 (ER stress marker) expression was quantified in the same RNA population in FIG. 11A in HEK293 (left) and Huh7 (right) cells. Levels were lower for ER stress sensor-controlled constructs compared to a BDD FVII construct with no ER stress sensor. Ordinary one-way ANOVA was applied to results.

[0017] FIG. 12A - FIG. 12C shows that incorporating XBP1F in front of anti-HIV antibody leronlimab reduced ER stress marker expression in HEK293 and Huh7 cells. FIG. 12A shows HEK293 (top) and Huh7 (bottom) cells that were transfected with CBA-XBPIF-leronlimab plasmids. 48 hours aftertransfection, RNA was collected and converted to cDNA. RT-qPCR was performed to calculate leronlimab mRNA abundance for all constructs. FIG. 12B shows BiP / GRP78 (ER stress marker) expression was quantified in the same RNA population in FIG. 12A in HEK293 (left) and Huh7 (right) cells. Levels were lower for ER stress sensor-controlled constructs compared to a leronlimab construct with no ER stress sensor. Ordinary one-way ANOVA was applied to results.

[0018] FIG. 13A shows that unconventional XBP1F splicing with BDD FVIII and leronlimab overexpression was validated by RNA-seq in HEK293 cells. FIG. 13A provides a diagram indicating two expected RNA populations with ER stress causing protein overexpression: unspliced (SEQ ID NO:58, SEQ ID NO:59) and spliced (SEQ ID NO:60) forms ofXBPIF-BDD FVIII or XBPIF-leronlimab. FIG. 13B shows that HEK293 cells were transfected with the constructs shown transcribed via CBA promoter. After RNA extraction and cDNA conversion 48 hours after transfection, cDNA was PCR amplified and sent for Illumina sequencing. Percent spliced XBP1F was calculated by dividing number of correctly spliced transcripts by number of total transcripts and multiplying by 100. A custom Python code was written for transcript analysis. FIG. 13C shows that experiment in FIG. 13B was repeated for leronlimab constructs.

[0019] FIG. 14A - FIG. 14E show XBP1F2 in front of leronlimab lowered ER stress marker transcript abundance in vivo. FIG. 14A shows that CBA-XBP1F2-Ieronlimab and CBA- leronlimab were packaged into AAV8. Then, 8-week-old - 10-week-old C57 / BL6 mice dosed IV with 1E11 vector genomes per mouse. Liver tissue and serum were harvested 28 days after injection. FIG. 14B shows the percent of spliced XBP1F2-Ieronlimab mRNA in liver was calculated . RNA was extracted from liver tissue, converted to cDNA, PCR amplified and Illumina sequenced. Percent spliced transcripts were quantified as described in FIG. 14A - FIG. 14C.FIG. 14C shows that leronlimab mRNA expression in livers was quantified by RT-qPCR. FIG. 14D shows that BiP / GRP78 expression in livers was quantified by RT-qPCR. FIG. 14E shows serum leronlimab protein levels was measured by an ELISA assay. Two-tailed unpaired t-test was applied to results.V. BRIEF SUMMARY

[0020] Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in any one SEQ ID NO:01 - SEQ ID NO:06. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F1, XBP1F2, XBP1F3, XBP1F4, XBP1F5, or XBP1F6. Disclosed herein is a nucleic acid molecule comprising an X- box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in in any one SEQ ID NO:01 - SEQ ID NO:06. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the RNA structure known as XBP1F1, XBP1F2, XBP1F3, XBP1F4, XBP1F5, or XBP1F6. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in any one SEQ ID NO:01 - SEQ ID NO:06, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F1, XBP1F2, XBP1F3, XBP1F4, XBP1F5, or XBP1F6, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in in any one SEQ ID NO:01 - SEQ ID NO:06, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the RNA structure known as XBP1F1, XBP1F2, XBP1F3, XBP1F4, XBP1F5, orXBPlF6, and a sequence encoding a transgene.

[0021] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:06, and a sequence encoding a transgene, wherein, following expression of the encoded transgene, the endoplasmic reticulum (ER) stress sensor variant ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0022] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F1, XBP1F2, XBP1F3, XBP1F4, XBP1F5,or XBP1F6, and a sequence encoding a transgene, wherein, following expression of the encoded transgene, the endoplasmic reticulum (ER) stress sensor variant ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0023] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:06, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBP1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0024] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F1. XBP1F2, XBP1F3, XBP1F4, XBP1F5, and XBP1F6, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBP 1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0025] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:06, and a sequence encoding a transgene, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBP 1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0026] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP IF 1, XBP1F2, XBP1F3, XBP1F4, XBP1F5, or XBP1F6, and a sequence encoding a transgene, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBP1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.VI. DETAILED DESCRIPTION

[0027] The present disclosure describes formulations, compounded compositions, kits, capsules, containers, and / or methods thereof. It is to be understood that the inventive aspects of which are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any method sand materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0028] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.A. Definitions

[0029] Before the present compounds, compositions, articles, systems, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0030] This disclosure describes inventive concepts with reference to specific examples. However, the intent is to cover all modifications, equivalents, and alternatives of the inventive concepts that are consistent with this disclosure.

[0031] As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0032] The phrase “consisting essentially of’ limits the scope of a claim to the recited components in a composition or the recited steps in a method as well as those that do not materially affect the basic and novel characteristic or characteristics of the claimed composition or claimed method. The phrase “consisting of’ excludes any component, step, or element that is not recited in the claim. The phrase “comprising” is synonymous with “including”, “containing”, or “characterized by”, and is inclusive or open-ended. “Comprising” does not exclude additional, unrecited components or steps.

[0033] As used herein, when referring to any numerical value, the term “about” means a value falling within a range that is ± 10% of the stated value.

[0034] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0035] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0036] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. In an aspect, a disclosed method can optionally comprise one or more additional steps, such as, for example, repeating an administering step or altering an administering step.

[0037] As used herein, “isolated” refers to a nucleic acid molecule or a nucleic acid sequence that has been substantially separated, produced apart from, or purified away from other biological components in the cell or tissue of an organism in which the component occurs, such as other cells, chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins.

[0038] As used herein, the term “subject” refers to the target of administration, e.g., a human being. The term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.). Thus, the subject of the herein disclosed methods can be a vertebrate, such asa mammal, a fish, a bird, a reptile, or an amphibian. Alternatively, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not denote a particular age or sex, and thus, adult and child subjects, as well as fetuses, whethermale orfemale, are intended tobe covered. In an aspect, a subject can be a human patient. In an aspect, a subject can have a disease or disorder, be suspected of having a disease or disorder, or be at risk of developing a disease or disorder (e.g., a genetic disease or disorder). In an aspect, a subject can be treatment-naive.

[0039] As used herein, a “regulatory element” can refer to promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyad enylation signals and poly-U sequences). Regulatory elements can include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).

[0040] As used herein, the term “diagnosed” means having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods. For example, “diagnosed with a disease or disordef’ means having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition (such as a genetic disease or disorder) that can be treated by one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods. For example, “suspected of having a disease or disordef’ can mean having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition (such as a genetic disease or disorder) that can likely be treated by one or more of by one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods. In an aspect, an examination can be physical, can involve various tests (e.g., blood tests, genotyping, biopsies, etc.) and assays (e.g., enzymatic assay), or a combination thereof.

[0041] A “patient” refers to a subject afflicted with a disease or disorder (e.g., a genetic disease or disorder). In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder. In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder and is seeking treatment or receiving treatment for a disease or disorder.

[0042] As used herein, the phrase “identified tobe in need of treatment for a disease or disorder,” or the like, refers to selection of a subject based upon need for treatment of the disease or disorder.For example, a subject can be identified as having a need for treatment of a disease or disorder (e.g., a genetic disease or disorder) based upon an earlier diagnosis by a person of skill and thereafter subjected to treatment for the genetic disease or disorder. In an aspect, the identification can be performed by a person different from the person making the diagnosis. In an aspect, the administration can be performed by one who performed the diagnosis.

[0043] As used herein, “inhibit,” “inhibiting”, and “inhibition” mean to diminish or decrease an activity, level, response, condition, severity, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, level, response, condition, severity, disease, or other biological parameter. This can also include, for example, a 10% inhibition or reduction in the activity, level, response, condition, severity, disease, or other biological parameter as compared to the native or control level (e.g., a subject not having a disease or disorder such as a genetic disease or disorder). Thus, in an aspect, the inhibition or reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of reduction in between as compared to native or control levels. In an aspect, the inhibition or reduction can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% as compared to native or control levels. In an aspect, the inhibition or reduction can be 0-25%, 25- 50%, 50-75%, or 75-100% as compared to native or control levels. In an aspect, a native or control level can be a pre-disease or pre-disorder level.

[0044] The words “treat” or “treating” or “treatment” include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In an aspect, the terms cover any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the undesired physiological change, disease, pathological condition, or disorder from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the physiological change, disease, pathological condition, or disorder, i.e., arresting its development; or (iii) relieving the physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease. For example, in an aspect, treating a disease or disorder can reduce the severity of an established a disease or disorder in a subject by l%-100% as compared to a control (such as, for example, an individual not having a genetic disease or disorder). In an aspect, treating can refer to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of a disease or disorder (such as a genetic disease or disorder). For example, treatinga disease or disorder can reduce one or more symptoms of a disease or disorder in a subject by l%-100% as compared to a control (such as, for example, an individual not having a genetic disease or disorder). In an aspect, treating can refer to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% reduction of one or more symptoms of an established a disease or disorder. It is understood that treatment does not necessarily refer to a cure or complete ablation or eradication of a disease or disorder. However, in an aspect, treatment can refer to a cure or complete ablation or eradication of a disease or disorder.

[0045] As used herein, the term “prevent” or “preventing” or “prevention” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. In an aspect, preventing a disease or disorder having chromatin deregulation and / or chromatin dysregulation is intended. The words “prevent”, “preventing”, and “prevention” also refer to prophylactic or preventative measures for protecting or precluding a subject (e.g., an individual) not having a given a disease or disorder (such as a genetic disease or disorder) or related complication from progressing to that complication.

[0046] As used herein, the terms “administering” and “administration” refer to any method of providing one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, the following: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can also include hepatic intra-arterial administration or administration through the hepatic portal vein (HPV). Administration of a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical composition, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed small molecule, a disclosed endonuclease, a disclosed oligonucleotide, and / or a disclosed RNA therapeutic can comprise administration directly into the CNS or thePNS. Administration can be continuous or intermittent. Administration can comprise a combination of one or more route.

[0047] In an aspect, the skilled person can determine an efficacious dose, an efficacious schedule, and an efficacious route of administration for one or more of the disclosed nucleic acid molecules,disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof to treat or prevent a disease or disorder (such as genetic disease or disorder). In an aspect, the skilled person can also alter, change, or modify an aspect of an administering step to improve efficacy of one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof.

[0048] By “determining the amount” is meant both an absolute quantification of a particular analyte (e.g., an mRNA sequence containing a particular tag) or a determination of the relative abundance of a particular analyte (e.g., an amount as compared to a mRNA sequence including a different tag). The phrase includes both direct or indirect measurements of abundance (e.g., individual mRNA transcripts may be quantified or the amount of amplification of an mRNA sequence under certain conditions for a certain period may be used a surrogate for individual transcript quantification) or both.

[0049] As used herein, “modifying the method” can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof administered to a subject, or by changing the frequency of administration of one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof to a subject, by changing the duration of time one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination are administered to a subject, or by substituting for one or more of the disclosed components and / or reagents with a similar or equivalent component and / or reagent. The same applies to all disclosed therapeutic agents, immune modulators, immunosuppressive agents, proteosome inhibitors, etc.

[0050] As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. In an aspect, a pharmaceutical carrier employed can be a solid, liquid, or gas. In an aspect, examples of solid carriers can include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. In an aspect, examples of liquid carriers can include sugar syrup, peanut oil, olive oil, and water. In an aspect, examples of gaseous carriers can include carbon dioxide and nitrogen. In preparing a disclosed composition for oral dosageform, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-poly glycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0051] As used herein, the term “excipient” refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.). See, also, for reference, Remington’s Pharmaceutical Sciences, (1990) Mack Publishing Co., Easton, Pa., which is hereby incorporated by reference in its entirety.

[0052] As used herein, “concurrently” means (1) simultaneously in time, or (2) at different times during the course of a common treatment schedule.

[0053] The term “contacting” as used herein refers to bringing one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof together with a target area or intended target area in such a manner that the one or more of the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof exert an effect on the intended target or targeted area either directly or indirectly. A target area can comprise one or more cells, and in an aspect, one or more cells can be in a subject. A target area or intended target area can be one or more of a subject’s organs (e.g., lungs, heart, liver, kidney, brain, etc.). In an aspect, a target area or intended target area can be any cell or any organ infected by a disease or disorder (such as a genetic disease or disorder). In an aspect, a target area or intended target area can be any organ, tissue, or cells that are affected by a disease or disorder (such as a genetic disease or disorder).

[0054] As used herein, “determining” can refer to measuring or ascertaining the presence and severity of a disease or disorder, such as, for example, a genetic disease or disorder. Methods and techniques used to determine the presence and / or severity of a disease or disorder are typically known to the medical arts. For example, the art is familiar with the ways to identify and / or diagnose the presence, severity, or both of a disease or disorder (such as, for example, a genetic disease or disorder).

[0055] As used herein, “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired result such as, for example, the treatment and / or prevention of a disease or disorder (e.g., a genetic disease or disorder) or a suspected disease or disorder. As used herein, the terms “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired an effect on an undesired condition e.g., a disease or disorder). For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. In an aspect, “therapeutically effective amount” means an amount of a disclosed nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation; that (i) treats the particular disease, condition, or disorder (e.g., a genetic disease or disorder), (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder e.g., a genetic disease or disorder), or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein (e.g., a genetic disease or disorder). The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceuticalformulations employed; the disclosed methods employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations employed; the duration of the treatment; drugs used in combination or coincidental with the disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations employed, and other like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, then the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, a single dose of the disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition, such as, for example, a disease or disorder due to a missing, deficient, and / or mutant protein or enzyme.

[0056] As used herein, “RNA therapeutics” can refer to the use of oligonucleotides to targetRNA. RN A therapeutics can offer the promise of uniquely targeting the precise nucleic acids involved in a particular disease with greater specificity, improved potency, and decreased toxicity. This could be particularly powerful for genetic diseases where it is most advantageous to aim for the RNA as opposed to the protein. In an aspect, a therapeutic RNA can comprise one or more expression sequences. As known to the art, expression sequences can comprise an RNAi, shRNA, mRNA, non-coding RNA (ncRNA), an antisense such as an antisense RNA, miRNA, morpholino oligonucleotide, peptide-nucleic acid (PNA) or ssDNA (with natural, and modified nucleotides, including but not limited to, LNA, BNA, 2’-0-Me-RNA, 2’-ME0-RNA, 2’-F-RNA), or analog or conjugate thereof. In an aspect, a disclosed therapeutic RNA can comprise one or more long non-coding RNA (IncRNA), such as, for example, a long intergenic non-coding RNA (lincRNA), pre-transcript, pre-miRNA, pre-mRNA, competing endogenous RNA (ceRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), pseudo-gene, rRNA, or tRNA. In an aspect, ncRNA can be piwi-interacting RNA (piRNA), primary miRNA (pri-miRNA), or premature miRNA (pre-miRNA). In an aspect, a disclosed therapeutic RNA or an RNA therapeutic can comprise antisense oligonucleotides (ASOs) that inhibit mRNA translation, oligonucleotides thatfunction via RNA interference (RNAi) pathway, RNA molecules that behave like enzymes (ribozymes), RNA oligonucleotides that bind to proteins and other cellular molecules, and ASOs that bind to mRNA and form a structure that is recognized by RNase H resulting in cleavage of the mRNA target. In an aspect, RNA therapeutics can comprise RNAi and ASOs that inhibit mRNA translation. Generally speaking, as known to the art, RNAi operates sequence specifically and post-transcriptionally by activating ribonucleases which, along with other enzymes and complexes, coordinately degrade the RNA after the original RNA target has been cut into smaller pieces while antisense oligonucleotides bind to their target nucleic acid via Watson-Crick base pairing, and inhibit or alter gene expression via steric hindrance, splicing alterations, initiation of target degradation, or other events.

[0057] As used herein, “small molecule” can refer to any organic or inorganic material that is not a polymer. Small molecules exclude large macromolecules, such as large proteins (e.g., proteins with molecular weights over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000), large nucleic acids (e.g., nucleic acids with molecular weights of over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000), or large polysaccharides (e.g., polysaccharides with a molecular weight of over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000). In an aspect, a “small molecule”, for example, can be a drug that can enter cells easily because it has a low molecular weight. In an aspect, a small molecule can be used in conjunction with a disclosed composition in a disclosed method.

[0058] In an aspect, the term “ex vivo” can refer generally to activities that take place outside an organism or subject such as experimentation, modification, differentiation, manipulation, and / or measurement done in or on living tissue in an artificial environment outside the organism. In an aspect, ex vivo experimentation, ex vivo modification, ex vivo differentiation, ex vivo manipulation, and / or ex vivo measurement can occur with a minimum alteration of the natural conditions. In an aspect, “ex vivo” can comprise living cells, tissues, or organs (e.g., cells in need of trans-splicing for one or more protein coding genes) taken from a subject in need thereof or a donor subject and cultured and / or maintained and / or perfused in a laboratory apparatus, usually under sterile conditions, and typically for a limited duration of time (e.g., a few hours or up to about 24 hours, up to about 48 hours, up to about 72 hours, up to about 96 hours, up to about 120 hours, up to about 144 hours, up to about 168 hours, or more depending on the circumstances and / or the desired characteristics. In an aspect, tissues, cells, or organs can be collected, frozen, and later thawed for ex vivo treatment.

[0059] As used herein, “operably linked” means that expression of a gene or a transgene is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5’ (upstream) or 3’ (downstream) of a gene under its control. The distance between the promoterand a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance can be accommodated without loss of promoter function.

[0060] As used herein, “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein must contain at least two amino acids and there is no limitation on the maximum number of amino acids that can comprise a protein’s sequence. The term “peptide” can refer to a short chain of amino acids including, for example, natural peptides, recombinant peptides, synthetic peptides, or any combination thereof. Proteins and peptides can include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others.

[0061] “Nucleic acid” or “oligonucleotide” or “polynucleotide” as used herein means at least two nucleotides covalently linked together. The depiction of a single strand can also define the sequence of the complementary strand. Thus, a nucleic acid can encompass the complementary strand of a depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid can encompass substantially identical nucleic acids and complements thereof. A single strand can provide a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid can encompass a probe that hybridizes under stringent hybridization conditions. A nucleic acid can be single-stranded, or double-stranded, or can contain portions of both double-stranded and single-stranded sequence. The nucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods. Also as used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid construct,” “nucleotide sequence”, and “polynucleotide” can refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term can encompass RNA / DNA hybrids. When dsRNA is produced synthetically, less common bases, such as inosine, 5- methylcytosine, 6-methyladenine, hypoxanthine and others can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modification to the phosphodiester backbone, or the 2’-hydroxy in the ribose sugar group of the RNA can also be made. A“synthetic”nucleic acid or polynucleotide, as used herein, refers to a nucleic acid or polynucleotide that is not found in nature but is constructed by the hand of man and therefore is not a product of nature.

[0062] A “polynucleotide” is a sequence of nucleotide bases, and may be RNA, DNA, or DNA- RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotides).

[0063] A “fragment” or “portion” of a nucleotide sequence can be understood to mean a nucleotide sequence of reduced length relative (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides) to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of, or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence. Such a nucleic acid fragment or portion according to the disclosure can be, where appropriate, included in a larger polynucleotide of which it is a constituent. In an aspect, a fragment or portion of a nucleotide sequence or nucleic acid sequence can comprise the sequence encoding an exon having one or more mutations.

[0064] A “fragment” or “portion” of an amino acid sequence can be understood to mean an amino acid sequence of reduced length relative (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or more amino acids) to a reference amino acid sequence and comprising, consisting essentially of, or consisting of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the reference amino acid sequence. Such an amino acid fragment or portion according to the disclosure can be, where appropriate, included in a larger amino acid sequence of which it is a constituent.

[0065] A “heterologous” or a “recombinant” nucleotide or amino acid sequence as used interchangeably herein can refer to a nucleotide or an amino acid sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleotide or amino acid sequence.

[0066] As used herein, the term “endogenous” can refer to a gene, protein, compound, or activity that is normally present in a host cell (e.g., a pre-mRNA). As used herein, an “exogenous” nucleic acid molecule, construct, or sequence (e.g., an RNA sequence to be trans-spliced) can refer to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to a host cell, but may be homologous to a nucleic acid molecule or portion of a nucleic acid molecule from the host cell.

[0067] As used herein, “promoted’ or “promoters” are known to the art. Depending on the level and tissue-specific expression desired, a variety of promoter elements can be used. A promoter can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the gene expression desired. A promoter can be native (endogenous) or foreign (exogenous) and can be a natural or a synthetic sequence. By foreign or exogenous, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

[0068] “Tissue-specific promoters” are known to the art and include, but are not limited to, neuron-specific promoters, muscle-specific promoters, liver-specific promoters, skeletal muscle - specific promoters, and heart-specific promoters.

[0069] “Liver-specific promoters” are known to the art and include, but are not limited to, the thyroxin binding globulin (TBG) promoter, the al-microglobulin / bikunin enhancer / thyroid hormone-binding globulin promoter, the human albumin (hALB) promoter, the thyroid hormone- binding globulin promoter, the a- 1 -anti-trypsin promoter, the bovine albumin (bAlb) promoter, the murine albumin (mAlb) promoter, the human al -antitrypsin (hAAT) promoter, the ApoEhAAT promoter comprising the ApoE enhancer and the hAAT promoter, the transthyretin (TTR) promoter, the liver fatty acid binding protein promoter, the hepatitis B virus (HBV) promoter, the DC 172 promoter comprising the hAAT promoter and the al -microglobulin enhancer, the DC 190 promoter comprising the human albumin promoter and the prothrombin enhancer, or any other natural or synthetic liver-specific promoter. In an aspect, a liver specific promoter can comprise about 845-bp and comprise the thyroid hormone-binding globulin promoter sequences (2382 to 13), two copies of al-microglobulin / bikunin enhancer sequences (22,804 through 22,704), and a 71 -bp leader sequence as described by Ill CR, et al. (1997).

[0070] Ubiquitous / constitutive promoters” are known to the art and include, but are not limited to, a CMV major immediate-early enhancer / chicken beta-actin promoter, a cytomegalovirus (CMV) major immediate-early promoter, an Elongation Factor 1-a (EFl -a) promoter, a simian vacuolating virus 40 (SV40) promoter, an AmpR promoter, aPyK promoter, a human ubiquitin C gene (Ubc) promoter, a MFG promoter, a human beta actin promoter, a C AG promoter, a EGR1 promoter, a FerH promoter, a FerL promoter, a GRP78 promoter, a GRP94 promoter, a HSP70 promoter, a [3-kin promoter, a murine phosphoglycerate kinase (mPGK) or human PGK (hPGK) promoter, a ROSA promoter, human Ubiquitin B promoter, a Rous sarcoma virus promoter, or any other natural or synthetic ubiquitous / constitutive promoters.

[0071] As used herein, an “inducible promoter” refers to a promoter that can be regulated by positive or negative control. Factors that can regulate an inducible promoter include, but are notlimited to, chemical agents (e.g., the metallothionein promoter or a hormone inducible promoter), temperature, and light.

[0072] “Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as “substantially identical” or “essentially similar” when they are optimally aligned. For example, sequence similarity or identity can be determined by searching against databases such as FASTA, BLAST, etc., but hits should be retrieved and aligned pairwise to compare sequence identity. Two proteins or two protein domains, or two nucleic acid sequences can have “substantial sequence identity” if the percentage sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, preferably 90%, 95%, 98%, 99% or more. Such sequences are also referred to as “variants” herein, e.g., other variants of a missing, deficient, and / or mutant protein or enzyme. It should be understood that sequence with substantial sequence identity do not necessarily have the same length and may differin length. For example, sequences that have the same nucleotide sequence but of which one has additional nucleotides on the 3’- and / or 5 ’-side are 100% identical.

[0073] As used herein, “codon optimization” can refer to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing one or more codons or more of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. As contemplated herein, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Cod on Usage Database.” Many methods and software tools for codon optimization have been reported previously. (See, for example, genomes . urv . es / OP TIMIZER / ) .

[0074] In an aspect, “RNA editing” can be a post -transcriptional modification where a precursor mRNA (pre-mRNA) nucleotide sequence is changed by base insertion, deletion, or modification. The extent of RNA editing varies from a few hundred bases, in mitochondrial DNA of trypanosomes, to a just single base, in nuclear genes of mammals. Even a single base change in the pre-mRNA can convert a cod on for one amino acid into the cod on for another amino acid or a stop codon. This type of re-coding can significantly affect the structure and function of a protein and may lead to the production of multiple variants of a protein from a single gene.

[0075] In an aspect, insertional and deletional RNA editing can involve the addition and deletion of specific nucleotides or sequences of nucleotides from pre-mRNA. In an aspect, substitutional RNA editing by base modifications is observed in higher eukaryotes, where the base is modified without changing the length of the pre-mRNA.

[0076] As known to the art, antibodies (Abs) can mitigate AAV infection through multiple mechanisms by binding to AAV capsids and blocking critical steps in transduction such as cell surface attachment and uptake, endosomal escape, productive trafficking to the nucleus, or uncoating as well as promoting AAV opsonization by phagocytic cells, thereby mediating their rapid clearance from the circulation. For example, in humans, serological studies reveal a high prevalence of NAbs in the worldwide population, with about 67% of people having antibodies against AAV1, 72% against AAV2, and approximately 40% against AAV serotypes 5 through 9. Vector immunogenicity represents a major challenge in re-administration of AAV vectors.

[0077] In an aspect, also disclosed herein are partial self-complementary parvovirus (e.g., a disclosed AAV) genomes, plasmid vectors encoding the parvovirus genomes, and parvovirus (e.g., a disclosed AAV) particles including such genomes. In an aspect, provided herein is a plasmid vector comprising a nucleotide sequence encoding a disclosed parvovirus genome such as for example, a disclosed AAV. In an aspect, provided herein is a partial self-complementary parvovirus genome including a payload construct, parvovirus ITRs flanking the payload construct, and a self-complementary region flanking one of the ITRs. A self-complementary region can comprise a nucleotide sequence that is complementary to the payload construct. A disclosed self- complementary region can have a length that is less the entire length of the payload construct.

[0078] In an aspect, a disclosed self-complementary region of a disclosed parvovirus genome can comprise a minimum length, while still having a length that is less the entire length of the payload construct. In an aspect, a disclosed self-complementary region can comprise at least 50 bases in length, at least 100 bases in length, at least 200 in length, at least 300 bases in length, at least 400 bases in length, at least 500 bases in length, at least 600 bases in length, at least 700 bases in length, at least 800 bases in length, at least 900 bases in length, or at least 1,000 bases in length.

[0079] In an aspect, a “self -complementary parvovirus genome” can be a single stranded polynucleotide having, in the 5’ to 3’ direction, a first parvovirus ITR sequence, a heterologous sequence (e.g., payload construct comprising, for example, a desired gene), a second parvovirus ITR sequence, a second heterologous sequence, wherein the second heterologous sequence is complementary to the first heterologous sequence, and a third parvovirus ITR sequence. In contrast to a self-complementary genome, a “partial self-complementary genome” does not include three parvovirus ITRs and the second heterologous sequence that is complementary to the first heterologous sequence has a length that is less than the entire length of the first heterologous sequence (e.g., payload construct). Accordingly, a partial self-complementary genome is a single stranded polynucleotide having, in the 5’ to 3’ direction or the 3’ to 5’ direction, a first parvovirus ITR sequence, a heterologous sequence (e.g., payload construct), a second parvovirus ITRsequence, and a self-complementary region that is complementary to a portion of the heterologous sequence and has a length that is less than the entire length the heterologous sequence.

[0080] As used herein, “immune-modulating” refers to the ability of a disclosed nucleic acid molecules, a disclosed vector, a disclosed pharmaceutical formulation, or a disclosed agent to alter (modulate) one or more aspects of the immune system. The immune system functions to protect the organism from infection and from foreign antigens by cellular and humoral mechanisms involving lymphocytes, macrophages, and other antigen-presenting cells that regulate each other by means of multiple cell-cell interactions and by elaborating soluble factors, including lymphokines and antibodies, that have autocrine, paracrine, and endocrine effects on immune cells.

[0081] As used herein, “immune modulator” refers to an agent that is capable of adjusting a given immune response to a desired level (e.g., as in immunopotentiation, immunosuppression, or induction of immunologic tolerance). Examples of immune modulators include but are not limited to, a disclosed immune modulator can comprise aspirin, azathioprine, belimumab, betamethasone dipropionate, betamethasone valerate, bortezomib, bredinin, cyazathioprine, cyclophosphamide, cyclosporine, deoxyspergualin, didemnin B, fluocinolone acetonide, folinic acid, ibuprofen, IL6 inhibitors (such as sarilumab) indomethacin, inebilizumab, intravenous gamma globulin (IVIG), methotrexate, methylprednisolone, mycophenolate mofetil, naproxen, prednisolone, prednisone, prednisolone indomethacin, rapamycin, rituximab, sirolimus, sulindac, synthetic vaccine particles containing rapamycin (SVP -Rapamycin or ImmTOR), thalidomide, tocilizumab, tolmetin, triamcinolone acetonide, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD19 antibodies, anti- CD20 antibodies, anti-CD22 antibodies, anti-CD40 antibodies, anti-FcRN antibodies, anti-IL6 antibodies, anti-IGFIR antibodies, an IL2 mutein, a BTK inhibitor, or a combination thereof. In an aspect, a disclosed immune modulator can comprise one or more Treg (regulatory T cells) infusions (e.g., antigen specific Treg cells to AAV). In an aspect, a disclosed immune modulator can be bortezomib or SVP -Rapamycin. In an aspect, an immune modulator can be administered by any suitable route of administration including, but not limited to, in utero, intra-CSF, intrathecally, intravenously, subcutaneously, transdermally, intradermally, intramuscularly, orally, transcutaneously, intraperitoneally (IP), or intravaginally. In an aspect, a disclosed immune modulator can be administered using a combination of routes. Administration can also include hepatic intra-arterial administration or administration through the hepatic portal vein (HPV). Administration of an immune modulator can be continuous or intermittent, and administration can comprise a combination of one or more routes.

[0082] As used herein, the term “immunotolerant” refers to unresponsiveness to an antigen (e.g., a vector, a therapeutic protein, a transgene product, etc.). An immunotolerant promoter canreduce, ameliorate, or prevent transgene-induced immune responses that can be associated with gene therapy. Assays known in the art to measure immune responses, such as immunohistochemical detection of cytotoxic T cell responses, can be used to determine whether one or more promoters can confer immunotolerant properties.

[0083] As used herein, the term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and / or warnings concerning the use of such therapeutic products.

[0084] As used herein, the term “in combination” in the context of the administration of other therapies (e.g., other agents) includes the use of more than one therapy (e.g., drug therapy). Administration “in combination with” one or more further therapeutic agents includes simultaneous (e.g., concurrent) and consecutive administration in any order. The use of the term “in combination” does not restrict the order in which therapies are administered to a subject. By way of non-limiting example, a first therapy (e.g., a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof) may be administered prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks), concurrently, or after (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or longer) the administration of a second therapy (e.g., agent) to a subject having or diagnosed with a disease or disorder (such as a genetic disease or disorder).

[0085] Disclosed are the components to be used to prepare the disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations as well as the disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an exampleof a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C- D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention.B. Unfolded Protein Response (UPR)

[0086] The unfolded protein response is a regulatory mechanism that enhances the expression of proteins involved in the function of the endoplasmic reticulum (ER), including ER chaperones as well as components of ER-associated degradation, when eukaryotic cells increase the production of secretory proteins and the capacity of the ER function is overwhelmed. Without proper functioning of the unfolded protein response, secretory recombinant proteins produced in the ER cannot be correctly folded, are detained in the ER, and evoke ER stress, resulting in apoptotic cell death. Thus, the unfolded protein response is one of the most critical elements for efficient production of recombinant proteins in eukaryotic cells.

[0087] IRE la is a single-pass type I membrane protein which localizes to ER lumen, and it interacts with several other proteins including GRP78,DAB2IP, TRAF2, and TAOK3 / JIK. Upon UPR activation, IREla undergoes dimerization / auto-phosphorylation mediated activation (phospho-Ser724 IREla). The active form of IRElathen induces the splicing of mRNA encoding the transcription factor, XBP1. Removal of an intron from XBP1 leads to the expression of the active form of XBP1 (XBP1-S, the spliced form) which positively regulates ER chaperones, as well as genes coding for the ER-associated protein degradation (ERAD) pathway and lipid metabolism. Through XBP1 -independ ent pathways, IRE1 binds to tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) and induces JUN amino-terminal kinase (JNK) activation. This interaction is known to modulate autophagic and apoptotic cell death. In addition to regulating cell survival and apoptosis, IREl’s endo-ribonuclease activity has been demonstrated to induce Regulated IRE 1 -depend ent mRNA Decay (RIDD) which is implicated in lipid anabolism and apoptosis. Moreover, IREla is involved in the processes of cell cycle arrest, response to glucose stimulus / insulin metabolism, transcriptional regulation, response to VEGF / angiogenesis, and regulation of macro-autophagy.

[0088] ATF6 is a transmembrane glycoprotein and transcription activator, which functions to initiate the UPR signaling during ER stress. Upon sensing of unfolded proteins, the full length ATF6 (p90) gets transported to the Golgi apparatus, where it is processed / cleaved through site 1protease (SIP) and S2P protease. The cleavage of p90 releases the N-terminal processed cAMP- dependent ATF-6 alpha form (p50) into the cytosol. Thereafter, p50 translocates to the nucleus of the cell, where it binds DNA on the ER stress response element (ERSE) and regulates ER- associated protein degradation (ERAD) and UPR genes. In addition to mediating the stress response through ERSE and ERAD, p50 also regulates transcription of the XBP1 protein, as well the induction of apoptosis, regulation of transcription from RNA polymerase II promoter, eye development and visual perception.

[0089] PERK is a transmembrane protein kinase belonging to the PEK family of proteins and is best known for its role in insulin processing. During ER stress responses and activation of the UPR, PERK functions to inhibit translation of new proteins. Specifically, ER stress causes oligomerization of the ER luminal domain (N-terminal) of PERK, which facilitates the transautophosphorylation of PERK’s cytoplasmic kinase domain (C -terminal) at Thr-982 (phospho- Thr982 PERK). Thus, the phosphorylated form of PERK at the Thr-982 site is often assessed as a measure of ER stress. In addition, activated PERK phosphorylates eukaryotic translation initiation factor 2 alpha (eIF2 alpha), which inhibits translation of proteins to maintain homeostasis. However, phospho-eIF2 alpha does not block the translation of ATF4. Upon accumulation, ATF4 translocates to the nucleus, where it induces the expression of ER chaperones, autophagy / apoptosis genes (especially CHOP), oxidative response genes, as well as amino acid metabolism signaling pathways.

[0090] UPR signaling, the Golgi apparatus facilitates the cleavage of ATF6 alpha. ATF6 is an ER stress sensor which regulates genes responsible for increasing ER protein folding capacity and restoring ER homeostasis. Upon sensing ER stress, ATF6 alpha is transported to the Golgi apparatus, where it undergoes cleavage through site-1 protease (SIP) and site-2 protease (S2P), respectively. The cleavage of ATF6 alpha releases its N-terminal domain from the membrane as a functional b-Zip transcription factor (ATF6 alpha -N) which then gets translocated to the nucleus of cell for activating the transcription of ATF6’s target genes.C. Compositions1. ER Stress Sensor Variants

[0091] Disclosed herein is an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:61.

[0092] Disclosed herein is an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:01. Disclosed herein is an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:02. Disclosed herein is an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:03. Disclosed herein is an endoplasmic reticulum (ER) stress sensor variant comprising thesequence set forth in SEQ ID NO:04. Disclosed herein is an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:05. Disclosed herein is an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:06.

[0093] Disclosed herein is an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F1. Disclosed herein is an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F2. Disclosed herein is an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F3. Disclosed herein is an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F4. Disclosed herein is an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F5. Disclosed herein is an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F6.

[0094] Disclosed herein is an endoplasmic reticulum (ER) stress sensor variant comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof. Disclosed herein is an endoplasmic reticulum (ER) stress sensor variant comprising (i) a HR and an intronic sequence, (ii) a HR1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, a HR2, and an AP. Disclosed herein is an endoplasmic reticulum (ER) stress sensor variant comprising an RNA structure provided in Table 4.

[0095] In an aspect, a disclosed ER stress sensor variant can confer UPR-mediated regulation to the transgene mRNA and / or mRNA therapy. In an aspect, a disclosed ER stress sensor variant can confer UPR-mediated regulation to the encoded protein or encoded RNA. In an aspect, a disclosed ER stress sensor variant can render the expression of the encoded transgene amenable to UPR-mediated regulation. In an aspect, a disclosed ER stress sensor variant can provide control over gene therapy delivered to a cell. In an aspect, a disclosed ER stress sensor variant can provide control over gene therapy delivered to the cell via an AAV vector. In an aspect, a disclosed ER stress sensor variant can provide control over gene therapy delivered to the cell via lipid nanoparticles (LNPs).

[0096] In an aspect, a disclosed ER stress sensor variant can facilitate ER homeostasis, thereby avoiding activation of terminal UPR in a cell. In an aspect, a disclosed ER stress sensor variant can facilitate ER homeostasis, thereby avoiding activation of the apoptotic UPR in a cell. In an aspect, wherein, under ER stress, a disclosed ER stress sensor variant can generate a nonfunctional transgene mRNA and / or non-functional mRNA therapy. In an aspect, wherein, underER stress, a disclosed ER stress sensor variant can decrease the expression and / or abundance of the full-length mRNA transcript for a disclosed transgene. In an aspect, a disclosed ER stress sensor variant can minimize and / or reduce ER stress. In an aspect, a disclosed ER stress sensor variant can increase and / or improve the likelihood of survival in the one or more cells of a subject. In an aspect, a disclosed ER stress sensor variant can increase and / or improve long term expression of the transgene in the one or more cells of a subject. In an aspect, a disclosed ER stress sensor variant can decrease and / or minimize the likelihood of apoptosis in the one or more cells of a subject. In an aspect, a disclosed ER stress sensor variant can confer UPR-mediated regulation to the transgene mRNA and / or mRNA therapy. In an aspect, a disclosed ER stress sensor variant can improve and / or enhance proteostasis (e.g., the network of interconnected quality-control processes in the cell that maintains the functional proteome).

[0097] In an aspect, a disclosed ER stress sensor variant can reduce the expression of one or more markers of ER stress. In an aspect, markers of ER stress can comprise HSPA5 (aka GRP78 / BiP), ERN (aka IRE la), XBP1, PERK, ATF6, DDIT3 (aka CHOP), and any combination thereof.Table 1 - ER Stress Marker and Description

[0098] In an aspect, ER stress can be measured using RNA based methods are employed to analyze XBP1 splicing or the expression of other UPR genes. In an aspect, as it relates to proteins, one or more antibodies can be used to determine the expression of UPR markers. In an aspect, markers of ER stress can comprise HSPA5 (aka GRP78 / BiP), IREla, XBP1, PERK, ATF6, CHOP, and any combination thereof. In an aspect, expression of one or more markers of ER stress can be determined by Western blot or staining based applications (e.g., immunocytochemistry or immunohistochemistry).

[0099] In an aspect, a disclosed ER stress sensor variant can improve protein folding capacity of the ER.

[0100] In an aspect, a disclosed ER stress sensor variant can be incorporated into a disclosed nucleic acid molecule. In an aspect, a disclosed ER stress sensor variant can be incorporated into a disclosed non-viral vector. In an aspect, a disclosed ER stress sensor variant can be incorporated into a disclosed viral vector. In an aspect, a disclosed ER stress sensor variant can be incorporated into a disclosed pharmaceutical formulation.

[0101] In an aspect, under ER stress, ERN (aka IREla) splices a fragment comprising the sequence of SEQ ID NO:61 from a disclosed nucleic acid molecule.

[0102] In an aspect, when ER stress is minimized and / or decreased, transgene expression can increase.2. X-Box Binding Protein 1 (XBP1) Fragments

[0103] Disclosed herein is an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO:61.

[0104] Disclosed herein is an X-b ox Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO:01. Disclosed herein is an X-box Binding Protein 1 (XBP1) fragmentcomprising the sequence set forth in SEQ ID NO:02. Disclosed herein is an X-box Binding Protein 1 (XBP1) fragment comprising thesequence set forthin SEQ ID NO:03. Disclosed herein is an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO:04. Disclosed herein is an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO:05. Disclosed herein is an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO:06.

[0105] Disclosed herein is an X-box Binding Protein 1 (XBP1) fragment comprising the RNA structure known as XBP1F1. Disclosed herein is an X-box Binding Protein 1 (XBP1) fragment comprising the RNA structure known as XBP1F2. Disclosed herein is an X-box Binding Protein 1 (XBP1) fragment comprising the RNA structure known as XBP1F3. Disclosed herein is an X- box Binding Protein 1 (XBP1) fragment comprising the RNA structure known as XBP1F4. Disclosed herein is an X-box Binding Protein 1 (XBP1) fragment comprising RNA structure known as XBP1F5. Disclosed herein is an X-box Binding Protein 1 (XBP1) fragment comprising RNA structure known as XBP1F6.

[0106] Disclosed herein is an X-box Binding Protein 1 (XBP1) fragment comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof. Disclosed herein is an X-box Binding Protein 1 (XBP1) fragment comprising (i) a HR and an intronic sequence, (ii) a HR1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, a HR2, and an AP. Disclosed herein is an X-box Binding Protein 1 (XBP1) fragment comprising an RNA structure provided in Table 4.

[0107] In an aspect, a disclosed XBP1 fragment can confer UPR-mediated regulation to the transgene mRNA and / or mRNA therapy. In an aspect, a disclosed XBP1 fragment can confer UPR-mediated regulation to the encoded protein or encoded RNA. In an aspect, a disclosed XBP1 fragment can render the expression of the encoded transgene amenable to UPR-mediated regulation. In an aspect, a disclosed XBP1 fragment can provide control over gene therapy d elivered to a cell . In an aspect, a d isclosed XBP 1 fragment can provid e control over gene therapy delivered to a cell via an AAV vector. In an aspect, a disclosed XBP1 fragment can provide control over gene therapy delivered to a cell via lipid nanoparticles (LNPs). In an aspect, a disclosed XBP1 fragment can facilitate ER homeostasis, thereby avoiding activation of terminal UPR in a cell. In an aspect, a disclosed XBP1 fragment can facilitate ER homeostasis, thereby avoiding activation of the apoptotic UPR in a cell. In an aspect, wherein, under ER stress, a disclosed XBP1 fragment can generate a non-functional transgene mRNA and / or non-functional mRNA therapy. In an aspect, a disclosed XBP1 fragment can improve and / or enhance proteostasis(e.g., the network of interconnected quality-control processes in the cell that maintains the fun cti onal prot eom e) ,

[0108] In an aspect, wherein, under ER stress, a disclosed XBP1 fragment can decrease the expression and / or abundance of the full-length mRNA transcript for a disclosed transgene. In an aspect, a disclosed XBP1 fragment can minimize and / or reduce ER stress. In an aspect, a disclosed XBP1 fragment can increase and / or improve the likelihood of survival in the one or more cells of a subject. In an aspect, a disclosed XBP1 fragment can increase and / or improve long term expression of the transgene in the one or more cells of a subject. In an aspect, a disclosed XBP1 fragment can decrease and / or minimize the likelihood of apoptosis in the one or more cells of a subject. In an aspect, a disclosed XBP1 fragment can confer UPR-mediated regulation to the transgene mRNA and / or mRNA therapy.

[0109] In an aspect, a disclosed XBP1 fragment can reduce the expression of one or more markers of ER stress. In an aspect, markers of ER stress are discussed supra and can comprise HSPA5 (aka GRP78 / BiP), ERN (aka IREla), XBP1, PERK, ATF6, DDIT3 (aka CHOP), and any combination thereof. In an aspect, ER stress can be measured using RNA based methods are employed to analyze XBP1 splicing or the expression of other UPR genes. In an aspect, as it relates to proteins, one or more antibodies can be used to determine the expression of UPR markers. In an aspect, markers of ER stress can comprise HSPA5 (aka GRP78 / BiP), IREla, XBP1, PERK, ATF6, CHOP, and any combination thereof. In an aspect, expression of one or more markers of ER stress can be determined by Western blot or staining based applications (e.g., immunocytochemistry or immunohistochemistry).

[0110] In an aspect, a disclosed XBP1 fragment can improve protein folding capacity of the ER.

[0111] In an aspect, a disclosed XBP1 fragment can be incorporated into a disclosed nucleic acid molecule. In an aspect, a disclosed XBP1 fragment can be incorporated into a disclosed non-viral vector. In an aspect, a disclosed XBP1 fragment can be incorporated into a disclosed viral vector. In an aspect, a disclosed XBP1 fragment can be incorporated into a disclosed pharmaceutical formulation. In an aspect, under ER stress, ERN (aka IREla) splices a fragment comprising the sequence of SEQ ID NO:61 from a disclosed nucleic acid molecule.

[0112] In an aspect, when ER stress is minimized and / or decreased, transgene expression can increase.3. Nucleic Acid Molecules

[0113] Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:61.

[0114] Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:01. Disclosed herein is anucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:02. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:03. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:04. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:05. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:06.

[0115] Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F1. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F2. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F3. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F4. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F5. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F6.

[0116] Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising (i) a HR and an intronic sequence, (ii) a HR1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, a HR2, and an AP. Disclosed herein is an endoplasmic reticulum (ER) stress sensor variant comprising an RNA structure provided in Table 4.

[0117] Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO:61. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO:01. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO:02. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1)fragment comprising the sequence set forth in SEQ ID NO:03. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ IDNO:04. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forthin SEQ ID NO:05. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO: 06.

[0118] Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP 1) fragment comprising the RNA structure known as XBP IF 1. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the RNA structure known as XBP1F2. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the RNA structure known as XBP 1F3. Disclosed herein is a nucleic acid molecule comprising an X-Box Binding Protein 1 (XBP1) fragment comprising the RNA structure known as XBP1F4. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising RNA structure known as XBP1F5. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising RNA structure known as XBP1F6.

[0119] Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising (i) a HR and an intronic sequence, (ii) a HR1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, a HR2, and an AP.

[0120] Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP 1) fragment comprising an RNA structure provided in Table 4. Disclosed herein is anucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:61, and a sequence encoding a transgene.

[0121] Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forthin SEQ IDNO:01, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:02, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:03, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ IDNO:04, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:05, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:06, and a sequence encoding a transgene.

[0122] Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising theRNA structure known as XBP1F1, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F2, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F3, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F4, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F5, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F6, and a sequence encoding a transgene.

[0123] Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising (i) a HR and an intronic sequence, (ii) a HR1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, a HR2, and an AP.

[0124] Disclosed herein is an endoplasmic reticulum (ER) stress sensor variant comprising an RNA structure provided in Table 4, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO:61, and a sequence encoding a transgene.

[0125] Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO:01, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO:02, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1(XBP1) fragment comprising the sequence set forth in SEQ ID NO:03, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO:04, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO:05, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO:06, and a sequence encoding a transgene.

[0126] Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the RNA structure known as XBP1F1, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the RNA structure known as XBP1F2, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the RNA structure known as XBP1F3, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the RNA structure known as XBP1F4, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising RNA structure known as XBP1F5, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising RNA structure known as XBP1F6, and a sequence encoding a transgene.

[0127] Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof, and a sequence encoding a transgene. Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising (i) a HR and an intronic sequence, (ii) a HR1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, a HR2, and an AP, and a sequence encoding a transgene.

[0128] Disclosed herein is a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising an RNA structure provided in Table 4, and a sequence encoding a transgene. In an aspect, a disclosed ER stress sensor variant can comprise a X-Box Binding Protein 1 (XBP1) fragment. In an aspect, a disclosed XBP1 fragment can comprise the sequence set forth in SEQ ID NO:61.

[0129] In an aspect, a disclosed XBP1 fragment can comprise the sequence set forth in any of SEQ ID NO:01 - SEQ ID NO:06.

[0130] In an aspect, a disclosed nucleic acid molecule can comprise the sequence for one or more regulatory elements For example, in an aspect, disclosed regulatory elements can comprise promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyad enylati on signals and poly-U sequences, Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulator Element (WPRE), triplex from MALAT1, the PRE of Hepatitis B virus (HPRE), and an iron response element). Regulatory elements can include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).

[0131] For example, in an aspect, a disclosed nucleic acid molecule can further comprise a promoter operably linked to the ER stress sensor variant. In an aspect, a disclosed promoter can comprise a chicken [3-actin promoter (CBA). As used herein, a CBA promoter is incorporated into each of SEQ ID NO :07 - SEQ ID NO : 12 and SEQ ID NO :24 - SEQ ID NO :29. In an aspect, a disclosed promoter can be a CMV promoter or a CMV promoter / enhancer. CMV promoters and CMV promoters / enhancers are well known to the art. In an aspect, a disclosed promoter can be any eukaryotic RNA polymerase II promoter.

[0132] In an aspect, a disclosed promoter can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the expression desired. A promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild -type host into which the transcriptional initiation region is introduced. In an aspect, a disclosed promoter can be a promoter / enhancer. In an aspect, a disclosed promoter for the disclosed nucleic acid molecule can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter / enhancer. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter / enhancer can generally be obtained from a non-coding region upstream of a transcription initiation site of a gene of interest. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter / enhancer can be used for constitutive and efficient expression of a disclosed gene.

[0133] Tissue-specific promoters are known to the art and include, but are not limited to, neuronspecific promoters, muscle-specific promoters, liver-specific promoters, skeletal muscle-specific promoters, and heart-specific promoters. Ubiquitous / constitutive promoters are known to the art and include, but are not limited to, a CMV major immediate-early enhancer / chicken beta-actin promoter, a cytomegalovirus (CMV) major immediate-early promoter, an Elongation Factor 1-a(EFl -a) promoter, a simian vacuolating virus 40 (SV40) promoter, an AmpR promoter, a PyK promoter, a human ubiquitin C gene (Ubc) promoter, a MFG promoter, a human beta actin promoter, a CAG promoter, a EGR1 promoter, a FerH promoter, a FerL promoter, a GRP78 promoter, a GRP94 promoter, a HSP70 promoter, a P-kin promoter, a murine phosphoglycerate kinase (mPGK) or human PGK (hPGK) promoter, a ROSA promoter, human Ubiquitin B promoter, a Rous sarcoma virus promoter, or any other natural or synthetic ubiquitous / constitutive promoters.

[0134] In an aspect, a disclosed linker sequence can separate the ER stress sensor variant from the sequence encoding the transgene or the payload. In an aspect, a disclosed linker sequence can comprise a non-self-cleaving peptide sequence or a self-cleaving peptide sequence. In an aspect, a disclosed linker sequence can comprise the sequence set forth in any one of SEQ ID NO:62 - SEQ ID NO:66. In an aspect, a disclosed encoded linker sequence can comprise the sequence set forth in any one of SEQ ID NO:67 - SEQ ID NO:71. In an aspect, a disclosed self-cleaving peptide sequence can comprise a self-cleaving viral 2A peptide. In an aspect, a disclosed selfcleaving viral 2A peptide can comprise a porcine teschovirus-1 peptide (P2A), a Thosea asigna virus peptide (T2A), an equine rhinitis A virus 2A peptide (E2A), a foot-and-mouth disease virus 2A peptide (F2A), or a TPT2A peptide.Table 2 - Exemplary 2A Peptides

[0135] In an aspect, a disclosed nucleic acid molecule can further comprise a sequence encoding one or more signal peptides. Signal peptides are known to the art. In an aspect, a disclosed signal / leader peptide can comprise any known signal / leader peptide or any known signal / leader peptide known to function in mammalian cells. For example, in an aspect, a disclosed signal / leader peptide can be the signal / leader peptide of Human Oncostatin (OSM) (SEQ ID NO:37), Human IgKV III (SEQ ID NO:38), Human Chymotrypsinogen (SEQ ID NO:39), Human Trypsinogen-2 (SEQ IDNO:40), Human Insulin (SEQ ID NO:41), Human IL-2 (SEQ IDNO:42), Human BM40 (osteonectin SPARC) (SEQ ID NO:43), Human Serum Albumin (SEQ ID NO:44), Human Tissue Plasminogen Activator (SEQ ID NO:45), Secrecon (SEQ ID NO:46), CD33 (SEQ ID NO:47), Vesicular stomatitis virus G protein (VSV-G) (SEQ ID NO:48), Gaussia luc (SEQ IDNO:49), Influenza Haemagglutinin (SEQ ID NO:50), Silkworm Fibroin LC (SEQ ID N0:51), Mouse Ig Kappa (SEQ ID NO:52), or Mouse Ig Heavy (SEQ ID NO:53).

[0136] In an aspect, a disclosed nucleic acid molecule can comprise a sequence encoding the signal peptide set forth in any one of SEQ ID NO:37 - SEQ ID NO:53.

[0137] In an aspect of a disclosed nucleic acid molecule, a disclosed XBP1 fragment can further comprise one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof. For example, in an aspect, a disclosed XBP1 fragment can comprise (i) a HR and an intronic sequence, (ii) a HR1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, a HR2, and an AP. In an aspect, a disclosed nucleic acid molecule can further comprise a poly adenylation (poly A) sequence.

[0138] In an aspect of a disclosed nucleic acid molecule, a disclosed transgene can encode a desired cargo or payload. In an aspect, a disclosed transgene can encode a polypeptide, a recombinant polypeptide, or a therapeutic polypeptide. In an aspect, a disclosed transgene can encode a missing, deficient, and / or mutant protein or enzyme.

[0139] In an aspect, a disclosed transgene can encode the protein or a fragment thereof associated with ABCA1, ABCA12, ABCA13, ABCA2, ABCA3, ABCA4, ABCA5, ABCC1, ABCC2, ABCC6, ABCC8, ABCC9, ACAN, AD AMTS 13, 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, AIR, ATRX, BAZ1A, BAZ2B,BCOR,BCORL1,BDP1, BLM, BPTF, BRCA1,BRCA2,BRD4, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD, CAMTAI, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44, CFAP65, CFTR / ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1, CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1, CP AMD 8, CPLANE1, CPS1, CPSF1, CRB 1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1, DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11, DNAH17, DNAH2, DNAH5, DNAH7, DNAH8, DNAH9, DNMBP, DNMT1, DOCK2, DOCK3, DOCK6, DOCK7, DOCK8, DSCAM, DSP, DST, DUOX2, DYNC1H1, DYNC2H1, DYSF, EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, EYS, F5, F8, FANCA, FANCD2, FANCM, FAT1,FAT4, FBN1, FBN2, FLG, FLG2, FLNA, FLNB, FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2, FSIP2, FYC01, GLI2, GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1, HERC2, HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R, IGF2R, IGSF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A, KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220, KIF14, KIF1A, KIF1B, KIF21A, KIF26B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1, LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMC3, LCT, L0XHD1, LPA, LRBA, LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD, MAGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5, MCM3AP, MED12, MED12L, MED13, MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MYH11, MYH14, MYH2, MYH3, MYH6, MYH7, MYH7B, MYH8, MYH9, MYLK, MYO 15 A, MYO 18B, MY03 A, MY05 A, MYO5B, MYO7A, MYO9A, NALCN, NBAS, NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF, NF1, NFASC, NHS, NIN, NIPBL, NLRP1, NOTCH1, NOTCH2, NOTCH3, NPHP4, NRXN1, NRXN3,NSD1,NSD2,NUP155, NUP188,NUP205, OBSCN, OBSL1,OTOF, OTOG, OTOGL, PARD3, PBRM1, PCDH15, PCLO, PONT, PHIP, PI4KA, PIEZO1, PIEZO2, PIK3C2A, PIKFYVE, PKD1, PKD1L1, PKHD1, PLCE1, PLEC, PLEKHG2, PNPLA6, POGZ, POLA1, POLE, POLR1 A, POLR2A, POLR3A, PRG4, PRKDC, PRPF8, PRR12, PRX, PTCHI, PTPN23, PTPRF, PTPRJ, PTPRQ, PXDN, QRICH2, RAB3GAP2, RAI1, RALGAPA1, RANBP2, RB ICC 1, RELN, RERE, REV3L, RIC1, RIMS1, RIMS2, RNF213, ROBO1, ROBO2, ROBO3, ROS1, RP1, RP1L1, RTTN, RUSC2, RYR1, RYR2, SACS, SAMD9, SAMD9L, SBF2, SCAPER, SCN10A, SCN11A, SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SETBP1, SETD1A, SETD1B, SETD2, SETD5, SETX, SHANK2, SHANK3, SHROOM4, SI, SIPA1L3, SLIT2, SLX4, SMARCA2, SMARCA4, SMCHD1, SNRNP200, SON, SPEF2, SPEG, SPG11, SPTA1, SPTAN1, SPTB, SPTBN2, SPTBN4, SRCAP, STRC, SVIL, SYNE1, SYNGAP1, SYNJ1, SZT2, TAF1, TANC2, TCF20, TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B, TNXB, TOGARAMI, TONSL, TRIO, TRIOBP, TRIP11, TRIP12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, TTN, TUBGCP6, UBR1, UNC80, USH2A, USP9X, VCAN, VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62, WDR81, WNK1, WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292, ZNF335, ZNF407, ZNF462, ZNF469, or a portion thereof.

[0140] In an aspect, a disclosed transgene can encode dystrophin including mini- and microdystrophins (DMD), titin (TTN), titin cap (TCAP) a-sarcoglycan (SGCA), P-sarcoglycan(SGCB), y-sarcoglycan (SGCG) or 5-sarcoglycan (SGCD), alpha- 1 -antitrypsin (Al-AT), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), myosin heavy chain 11 (MYH11), myosin light chain 2 (ML2), myosin light chain 3 (ML3), myosin light chain kinase 2 (MYLK2), myosin binding protein C (MYBPC3), desmin (DES), dynamin 2 (DNM2), laminin a2 (LAMA2), lamin A / C (LMNA), lamin B (LMNB), lamin B receptor (LBR), dysferlin (DYSF), emerin (EMD), insulin, blood clotting factors, including but not limited to, factor VIII and factor IX, erythropoietin (EPO), lipoprotein lipase (LPL), sarcoplasmic reticulum Ca2++-ATPase (SERCA2A), S100 calcium binding protein Al (S100A1), myotubularin (MTM), DM1 protein kinase (DMPK), glycogen phosphorylase L (PYGL), glycogen phosphorylase, muscle associated (PYGM), glycogen synthase 1 (GYSI), glycogen synthase 2 (GYS2), a-galactosidase A (GLA), a-N-acetylgalactosaminidase (NAGA), acid a-glucosidase (GAA), sphingomyelinase phosphodiesterase 1 (SMPD 1), lysosomal acid lipase (LIPA), collagen type I al chain (COL 1 Al), collagen type I a2 chain (COL1A2), collagen type III al chain (COL3A1), collagen type V al chain (COL5A1), collagen type V a2 chain (COL5A2), collagen type VI al chain (COL6A1), collagen type VI a2 chain (COL6A2), collagen type VI a3 chain (COL6A3), procollagen-lysine 2-oxoglutarate 5-dioxygenase (PLOD1), lysosomal acid lipase (LIPA), frataxin (FXN), myostatin (MSTN), 0-N-acetyl hexosaminidase A (HEXA), 0-N -acetylhexosaminidase B (HEXB), 0- glucocerebrosidase (GBA), adenosine monophosphate deaminase 1 (AMPD1), 0-globin (HBB), iduronidase (IDUA), iduronate 2-sulfate (IDS), troponin 1 (TNNI3), troponin T2 (TNNT2), troponin C (TNNC1), tropomyosin 1 (TPM1), tropomyosin 3 (TPM3), N-acetyl-a- glucosaminidase (NAGLU), N-sulfoglucosamine sulfohydrolase (SGSH), heparan-a- glucosaminide N-acetyltransf erase (HGSNAT), integrin a 7 (IGTA7), integrin a 9 (IGTA9), glucosamine(N-acetyl)-6-sulfatase (GNS), galactosamine(N-acetyl)-6-sulfatase (GALNS), 0- galactosidase (GLB 1), 0-glucuronidase (GUSB), hyaluronoglucosaminidase 1 (HYAL1), acid ceramidase (ASAHI), galactosylcermidase (GALC), cathepsin A (CTSA), cathepsin D (CTSA), cathepsin K (CTSK), GM2 ganglioside activator (GM2A), arylsulfatase A (ARSA), arylsulfatase B (ARSB), formylgly cine-generating enzyme (SUMFI), neuraminidase 1 (NEU1), N- acetylglucosamine-1 -phosphate transferase a (GNPTA), N-acetylglucosamine-1 -phosphate transferase 0 (GNPTB), N-acetylglucosamine-1 -phosphate transferase y (GNPTG), mucolipin-1 (MCOLN1), NPC intracellular transporter 1 (NPC1), NPC intracellular transporter 2 (NPC2), ceroid lipofuscinosis 5 (CLN5), ceroid lipofuscinosis 6 (CLN6), ceroid lipofuscinosis 8 (CLN8), palmitoyl protein thioesterase 1 (PPT1), tripeptidyl peptidase 1 (TPP1), battenin (CLN3), DNAJ heat shock protein family 40 member C5 (DNAJC5), major facilitator superfamily domain containing 8 (MFSD8), mannosidase a class 2B member 1 (MAN2B1), mannosidase R (MANBA), aspartylglucosaminidase (AGA), a-L-fucosidase (FUCA1), cystinosin, lysosomalcysteine transporter (CTNS), sialin, solute carrier family 2 member 10 (SLC2A10), solute carrier family 17 member 5 (SLC17A5), solute carrier family 6 member 19 (SLC6A19), solute carrier family 22 member 5 (SLC22A5), solute carrier family 37 member 4 (SLC37A4), lysosomal associated membrane protein 2 (LAMP2), sodium voltage-gated channel a subunit 4 (SCN4A), sodium voltage-gated channel P subunit 4 (SCN4B), sodium voltage-gated channel a subunit 5 (SCN5A), sodium voltage-gated channel a subunit 4 (SCN4A), calcium voltage-gated channel subunit ale (CACNA1C), calcium voltage-gated channel subunit als (CACNA1S), phosphoglycerate kinase 1 (PGK1), phosphoglycerate mutase 2 (PGAM2), amylo-a-1,6- glucosidase,4-a-glucanotransferase (AGL), potassium voltage-gated channel ISK-related subfamily member 1 (KCNE1), potassium voltage-gated channel ISK-related subfamily member 2 (KCNE2), potassium voltage-gated channel subfamily J member 2 (KCNJ2), potassium voltagegated channel subfamily J member 5 (KCNJ5), potassium voltage-gated channel subfamily H member 2 (KCNH2), potassium voltage-gated channel KQT-like subfamily member 1 (KCNQ1), hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4), chloride voltage-gated channel 1 (CLCN1), carnitine palmitoyltransferase 1A (CPT1A), ryanodine receptor 1 (RYR1), ryanodine receptor 2 (RYR2), bridging integrator 1 (BIN 1), LARGE xylosyl- and glucuronyltransf erase 1 (LARGE1), docking protein 7 (D0K7), fukutin (FKTN), fukutin related protein (FKRP), selenoprotein N (SELENON), protein O-mannosyltransferase 1 (POMTl), protein O-mannosyltransferase 2 (POMT2), protein O-linked mannose N- acetylglucosaminyltransf erase 1 (POMGNT1), protein O-linked mannose N- acetylglucosaminyltransf erase 2 (POMGNT2), protein-O-mannose kinase (POMK), isoprenoid synthase domain containing (ISPD), plectin (PLEC), cholinergic receptor nicotinic epsilon subunit (CHRNE), choline O-acetyltransferase (CHAT), choline kinase P (CHKB), collagen like tail subunit of asymmetric acetylcholinesterase (COLQ), receptor associated protein of the synapse (RAPSN), four and a half LIM domains 1 (FHL1), P-1, 4-glucuronyltransf erase 1 (B4GAT1), P-l,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2), dystroglycan 1 (DAGI), transmembrane protein 5 (TMEM5), transmembrane protein 43 (TMEM43), SECIS binding protein 2 (SECISBP2), glucosamine (UDP-N-acetyl)-2-epimerase / N-acetylmannosamine kinase (GNE), anoctamin 5 (AN05), structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1), lactate dehydrogenase A (LDHA), lactate dehydrogenase B (LHDB), calpain 3 (CAPN3), caveolin 3 (CAV3), tripartite motif containing 32 (TRIM32), CCHC-type zinc finger nucleic acid binding protein (CNBP), nebulin (NEB), actin, al, skeletal muscle (ACTA1), actin, al, cardiac muscle (ACTC1), actinin a2 (ACTN2), poly(A)-binding protein nuclear 1 (PABPN 1), LEM domain-containing protein 3 (LEMD3), zinc metalloproteinase STE24 (ZMPSTE24), microsomal triglyceride transfer protein (MTTP), cholinergic receptor nicotinic alsubunit (CHRNA1), cholinergic receptor nicotinic a2 subunit (CHRNA2), cholinergic receptor nicotinic a3 subunit (CHRNA3), cholinergic receptor nicotinic a4 subunit (CHRNA4), cholinergic receptor nicotinic a5 subunit (CHRNA5), cholinergic receptor nicotinic a6 subunit (CHRNA6), cholinergic receptor nicotinic a7 subunit (CHRNA7), cholinergic receptor nicotinic a8 subunit (CHRNA8), cholinergic receptor nicotinic a9 subunit (CHRNA9), cholinergic receptor nicotinic alO subunit (CHRNA10), cholinergic receptor nicotinic pi subunit (CHRNB1), cholinergic receptor nicotinic P2 subunit (CHRNB2), cholinergic receptor nicotinic P3 subunit (CHRNB3), cholinergic receptor nicotinic P4 subunit (CHRNB4), cholinergic receptor nicotinic y subunit (CHRNG1), cholinergic receptor nicotinic a subunit (CHRND), cholinergic receptor nicotinic E subunit (CHRNE1), ATP binding cassette subfamily A member 1 (ABCA1), ATP binding cassette subfamily C member 6 (ABCC6), ATP binding cassette subfamily C member 9 (ABCC9), ATP binding cassette subfamily D member 1 (ABCD1), ATPase sarcoplasmic / endoplasmic reticulum Ca2+ transporting 1 (ATP2A1), ATM serine / threonine kinase (ATM), a tocopherol transferase protein (TTP A), kinesin family member 21A (KIF21A), paired-like homeobox 2a (PH0X2A), heparan sulfate proteoglycan 2 (HSPG2), stromal interaction molecule 1 (SUMI), notch 1 (NOTCHI), notch 3 (N0TCH3), dystrobrevin a(DTNA), protein kinase AMP -activated, noncatalytic y2 (PRKAG2), cysteine- and glycine-rich protein 3 (CSRP3), viniculin (VCL), myozenin 2 (MyoZ2), myopalladin (MYPN), junctophilin 2 (JPH2), phospholamban (PLN), calreticulin 3 (CALR3), nexilin F-actin-binding protein (NEXN), LIM domain binding 3 (LDB3), eyes absent 4 (EYA4), huntingtin (HTT), androgen receptor (AR), protein tyrosine phosphate non-receptor type 11 (PTPN11), junction plakoglobin (JUP), desmoplakin (DSP), plakophilin 2 (PKP2), desmoglein 2 (DSG2), desmocollin 2 (DSC2), catenin a3 (CTNNA3), NK2 homeobox 5 (NKX2-5), A-kinase anchor protein 9 (AKAP9), A-kinase anchor protein 10 (AKAP10), guanine nucleotide-binding protein a-inhibiting activity polypeptide 2 (GNAI2), ankyrin 2 (ANK2), syntrophin a-1 (SNTAT), calmodulin 1 (CALM1), calmodulin 2 (CALM2), HTRA serine peptidase 1 (HIRAI), fibrillin 1 (FBN1), fibrillin 2 (FBN2), xylosy Itransf erase 1 (XYLT1), xylosy Itransf erase 2 (XYLT2), tafazzin (TAZ), homogentisate 1,2-dioxygenase (HGD), glucose-6-phosphatase catalytic subunit (G6PC), 1,4- alpha-glucan enzyme 1 (GBE1), phosphofructokinase, muscle (PFKM), phosphorylase kinase regulatory subunit alpha 1 (PHKA1), phosphorylase kinase regulatory subunit alpha 2 (PHKA2), phosphorylase kinase regulatory subunit beta (PHKB), phosphorylase kinase catalytic subunit gamma 2 (PHKG2), phosphoglycerate mutase 2 (PGAM2), cystathionine-beta-synthase (CBS), methylenetetrahydrofolate reductase (MTHFR), 5-methyltetrahydrofolate-homocysteine methyltransferase (MIR), 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR), methylmalonic aciduria and homocystinuria, cblD type (MMADHC), mitochondrialDNA, including, but not limited to mitochondrially encoded NADH ubiquinone oxidoreductase core subunit 1 (MT-ND1), mitochondrially encoded NADH ubiquinone oxidoreductase core subunit 5 (MT-ND5), mitochondrially encoded tRNA glutamic acid (MT-TE), mitochondrially encoded tRNA histadine (MT-TH), mitochondrially encoded tRNA leucine 1 (MT-TL1), mitochondrially encoded tRNA lysine (MT-TK), mitochondrially encoded tRNA serine 1 (MT- TS1), mitochondrially encoded tRNA valine (MT-TV), mitogen-activated protein kinase 1 (MAP2K1), B-Raf proto-oncogene, serine / threonine kinase (BRAF), raf-1 proto-oncogene, serine / threonine kinase (RAFI), growth factors, including, but not limited to insulin growth factor 1 (IGF-1), transforming growth factor P3 (TGFP3), transforming growth factor P receptor, type I (TGFpRl), transforming growth factor P receptor, type II (TGFPR2), fibroblast growth factor 2 (FGF2), fibroblast growth factor 4 (FGF4), vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor B (VEGF-B), vascular endothelial growth factorC (VEGF-C), vascular endothelial growth factor D (VEGF-D), vascular endothelial growth factor receptor 1 (VEGFR1), or vascular endothelial growth factor receptor 2 (VEGFR2), or any fragment thereof.

[0141] In an aspect, a disclosed transgene can encode an RNA, a recombinant RNA, or a therapeutic RNA. In an aspect, a disclosed RNA can comprise ribosomal RNA (rRNA), viral RNA, transfer RNA(tRNA), antisense RNA, heterogeneous nuclear RNA(hnRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), micro RNA (miRNA), Piwi-interacting RNA (piRNA), CRISPR RNA, immunostimulating RNA, small interfering RNA(siRNA), short hairpin RNA (shRNA), singe guide RNA (sgRNA), non-coding RNA (ncRNA), long non-coding RNA (IncRNA), 7SL RNA, X-inactive specific transcript (Xist) RNA, short enhancer RNA (eRNA), circular RNA, intergenic RNA, or any combination thereof.

[0142] In an aspect, a disclosed transgene can encode a monoclonal antibody (mAB), a recombinant monoclonal antibody, an engineered monoclonal antibody, or a therapeutic monoclonal antibody. In an aspect, a disclosed monoclonal antibody can comprise a naked antibody. In an aspect, a disclosed monoclonal antibody can be used to generate a conjugated antibody and / or a bispecific antibody. In an aspect, a disclosed conjugated antibody can comprise a disclosed mAB and one or more attached chemotherapeutic and / or radiolabeled drugs. In an aspect, a disclosed bispecific antibody can comprise an antibody having parts of 2 different mAbs, conferring the ability to attach to 2 different proteins / eptitopes at the same time.

[0143] In an aspect, a disclosed transgene can encode abagovomab, abciximab, abituzumab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afelimomab, afutuzumab, alacizumab pegol, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, anatumomab mafenatox, anetumab ravtansine, anifrolumab, anrukinzumab, apolizumab, arcitumomab, ascrinvacumab, aselizumab, atezolizumab, atinumab, atlizumab (tocilizumab), atorolimumab ,bapineuzumab, basiliximab, bavituximab, bectumomab, begelomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, bivatuzumab mertansine, blinatumomab, blosozumab, bococizumab, brentuxim abvedotin, briakinumab, brodalumab, brolucizumab, brontictuzumab, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cbr96- doxombicin immunoconjugate, cedelizumab, certolizumab pegol, cetuximab, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, codrituzumab, coltuximab ravtansine, conatumumab, concizumab, crenezumab, dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, daratumumab, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, derlotuximab biotin, detumomab, dinutuximab, diridavumab, dorlimomab aritox, drozitumab, duligotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab, enavatuzumab, enfortumab vedotin, enlimomab pegol, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, etanercept, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, felvizumab, fezakinumab, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, fontolizumab, foralumab, foravirumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, guselkumab, ibalizumab, ibritumomab tiuxetan, icrucumab, idarucizumab, igovomab, imalumab, imciromab, imgatuzumab, inclacumab, indatuximab ravtansine, indusatumab vedotin, infliximab, inolimomab, inotuzumab ozogamicin, intetumumab, ipilimumab, iratumumab, isatuximab, itolizumab, ixekizumab, keliximab, labetuzumab, lambrolizumab, lampalizumab, lebrikizumab, lemalesomab, lenzilumab, lerdelimumab, lexatumumab, libivirumab, lifastuzumab vedotin, ligelizumab, lilotomab satetraxetan, lintuzumab, lirilumab, lodelcizumab, lokivetmab, lorvotuzumab mertansine, lucatumumab, lulizumab pegol, lumiliximab, lumretuzumab, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mirvetuximab soravtansine, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-cd3, nacolomab tafenatox, namilumab, naptumomab estafenatox, namatumab, natalizumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, ontuxizumab, opicinumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, otlertuzumab,oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, pankomab, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, placulumab, polatuzumab vedotin, ponezumab, priliximab, pritoxaximab, pritumumab, quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab , ramucirumab, ranibizumab, raxibacumab, refanezumab, regavirumab, reslizumab, rilotumumab, rinucumab, rituximab, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, sacituzumab govitecan, samalizumab, sarilumab, satumomab pend etide, secukinumab, seribantumab, setoxaximab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tarextumab, tefibazumab, telimomab aritox, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tetulomab, ticilimumab, tigatuzumab, tildrakizumab, tocilizumab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, tregalizumab, tremelimumab, trevogrumab, tucotuzumab celmoleukin, tuvirumab, ublituximab, ulocuplumab, urelumab, urtoxazumab, ustekinumab, vandortuzumab vedotin, vantictumab, vanucizumab, vapaliximab, varlilumab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, zolimomab aritox, or any combination thereof.

[0144] In an aspect, a disclosed transgene can encode leronlimab (a humanized immunoglobulin G4 monoclonal antibody that targets C-C chemokine receptor type 5 (CCR5)). In an aspect, the heavy chain for leronlimab can comprise the sequence of SEQ ID NO: 70. In an aspect, the light chain for leronlimab can comprise the sequence of SEQ ID NO:71. In an aspect, a disclosed leronlimab can comprise disulfide bridges at H22-H96, H-136-L219, H149-H205, H228-H'228, H231-H'231, H263-H323, H369-H427, H'22-H'96, H'136-L'219, H'149-H'2O5, H'263-H'323, H'369-H'427, L23-L93, L139-L199, L'23-L'93, and L'139-L'199).

[0145] In an aspect, a disclosed transgene can encode a component of a gene-editing system. Gene-editing systems are known to the art and include, but are not limited to, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases, or clustered regularly interspaced short palindromic repeats (CRISPR). In an aspect, CRISPR can comprise CRISPR / Cas9.

[0146] In an aspect, a disclosed component of a gene-editing system can comprise an endonuclease. In an aspect, a disclosed endonuclease can comprise Cas9. In an aspect, a disclosed CRISPR-based endonuclease can be derived from a CRISPR / Castype I, type II, or type III system.

[0147] In an aspect, a disclosed component of a gene-editing system can comprise a guide RNA (gRNA) or a single guide RNA (sgRNA). In an aspect, a disclosed gRNA or sgRNA can target an endogenous gene.

[0148] In an aspect, a disclosed transgene can encode a reporter gene. In an aspect, an encoded reporter gene can be used in methods of validating a disclosed ER stress sensor variant, a disclosed XBP1 fragment, a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed plasmid, or any combination thereof.

[0149] In an aspect, a disclosed targeted endogenous gene can comprise one or more mutations. In an aspect, a disclosed targeted endogenous gene can encode a dysfunctional or mutant enzyme or protein. In an aspect, a disclosed targeted endogenous gene can be the reason for a subject's disease or disorder.

[0150] In an aspect, one or more disclosed mutations can contribute to pathogenesis of one or more cells, for example, cells in a subject. In an aspect, one or more disclosed mutations can inhibit translation of the encoded protein. In an aspect, one or more disclosed mutations can modify translation of the encoded protein. In an aspect, one or more disclosed mutations can generate an encoded protein having a non-sense mutation or a missense mutation.

[0151] In an aspect, expression of a disclosed targeted endogenous gene having one or more mutations can be restored and / or returned to a wild-type, normal, or control expression level. In an aspect, a disclosed nucleic acid molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation. In an aspect, a disclosed nucleic acid molecule can restore the functionality and / or structural integrity of a missing, deficient, and / or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra). In an aspect, restoring one or more aspects of cellular homeostasis and / or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and / or ameliorating autophagy); (iii) improving, enhancing, restoring, and / or preserving mitochondrial functionality and / or structural integrity; (iv) improving, enhancing, restoring, and / or preserving organelle functionality and / or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of the multi-systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and / or preserving one or more aspects of cellular structural and / or functional integrity.

[0152] In an aspect, restoring the activity and / or functionality of a missing, deficient, and / or mutant protein or enzyme can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and / or mutant protein or enzyme). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and / or functionality is similar to that of a wild-type or control level.

[0153] In an aspect, a disclosed nucleic acid molecule can be used in a delivery agnostic method. In an aspect, a disclosed nucleic acid molecule can be used in a modular method.

[0154] In an aspect, a disclosed nucleic acid molecule can confer UPR-mediated regulation to the transgene mRNA and / or mRNA therapy. In an aspect, a disclosed nucleic acid molecule can confer UPR-mediated regulation to the encoded protein or encoded RNA. In an aspect, a disclosed nucleic acid molecule can render the expression of the encoded transgene amenable to UPR- mediated regulation. In an aspect, a disclosed nucleic acid molecule can provide control over gene therapy delivered to a cell. In an aspect, a disclosed nucleic acid molecule can provide control over gene therapy delivered to a cell via a AAV vector. In an aspect, a disclosed nucleic acid molecule can provide control over gene therapy delivered to a cell via lipid nanoparticles (LNPs). In an aspect, a disclosed nucleic acid molecule can facilitate ER homeostasis, thereby avoiding activation of terminal UPR in a cell. In an aspect, a disclosed nucleic acid molecule can facilitate ER homeostasis, thereby avoiding activation of the apoptotic UPR in a cell. In an aspect, a disclosed nucleic acid molecule, under ER stress, can generate a non-functional transgene mRNA and / or non-functional mRNA therapy. In an aspect, wherein, under ER stress, a disclosed nucleic acid molecule can decrease the expression and / or abundance of the full-length mRNA transcript for a disclosed transgene. In an aspect, a disclosed nucleic acid molecule can minimize and / or reduce ER stress. In an aspect, a disclosed nucleic acid molecule can increase and / or improve the likelihood of survival in the one or more cells of the subject. In an aspect, a disclosed nucleic acid molecule can increase and / or improve long term expression of the transgene in the one or more cells of the subject. In an aspect, a disclosed nucleic acid molecule can decrease and / or minimize the likelihood of apoptosis in the one or more cells of the subject. In an aspect, a disclosed nucleic acid molecule can confer UPR-mediated regulation to the transgene mRNA and / or mRNA therapy. In an aspect, a disclosed nucleic acid moelcule can improve and / orenhance proteostasis (e.g., the network of interconnected quality-control processes in the cell that maintains the functional proteome).

[0155] In an aspect, a disclosed nucleic acid molecule can reduce the expression of one or more markers of ER stress. In an aspect, markers of ER stress can comprise HSPA5 (aka GRP78 / BiP), ERN (IRE la), XBP1, PERK, ATF6, DDIT3 (aka CHOP), and any combination thereof.

[0156] In an aspect, a disclosed nucleic acid molecule can improve protein folding capacity of the ER.In an aspect, a disclosed nucleic acid molecule can incorporate a disclosed ER stress sensor variant. In an aspect, a disclosed nucleic acid molecule can be incorporated into a disclosed non- viral vector. In an aspect, a disclosed nucleic acid molecule can be incorporated into a disclosed viral vector. In an aspect, a disclosed nucleic acid molecule can be incorporated into a disclosed pharmaceutical formulation.

[0157] Disclosed herein is a nucleic acid molecule comprising a promoter operably linked to a disclosed endoplasmic reticulum (ER) stress sensor variant, a linker, a sequence encoding a transgene, and a poly A sequence. Disclosed herein is a nucleic acid molecule comprising a promoter operably linked to a disclosed XBP1 fragment, a linker, a sequence encoding a transgene, and a poly A sequence.

[0158] Disclosed herein is a nucleic acid molecule comprising a promoter operably linked to an XBP1 fragment comprising the sequence of SEQ ID NO:01, a linker, a sequence encoding a transgene, and a poly A sequence. Disclosed herein is a nucleic acid molecule comprising a promoter operably linked to an XBP1 fragment comprising the sequence of SEQ ID NO:02, a linker, a sequence encoding a transgene, and a poly A sequence. Disclosed herein is a nucleic acid molecule comprising a promoter operably linked to an XBP1 fragment comprising the sequence of SEQ ID NO:03, a linker, a sequence encoding a transgene, and a poly A sequence. Disclosed herein is a nucleic acid molecule comprising a promoter operably linked to an XBP1 fragment comprising the sequence of SEQ ID NO:04, a linker, a sequence encoding a transgene, and a poly A sequence. Disclosed herein is a nucleic acid molecule comprising a promoter operably linked to an XBP1 fragment comprising the sequence of SEQ ID NO:05, a linker, a sequence encoding a transgene, and a poly A sequence. Disclosed herein is a nucleic acid molecule comprising a promoter operably linked to an XBP1 fragment comprising the sequence of SEQ ID NO:06, a linker, a sequence encoding a transgene, and a poly A sequence.

[0159] In an aspect, ER stress can be measured using RNA based methods are employed to analyze XBP1 splicing or the expression of other UPR genes. In an aspect, as it relates to proteins, one or more antibodies can be used to determine the expression of UPR markers. In an aspect, markers of ER stress can comprise HSPA5 (aka GRP78 / BiP), IREla, XBP1, PERK, ATE6, CHOP, and any combination thereof. In an aspect, expression of one or more markers of ER stresscan be determined by Western blot or staining based applications (e.g., immunocytochemistry or immunohi stochemi stry ) .

[0160] In an aspect, under ER stress, ERN (aka IRE la) splices a fragment comprising the sequence of SEQ ID NO:61 from a disclosed nucleic acid molecule.

[0161] Disclosed herein is an expression cassette comprising a disclosed nucleic molecule. Disclosed herein is an expression cassette comprising a promoter operably linked to an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:06. Disclosed herein is an expression cassette comprising a promoter operably linked to an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F1. XBP1F2, XBP1F3, XBP1F4, XBP1F5, and XBP1F6. Disclosed herein is an expression cassette comprising a promoter operably linked to an endoplasmic reticulum (ER) stress sensor variant comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof. Disclosed herein is an expression cassette comprising a promoter operably linked to an endoplasmic reticulum (ER) stress sensor variant comprising (i) a HR and an intronic sequence, (ii) a HR1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, a HR2, and an AP.

[0162] In an aspect, when ER stress is minimized and / or decreased, transgene expression can increase.4. Vectors

[0163] Disclosed herein is a vector comprising a disclosed nucleic acid molecule or any disclosed nucleic acid molecule. Disclosed herein is a non-viral vector comprising a disclosed nucleic acid molecule or any disclosed nucleic acid molecule. Disclosed herein is a non-viral vector comprising one or more disclosed nucleic acid molecules. Disclosed herein is a viral vector comprising a disclosed nucleic acid molecule or any disclosed nucleic acid molecule. Disclosed herein is a viral vector comprising one or more disclosed nucleic acid molecules.

[0164] Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:61. Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:06. Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F1. XBP1F2, XBP1F3, XBP1F4, XBP1F5, and XBP1F6.

[0165] Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof. Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising (i) a HR and an intronic sequence, (ii) a HR1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, a HR2, and an AP. In an aspect, disclosed endoplasmic reticulum (ER) stress sensor variant can comprise an RNA structure provided in Table 4.

[0166] Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO:61.

[0167] Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:06. Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the RNA structure known as XBP1F1, XBP1F2. XBP1F3, XBP1F4, XBP1F5, or XBP1F6.

[0168] Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof. Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising (i) a HR and an intronic sequence, (ii) a HR1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, a HR2, and an AP.

[0169] In an aspect, a disclosed X-box Binding Protein 1 (XBP1) fragment can comprise an RNA structure provided in Table 4.

[0170] Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:61, and a sequence encoding a transgene.

[0171] Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:06, and a sequence encoding a transgene. Disclosedherein is anon-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F1, XBP1F2, XBP1F3, XBP1F4, XBP1F5, or XBP1F6, and a sequence encoding a transgene.

[0172] Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof, and a sequence encoding a transgene. Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising (i) a HR and an intronic sequence, (ii) a HR1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, a HR2, and an AP.

[0173] Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO:61, and a sequence encoding a transgene.

[0174] Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:06, and a sequence encoding a transgene. Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the RNA structure known as XBP1F1, XBP1F2, XBP1F3, XBP1F4, XBP1F5, or XBP1F6, and a sequence encoding a transgene.

[0175] Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof, and a sequence encoding a transgene. Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising (i) a HR and an intronic sequence, (ii) a HR1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, a HR2, and an AP, and a sequence encoding a transgene.

[0176] In an aspect, a disclosed pharmaceutical formulation can confer UPR-mediated regulation to the transgene mRNA and / or mRNA therapy. In an aspect, a disclosed pharmaceutical formulation can confer UPR-mediated regulation to the encoded protein or encoded RNA. In an aspect, a disclosed pharmaceutical formulation can rend er the expression of the encoded transgene amenable to UPR-mediated regulation. In an aspect, a disclosed pharmaceutical formulation canprovide control over gene therapy delivered to a cell. In an aspect, a disclosed pharmaceutical formulation can provide control over gene therapy delivered to a cell via a AAV vector. In an aspect, a disclosed pharmaceutical formulation can provide control over gene therapy delivered to a cell via lipid nanoparticles (LNPs). In an aspect, a disclosed pharmaceutical formulation can facilitate ER homeostasis, thereby avoiding activation of terminal UPR in a cell. In an aspect, a disclosed pharmaceutical formulation can facilitate ER homeostasis, thereby avoiding activation of the apoptotic UPR in a cell. In an aspect, a disclosed pharmaceutical formulation, under ER stress, can generate a non-functional transgene mRNA and / or non-functional mRNA therapy. In an aspect, wherein, under ER stress, a disclosed pharmaceutical formulation can decrease the expression and / or abundance of the full-length mRNA transcript for a disclosed transgene. In an aspect, a disclosed pharmaceutical formulation can minimize and / or reduce ER stress. In an aspect, a disclosed pharmaceutical formulation can increase and / or improve the likelihood of survival in the one or more cells of the subject. In an aspect, a disclosed pharmaceutical formulation can increase and / or improve long term expression of the transgene in the one or more cells of the subject. In an aspect, a disclosed pharmaceutical formulation can decrease and / or minimize the likelihood of apoptosis in the one or more cells of the subject. In an aspect, a disclosed pharmaceutical formulation can confer UPR-mediated regulation to the transgene mRNA and / or mRNA therapy. In an aspect, a disclosed vector can improve and / or enhance proteostasis (e.g., the network of interconnected quality-control processes in the cell that maintains the functional proteome).

[0177] In an aspect, a disclosed pharmaceutical formulation can reduce the expression of one or more markers of ER stress. In an aspect, markers of ER stress can comprise HSPA5 (aka GRP78 / BiP), ERN (IRE la), XBP1, PERK, ATF6, DDIT3 (aka CHOP), and any combination thereof.

[0178] In an aspect, a disclosed pharmaceutical formulation can incorporate a disclosed ER stress sensor variant or a disclosed XBP1 fragment. In an aspect, a disclosed pharmaceutical formulation can incorporate a disclosed non-viral vector. In an aspect, a disclosed pharmaceutical formulation can incorporate a disclosed viral vector. In an aspect, a disclosed pharmaceutical formulation can incorporate a disclosed nucleic acid molecule.

[0179] Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising a promoter operably linked to a disclosed endoplasmic reticulum (ER) stress sensor variant, a linker, a sequence encoding a transgene, and a poly A sequence. Disclosed herein is a non-viral or viral vector comprising a nucleic acid molecule comprising a promoter operably linked to a disclosed XBP1 fragment, a linker, a sequence encoding a transgene, and a poly A sequence. Disclosed herein is a non-viral or viral vector comprising a nucleic acid moleculecomprising a promoter operably linked to an XBP1 fragment comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:06, a linker, a sequence encoding a transgene, and a poly A sequence.

[0180] In an aspect, ER stress can be measured using RNA based methods are employed to analyze XBP1 splicing or the expression of other UPR genes. In an aspect, as it relates to proteins, one or more antibodies can be used to determine the expression of UPR markers. In an aspect, markers of ER stress can comprise HSPA5 (aka GRP78 / BiP), IREla, XBP1, PERK, ATF6, CHOP, and any combination thereof. In an aspect, expression of one or more markers of ER stress can be determined by Western blot or staining based applications (e.g., immunocytochemistry or immunohistochemistry).

[0181] In an aspect, a disclosed vector can be formulated for administration via one or more routes. Such methods are well known to those skilled in the art and include, but are not limited to, the following routes: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can also include hepatic intraarterial administration or administration through the hepatic portal vein (HPV). Administration of a disclosed therapeutic agent, a disclosed pharmaceutical composition, or a combination thereof can comprise administration directly into the CNS (e.g., intraparenchymal, intracerebroventricular, intrathecal cisternal, intrathecal (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or the PNS. Administration of a disclosed AAV particle or a disclosed AAV vector can be continuous or intermittent. In an aspect, a disclosed AAV particle or a disclosed AAV vector can be administered via one or more ex vivo methods such as, for example, an ex vivo perfusion protocol. In an aspect, a disclosed ex vivo perfusion protocol employing a disclosed AAV particle and / or a disclosed AAV vector can be applied to other relevant tissues in the subject in need thereof.

[0182] In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x IO10vg / kg to about 2 x 1014.vg / kg. In an aspect, for example, a disclosed vector can be administered at a dose of about 1 x 1011to about 8 x 1013vg / kg or about 1 x 1012to about 8 x 1013vg / kg. In an aspect, a disclosed vector can be administered at a dose of about 1 x 1013to about 6 x 1013vg / kg. In an aspect, a disclosed vector can be administered at a dose of at least about 1 x IO10, at least about 5 x IO10, at least about 1 x1011, at least about 5 x IO11, at least about 1 x 1012, at least about 5 x 1012, at least about 1 x 1013, at least about 5 x 1013, or at least about 1 x 1014vg / kg. In an aspect, a disclosed vector can be administered at a dose of no more than about 1 x IO10, no more than about 5 x IO10, no more than about 1 x 1011, no more than about 5 x 1011, no more than about 1 x 1012, no more than about 5 x1012, no more than about 1 x 1013, no more than about 5 x 1013, or no more than about 1 x 1014vg / kg. In an aspect, a disclosed vector can be administered at a dose of about 1 x 1012vg / kg. In an aspect, a disclosed vector can be administered at a dose of about 1 x 1011vg / kg. In an aspect, a disclosed vector can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results.

[0183] In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 1012vg per subject total to about 1 x 1017vg per subject total. In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 1012vg per subject total, about 1 x 1013vg per subject total, about 1 x 1014vg per subject total, about 1 x 1015vg per subject total, about 1 x 1016vg per subject total, or about 1 x 1017vg per subject total. In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can be by delivered retrograde ureteral infusion and / or renal arterial administration and can comprise a range of about 1 x 1012vg per subject total to about 1 x 1017vg per subject total.

[0184] In an aspect, a therapeutically effective amount of a disclosed AAV particle can comprise about 1 x 106DRP / mL to about 1 x 1014DRP / mL. In an aspect, a disclosed pharmaceutical formulation can comprise about 1 x 106DRP / rnL, 1 x 107DRP / rnL, 1 x 108DRP / mL, 1 x 109DRP / rnL, 1 x IO10DRP / mL, 1 x 1011DRP / mL, 1 x 1012DRP / mL, 1 x 1013DRP / mL, or 1 x 1014DRP / mL. In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed vector can comprise a range determined by a skilled person.

[0185] In an aspect, a disclosed non-viral vector can be a polymer-based vector, a peptide-based vector, a lipid nanoparticle, a solid lipid nanoparticle, or a cationic lipid -based vector. In an aspect, a disclosed vector can comprise exosomes, extracellular vesicles, and virus like particles.

[0186] In an aspect, a disclosed viral vector can achieve stable transduction. In an aspect, a disclosed viral can achieve transient transduction. In an aspect, a disclosed viral vector can be a recombinant viral vector. In an aspect, a disclosed viral vector or a disclosed recombinant viral vector can comprise an adeno-associated virus vector, an adenovirus vector, herpes virus, and / or a lentiviral virus vector.

[0187] In an aspect, a disclosed viral vector can be an adenovirus vector, an AAV vector, a herpes simplex virus vector, a retrovirus vector, a lentivirus vector, and alphavirus vector, a flavivirus vector, a rhabdovirus vector, a measles virus vector, a Newcastle disease viral vector, a poxvirusvector, or a picomavirus vector. In an aspect, a disclosed viral vector can be an adenovirus vector, an adeno-associated virus (AAV) vector, or a lentivirus vector. In an aspect, a disclosed AAV vector can be a recombinant viral vector.

[0188] In an aspect, a disclosed viral vector can be an AAV vector or a recombinant AAV vector. In an aspect, a disclosed nucleic acid sequence can have a coding sequence that is less than about 4.5 kilobases.

[0189] In an aspect, a disclosed AAV vector can include naturally isolated serotypes including, but not limited to, AAV1, AAV2, AAV3 (including 3a and 3b), AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV 10, AAVrhlO, A AVI 1, AAV 12, AAV13, AAVrh39, AAVrh43, AAVcy.7 as well as bovine AAV, caprine AAV, canine AAV, equine AAV, ovine AAV, avian AAV, primate AAV, non-primate AAV, and any other virus classified by the International Committee on Taxonomy of Viruses (ICTV) as an AAV. In an aspect, an AAV capsid can be a chimera either created by capsid evolution or by rational capsid engineering from a naturally isolated AAV variants to capture desirable serotype features such as enhanced or specific tissue tropism and / or a host immune response escape. Naturally isolated AAV variants include, but not limited to, AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV- 1829, AAV2 Y / F, AAV2 T / V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B 1, AAV-AS, AAV9.45A-String (e.g., AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep, and AAV9.47-AS, AAV-PHP.B, AAV -PHP. eB, AAV-PHP.S, AAV-F, AAVcc.47, and AAVcc.81. In an aspect, a disclosed AAV vector can be AAV-Rh74 or a related variant (e.g., capsid variants like RHM4-1). In an aspect, a disclosed AAV vector can be a self-complementary AAV. In an aspect, a disclosed AAV vector can comprise a variant capsid protein. In an aspect, a disclosed AAV vector can comprise one or more substitutions in the sequence of a disclosed capsid protein, which substitutions confer to the capsid less immunogenicity and / or improved tropism for a targeted cell, tissue, or organ.

[0190] In an aspect, a disclosed vector can comprise one or more ITRs (such as, for example, ITRs from AAV2). In an aspect, a disclosed vector can further comprise one or more nuclear localization signals (NLS). NLS are known to the skilled person in the art. In an aspect, a disclosed NLS can comprise any NLS known to the art. As known to the art (see, e.g., Lu J, et al. (2021) Cell Commun Signal. 19:60, which is incorporated herein by reference for its teachings of NLS), nuclear localization signals (NLS) are generally short peptides that act as a signal fragment that mediates the transport of proteins from the cytoplasm into the nucleus. In an aspect, a disclosed vector can further comprise one or more nuclear retention elements (NRE). NRE are known to the skilled person in the art.

[0191] In an aspect, a disclosed vector can comprise one or more promoters operably linked to a disclosed nucleic acid molecule (discussed supra) and / or any other nucleic acid sequence. In anaspect of a disclosed vector, a disclosed promoter can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the expression desired. A disclosed promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. In an aspect, a disclosed promoter can be a promoter / enhancer. In an aspect, a disclosed endogenous promoter can be an endogenous promoter / enhancer. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter / enhancer can generally be obtained from a non-coding region upstream of a transcription initiation site of a gene of interest. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter / enhancer can be used for constitutive and efficient expression of a disclosed protein coding gene. In an aspect, a disclosed promoter can be a CMV promoter or a CMV promoter / enhancer. CMV promoters and CMV promoters / enhancers are well known to the art. In an aspect, a disclosed promoter can be any eukaryotic RNA polymerase II promoter.

[0192] In an aspect, a disclosed AAV vector can be used to generate AAV particles. In an aspect, a disclosed AAV vector can be used to generate AAV particles comprising a disclosed nucleic acid molecule under its control.

[0193] In an aspect, a disclosed viral vector or a disclosed non-viral vector can improve protein folding capacity of the ER.

[0194] In an aspect, a disclosed viral vector or a disclosed non-viral vector can confer UPR- mediated regulation to the transgene mRNA and / or mRNA therapy. In an aspect, a disclosed viral vector or a disclosed non-viral vector can confer UPR-mediated regulation to the encoded protein or encoded RNA. In an aspect, a disclosed viral vector or a disclosed non-viral vector can render the expression of the encoded transgene amenable to UPR-mediated regulation. In an aspect, a disclosed viral vector or a disclosed non-viral vector can provide control over gene therapy delivered to a cell. In an aspect, a disclosed viral vector or a disclosed non-viral vector can provide control over gene therapy delivered to a cell via an AAV vector. In an aspect, a disclosed XBP1 fragment can provide control over gene therapy delivered to a cell via lipid nanoparticles (LNPs).

[0195] In an aspect, a disclosed viral vector or a disclosed non-viral vector can facilitate ER homeostasis, thereby avoiding activation of terminal UPR in a cell. In an aspect, a disclosed viral vector or a disclosed non-viral vector can facilitate ER homeostasis, thereby avoiding activation of the apoptotic UPR in a cell. In an aspect, wherein, under ER stress, a disclosed viral vector or a disclosed non-viral vector can generate a non-functional transgene mRNA and / or non-functional mRNA therapy. In an aspect, wherein, under ER stress, a disclosed viral vector or a disclosed non-viral vector can decrease the expression and / or abundance of the full-length mRNA transcript for a disclosed transgene. In an aspect, a disclosed viral vector or a disclosed non-viral vector canminimize and / or reduce ER stress. In an aspect, a disclosed viral vector or a disclosed non-viral vector can increase and / or improve the likelihood of survival in the one or more cells of a subject. In an aspect, a disclosed viral vector or a disclosed non-viral vector can increase and / or improve long term expression of the transgene in the one or more cells of a subject. In an aspect, a disclosed viral vector or a disclosed non-viral vector can decrease and / or minimize the likelihood of apoptosis in the one or more cells of a subject. In an aspect, a disclosed viral vector or a disclosed non-viral vector can confer UPR-mediated regulation to the transgene mRNA and / or mRNA therapy.

[0196] In an aspect, a disclosed viral vector or a disclosed non-viral vector can reduce the expression of one or more markers of ER stress. In an aspect, markers of ER stress are discussed supra and can comprise HSPA5 (aka GRP78 / BiP), ERN (aka IREla), XBP1, PERK, ATF6, DDIT3 (aka CHOP), and any combination thereof. In an aspect, ER stress can be measured using RNA based methods are employed to analyze XBP1 splicing or the expression of other UPR genes. In an aspect, as it relates to proteins, one or more antibodies can be used to determine the expression of UPR markers. In an aspect, markers of ER stress can comprise HSPA5 (aka GRP78 / BiP), IREla, XBP1, PERK, ATE6, CHOP, and any combination thereof. In an aspect, expression of one or more markers of ER stress can be determined by Western blot or staining based applications (e.g., immunocytochemistry or immunohistochemistry).

[0197] In an aspect, when ER stress is minimized and / or decreased, transgene expression can increase.5. Pharmaceutical Formulations

[0198] Disclosed herein is a pharmaceutical formulation comprising a disclosed nucleic acid molecule. Disclosed herein is a pharmaceutical formulation comprising a disclosed nucleic acid molecule and a pharmaceutically acceptable carrier. Disclosed herein is a pharmaceutical formulation comprising a disclosed vector. Disclosed herein is a pharmaceutical formulation comprising a disclosed vector and a pharmaceutically acceptable carrier. Disclosed herein is a pharmaceutical formulation comprising a disclosed AAV particle. Disclosed herein is a pharmaceutical formulation comprising a disclosed AAV particle and a pharmaceutically acceptable carrier. Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or a viral vector comprising a nucleic acid molecule. Disclosed herein is a pharmaceutical formulation comprising a non-viral vector or a viral vector comprising a nucleic acid molecule and a pharmaceutically acceptable carrier.

[0199] Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ IDN0:61. Disclosed herein is a pharmaceuticalformulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ IDNO:06. Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F1. XBP1F2, XBP1F3, XBP1F4, XBP1F5, and XBP1F6.

[0200] Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof. Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising (i) a HR and an intronic sequence, (ii) aHRl, an intronic sequence, and aHR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) aHRl, one or more intronic sequences, aHR2, and an AP. In an aspect, disclosed endoplasmic reticulum (ER) stress sensor variant can comprise an RNA structure provided in Table 4.

[0201] Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO:61.

[0202] Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:06. Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the RNA structure known as XBP1F1, XBP1F2. XBP1F3, XBP1F4, XBP1F5, or XBP1F6.

[0203] Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof. Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising (i) a HR and an intronic sequence, (ii) a HR1, an intronic sequence, and aHR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, a HR2, and an AP.

[0204] In an aspect, a disclosed X-box Binding Protein 1 (XBP1) fragment can comprise anRNA structure provided in Table 4.

[0205] Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ IDNO:61, and a sequence encoding a transgene.

[0206] Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:06, and a sequence encoding a transgene. Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F1, XBP1F2, XBP1F3, XBP1F4, XBP1F5, or XBP1F6, and a sequence encoding a transgene.

[0207] Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof, and a sequence encoding a transgene. Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising (i) a HR and an intronic sequence, (ii) a HR1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, a HR2, and an AP.

[0208] Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO:61, and a sequence encoding a transgene.

[0209] Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:06, and a sequence encoding a transgene. Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the RNA structure known as XBP1F1, XBP1F2, XBP1F3, XBP1F4, XBP1F5, or XBP1F6, and a sequence encoding a transgene.

[0210] Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or morecalreticulin peptides (CP), one or more intronic sequences, or any combination thereof, and a sequence encoding a transgene. Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising (i) a HR and an intronic sequence, (ii) a HR 1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, aHR2, and an AP, and a sequence encoding a transgene.

[0211] Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising a promoter operably linked to a disclosed endoplasmic reticulum (ER) stress sensor variant, a linker, a sequence encoding a transgene, and a poly A sequence. Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising a promoter operably linked to a disclosed XBP1 fragment, a linker, a sequence encoding a transgene, and a poly A sequence.

[0212] Disclosed herein is a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising a promoter operably linked to an XBP1 fragment comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:06, a linker, a sequence encoding a transgene, and a poly A sequence.

[0213] In an aspect, a disclosed pharmaceutical formulation can comprise (i) one or more active agents, (ii) biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In an aspect, a disclosed composition can comprise one or more immune modulators. In an aspect, a disclosed composition can comprise one or more proteasome inhibitors. In an aspect, a disclosed composition can comprise one or more immunosuppressives or immunosuppressive agents. In an aspect, an immunosuppressive agent can be anti-thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), or a combination thereof. In an aspect, a disclosed formulation can comprise an anaplerotic agent (such as, for example, C7 compounds like triheptanoin or MCT).

[0214] In an aspect, any disclosed pharmaceutical formulation can further comprise one or more excipients and / or diluents. Excipients and / or diluents known to the art and are discussed supra. In an aspect, any disclosed pharmaceutical formulation can further comprise one or more additional pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers known to the art and are discussed supra.

[0215] In an aspect, a therapeutically effective amount of a disclosed pharmaceutical formulation can comprise a range of about 1 x 1010vg / kg to about 2 x 1014vg / kg of a disclosed vector and / or a disclosed AAV particle. In an aspect, for example, a dose of a disclosed pharmaceuticalformulation can comprise about 1 x 1011to about 8 x 1013vg / kg or about 1 x 1012to about 8 x 1013vg / kg. In an aspect, a dose of a disclosed pharmaceutical formulation can comprise about l x 1013to about 6 x 1013vg / kg. In an aspect, a dose of a disclosed pharmaceutical formulation can comprise at least about 1 x IO10, at least about 5 x IO10, at least about 1 x 1011, at least about 5 x 1011, at least about 1 x 1012, at least about 5 x 1012, at least about 1 x 1013, at least about 5 x 1013, or at least about 1 x 1014vg / kg. In an aspect, a dose of a disclosed pharmaceutical formulation can comprise no more than about 1 x IO10, no more than about 5 x IO10, no more than about 1 x 1011, no more than about 5 x 1011, no more than about 1 x 1012, no more than about 5 x 1012, no more than about 1 x 1013, no more than about 5 x 1013, or no more than about 1 x 1014vg / kg. In an aspect, a dose of a disclosed pharmaceutical formulation can comprise about 1 x 1012vg / kg. In an aspect, a dose of a disclosed pharmaceutical formulation can comprise about 1 x 1011vg / kg. In an aspect, a dose of a disclosed pharmaceutical formulation can comprise a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results.

[0216] In an aspect, a therapeutically effective amount of a disclosed pharmaceutical formulation can comprise a range of about l x 1012vg per subject total to about 1 x 1017vg per subject total. In an aspect, a therapeutically effective amount of a disclosed pharmaceutical formulation can comprise a range of about 1 x 1012vg per subject total, about 1 x 1013vg per subject total, about 1 x 1014vg per subject total, about 1 x 1015vg per subject total, about 1 x 1016vg per subject total, or about 1 x 1017vgper subject total. In an aspect, a therapeutically effective amount of a disclosed pharmaceutical formulation can comprise about 1 x 106DRP / mL to about 1 x 1014DRP / mL. In an aspect, a therapeutically effective amount of a disclosed pharmaceutical formulation can comprise about 1 x 106DRP / mL, 1 x 107DRP / mL, 1 x 108DRP / mL, 1 x 109DRP / mL, l x IO10DRP / mL, 1 x 1011DRP / mL, 1 x 1012DRP / mL, 1 x 1013DRP / mL, or 1 x 1014DRP / mL. In an aspect, a therapeutically effective amount of a disclosed pharmaceutical formulation can comprise a range determined by a skilled person.

[0217] In an aspect, a disclosed pharmaceutical formulation can be used to restore and / or return expression of a disclosed protein coding gene to a wild-type, normal, or control expression level. In an aspect, a disclosed pharmaceutical formulation can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation. In an aspect, a disclosed nucleic acid molecule can restore the functionality and / or structural integrity of a missing, deficient, and / or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra). In an aspect, restoring one or more aspects of cellular homeostasis and / or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example,correcting, preventing, reducing, and / or ameliorating autophagy); (iii) improving, enhancing, restoring, and / or preserving mitochondrial functionality and / or structural integrity; (iv) improving, enhancing, restoring, and / or preserving organelle functionality and / or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of the multi-systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and / or preserving one or more aspects of cellular structural and / or functional integrity. In an aspect, restoring the activity and / or functionality of a missing, deficient, and / or mutant protein or enzyme can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and / or mutant protein or enzyme). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and / or functionality is similar to that of a wild-type or control level.

[0218] In an aspect, a disclosed pharmaceutical formulation can improve protein folding capacity of the ER. In an aspect, when ER stress is minimized and / or decreased, transgene expression can increase.

[0219] In an aspect, a disclosed pharmaceutical formulation can confer UPR-mediated regulation to the transgene mRNA and / or mRNA therapy. In an aspect, a disclosed pharmaceutical formulation can confer UPR-mediated regulation to the encoded protein or encoded RNA. In an aspect, a disclosed pharmaceutical formulation can render the expression of the encoded transgene amenable to UPR-mediated regulation. In an aspect, a disclosed pharmaceutical formulation can provide control over gene therapy delivered to a cell. In an aspect, a disclosed pharmaceutical formulation can provide control over gene therapy delivered to a cell via a AAV vector. In an aspect, a disclosed pharmaceutical formulation can provide control over gene therapy delivered to a cell via lipid nanoparticles (LNPs). In an aspect, a disclosed pharmaceutical formulation can facilitate ER homeostasis, thereby avoiding activation of terminal UPR in a cell. In an aspect, a disclosed pharmaceutical formulation can facilitate ER homeostasis, thereby avoiding activation of the apoptotic UPR in a cell. In an aspect, a disclosed pharmaceutical formulation, under ER stress, can generate a non-functional transgene mRNA and / or non-functional mRNA therapy. Inan aspect, wherein, under ER stress, a disclosed pharmaceutical formulation can decrease the expression and / or abundance of the full-length mRNA transcript for a disclosed transgene. In an aspect, a disclosed pharmaceutical formulation can minimize and / or reduce ER stress. In an aspect, a disclosed nucleic acid molecule can increase and / or improve the likelihood of survival in the one or more cells of the subject. In an aspect, a disclosed pharmaceutical formulation can increase and / or improve long term expression of the transgene in the one or more cells of the subject. In an aspect, a disclosed pharmaceutical formulation can decrease and / or minimize the likelihood of apoptosis in the one or more cells of the subject. In an aspect, a disclosed pharmaceutical formulation can confer UPR-mediated regulation to the transgene mRNA and / or mRNA therapy. In an aspect, a disclosed pharmaceutical formulation can improve and / or enhance proteostasis (e.g., the network of interconnected quality-control processes in the cell that maintains the functional proteonie).6. Plasmids

[0220] Disclosed herein is a plasmid comprising one or more disclosed nucleic acid molecules. Disclosed herein is a plasmid comprising one or more disclosed vectors. Disclosed here are plasmids used in methods of making a disclosed composition such as, for example, a disclosed nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. Plasmids and using plasmids are known to the art.

[0221] Disclosed herein is a plasmid comprising the sequence set forth in any one of SEQ ID NO:07 - SEQ ID NO:12 or a fragment thereof. Disclosed herein is a plasmid comprising a sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the sequence set forth in any one of SEQ ID NO:07 - SEQ ID NO: 12 or a fragment thereof. Disclosed herein is a plasmid comprising a sequence having at least 40%-60%, at least 60%-80%, at least 80%-90%, or at least 90%-100% identity to the sequence set forth in any one of SEQ ID NO:07 - SEQ ID NO: 12 or a fragment thereof.

[0222] Disclosed herein is a plasmid comprising the sequence set forth in any one of SEQ ID NO:24 - SEQ ID NO:29 or a fragment thereof. Disclosed herein is a plasmid comprising a sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the sequence set forth in any one of SEQ ID NO:24 - SEQ ID NO:29 or a fragment thereof. Disclosed herein is a plasmid comprising a sequence having at least 40%-60%, at least 60%-80%, at least 80%-90%, or at least 90%-100% identity to the sequence set forth in any one of SEQ ID NO:24 - SEQ ID NO:29 or a fragment thereof.7. Cells

[0223] Disclosed herein are cells comprising a disclosed nucleic acid molecule, a disclosed vector, and / or a disclosed plasmid (such as, for example, a plasmid having the sequence set forth in any one of SEQ ID NO:07 - SEQ ID N0:12 or SEQ ID NO:24 - SEQ ID NO:29. Disclosed herein are cells transduced by one or more disclosed viral vectors. Disclosed herein are cells transfected with one or more disclosed nucleic acid molecules. Techniques to achieve transfection and transduction are known to the art and using transfected or transduced cells are known to the art. In an aspect, disclosed herein are human cells lines transduced by one or more disclosed viral vectors or transfected with one or more disclosed nucleic acids, one or more disclosed non-viral vectors, or one or more disclosed plasmids. In an aspect, disclosed herein are human cells lines having one or more genetic diseases or genetic disorders contacted with one or more nucleic acid molecules, one or more disclosed vectors, and / or one or more disclosed pharmaceutical formulations. Disclosed herein are cells obtained for a subject treated with one or more disclosed nucleic acid molecules, one or more disclosed vectors, one or more disclosed plasmids, and / or one or more disclosed pharmaceutical formulations.8. Animals

[0224] Disclosed herein are animals treated with one or more disclosed nucleic acid molecules, one or more disclosed ER stress sensor variants, one or more disclosed XBP1 fragments, one or more disclosed vectors, one or more disclosed AAV particles, one or more disclosed pharmaceutical formulations, and / or one or more disclosed plasmids. Transgenic animals are known to the art as are the techniques to generate transgenic animals.9. Libraries

[0225] Disclosed herein is a library of one or more disclosed endoplasmic reticulum (ER) stress sensor variants. Disclosed herein is a library of one or more disclosed X-Box Binding Protein 1 (XBP1) fragments. Disclosed herein is a library of one or more ER stress sensor variants comprising the sequence set forth in SEQ ID NO:01 - SEQ ID NO:06. Disclosed herein is a library of one or more X-Box Binding Protein 1 (XBP1) fragments comprising the sequence set forth in SEQ ID NO:01 - SEQ ID NO:06. Disclosed herein is a library of one or more disclosed vectors.10. Kits

[0226] Disclosed herein is a kit comprising one or more disclosed ER stress sensor variants, disclosed XBP1 fragments, disclosed nucleic acid molecules, disclosed vectors or disclosed AAV particles, disclosed pharmaceutical formulations, or any combination thereof. Disclosed herein is a kit comprising one or more disclosed ER stress sensor variants, disclosed XBP1 fragments, disclosed nucleic acid molecules, one or more disclosed vectors, one or more disclosedpharmaceutical formulations, or any combination thereof. In an aspect, a kit can comprise a disclosed nucleic acid molecule, a disclosed vector or disclosed AAV particle, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, or a combination thereof, and one or more agents. “Agents” and “Therapeutic Agents” are known to the art and are described supra.

[0227] In an aspect, the one or more agents can treat, prevent, inhibit, and / or ameliorate one or more comorbidities in a subject. In an aspect, one or more active agents can treat, inhibit, prevent, and / or ameliorate cellular and / or metabolic complications related to a missing, deficient, and / or mutant protein or enzyme.

[0228] In an aspect, a disclosed kit can comprise at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose (such as, for example, treating a subject diagnosed with or suspected of having a genetic disease or genetic disorder). Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. In an aspect, a kit for use in a disclosed method can comprise one or more containers holding a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed RNA therapeutic, or a combination thereof, and a label or package insert with instructions for use. In an aspect, suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers can be formed from a variety of materials such as glass or plastic. The container can hold a disclosed ER stress sensor variant, a disclosed XBP1 fragment, a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof, and can have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert can indicate that a disclosed ER stress sensor variant, a disclosed XBP1 fragment, a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, a disclosed RNA therapeutic agent, or a combination thereof can be used for treating, preventing, inhibiting, and / or ameliorating a disease or disorder or complications and / or symptoms associated with a disease or disorder. A disclosed kit can comprise additional components necessary for administration such as, for example, other buffers, diluents, filters, needles, and syringes. In an aspect, a disclosed kit can be used in any disclosed method. In an aspect, a disclosed kit can be used to maintain homeostasis. In an aspect, a disclosed kit can be used to treat a genetic disease or genetic disorder. In an aspect, a disclosed kit can be used to inhibit and / or minimize disease progression.D. Methods1. Methods of Maintaining Homeostasis

[0229] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector, wherein, following expression of the encoded transgene, the endoplasmic reticulum (ER) stress sensor variant ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0230] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:61, wherein, following expression of the encoded transgene, the endoplasmic reticulum (ER) stress sensor variant ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0231] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:06, wherein, following expression of the encoded transgene, the endoplasmic reticulum (ER) stress sensor variant ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0232] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F1. XBP1F2, XBP1F3, XBP1F4,XBP1F5, and XBP1F6, wherein, following expression of the encoded transgene, the endoplasmic reticulum (ER) stress sensor variant ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0233] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof, wherein, following expression of the encoded transgene, the endoplasmic reticulum (ER) stress sensor variant ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0234] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising an endoplasmic reticulum (ER) stress sensor variant comprising (i) a HR and an intronic sequence, (ii) a HR1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, a HR2, and an AP, wherein, following expression of the encoded transgene, the endoplasmic reticulum (ER) stress sensor variant ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0235] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forthin SEQ IDNO:61, wherein, following expression of the encoded transgene, the XBP1 fragment ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0236] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:06, wherein, following expression of the encoded transgene, the XBP1 fragment ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0237] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the RNA structure known as XBP1F1, XBP1F2. XBP1F3, XBP1F4, XBP1F5, or XBP1F6, wherein, following expression of the encoded transgene, the XBP1 fragment ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0238] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vectorcomprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof, wherein, following expression of the encoded transgene, the XBPl fragment ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0239] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising (i) a HR and an intronic sequence, (ii) a HR1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, a HR2, and an AP, wherein, following expression of the encoded transgene, the XBP1 fragment ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0240] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO :61 , and a sequence encoding a transgene, wherein, following expression of the encoded transgene, the endoplasmic reticulum (ER) stress sensor variant ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained .

[0241] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:06, and a sequence encoding a transgene, wherein, following expression of the encoded transgene, the endoplasmic reticulum (ER) stress sensor variant ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0242] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F1, XBP1F2,XBP1F3, XBP1F4,XBP1F5, or XBP1F6, and a sequence encoding a transgene, wherein, following expression of the encoded transgene, the endoplasmic reticulum (ER) stress sensor variant ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0243] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof, and a sequence encoding a transgene, wherein, following expression of the encoded transgene, theendoplasmic reticulum (ER) stress sensor variant ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0244] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising an endoplasmic reticulum (ER) stress sensor variant comprising (i) a HR and an intronic sequence, (ii) a HR1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, a HR2, and an AP, wherein, following expression of the encoded transgene, the endoplasmic reticulum (ER) stress sensor variant ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0245] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO:61, and a sequence encoding a transgene, wherein, following expression of the encoded transgene, the XBP1 fragment ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0246] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:06, and a sequence encoding a transgene, wherein, following expression of the encoded transgene, the XBP1 fragment ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0247] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the RNA structure known as XBP1F1, XBP1F2, XBP1F3, XBP1F4, XBP1F5, or XBP1F6, and a sequence encoding a transgene, wherein, following expression of the encoded transgene, the XBP1 fragment ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0248] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof, and asequence encoding a transgene, wherein, following expression of the encoded transgene, the XBP1 fragment ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0249] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising an X-box Binding Protein 1 (XBP1) fragment comprising (i) a HR and an intronic sequence, (ii) a HR1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, aHR2, and an AP, and a sequence encoding a transgene, wherein, following expression of the encoded transgene, the XBP1 fragment ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0250] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising a promoter operably linked to a disclosed endoplasmic reticulum (ER) stress sensor variant, a linker, a sequence encoding a transgene, and a poly A sequence, wherein, following expression of the encoded transgene, the endoplasmic reticulum (ER) stress sensor variant ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0251] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising a promoter operably linked to a disclosed XBP1 fragment, a linker, a sequence encoding a transgene, and a poly A sequence, wherein, following expression of the encoded transgene, the endoplasmic reticulum (ER) stress sensor variant ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0252] Disclosed herein is a method of maintaining homeostasis, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector comprising a nucleic acid molecule comprising a promoter operably linked to an XBP1 fragment comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO: 06, a linker, a sequence encoding a transgene, and a poly A sequence, wherein, following expression of the encoded transgene, the XBP1 fragment ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0253] Disclosed herein is a method of maintaining homeostasis during a gene therapy protocol, the method comprising contacting one or more cells with a disclosed nucleic acid molecule, wherein, following expression of the encoded transgene, the endoplasmic reticulum (ER) stresssensor variant ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained .

[0254] Disclosed herein is a method of maintaining homeostasis during a gene therapy protocol, the method comprising contacting one or more cells with a disclosed viral vector or a disclosed non-viral vector, wherein, following expression of the encoded transgene, the endoplasmic reticulum (ER) stress sensor variant ensures that homeostasis in the ER of the one or more cells expressing the encoded transgene is maintained.

[0255] In an aspect of a disclosed method, wherein following expression, the encoded transgene restores one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation in the one or more cells. In an aspect, a disclosed method can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation. In an aspect, a disclosed method can restore the functionality and / or structural integrity of a missing, deficient, and / or mutant protein or enzyme (such as those, for example, encoded by one of the genes provided supra).

[0256] In an aspect, restoring one or more aspects of cellular homeostasis and / or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and / or ameliorating autophagy); (iii) improving, enhancing, restoring, and / or preserving mitochondrial functionality and / or structural integrity; (iv) improving, enhancing, restoring, and / or preserving organelle functionality and / or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of the multi-systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and / or preserving one or more aspects of cellular structural and / or functional integrity.

[0257] In an aspect, a disclosed method can restore the activity and / or functionality of a missing, deficient, and / or mutant protein or enzyme (e.g., a disclosed transgene) can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90- 100% more than a pre-existing level such as, for example, a pre-treatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and / or mutant protein or enzyme). In an aspect, restoration can be a partial or incomplete restoration. In an aspect,restoration can be complete or near complete restoration such that the level of expression, activity, and / or functionality is similar to that of a wild -type or control level. In an aspect, expression of a disclosed transgene can be restored and / or returned to a wild-type, normal, or control expression level.

[0258] In an aspect, the one or more cells can be in a subject. In an aspect, a subject can be diagnosed with or can be suspected of having a genetic disease or disorder. In an aspect, a disease or disorder can comprise any disease or disorder caused by a disclosed gene or a missing, deficient, and / or mutant gene. In an aspect, a subject can be a subject in need of treatment of a disclosed disease or disorder (e.g., a genetic disease or disorder). Genetic diseases and disorders are discussed extensively herein.

[0259] In an aspect, a disclosed method can be performed ex vivo. In an aspect, administration can be performed by one or more ex vivo methods such as, for example, an ex vivo perfusion protocol. In an aspect, an ex vivo perfusion protocol can be employed with a one or more cells, tissues, and / or organs affected by a genetic disease or disorder obtained for a subject. In an aspect, one or more cells and / or one or more tissues and / or one or more organs can be obtained from the subject in need thereof, can be subjected to an ex vivo perfusion and / or treatment and / or contact protocol, and can be returned to the subject in need thereof, wherein the one or more cells express an encoded transgene and maintain ER homeostasis.

[0260] In an aspect, a disclosed ER stress sensor variant can confer UPR-mediated regulation to the transgene mRNA and / or mRNA therapy. In an aspect, a disclosed ER stress sensor variant can confer UPR-mediated regulation to the encoded protein or encoded RNA. In an aspect, a disclosed ER stress sensor variant can render the expression of the encoded transgene amenable to UPR-mediated regulation. In an aspect, a disclosed ER stress sensor variant can provide control over gene therapy delivered to a cell. In an aspect, a disclosed ER stress sensor variant can provide control over gene therapy delivered to the cell via an AAV vector. In an aspect, a disclosed ER stress sensor variant can provide control over gene therapy delivered to the cell via lipid nanoparticles (LNPs).

[0261] In an aspect of a disclosed method, a disclosed ER stress sensor variant can facilitate ER homeostasis, thereby avoiding activation of terminal UPR in a cell. In an aspect of a disclosed method, a disclosed ER stress sensor variant can facilitate ER homeostasis, thereby avoiding activation of the apoptotic UPR in a cell. In an aspect of a disclosed method, wherein, under ER stress, a disclosed ER stress sensor variant can generate a non-functional transgene mRNA and / or non-functional mRNA therapy. In an aspect of a disclosed method, wherein, under ER stress, a disclosed ER stress sensor variant can decrease the expression and / or abundance of the full-length mRNA transcript for a disclosed transgene. In an aspect of a disclosed method, a disclosed ERstress sensor variant can minimize and / or reduce ER stress. In an aspect of a disclosed method, a disclosed ER stress sensor variant can increase and / or improve the likelihood of survival in the one or more cells of a subject. In an aspect of a disclosed method, a disclosed ER stress sensor variant can increase and / or improve long term expression of the transgene in the one or more cells of a subject. In an aspect of a disclosed method, a disclosed ER stress sensor variant can decrease and / or minimize the likelihood of apoptosis in the one or more cells of a subject. In an aspect of a disclosed method, a disclosed ER stress sensor variant can confer UPR-mediated regulation to the transgene mRNA and / or mRNA therapy. In an aspect of a disclosed method, a disclosed ER stress sensor variant can reduce the expression of one or more markers of ER stress. In an aspect, markers of ER stress can comprise HSPA5 (aka GRP78 / BiP), ERN (aka IREla), XBP1, PERK, ATF6, DDIT3 (aka CHOP), and any combination thereof.

[0262] In an aspect of a disclosed method, under ER stress, ERN (aka IREla) splices a fragment comprising the sequence of SEQ ID NO:61 from a disclosed nucleic acid molecule.

[0263] In an aspect, a disclosed method can improve protein folding capacity of the ER.

[0264] In an aspect, a disclosed method can further comprise generating and / or validating a disclosed ER stress sensor variant. In an aspect, a disclosed method can further comprise generating and / or validating a disclosed XBP1 fragment. In an aspect, a disclosed method can further comprise generating and / or validating a disclosed nucleic acid molecule. In an aspect, a disclosed method can further comprise generating and / or validating a disclosed vector. In an aspect, a disclosed method can further comprise generating and / or validating a disclosed pharmaceutical formulation. In an aspect, a disclosed method can further comprise generating and / or validating a disclosed plasmid.2. Methods of Treating a Genetic Disease or Genetic Disorder

[0265] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a disclosed nucleic acid molecule or any disclosed nucleic acid molecule, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBP1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0266] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a viral vector or a non-viral vector comprising a disclosed nucleic acid molecule or any disclosed nucleic acid molecule, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBPl fragmentensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained .

[0267] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a viral vector or a non-viral vector comprising one or more disclosed nucleic acid molecules, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBP1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0268] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a disclosed pharmaceutical formulation, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBP1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0269] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO :61, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBP1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0270] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:06, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBP1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0271] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:06, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and whereinthe XBP1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0272] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F1. XBP1F2, XBP1F3, XBP1F4, XBP1F5, and XBP1F6, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBP 1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0273] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBP1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0274] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising (i) a HR and an intronic sequence, (ii) a HR1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, a HR2, and an AP, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBP 1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0275] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ ID NO:61, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellularfunctionality and / or metabolic dysregulation is restored, and wherein the XBP1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0276] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBPl fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0277] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising (i) a HR and an intronic sequence, (ii) a HR 1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, a HR2, and an AP, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBP1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0278] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in SEQ ID NO:61, and a sequence encoding a transgene, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBP1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained .

[0279] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:06, and a sequence encoding a transgene, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / ormetabolic dysregulation is restored, and wherein the XBPl fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0280] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising the RNA structure known as XBP1F1, XBP1F2, XBP1F3, XBP1F4, XBP1F5, or XBP1F6, and a sequence encoding a transgene, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBP1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0281] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof, and a sequence encoding a transgene, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBP1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0282] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an endoplasmic reticulum (ER) stress sensor variant comprising (i) a HR and an intronic sequence, (ii) a HR1, an intronic sequence, and a HR2, (iii) a HR, an intronic sequence, and an AP, (iv) a HR, an intronic sequence, a CP, and an AP, or (v) a HR1, one or more intronic sequences, a HR2, and an AP, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBPl fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0283] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in SEQ IDNO:61, and a sequence encoding a transgene, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and whereinthe XBP1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0284] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:06, and a sequence encoding a transgene, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBPl fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0285] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising the RNA structure known as XBP1F1, XBP1F2, XBP1F3, XBP1F4, XBP1F5, or XBP1F6, and a sequence encoding a transgene, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBP1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0286] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof, and a sequence encoding a transgene, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBP1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0287] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising an X-box Binding Protein 1 (XBP1) fragment comprising one or more hydrophobic regions (HR), one or more arrest peptides (AP), one or more calreticulin peptides (CP), one or more intronic sequences, or any combination thereof, and a sequence encoding a transgene, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionalityand / or metabolic dysregulation is restored, and wherein the XBP1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0288] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising a promoter operably linked to a disclosed endoplasmic reticulum (ER) stress sensor variant, a linker, a sequence encoding a transgene, and a poly A sequence, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBPl fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0289] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising a promoter operably linked to a disclosed XBP1 fragment, a linker, a sequence encoding a transgene, and a poly A sequence, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBP1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0290] Disclosed herein is a method of treating a genetic disease or disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a non-viral or viral vector comprising a nucleic acid molecule comprising a promoter operably linked to an XBP1 fragment comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:06, a linker, a sequence encoding a transgene, and a poly A sequence, wherein, following expression the encoded transgene, one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation is restored, and wherein the XBP1 fragment ensures that homeostasis in the endoplasmic reticulum (ER) one of one or more cells is maintained.

[0291] In an aspect, restoring one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation comprises restoring the functionality and / or structural integrity of a missing, deficient, and / or mutant protein or enzyme.

[0292] In an aspect of a disclosed method, a disclosed ER stress sensor variant can facilitate ER homeostasis, thereby avoiding activation of terminal UPR in a cell. In an aspect of a disclosed method, a disclosed ER stress sensor variant can facilitate ER homeostasis, thereby avoiding activation of the apoptotic UPR in a cell. In an aspect of a disclosed method, wherein, under ER stress, a disclosed ER stress sensor variant can generate a non-functional transgene mRNA and / or non-functional mRNA therapy. In an aspect of a disclosed method, wherein, under ER stress, adisclosed ER stress sensor variant can decrease the expression and / or abundance of the full-length mRNA transcript for a disclosed transgene. In an aspect of a disclosed method, a disclosed ER stress sensor variant can minimize and / or reduce ER stress. In an aspect of a disclosed method, a disclosed ER stress sensor variant can increase and / or improve the likelihood of survival in the one or more cells of a subject. In an aspect of a disclosed method, a disclosed ER stress sensor variant can increase and / or improve long term expression of the transgene in the one or more cells of a subject. In an aspect of a disclosed method, a disclosed ER stress sensor variant can decrease and / or minimize the likelihood of apoptosis in the one or more cells of a subject. In an aspect of a disclosed method, a disclosed ER stress sensor variant can confer UPR-mediated regulation to the transgene mRNA and / or mRNA therapy. In an aspect of a disclosed method, a disclosed ER stress sensor variant can reduce the expression of one or more markers of ER stress. In an aspect, markers of ER stress can comprise HSPA5 (aka GRP78 / BiP), ERN (aka IRE la), XBP1, PERK, ATF6, DDIT3 (aka CHOP), and any combination thereof.

[0293] In an aspect of a disclosed method, under ER stress, ERN (aka IREla) splices a fragment comprising the sequence of SEQ ID NO:61 from a disclosed nucleic acid molecule.

[0294] In an aspect of a disclosed method of treating, a subject can have a genetic disease or disorder related to and / or caused by one or more disclosed genes including those disclosed in supra (Section VI(B)(3)). In an aspect of a disclosed method of treating, a subject can have a genetic disease or disorder related to and / or caused by one or more disclosed genes including those disclosed in Section VI(B)(3).

[0295] In an aspect, a disclosed method of treating a genetic disease or disorder can restore the functionality and / or structural integrity of a missing, deficient, and / or mutant protein or enzyme. In an aspect, a disclosed method of treating can comprise restoring one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation. In an aspect, restoring one or more aspect of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation comprises restoring the functionality and / or structural integrity of a missing, deficient, and / or mutant protein or enzyme. In an aspect, restoring one or more aspects of cellular homeostasis and / or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and / or ameliorating autophagy); (iii) improving, enhancing, restoring, and / or preserving mitochondrial functionality and / or structural integrity; (iv) improving, enhancing, restoring, and / or preserving organelle functionality and / or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of the multi-systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progressionof a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and / or preserving one or more aspects of cellular structural and / or functional integrity. In an aspect, restoring the activity and / or functionality of a missing, deficient, and / or mutant protein or enzyme (e.g., encoded by a protein coding gene described supra) can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and / or mutant protein or enzyme). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and / or functionality is similar to that of a wild-type or control level.

[0296] In an aspect, a disclosed method of treating can further comprise monitoring the subject’s metabolic and / or physiologic improvement following the administering step and / or following the administering steps. In an aspect, a clinician can measure and / or determine the subject’s metabolic and / or physiologic status over time to identify one or more improvements and / or one or more diminishments. In an aspect of a disclosed method, a clinician can use the subject’s metabolic and / or physiologic status and / or the trend of the subject’s metabolic and / or physiological status and / or trend to make a treatment decision and / or to modify an aspect of a disclosed method and / or to continue treating the subject and / or continue to administer a disclosed AAV particle, a disclosed vector, a disclosed nucleic acid molecule, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, and / or a disclosed immune modulator, or any combination thereof. In an aspect, metabolic and / or physiologic data can inform the clinician and a treatment plan.

[0297] In an aspect of a disclosed method of treating, techniques to monitor, measure, and / or assess the restoring one or more aspects of cellular homeostasis and / or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example, representative regulated variables and sensors relating to systemic homeostasis are provided below.

[0298] In an aspect, a disclosed method of treating can further comprise subjecting the subject to one or more invasive or non-invasive diagnostic assessments. Diagnostic assessments are known to the art. In an aspect, a disclosed non-invasive diagnostic assessment can comprise x-rays, computerized tomography (CT) scans, magnetic resonance imaging (MRI) scans, ultrasounds, positron emission tomography (PET) scans, or any combination thereof. In an aspect, a disclosed invasive diagnostic assessment can comprise a tissue biopsy or exploratory surgery.

[0299] In an aspect, a disclosed method of treating can further comprise diagnosing a subject as having a genetic disease and / or disorder. In an aspect, a disclosed method of treating can further comprise diagnosing a subject as having a need of a disclosed nucleic acid molecule, disclosed vector, disclosed pharmaceutical formulation, or any combination thereof.

[0300] In an aspect, a disclosed method of treating can be used to repair diseased and / or dysfunctional cell types in the one or more diseased and / or disordered cells, tissues, and / or organs.

[0301] In an aspect, a disclosed method of treating can be used to improve and / or can be used to enhance the quality of the subject’s life when compared to a pre-treatment level. In an aspect, a disclosed method of treating can be used to improve the subject’s quality of life by at least 50% when compared to the subject’s pre-treatment quality of life.

[0302] In an aspect, a disclosed method of treating can be used to improve and / or can be used to enhance the quality of the subject’s life when compared to a pre-treatment level. In an aspect, a disclosed method of treating can be used to improve the subject’s quality of life by at least 50% when compared to the subject’s pre-treatment quality of life.

[0303] In an aspect, a disclosed method of treating can be used to diminish and / or decrease one or more symptoms associated with and / or related to the subject’s genetic disease and / or genetic disorder. In an aspect, a disclosed method of treating can be used to prevent an undesired physiological change, disease, pathological condition, or disorder from occurring in the subject. In an aspect, a disclosed method of treating can be used to inhibit a physiological change, disease, pathological condition, or disorder, z.e., arresting its development, in the subject. In an aspect, adisclosed method of treating can be used to relieve a physiological change, disease, pathological condition, or disorder, z.e., causing regression of the disease, in the subject.

[0304] In an aspect of a disclosed method of treating, administering to the subject can comprise contacting one or more cells with one or more disclosed ER stress sensor variants, disclosed XBP1 fragments, disclosed nucleic acid molecules, disclosed vectors or disclosed AAV particles, disclosed pharmaceutical formulations, or any combination thereof.

[0305] In an aspect of a disclosed method of treating, administering of a disclosed ER stress sensor variant, a disclosed XBP1 fragment, a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof can comprise one or more routes. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can also include hepatic intraarterial administration or administration through the hepatic portal vein (HPV). Administration of a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination can comprise administration directly into the CNS (e.g., intraparenchymal, intracerebroventricular, intrathecal cisternal, intrathecal (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or the PNS. Administration can be continuous or intermittent.

[0306] In an aspect, a disclosed method of treating can employ multiple routes of administration to the subject. In an aspect, a disclosed method of treating can employ a first route of administration that can be the same or different as a second and / or subsequent routes of administration.

[0307] In an aspect of a disclosed method of treating a genetic disease or disorder, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a range of about 1 x 1010vg / kg to about 2 x 1014vg / kg. In an aspect, for example, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a dose of about 1 x 1011vg / kg to about 8 x 1013vg / kg or about 1 x 1012vg / kg to about 8 x 1013vg / kg. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a dose of about 1 x 1013vg / kg to about 6 x 1013vg / kg. In an aspect, a therapeutically effective amount of disclosed vector or a disclosedpharmaceutical formulation can comprise a dose of at least about 1 x IO10vg / kg, at least about 5 x IO10vg / kg, at least about 1 x 1011vg / kg, at least about 5 x 1011vg / kg, at least about 1 x 1012vg / kg, at least about 5 x 1012vg / kg, at least about 1 x 1013vg / kg, at least about 5 x 1013vg / kg, or at least about 1 x 1014vg / kg. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a dose of no more than about l x IO10vg / kg, no more than about 5 x 1010vg / kg, no more than about 1 x 1011vg / kg, no more than about 5 x 1011vg / kg, no more than about 1 x 1012vg / kg, no more than about 5 x 1012vg / kg, no more than about 1 x 1013vg / kg, no more than about 5 x 1013, or no more than about 1 x 1014vg / kg. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a dose of about 1 x 1012vg / kg or about 1 x 1011vg / kg. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results.

[0308] In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a range of about 1 x 1012vg per subject total to about 1 x 1017vg per subject total. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a range of about 1 x 1012vg per subject total, about 1 x 1013vg per subject total, about 1 x 1014vg per subject total, about 1 x 1015vg per subject total, about 1 x 1016vg per subject total, or about 1 x 1017vg per subject total.

[0309] In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise about 1 x 106DRP / mL to about 1 x 1014DRP / mL. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise about 1 x 106DRP / mL, 1 x 107DRP / mL, 1 x 108DRP / mL, 1 x 109DRP / mL, 1 x 1010DRP / rnL, 1 x 1011DRP / rnL, 1 x 1012DRP / rnL, 1 x 1013DRP / rnL, or 1 x 1014DRP / mL. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a range determined by a skilled person.

[0310] In an aspect, a disclosed method of treating a genetic disease or disorder can further comprise administering to the subject a therapeutically effective amount of a therapeutic agent. A therapeutic agent can be any disclosed agent that effects a desired clinical outcome.

[0311] In an aspect, a disclosed method of treating a genetic disease or disorder can further comprise monitoring the subject for adverse effects. In an aspect, in the absence of adverse effects, the method can further comprise continuing to treat the subject. In an aspect, in the presence of adverse effects, the method can further comprise modifying the treating step. Methods of monitoring a subject’s well-being can include both subjective and objective criteria (and are discussed supra). Such methods are known to the skilled person.

[0312] In an aspect, a disclosed method of treating can further comprise administering to the subject a therapeutically effective amount of an agent that can correct one or more aspects of a dysregulated metabolic or enzymatic pathway. In an aspect, such an agent can comprise an enzyme for enzyme replacement therapy. In an aspect, a disclosed enzyme can replace any enzyme in a dysregulated or dysfunctional metabolic or enzymatic pathway. In an aspect, a disclosed method of treating can comprise replacing one or more enzymes in a dysregulated or dysfunctional metabolic pathway.

[0313] In an aspect, a disclosed method of treating a genetic disease or disorder can further comprise administering one or more immune modulators. In an aspect, a disclosed immune modulator can be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or a combination thereof. In an aspect, a disclosed immune modulator can be bortezomib or SVP- Rapamycin. In an aspect, a disclosed immune modulator can be Tacrolimus. In an aspect, a disclosed immune modulator such as methotrexate can be administered at a transient low to high dose. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.1 mg / kg body weight to about 0.6 mg / kg body weight. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.4 mg / kg body weight. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg / kg body weight for 3 to 5 or greater cycles, with up to three days per cycle. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg / kg body weight for a minimum of 3 cycles, with three days per cycle. In an aspect, a person skilled in the art can determine the appropriate number of cycles. In an aspect, a disclosed immune modulator can be administered as many times as necessary to achieve a desired clinical effect.

[0314] In an aspect, a disclosed immune modulator can be administered orally about one hour before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered orally about one hour or a few days before a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before or a few days before a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof.

[0315] In an aspect, a disclosed method of treating a genetic disease or disorder can further comprise administering one or more proteasome inhibitors (e.g., bortezomib, carfilzomib, marizomib, ixazomib, and oprozomib). In an aspect, a proteasome inhibitor can be an agent that acts on plasma cells (e.g., daratumumab). In an aspect, an agent that acts on a plasma cell can be melphalan hydrochloride, melphalan, pamidronate disodium, carmustine, carfilzomib, carmustine, cyclophosphamide, daratumumab, doxorubicin hydrochloride liposome, doxorubicin hydrochloride liposome, elotuzumab, melphalan hydrochloride, panobinostat, ixazomib citrate, carfilzomib, lenalidomide, melphalan, melphalan hydrochloride, plerixafor, ixazomib citrate, pamidronate disodium, panobinostat, plerixafor, pomalidomide, pomalidomide, lenalidomide, selinexor, thalidomide, thalidomide, bortezomib, selinexor, zoledronic acid, or zoledronic acid.

[0316] In an aspect, a disclosed method of treating stability can further comprise administering one or more proteasome inhibitors or agents that act on plasma cells prior to administering a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof. In an aspect, a disclosed method of treating can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells concurrently with administering a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination of thereof. In an aspect, a disclosed method of treating can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells subsequent to administering a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof. In an aspect, a disclosed method of treating can further comprise administering one or more proteasome inhibitors more than 1 time. In an aspect, a disclosed method of treating can comprise administering one or more proteasome inhibitors repeatedly over time.

[0317] In an aspect, a disclosed method of treating a genetic disease or disorder can further comprise administering one or more immunosuppressive agents. In an aspect, an immunosuppressive agent can be, but is not limited to, azathioprine, methotrexate, sirolimus, antithymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), steroids, or a combination thereof. In an aspect, a disclosed method of treating can comprise administering one or more immunosuppressive agents more than 1 time. In an aspect, a disclosed method can comprise administering one or more one or more immunosuppressive agents repeatedly over time. In an aspect, a disclosed method of treating can comprise administering a compound that targets or alters antigen presentation or humoral or cell mediated or innate immune responses.

[0318] In an aspect, a disclosed method of treating a genetic disease or disorder can further comprise administering a compound that exerts a therapeutic effect against B cells and / or acompound that targets or alters antigen presentation or humoral or cell mediated immune response. In an aspect, a disclosed compound can be rituximab, methotrexate, intravenous gamma globulin, anti CD4 antibody, anti CD2, an anti-FcRN antibody, a BTK inhibitor, an anti-IGFIR antibody, a CD 19 antibody (e.g., inebilizumab), an anti-IL6 antibody (e.g., tocilizumab), an antibody to CD40, an IL2 mutein, or a combination thereof. Also disclosed herein are Treg infusions that can be administered as away to help with immune tolerance (e.g., antigen specific Treg cells to AAV).

[0319] In an aspect, a disclosed method of treating can further comprise administering lipid nanoparticles (LNPs). In an aspect, LNPs can be organ targeted. For example, in an aspect, mRNA therapy with LNP encapsulation for systemic delivery to a subject has the potential to restore the functionality and / or structural integrity of a missing, deficient, and / or mutant protein or enzyme.

[0320] In an aspect, a disclosed method of treating a genetic disease or disorder can further comprise treating a subject that has developed oris likely to develop neutralizing antibodies (ABs) to a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof. In an aspect, treating a subject that has developed or is likely to develop neutralizing antibodies can comprise plasmapheresis and immunosuppression. In an aspect, a disclosed method can comprise using immunosuppression to decrease the T cell, B cell, and / or plasma cell population, decrease the innate immune response, inflammatory response, and antibody levels in general. In an aspect, a disclosed method can comprise administering an IgG-d egrad ingagent that depletes pre-existing neutralizing antibodies. In an aspect, a disclosed method can comprise administering to the subject IdeS or IdeZ, rapamycin, and / or SVP-Rapamycin. In an aspect, a disclosed method of treating can comprise administering Tacrolimus. In an aspect, a disclosed IgG-degrading agent is bacteria-derived IdeS or IdeZ.

[0321] In an aspect, a disclosed method of treating can comprise repeating a disclosed administering step such as, for example, repeating the administering of a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed immunosuppressive agent, a disclosed compound that exerts a therapeutic effect against B cells and / or a disclosed compound that targets or alters antigen presentation or humoral or cell mediated immune response.

[0322] In an aspect, a disclosed method of treating can comprise modifying one or more of the disclosed steps. For example, modifying one or more of steps of a disclosed method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of one or more of thedisclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof administered to a subject, or by changing the frequency of administration of one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof to a subject, or by changing the duration of time one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof are administered to a subject.

[0323] In an aspect, a disclosed method of treating can be altered by changing the amount of one or more disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and / or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject, or by changing the frequency of administration of one or more of the disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and / or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject.

[0324] In as aspect, a disclosed method of treating can comprise concurrent administration of one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, , one or more disclosed therapeutic agents, one or more disclosed immune modulators, one or more disclosed proteasome inhibitors, one or more disclosed immunosuppressive agents, one or more disclosed compounds that exert therapeutic effect against B cells, one or more disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response, or any combination thereof. In an aspect, a disclosed immune modulator can be administered prior to or after the administration of a disclosed therapeutic agent.

[0325] In an aspect, a disclosed method of treating a genetic disease or disorder can further comprise generating a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof.

[0326] In an aspect, a disclosed method of treating can further comprise gene editing one or more relevant genes (such as, for example, a missing, deficient, and / or mutant protein or enzyme), wherein editing includes but is not limited to single gene knockout, loss of function screening of multiple genes at one, gene knockin, or a combination thereof.

[0327] In an aspect, a disclosed method of treating a genetic disease or disorder can further comprise administering to the subject an oligonucleotide therapeutic agent. A disclosed oligonucleotide therapeutic agent can comprise a single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), an antisense molecule, miRNA, a morpholino, a peptide-nucleic acid (PNA), or an analog or conjugate thereof. In an aspect, a disclosed oligonucleotide therapeutic agent can be an ASO or an RNAi. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise one or more modifications at any position applicable. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise a CRISPR- based endonuclease. In an aspect, a disclosed endonuclease can be Cas9. In an aspect, a disclosed Cas9 can be from Staphylococcus aureus or Streptococcus pyogenes. In an aspect, a disclosed method of treating can comprise administering the subject a disclosed RNA therapeutic.

[0328] In an aspect, a disclosed method of treating a genetic disease or disorder can further comprise generating and / or validating one or more disclosed ER stress sensor variants, one or more disclosed XBP1 fragments, one or more disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof. In an aspect, a disclosed method of treating can inhibit and / or minimize one or more aspects of disease progression in the subject (e.g., a genetic disease or disorder described supra). In an aspect, a disclosed method of treating can slow and / or diminish one or more aspects of disease progression in the subject (e.g., a genetic disease or genetic disorder described supra).

[0329] In an aspect, a disclosed method of treating can inhibit and / or minimize disease progression. In an aspect of a disclosed method of inhibiting and / or minimizing disease progression, expression of a disclosed protein coding gene can be restored and / or returned to a wild-type, normal, or control expression level. In an aspect, a disclosed method of inhibiting and / or minimizing disease progression of a genetic disease or disorder can restore the functionality and / or structural integrity of a missing, deficient, and / or mutant protein or enzyme. In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can comprise restoring one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation. In an aspect, restoring one or more aspect of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation comprises restoring the functionality and / or structural integrity of a missing, deficient, and / or mutant protein or enzyme.

[0330] In an aspect, restoring one or more aspects of cellular homeostasis and / or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and / or ameliorating autophagy); (iii) improving, enhancing,restoring, and / or preserving mitochondrial functionality and / or structural integrity; (iv) improving, enhancing, restoring, and / or preserving organelle functionality and / or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of the multi-systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and / or preserving one or more aspects of cellular structural and / or functional integrity. In an aspect, restoring the activity and / or functionality of a missing, deficient, and / or mutant protein or enzyme (e.g., encoded by a protein coding gene described supra) can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and / or mutant protein or enzyme). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and / or functionality is similar to that of a wild -type or control level.

[0331] In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can further comprise monitoring the subject’s metabolic and / or physiologic improvement following the administering step and / or following the administering steps. In an aspect, a clinician can measure and / or determine the subject’s metabolic and / or physiologic status over time to identify one or more improvements and / or one or more diminishments. In an aspect of a disclosed method, a clinician can use the subject’s metabolic and / or physiologic status and / or the trend of the subject’s metabolic and / or physiological status and / or trend to make a treatment decision and / or to modify an aspect of a disclosed method and / or to continue treating the subject and / or continue to administer a disclosed AAV particle, a disclosed vector, a disclosed nucleic acid molecule, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, and / or a disclosed immune modulator, or any combination thereof. In an aspect, metabolic and / or physiologic data can inform the clinician and a treatment plan.

[0332] In an aspect of a disclosed method of inhibiting and / or minimizing disease progression, techniques to monitor, measure, and / or assess the restoring one or more aspects of cellular homeostasis and / or cellular functionality can comprise qualitative (or subjective) means as wellas quantitative (or objective) means. These means are known to the skilled person. For example, representative regulated variables and sensors relating to systemic homeostasis are provided supra.

[0333] In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can further comprise subjecting the subject to one or more invasive or non-invasive diagnostic assessments. Diagnostic assessments are known to the art. In an aspect, a disclosed non-invasive diagnostic assessment can comprise x-rays, computerized tomography (CT) scans, magnetic resonance imaging (MRI) scans, ultrasounds, positron emission tomography (PET) scans, or any combination thereof. In an aspect, a disclosed invasive diagnostic assessment can comprise a tissue biopsy or exploratory surgery.

[0334] In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can be used to repair diseased and / or dysfunctional cell types in the one or more diseased and / or disordered cells, tissues, and / or organs.

[0335] In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can be used to improve and / or can be used to enhance the quality of the subject’s life when compared to a pre-treatment level. In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can be used to improve the subject’s quality of life by at least 50% when compared to the subject’s pre-treatment quality of life. In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can be used to improve and / or can be used to enhance the quality of the subject’s life when compared to a pre-treatment level. In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can be used to improve the subject’s quality of life by at least 50% when compared to the subject’s pre-treatment quality of life.

[0336] In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can be used to diminish and / or decrease one or more symptoms associated with and / or related to the subject’s genetic disease and / or genetic disorder. In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can be used to prevent an undesired physiological change, disease, pathological condition, or disorder from occurring in the subject. In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can be used to inhibit a physiological change, disease, pathological condition, or disorder, i.e., arresting its development, in the subject. In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can be used to relieve a physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease, in the subject.

[0337] In an aspect of a disclosed method of inhibiting and / or minimizing disease progression, administering to the subject can comprise contacting one or more cells with one or more disclosed ER stress sensor variants, one or more disclosed XBP1 fragments, one or more disclosed nucleicacid molecules, disclosed vectors or disclosed AAV particles, disclosed pharmaceutical formulations, or any combination thereof.

[0338] In an aspect of a disclosed method of inhibiting and / or minimizing disease progression, administering of a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof can comprise one or more routes. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can also include hepatic intraarterial administration or administration through the hepatic portal vein (HPV). Administration of a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination can comprise administration directly into the CNS (e.g., intraparenchymal, intracerebroventricular, intrathecal cisternal, intrathecal (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or the PNS. Administration can be continuous or intermittent.

[0339] In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can employ multiple routes of administration to the subject. In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can employ a first route of administration that can be the same or different as a second and / or subsequent routes of administration.

[0340] In an aspect of a disclosed method of inhibiting and / or minimizing disease progression of a genetic disease or disorder, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a range of about 1 x IO10vg / kg to about 2 x 1014vg / kg. In an aspect, for example, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a dose of about 1 x 1011vg / kg to about 8 x 1013vg / kg or about 1 x 1012vg / kg to about 8 x 1013vg / kg. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a dose of about 1 x 1013vg / kg to about 6 x 1013vg / kg. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a dose of at least about 1 x IO10vg / kg, at least about 5 x IO10vg / kg, at least about 1 x 1011vg / kg, at least about 5 x 1011vg / kg, at least about 1 x 1012vg / kg, at least about 5 x 1012vg / kg, at least about 1 x 1013vg / kg, at least about 5 x 1013vg / kg, or at least about 1 x 1014vg / kg. In an aspect, a therapeutically effective amountof disclosed vector or a disclosed pharmaceutical formulation can comprise a dose of no more than about 1 x IO10vg / kg, no more than about 5 x IO10vg / kg, no more than about 1 x 1011vg / kg, no more than about 5 x 1011vg / kg, no more than about 1 x 1012vg / kg, no more than about 5 x 1012vg / kg, no more than about l x 1013vg / kg, no more than about 5 x 1013, or no more than about 1 x 1014vg / kg. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a dose of about 1 x 1012vg / kg or about 1 x 1011vg / kg. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results.

[0341] In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a range of about 1 x 1012vg per subject total to about 1 x 1017vg per subject total. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a range of about 1 x 1012vg per subject total, about 1 x 1013vg per subject total, about 1 x 1014vg per subject total, about 1 x 1015vg per subject total, about 1 x 1016vg per subject total, or about 1 x 1017vg per subject total.

[0342] In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise about 1 x 106DRP / mL to about 1 x 1014DRP / mL. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise about 1 x 106DRP / mL, 1 x 107DRP / mL, 1 x 108DRP / mL, 1 x 109DRP / mL, 1 x IO10DRP / rnL, 1 x 1011DRP / rnL, 1 x 1012DRP / rnL, 1 x 1013DRP / rnL, or 1 x 1014DRP / mL. In an aspect, a therapeutically effective amount of disclosed vector or a disclosed pharmaceutical formulation can comprise a range determined by a skilled person.

[0343] In an aspect, a disclosed method of inhibiting and / or minimizing disease progression of a genetic disease or disorder can further comprise administering to the subject a therapeutically effective amount of a therapeutic agent. A therapeutic agent can be any disclosed agent that effects a desired clinical outcome.

[0344] In an aspect, a disclosed method of inhibiting and / or minimizing disease progression of a genetic disease or disorder can further comprise monitoring the subject for adverse effects. In an aspect, in the absence of adverse effects, the method can further comprise continuing to treat the subject. In an aspect, in the presence of adverse effects, the method can further comprise modifying the treating step. Methods of monitoring a subject’s well-being can include both subjective and objective criteria (and are discussed supra). Such methods are known to the skilled person.

[0345] In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can further comprise administering to the subject a therapeutically effective amount of an agent thatcan correct one or more aspects of a dysregulated metabolic or enzymatic pathway. In an aspect, such an agent can comprise an enzyme for enzyme replacement therapy. In an aspect, a disclosed enzyme can replace any enzyme in a dysregulated or dysfunctional metabolic or enzymatic pathway. In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can comprise replacing one or more enzymes in a dysregulated or dysfunctional metabolic pathway.

[0346] In an aspect, a disclosed method of inhibiting and / or minimizing disease progression of a genetic disease or disorder can further comprise administering one or more immune modulators. In an aspect, a disclosed immune modulator can be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or a combination thereof. In an aspect, a disclosed immune modulator can be bortezomib or SVP-Rapamycin. In an aspect, a disclosed immune modulator can be Tacrolimus. In an aspect, a disclosed immune modulator such as methotrexate can be administered at a transient low to high dose. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.1 mg / kg body weight to about 0.6 mg / kg body weight. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.4 mg / kg body weight. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg / kg body weight for 3 to 5 or greater cycles, with up to three days per cycle. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg / kg body weight for a minimum of 3 cycles, with three days per cycle. In an aspect, a person skilled in the art can determine the appropriate number of cycles. In an aspect, a disclosed immune modulator can be administered as many times as necessary to achieve a desired clinical effect.

[0347] In an aspect, a disclosed immune modulator can be administered orally about one hour before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered orally about one hour or a few days before a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before or a few days before a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof.

[0348] In an aspect, a disclosed method of inhibiting and / or minimizing disease progression of a genetic disease or disorder can further comprise administering one or more proteasome inhibitors(e.g., bortezomib, carfilzomib, marizomib, ixazomib, and oprozomib). In an aspect, aproteasome inhibitor can be an agent that acts on plasma cells (e.g., daratumumab). In an aspect, an agent that acts on a plasma cell can be melphalan hydrochloride, melphalan, pamidronate disodium, carmustine, carfilzomib, carmustine, cyclophosphamide, daratumumab, doxorubicin hydrochloride liposome, doxorubicin hydrochloride liposome, elotuzumab, melphalan hydrochloride, panobinostat, ixazomib citrate, carfilzomib, lenalidomide, melphalan, melphalan hydrochloride, plerixafor, ixazomib citrate, pamidronate disodium, panobinostat, plerixafor, pomalidomide, pomalidomide, lenalidomide, selinexor, thalidomide, thalidomide, bortezomib, selinexor, zoledronic acid, or zoledronic acid.

[0349] In an aspect, a disclosed method of inhibiting and / or minimizing disease progression stability can further comprise administering one or more proteasome inhibitors or agents that act on plasma cells prior to administering a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof. In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells concurrently with administering a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination of thereof. In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells subsequent to administering a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof. In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can further comprise administering one or more proteasome inhibitors more than 1 time. In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can comprise administering one or more proteasome inhibitors repeatedly over time.

[0350] In an aspect, a disclosed method of inhibiting and / or minimizing disease progression of a genetic disease or disorder can further comprise administering one or more immunosuppressive agents. In an aspect, an immunosuppressive agent can be, but is not limited to, azathioprine, methotrexate, sirolimus, anti -thymocyte globulin (ATG), cyclosporine (CSP), my cophenolate mofetil (MMF), steroids, or a combination thereof. In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can comprise administering one or more immunosuppressive agents more than 1 time. In an aspect, a disclosed method can comprise administering one or more one or more immunosuppressive agents repeatedly over time. In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can compriseadministering a compound that targets or alters antigen presentation or humoral or cell mediated or innate immune responses.

[0351] In an aspect, a disclosed method of inhibiting and / or minimizing disease progression of a genetic disease or disorder can further comprise administering a compound that exerts a therapeutic effect against B cells and / or a compound that targets or alters antigen presentation or humoral or cell mediated immune response. In an aspect, a disclosed compound can be rituximab, methotrexate, intravenous gamma globulin, anti CD4 antibody, anti CD2, an anti-FcRN antibody, a BTK inhibitor, an anti-IGFIR antibody, a CD19 antibody (e.g., inebilizumab), an anti-IL6 antibody (e.g., tocilizumab), an antibody to CD40, an IL2 mutein, or a combination thereof. Also disclosed herein are Treg infusions that can be administered as a way to help with immune tolerance (e.g., antigen specific Treg cells to AAV).

[0352] In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can further comprise administering lipid nanoparticles (LNPs). In an aspect, LNPs can be organ- targeted. In an aspect, LNPs can comprise one or more disclosed nucleic acid molecules, one or more disclosed vectors, and / or one or more disclosed pharmaceutical formulations. For example, in an aspect, mRNA therapy with LNP encapsulation for systemic delivery to a subject has the potential to restore the functionality and / or structural integrity of a missing, deficient, and / or mutant protein or enzyme.

[0353] In an aspect, a disclosed method of inhibiting and / or minimizing disease progression of a genetic disease or disorder can further comprise treating a subject that has developed or is likely to develop neutralizing antibodies (ABs) to a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof. In an aspect, treating a subject that has developed or is likely to develop neutralizing antibodies can comprise plasmapheresis and immunosuppression. In an aspect, a disclosed method can comprise using immunosuppression to decrease the T cell, B cell, and / or plasma cell population, decrease the innate immune response, inflammatory response, and antibody levels in general. In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can comprise administering an IgG-degrading agent that depletes pre-existing neutralizing antibodies. In an aspect, a disclosed method can comprise administering to the subject IdeS or IdeZ, rapamycin, and / or SVP -Rapamycin. In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can comprise administering Tacrolimus. In an aspect, a disclosed IgG-degrading agent is bacteria-derived IdeS or IdeZ.

[0354] In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can comprise repeating a disclosed administering step such as, for example, repeating the administering of a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle,a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed immunosuppressive agent, a disclosed compound that exerts a therapeutic effect against B cells and / or a disclosed compound that targets or alters antigen presentation or humoral or cell mediated immune response.

[0355] In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can comprise modifying one or more of the disclosed steps. For example, modifying one or more of steps of a disclosed method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof administered to a subject, or by changing the frequency of administration of one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof to a subject, or by changing the duration of time one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof are administered to a subject.

[0356] In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can be altered by changing the amount of one or more disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and / or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject, or by changing the frequency of administration of one or more of the disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and / or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject.

[0357] In as aspect, a disclosed method of inhibiting and / or minimizing disease progression can comprise concurrent administration of one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, , one or more disclosed therapeutic agents, one or more disclosed immune modulators, one or more disclosed proteasome inhibitors, one or more disclosed immunosuppressive agents, one or more disclosed compounds that exert therapeutic effect against B cells, one or more disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response, or any combination thereof. In an aspect, a disclosed immunemodulator can be administered prior to or after the administration of a disclosed therapeutic agent. In an aspect, a disclosed method of inhibiting and / or minimizing disease progression of a genetic disease or disorder can further comprise generating a disclosed nucleic acid molecule, a disclosed vector, a disclosed AAV particle, a disclosed pharmaceutical formulation, or any combination thereof.

[0358] In an aspect, a disclosed method of inhibiting and / or minimizing disease progression can further comprise gene editing one or more relevant genes (described supra).

[0359] In an aspect, under ER stress, ERN (aka IRE la) splices a fragment comprising the sequence of SEQ ID NO:61 from a disclosed nucleic acid molecule.

[0360] In an aspect, a disclosed method can further comprise administering one or more inhibitors of UPR / ER stress (e.g., Eeyarestatin I, GSK 2606414, Salubrinal, or any combination thereof).

[0361] In an aspect, a disclosed method can further comprise generating and / or validating a disclosed ER stress sensor variant. In an aspect, a disclosed method can further comprise generating and / or validating a disclosed XBP1 fragment. In an aspect, a disclosed method can further comprise generating and / or validating a disclosed nucleic acid molecule. In an aspect, a disclosed method can further comprise generating and / or validating a disclosed vector. In an aspect, a disclosed method can further comprise generating and / or validating a disclosed pharmaceutical formulation. In an aspect, a disclosed method can further comprise generating and / or validating a disclosed plasmid.

[0001] Use of one or more disclosed compositions (e.g., disclosed ER stress sensor variants, disclosed XBP1 fragments, disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or any combination thereof) for the manufacture of a medicament for the treatment of a genetic disease or genetic disorder. Use of one or more disclosed compositions (e.g., disclosed ER stress sensor variants, disclosed XBP1 fragments, disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or any combination thereof) for the manufacture of a medicament for the treatment of a disease or disorder requiring gene therapy. A disclosed pharmaceutical composition comprising one or more disclosed ER stress sensor variants, disclosed XBP1 fragments, disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or any combination thereof for use in the treatment of a genetic disease or genetic disorder. A disclosed pharmaceutical composition comprising one or more disclosed ER stress sensor variants, disclosed XBP1 fragments, disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or any combination thereof for use in the treatment of a disease or disorder requiring gene therapy.E. Miscellaneous

[0362] Disclosed herein is an isolated nucleic acid molecule comprising a promoter operably linked to one or more sequences; a variant XBP1 sequence; and a sequence encoding a transgene. In an aspect, a disclosed isolated nucleic acid molecule can further comprise a linker sequencer. In an aspect, a disclosed isolated nucleic acid molecule can further comprise a polyad enylation (poly A) sequence. In an aspect, a disclosed linker sequence can separate the variant XBP1 sequence from the transgene. In an aspect, a disclosed linker sequence can comprise a non-selfcleaving peptide sequence. In an aspect, a disclosed linker sequence can comprise a self-cleaving peptide sequence. In an aspect, a disclosed self-cleaving peptide sequence can comprise a selfcleaving viral 2A peptide. In an aspect, a disclosed viral 2A peptide can comprise a porcine teschovirus-1 (P2A) peptide, a Thosea asigna virus (T2A) peptide, an equine rhinitis A virus (E2A) peptide, or a foot-and-mouth disease virus (F2A) peptide. In an aspect, a disclosed variant XBP1 sequence can be truncated when compared to the wild -type XBP1 sequence. In an aspect, a disclosed variant XBP1 sequence can comprise the sequence set forth in any of SEQ ID NO:01 - SEQ ID NO:06. In an aspect, a disclosed isolated nucleic acid molecule can further comprise one or more signal peptides. In an aspect, one or more disclosed signal peptides can comprise human signal peptides. In an aspect, the one or more disclosed signal peptides can comprise OSM, VSV-G, BM40, secrecon, IgKVIII, CD33, tPA, chymotrypsinogen, trypsinogen-2, IL-2, gaussia luc, albumin (HSA), influenza haemagglutinin, insulin, Silkworm Fibroin LC, or any combination thereof. In an aspect, a disclosed XBP1 variant can comprise (i) an HR1 -intron sequence, (ii) an HRl-intron-HR2 sequence, (iii) an HR l-intron-HR2 -arrest sequence, (iv) an HRl-intron- calrecticulin signal peptide-arrest sequence, or(v) an HRl-intron-intron-HR2-arrest sequence. In an aspect, a disclosed nucleic acid molecule can comprise from 5’ to 3’ the promoter operably linked to the one or more sequences, the XBP1 variant sequence, the self-cleaving peptide sequence, the transgene, and the poly A sequence. In an aspect, a disclosed variant XBP 1 sequence can render the expression of the encoded transgene amenable to unfolded protein response- mediated regulation. In an aspect, a disclosed transgene can encode a desired cargo, a polypeptide or an RNA, a monoclonal antibody, a recombinant monoclonal antibody, an engineered monoclonal antibody, a polypeptide or an RNA, a recombinant polypeptide or a recombinant RNA, or a therapeutic polypeptide or a therapeutic RNA. In an aspect, a disclosed encoded RNA can comprise ribosomal RNA (rRNA), transfer RNA (tRNA), heterogeneous nuclear RNA (hnRNA), small nuclear RNA(snRNA), small nucleolar RNA(snoRNA), micro RNA(miRNA), Piwi-interacting RNA (piRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), singe guide RNA (sgRNA), non-coding RNA (ncRNA), long non-coding RNA (lncRNA), 7SL, Xist, short enhancer RNA (eRNA), circular RNA, intergenic RNA, or any combination thereof.In an aspect, a disclosed encoded RNA can comprise IncRNA, siRNA, shRNA, sgRNA, circular RNA, snoRNA, miRNA, or any combination thereof. In an aspect, a disclosed transgene can encode a missing, deficient, and / or mutant protein or enzyme or can encode a gene-editing molecule. In an aspect, a disclosed gene-editing molecule can comprise a nuclease (such as, for example, a Cas9 nuclease). In an aspect, a gene-editing molecule can comprise a single guide RNA (sgRNA), such as, for example a single guide RNA (sgRNA) targeting an endogenous gene in one or more organs. In an aspect, a disclosed transgene can encode the protein or a portion thereof associated with any gene disclosed herein. In an aspect, a disclosed isolated nucleic acid molecule can be used in a delivery agnostic method. In an aspect, a disclosed isolated nucleic acid molecule can be used in a modular method.

[0363] Disclosed herein is a non-viral vector comprising a disclosed isolated nucleic acid molecule. Disclosed herein is a viral vector comprising a disclosed isolated nucleic acid molecule. Disclosed herein is a recombinant adeno-associated virus (AAV) vector comprising a disclosed isolated nucleic acid molecule. Disclosed herein is a pharmaceutical formulation comprising a disclosed isolated nucleic acid molecule and a pharmaceutically acceptable carrier. Disclosed herein is a pharmaceutical formulation comprising a disclosed vector and a pharmaceutically acceptable carrier. Disclosed herein is a method of maintaining homeostasis the method comprising administering to a subject in need thereof a disclosed pharmaceutical formulation, wherein, following expression, the encoded transgene restores one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation in one or more cells of the subject, and wherein, following expression the XBPl variant, homeostasis in the endoplasmic reticulum of the one or more cells of the subject expressing the encoded transgene is maintained.

[0364] In an aspect, restoring one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation can comprise restoring the functionality and / or structural integrity of a missing, deficient, and / or mutant protein or enzyme. In an aspect, following expression of the XBP1 variant, ER stress can be minimized and / or reduced. In an aspect, following expression of the XBP1 variant, the likelihood of survival in the one...

Claims

VIII. CLAIMSWhat is claimed is:

1. A nucleic acid molecule, comprising: an endoplasmic reticulum (ER) stress sensor variant; a promoter operably linked to the ER stress sensor variant; and a sequence encoding a transgene.

2. The nucleic acid molecule of Claim 1, wherein the ER stress sensor variant comprises a X-BoxBinding Protein 1 (XBP1) fragment.

3. The nucleic acid molecule of Claim 2, wherein the XBPl fragment comprises the sequence set forth in any of SEQ ID NO:01 - SEQ ID NO:06.

4. The nucleic acid molecule of Claim 2, wherein the XBPl fragment comprises the sequence set forth in SEQ ID NO:61.

5. The nucleic acid molecule of any preceding claim, further comprising a linker sequence.

6. The nucleic acid molecule of Claim 5, wherein the linker sequence separates the ER stress sensor variant from the sequence encoding the transgene.

7. The nucleic acid molecule of Claim 5, wherein the linker sequence comprises a non-selfcleaving peptide sequence or a self -cleaving peptide sequence.

8. The nucleic acid molecule of Claim 5, wherein the linker sequence comprises the sequence set forth in any one of SEQ ID NO:62 - SEQ ID NO:66.

9. The nucleic acid molecule of any preceding claim, further comprising one or more signal peptides.

10. The nucleic acid molecule of Claim 9, wherein the signal peptides comprise the sequence set forth in any one of SEQ ID NO:37 - SEQ ID NO:53.

11. The nucleic acid molecule of any preceding claim, further comprising a polyad enylation(poly A) sequence.

12. The nucleic acid molecule of any one of Claims 1 - 11, wherein the transgene encodes a polypeptide, a recombinant polypeptide, or a therapeutic polypeptide.

13. The nucleic acid molecule of any one of Claims 1 - 11, wherein the transgene encodes anRNA, a recombinant RNA, or a therapeutic RNA.

14. The nucleic acid molecule of any one of Claims 1 - 11, wherein the transgene encodes a monoclonal antibody, a recombinant monoclonal antibody, an engineered monoclonal antibody, or a therapeutic monoclonal antibody.

15. The nucleic acid molecule of any one of Claims 1 - 11, wherein the transgene encodes a component of a gene-editing system.

16. The nucleic acid molecule of Claim 15, wherein the gene-editing system comprises zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases, or clustered regularly interspaced short palindromic repeats (CRISPR).

17. The nucleic acid molecule of Claim 15, wherein the component of a gene-editing system comprises a guide RNA (gRNA) or a single guide RNA (sgRNA).

18. The nucleic acid molecule of Claim 17, wherein the gRNA or the sgRNA targets a gene comprising one or more mutations.

19. The nucleic acid molecule of Claim 18, wherein the gene comprising one or more mutations encodes a dysfunctional or mutant enzyme or protein.

20. The nucleic acid molecule of any preceding claim, wherein, upon expression in a cell, protein folding capacity in the cell's endoplasmic reticulum is improved and / or enhanced.

21. The nucleic acid molecule of any one of Claims 1 - 20, encapsulated in a lipid nanoparticle.

22. A viral vector, comprising: the nucleic acid molecule of any one of Claims 1 - 20.

23. An adeno-associated virus (AAV) vector, comprising: the nucleic acid molecule of any one ofClaims 1 - 20.

24. A pharmaceutical formulation, comprising: the nucleic acid molecule of any one of Claims 1 - 21 and a pharmaceutically acceptable carrier.

25. A pharmaceutical formulation, comprising: the vector of Claim 22 or Claim 23 and a pharmaceutically acceptable carrier.

26. A method of treating a subject, the method comprising: administering to a subject in need thereof a pharmaceutical formulation of Claim 24 or 25, wherein, following expression, the encoded transgene restores one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation in one or more cells of the subject, and wherein the ER stress sensor variant ensures that homeostasis in the endoplasmic reticulum of the one or more cells of the subject expressing the encoded transgene is maintained.

27. The method of Claim 26, wherein restoring one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation comprises restoring the functionality and / or structural integrity of a missing, deficient, and / or mutant protein or enzyme.

28. The method of Claim 26 or Claim 27, wherein the ER stress sensor variant (i) grants UPR- mediated regulation to the transgene mRNA, (ii) grants UPR-mediated regulation to the encoded protein or encoded RNA, (iii) renders the expression of the encoded transgeneamenable to UPR-mediated regulation, (iv) avoids activation of a terminal UPR, (v) avoids activation of an apoptotic UPR, or (vi) any combination thereof.

29. The method of any one of Claims 26 - 28, wherein, under ER stress, the ER stress sensor variant (i) generates a non-functional transgene mRNA, (ii) increases and / or improves the likelihood of survival in the one or more cells of the subject, (iii) increases and / or improves long term expression of the transgene in the one or more cells of the subject, (iv) decreases and / or minimizes the likelihood of apoptosis in the one or more cells of the subject, or (v) any combination thereof.

30. The method of any one of Claims 26 - 29, wherein the subject has a genetic disease or genetic disorder.

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