WO2024137857A1 - Conditional expression of a gene of interest by convergent promoters and uses thereof - Google Patents

Abstract

The disclosure relates generally to nucleic acid compositions, e.g., expression vectors, for conditionally expressing a gene of interest, e.g., a non-coding gene, using convergently-aligned promoters, and methods of use thereof.

Description

Attorney Docket No.: TVD-009WO CONDITIONAL EXPRESSION OF A GENE OF INTEREST BY CONVERGENT PROMOTERS AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63/476851, filed December 22, 2022, the entire disclosure of which is hereby incorporated by reference in its entirely for all purposes. FIELD OF THE DISCLOSURE [0002] The disclosure relates generally to nucleic acid compositions, e.g., expression vectors and expression systems, for conditionally expressing a gene of interest, e.g., a non-coding gene, by convergently-aligned promoters, and methods of use thereof. BACKGROUND [0003] Protein synthesis is directed by a genetic code that includes 61 three-base-pair codons encoding amino acids that are incorporated into the protein being synthesized and 3 three-base- pair codons (referred to as stop or termination codons) that terminate the synthesis of a protein. When a nucleic acid sequence encoding a protein is mutated to contain a premature termination codon rather than a codon for the next amino acid, the resulting protein is prematurely terminated, which is often nonfunctional or less functional than the untruncated or full length protein. Such mutations, termed nonsense mutations, are often associated with, or are a causative agent in numerous different genetic diseases. [0004] A number of disorders are associated with, or are caused by, nonsense mutations. These include epilepsies, for example, Dravet Syndrome, Genetic Epilepsy with Febrile Seizures (GEFS), Benign Familial Infantile Epilepsy (BFIE), Early Infantile Epileptic Encephalopathy (EIEE), Lennox-Gastaut Syndrome, Rett Syndrome, PPM-X Syndrome, Ohtahara Syndrome, Episodic Ataxia, Hemiplegic Migraine, Idiopathic Generalized Epilepsy, FOXG1 Syndrome, Familial Focal Epilepsy with Variable Foci (FFEVF), Childhood-Onset Epileptic Encephalopathy, SYNGAP1-Related Intellectual Disability, Pyridoxine-Dependent Epilepsy, Familial Infantile Myoclonic Epilepsy (FIME), Myoclonic Astatic Epilepsy, X-Linked Intellectual Disability, Partial Epilepsy and Episodic Ataxia, Febrile Seizures, Autosomal Dominant Partial Epilepsy with Auditory Features (ADPEAF), PNPO-Deficiency, Progressive Myoclonus Epilepsy, Action Myoclonus – Renal Failure (AMRF), CDKL5 deficiency disorder, and Benign Familial Infantile Seizures (BFIS). Attorney Docket No.: TVD-009WO [0005] By way of example, Dravet Syndrome is a rare and catastrophic form of intractable epilepsy that begins in infancy. Initially, patients experience prolonged seizures. In their second year, additional types of seizure begin to occur, which typically coincide with a developmental decline, possibly due to repeated cerebral hypoxia. This leads to poor development of language and motor skills. Mutations in SCN1A (encoding the voltage-gated sodium channel α subunit Nav1.1), SCN1B (encoding the voltage-gated sodium channel β1 subunit), SCN2A (encoding Nav1.2), SCN3A (encoding Nav1.3), SCN9A (encoding Nav1.7), GABRG2 (encoding the γ- aminobutyric acid receptor γ2 subunit), GABRD (encoding the γ-aminobutyric acid receptor Δ subunit) and/or PCDH19 (encoding Protocadherin-19) genes have been linked to Dravet Syndrome. [0006] Dravet syndrome may be caused by a nonsense mutation in, for example, the SCN1A gene, resulting in a premature termination codon and a lack of or reduced amount of untruncated or functional protein. The SCN1A gene normally codes for the neuronal voltage-gated sodium channel α subunit, Na(V)1.1. In mouse models, loss-of-function mutations in SCN1A have been observed to result in a decrease in sodium currents and impaired excitability of GABAergic interneurons of the hippocampus. [0007] Despite the efforts made to date, there is a need in the art for improved compositions and methods for treating disorders mediated by premature termination codons, such as Dravet syndrome. SUMMARY OF THE DISCLOSURE [0008] Although approaches have been developed for treating certain genetic disorders, such as premature stop codon-mediated disorders, in order to make such approaches clinically viable there exists an ongoing need for expression systems that reduce or eliminate off-target toxicity by regulating expression of genes of interest in specific environments, for example, in specific tissues of interest in a subject. In addition, there is an ongoing need for improved viral-based delivery approaches including enhanced viral packaging techniques, so that sufficient amounts of genes of interest (e.g., a gene encoding a non-coding RNA (ncRNA)) can be packaged in a given viral particle to permit the delivery of enough genes to treat the genetic disorder in the subject, where the genes of interest can also be expressed in a tissue specific manner. Described herein is an expression system using, among other things, convergently-aligned promoters, that can be used in reducing off-target toxicity by regulation of the GOI when utilizing a tissue- or cell type-specific promoter or a stress-responsive-promoter and/or facilitates efficient packaging of genes of interest (e.g., a gene encoding a ncRNA) into a viral particle for gene therapy. Attorney Docket No.: TVD-009WO [0009] In one aspect, the disclosure provides an expression vector comprising: (a) a first promoter; (b) a second promoter; and (c) a gene of interest comprising an antisense strand encoding a non-coding RNA (ncRNA) and a complementary sense strand. The complementary strand encodes a complementary non-functional antisense ncRNA. The first promoter is transcriptionally operative in a first direction to transcribe the antisense strand of the gene of interest and produce the ncRNA. The second promoter is transcriptionally operative in a second direction opposite to the first direction of the first promoter to transcribe the sense strand of the gene of interest. The second promoter is a tissue- or cell type-specific promoter, a stress- responsive promoter, and/or a human promoter. The transcriptional activity of the second promoter can be regulated to interfere with transcriptional activity of the first promoter and reduce production of the ncRNA. [0010] When the expression vector is to be incorporated into a viral particle, the expression vector may also include additional elements to facilitate the integration of the expression vector into the viral particle. For example, when the expression vector is incorporated in an adeno- associated virus (AAV), the expression vector can further comprise AAV inverted terminal repeats (ITRs) flanking the first promoter and the second promoter. [0011] In another aspect, the disclosure provides an expression vector (e.g., an AAV viral expression vector) comprising (a) a first promoter; (b) a second promoter; and (c) a gene of interest comprising an antisense strand encoding a non-coding RNA (ncRNA) and a complementary sense strand. The complementary strand encodes a complementary non- functional antisense ncRNA. The first promoter is transcriptionally operative in a first direction to transcribe the antisense strand of the gene of interest and produce the ncRNA. The second promoter is transcriptionally operative in a second direction opposite to the first direction of the first promoter to transcribe the sense strand of the gene of interest. The transcriptional activity of the second promoter can be regulated to interfere with transcriptional activity of the first promoter and reduce production of the ncRNA. The expression vector is an AAV vector that further comprises AAV ITRs flanking the first promoter and the second promoter. [0012] In certain embodiments of the foregoing expression vectors, the second promoter can comprise a conditional promoter, e.g., a tissue- or cell type-specific promoter. The tissue- specific promoter can comprise, e.g., a liver-specific, heart-specific, muscle-specific, retinal- specific, inner ear-specific, spinal cord-specific, or dorsal root ganglion-specific promoter. In certain embodiments, the second promoter can comprise a plurality of tissue-specific promoters. Attorney Docket No.: TVD-009WO [0013] In certain embodiments of the foregoing expression vectors, the second promoter comprises a stress-responsive promoter and/or an endogenous human promoter. [0014] In certain embodiments of the foregoing expression vectors, the second promoter is a conditional promoter, and the conditional promoter comprises an inducible promoter. The inducible promoter can be selected from, for example, a tetracycline-inducible promoter (e.g., a Tet-On or Tet-Off promoter), a Lac-inducible promoter, a Bad-inducible promoter, a temperature-inducible promoter, a light-inducible promoter, and a CRISPR/Cas-based promoter. [0015] In certain embodiments of any of the foregoing expression vectors, the second promoter comprises an RNA polymerase II promoter. [0016] In certain embodiments of any of the foregoing expression vectors, the first promoter comprises an RNA Polymerase III promoter e.g., a gene-internal type 1 RNA Polymerase III promoter, a gene-internal type 2 RNA Polymerase III promoter or a gene-external type 3 RNA Polymerase III promoter. Additionally or alternatively, the RNA Polymerase III promoter comprises a synthetic hybrid promoter. [0017] In certain embodiments, the expression vector further comprises a third promoter. In certain embodiments, the third promoter is disposed upstream of the first promoter and transcriptionally operative in the first direction opposite to the second direction of the second promoter. In certain embodiments, the third promoter comprises an RNA Polymerase III promoter. [0018] In certain embodiments, the expression vector further comprises a second gene of interest (or a plurality of genes of interest) disposed between the first promoter and the second promoter. Each gene of interest can be operatively linked to a Polymerase III promoter transcriptionally operative to transcribe the gene of interest. [0019] In certain embodiments, the ncRNA is selected from the group consisting of a tRNA, an siRNA, an shRNA, an sgRNA, an miRNA, a piRNA, a snoRNA, an snRNA, and a lncRNA. In some embodiments, the ncRNA is a tRNA, e.g., a suppressor tRNA. Exemplary tRNA suppressors comprise a nucleotide sequence set forth in TABLE 4 or TABLE 5. Alternatively or in addition, an exemplary tRNA suppressor comprises (i) a nucleotide sequence selected from any one of SEQ ID NOs: 6-9, 11, 16-22, and 35 (e.g., SEQ ID NOs: e.g., 6, 8, 17, 18, and 22), (ii) a nucleotide sequence selected from any one of SEQ ID NOs: 178-182, 186, and 187 (e.g., SEQ ID NOs: 178 and 181), or (iii) a nucleotide sequence selected from any one of SEQ ID NOs: 36-40, 44, and 45 (e.g., SEQ ID NOs: 36 and 39). In certain embodiments, the expression Attorney Docket No.: TVD-009WO vector comprises 1, 2, 3, 4, or more than 4 nucleotide sequences each encoding the same suppressor tRNA. [0020] In certain embodiments, the expression vector further comprises a nucleotide sequence set forth in TABLE 6 disposed immediately upstream of the suppressor tRNA, e.g., a nucleotide sequence selected from any one of SEQ ID NOs: 869-888. [0021] In certain embodiments, a suppressor tRNA is flanked by a nucleotide sequence set forth in TABLE 6, for example, a combination of a sequence positioned 5’ to the tRNA and a sequence position 3’ to the tRNA. In some embodiments, the suppressor tRNA is flanked by a nucleotide sequence selected from any one of SEQ ID NOs: 869-888. [0022] In certain embodiments, the expression vector is a viral vector (e.g., a DNA virus vector, e.g., an AAV vector). [0023] In another aspect, provided herein is a virus (e.g., an AAV) comprising the expression vector of any one of the foregoing aspects and embodiments. [0024] In another aspect, provided herein is a system comprising the expression vector or the virus of any one of the above aspects and embodiments. In certain embodiments, the system further comprises an agent for regulating the second promoter (e.g., an activator), which can act in cis or trans. Depending upon the circumstances, the system is a cell (e.g., a producer cell for AAV production, including for example, a human embryonic kidney (HEK) cell or SF9 (Spodoptera frugiperda) insect cell). [0025] In another aspect, provided herein is a pharmaceutical composition comprising the expression vector or the virus of any one of the above aspects and embodiments, and a pharmaceutically acceptable excipient. [0026] In another aspect, provided herein is a method of expressing in a mammalian cell (e.g., a human cell) a functional gene product encoded by a gene of interest containing a premature termination codon. The method comprises contacting or exposing the cell with an effective amount of the expression vector, the virus, or the pharmaceutical composition of any one of the above aspects and embodiments, thereby permitting an amino acid to be incorporated into the gene product at a position that would otherwise result in a truncated gene product caused by the premature termination codon The gene of interest can be, for example, SCN1A or dystrophin. Under certain circumstances, the tRNA becomes aminoacylated in the cell. [0027] In another aspect, provided herein is a method of treating a premature termination codon (PTC)-mediated disorder in a subject (e.g., human) in need thereof wherein the subject has a Attorney Docket No.: TVD-009WO gene with a premature termination codon, the method comprising administering to the subject a therapeutically effective amount of the expression vector, the virus, or the pharmaceutical composition of any one of the above aspects and embodiments, thereby to treat the disorder in the subject. In certain embodiments, the disorder is Dravet Syndrome or Duchenne Muscular Dystrophy. [0028] In another aspect, provided herein is a method of reducing off-target toxicity in a subject (e.g., in a tissue of the subject), the method comprising administering to the subject a therapeutically effective amount of the expression vector, the virus, or the pharmaceutical composition of any one of the above aspects and embodiments, thereby to reduce off-target toxicity in the subject. In another aspect, provided herein is a method of reducing expression of a gene of interest in a tissue of a subject, the method comprising administering to the subject a therapeutically effective amount of the expression vector, the virus, or the pharmaceutical composition of any one of the above aspects and embodiments, thereby to reduce the expression of the gene of interest in the tissue of the subject. In certain embodiments of each method, the tissue (e.g., human tissue) is liver, heart, muscle, retina, inner-ear, spinal cord, or dorsal root ganglion. In some embodiments of each method, the second promoter comprises a liver-specific promoter, heart-specific promoter, muscle-specific promoter, retinal-specific promoter, inner ear-specific promoter, spinal cord-specific promoter, or dorsal root ganglion-specific promoter. [0029] In another aspect, provided herein is a method of producing an AAV particles from a producer cell. The method comprises contacting a producer cell with an effective amount of the expression vector of any one of the above embodiments, thereby to produce the AAV. The AAV can be a high titer AAV. A variety of producer cells can be used including for example, HEK cells or SF9 insect cells. It is understood that the contacting can comprise transfecting the producer cell with the expression vector. In some embodiments, the second promoter is transcriptionally active in the producer cell. In another aspect, provided herein is a high-titer AAV produced by any of the foregoing methods. [0030] These and other aspects and features of the disclosure are described in the following detailed description and claims. DESCRIPTION OF THE DRAWINGS [0031] The disclosure can be more completely understood with reference to the following drawings. Attorney Docket No.: TVD-009WO [0032] FIGs.1A-1C are schematic representations depicting an exemplary type 1 RNA polymerase III promoter (FIG.1A), an exemplary type 2 RNA polymerase III promoter (FIG. 1B), and an exemplary type 3 RNA polymerase III promoter (FIG.1C), all of which can bind an RNA polymerase III (hatched polygons) and which can transcribe the mRNA of a downstream gene of interest (“GOI”). In each instance, a polymerase III termination sequence (“Pol III term”) is present at the 3′ end of the GOI. In FIG.1A, the type 1 RNA polymerase III promoter (light gray rectangle) can typically be subdivided into an A-box (“A”; gray rectangle), an intermediate element (“IE”; gray rectangle), and a C-box (“C”; gray rectangle). Together, these three units constitute the internal control region (ICR). In addition to binding an RNA polymerase III, the type 1 RNA polymerase III promoter recruits directly or indirectly (through an intermediate) transcription factor IIIA (“TFIIIA”; dark gray oval), transcription factor IIIC (“TFIIIC”; dark gray polygon), TATA-binding protein (“tbp”; dark gray oval), BRF1 RNA Polymerase III Transcription Initiation Factor Subunit (“BRF1”; dark gray oval), and B double prime 1, subunit of RNA polymerase III transcription initiation factor IIIB (“BDP1”; dark gray oval) to facilitate transcription. FIG.1B depicts a type 2 RNA polymerase III promoter that can bind an RNA polymerase III, TFIIIC, tbp, BRF1, and BDP1, though the type 2 RNA polymerase III promoter, which typically comprises an A-box (“A”) and a B-box (“B”). FIG.1C depicts a type 3 RNA polymerase III promoter, which typically contains a proximal sequence element (“PSE”) and a TATA box (“TATA”) to which tbp and BDP1, as well as a BRF2 RNA Polymerase III Transcription Initiation Factor Subunit (“BRF2”) and a small nuclear RNA (snRNA) activating protein complex (“SNAPc”), can bind directly or indirectly through an intermediate. [0033] FIGs.2A-2B depict a schematic representation of a standard expression system with a single promoter (promoter 1) (FIG.2A), and an expression system comprising two convergently-aligned promoters (promoter 1 and promoter 2) (FIG 2B). FIG.2A depicts the expression of a GOI (e.g., a non-coding RNA (“ncRNA”), such as a tRNA) expressed from promoter 1. FIG.2B depicts a system when a second promoter, promoter 2, is convergently- aligned to promoter 1. Promoter 1, when operative, transcribes the antisense strand of the GOI to produce the ncRNA, and promoter 2, when operative, transcribes the sense strand of the GOI to produce complimentary non-functional antisense ncRNA. However, when promoter 2 is active, its activity collides with the activity of promoter 1 and reduces or inhibits transcription of the antisense strand of the GOI thereby reducing or inhibiting transcription of the ncRNA GOI. [0034] FIGs.3A-3E are schematic representations of exemplary promoter systems disclosed Attorney Docket No.: TVD-009WO herein. FIGs.3A-3D highlight the concept of convergently-aligned promoters as described in FIG.2. FIG.3A depicts an RNA polymerase II promoter convergently-aligned to a type 2 RNA polymerase III promoter that regulates the transcription of a GOI, such as a tRNA. FIG. 3B depicts an RNA polymerase II promoter convergently-aligned to a type 2 RNA polymerase III promoter that regulates the transcription of a GOI, such as a tRNA, though further includes a type 3 RNA polymerase III promoter upstream of the type 2 RNA polymerase III promoter. FIG.3C depicts a second type 3 RNA polymerase III promoter upstream of the type 2 RNA polymerase III promoter that regulates the transcription of a GOI. FIG.3D depicts a second RNA polymerase II promoter, which, for example, can be a cell- or tissue-specific promoter or a conditional promoter, such as a tetracycline (Tet)-Off or Tet-On promoter. FIG.3E depicts an exemplary expression system in a host cell (e.g., a HEK293 cell line) including an expression vector containing two convergently-aligned promoters (a CMV promoter and a U6 promoter) and multiple genes of interest (each denoted as tr0115) under the control of the U6 promoter, and a separate modulator (activator) of the CMV promoter. [0035] FIG.4 is a schematic representation of an exemplary tRNA, which is transcribed by a type 2 RNA polymerase III due to the presence of an intragenic promoter, including an A-box and a B-box, as described in FIG.1. In the figure, thymines and uracils are used interchangeably. Based upon PMID 16600899, the A-box signature is 11 nucleotides (nt) and consists of TRGYnnAnnnG (SEQ ID NO: 901), where: T is thymine; R is a purine (e.g., guanine or adenine); G is guanine; Y is a pyrimidine (e.g., cytosine or thymine); n is adenine, cytosine, guanine, or thymine/uracil; and A is adenine. The B-box signature is 9 nt and consists of GWTCRANNC (SEQ ID NO: 902), where G is guanine; where: W is adenine or thymine; T is thymine; C is cytosine; R is a purine (guanine or adenine); A is adenine; and n is adenine, cytosine, guanine, or thymine/uracil. The paired bases located in the D-stem and the T-stem are underlined in the A-box (circled nucleotides) and B-box (circled nucleotides), respectively, excluding the final underlined n in the A-box signature which is not necessarily paired. In the exemplary tRNA depicted, the A-box consists of 5′-UGGCGCAAUGG-3′ and the B-box consists of 5′-GUUCGAGUC-3′ (e.g., 5′-UGGCGCAAUGG-3′ and 5′-GUUCGAGUC-3′, respectively). Also depicted in the tRNA is an anticodon, which can be modified such that the modified anticodon hybridizes with a different codon than the corresponding naturally-occurring anticodon. [0036] FIG.5 is a bar chart of the normalized adeno-associated viral (AAV) vector yield (measured as number of genome copies (GC)) following transfection. The AAV comprises a Attorney Docket No.: TVD-009WO nucleic acid molecule including convergently-aligned promoters, wherein the type 2 RNA polymerase III promoter regulates the transcription of a suppressor tRNA gene, as described in FIG.4 (“Suppressor tRNA + Pol II”), a nucleic acid molecule including a suppressor tRNA gene that is not paired with a convergently-aligned promoter (“Suppressor tRNA”), or a nucleic acid molecule that does not contain a suppressor tRNA gene or any other gene transcribed from an RNA polymerase III promoter (“Control”). The composition of each vector is summarized in TABLE 10. [0037] FIG.6 is a bar chart of the relative production scale required to produce at least 2e¹³ genome copies (GCs). A value of 1 on the y-axis indicates the normal production scale required to produce at least 2e¹³ GCs, and values greater than 1 indicate the fold-increase in production scale required to produce at least 2e¹³ GCs. Data are presented from AAV vectors including a nucleic acid molecule including convergently-aligned promoters, wherein the type 2 RNA polymerase III promoter regulates the transcription of a suppressor tRNA gene, as described in FIG.4 (“Suppressor tRNA + Pol II”), a nucleic acid molecule including a suppressor tRNA gene that is not paired with a convergently-aligned promoter (“Suppressor tRNA”), or a nucleic acid molecule that does not contain a suppressor tRNA gene or any other gene transcribed from an RNA polymerase III promoter (“Control”). The composition of each vector is summarized in TABLE 10. [0038] FIG.7 is a dot plot showing the intensity of MECP2 expression in the brains of MECP2 hemizygous mice (such mice have a premature termination codon (PTC) and do not express full- length wildtype MECP2 protein) when transfected with an AAV vector including a nucleic acid molecule including convergently-aligned promoters, when 50 ng/mL of doxycycline (Dox) was present or absent for inducible expression. The x-axis depicts two different experiments, with AAV provided at 7.5 x 10³ (7.5e³) or 2.5 x 10⁴ (2.5e⁴) viral genomes (vg) per cell, respectively. [0039] FIG.8 is a bar chart of the same experiment described in FIG.7, presented as the percentage of MECP2-positive (rescued) neurons as function of virus dose. [0040] FIG.9 is a bar chart of the same experiment described in FIG.7, presented as the number of neurons, where the results show no toxicity based on neuron counts as a function of virus dose. [0041] FIG.10 is a schematic representation of an AAV vector encoding a Tet-On TRE3GV promoter linked to a nucleic acid sequence encoding a tRNA convergently aligned to a CMV promoter and Tet-Off Tet3G tTA element. Each tRNA gene comprises an internal RNA Polymerase III promoter. The shaded arrows and rectangles represent a nucleic acid molecule Attorney Docket No.: TVD-009WO including from 5′-to-3′ a first AAV inverted terminal repeat (ITR), the Tet-On TRE3GV promoter operably linked to three reading frames each encoding a tRNA (“TCA-115”, also referred to herein as “tr0115”), a bovine growth hormone polyadenylation signal (“bgh poly(A) signal”), as well as a convergently-aligned CMV enhancer and promoter, a Tet-Off Tet3G tTA element, and a second AAV ITR. [0042] FIGs.11A-11C are schematic representations of exemplary AAV vectors encoding different promoter systems disclosed herein. FIG.11A depicts a tetracycline response element convergently-aligned to three type 2 RNA polymerase III promoters that each regulate the transcription of a suppressor tRNA ("Sup tRNA”), all disposed between a pair of AAV2 ITRs. FIGs.11B and 11C depict the human liver tissue-specific alpha-1-antitrypsin (hAAT) and thyroxine binding globulin (TBG) promoters, respectively, upstream of a Tet-Off tetracycline transactivator (tTA) element, which when expressed binds the tetracycline response element convergently-aligned to three type 2 RNA polymerase III promoters that each regulate the transcription of a suppressor tRNA (Sup tRNA). Such a system, allows the tissue-specific promoter to interfere with transcriptional activity of the Sup tRNA promoters and reduce production of the Sup tRNA. FIG.11A depicts an experimental system (lacks a tissue specific promoter that modulates expression of an agent that modulates the activity of the tetracycline response element) that acts as a control for the promoter systems described in FIGs.11B and 11C. [0043] FIG.12 is a graph showing the level of suppressor tRNA expression, as quantified by droplet digital polymerase chain reaction (ddPCR) and measured in the heart or liver of C57BL/6 mice transduced with one of the three AAVs described in FIGs.11A-11C. [0044] FIG.13 provides schematic representations of AAV vectors encoding exemplary promoter systems disclosed herein, including one or more tissue-specific promoters convergently-aligned to one or more type 2 RNA polymerase III promoters that each regulate the transcription of a tRNA, such as a suppressor tRNA. [0045] FIG.14 provides schematic representations of exemplary promoter systems disclosed herein, including a stress-responsive promoter convergently-aligned to one or more type 2 RNA polymerase III promoters that each regulate the transcription of a tRNA, such as a suppressor tRNA. The stress-responsive promoters consist of a stress-responsive response element (e.g., ERSE1, ERSE1, AARE, ATF4RE, ATF6RE, and CARE) operably linked to a minimal promoter, such as MinP (Promega, Madison WI). Attorney Docket No.: TVD-009WO DETAILED DESCRIPTION [0046] The disclosure utilizes the phenomenon of convergent transcription for transcriptional regulation (e.g., by a conditional promoter) of a gene of interest (GOI) (e.g., a non-coding RNA (ncRNA), e.g., a tRNA) and is based, in part, upon an expression vector and expression system using, among other things, convergently-aligned promoters, that facilitate impaired transcription of one or both transcripts. Such conditional regulation can be used in reducing off-target toxicity by regulation of the GOI when utilizing a tissue-specific promoter. [0047] Furthermore, such conditional regulation can be used for example, in reducing toxicity of producer cells during production of viral particles (e.g., adeno-associated virus (AAV) particle) when utilizing an inducible promoter. For example, the convergently-aligned promoters described herein facilitate efficient packaging of genes of interest (e.g., a gene encoding a ncRNA) into a viral particle for gene therapy. As a result, a gene of interest, e.g., a tRNA, e.g., a suppressor tRNA, is expressed using a single expression vector displaying convergent transcription, which can impair transcription of one or both transcripts. I. Expression Vectors Containing Convergent Promoters [0048] As used herein, the term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression. [0049] In certain embodiments, the expression vector comprises one or more regulatory sequences (e.g., one or more promoters) operably linked to the nucleotide sequence encoding the gene of interest. The term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid sequence is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a gene if it affects the transcription of the gene. Operably linked nucleotide sequences are typically contiguous. However, as enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not directly flanked and may even function in trans from a different allele or chromosome. Convergent Promoters [0050] FIG.2A depicts a schematic representation of a standard expression system with a single promoter (promoter 1). The antisense strand of the GOI (e.g., a non-coding RNA (“ncRNA”), Attorney Docket No.: TVD-009WO such as a tRNA) is expressed from promoter 1. FIG 2B depicts an expression system comprising two convergently-aligned promoters (promoter 1 and promoter 2). When a second promoter, promoter 2, is convergently-aligned to promoter 1, the complementary sense strand of GOI is transcribed in the opposite direction to produce complementary non-functional antisense ncRNA, and the transcriptional activity of promoter 2 regulates (interferes with, reduces or inhibits) transcriptional activity of promoter 1 to express the antisense strand of the GOI. [0051] In one aspect, the disclosure provides an expression vector comprising: (a) a first promoter; (b) a second, regulatable promoter; and (c) a gene of interest comprising an antisense strand encoding a non-coding RNA (ncRNA) and a complementary sense strand. The complementary strand encodes a complementary non-functional antisense ncRNA. The first promoter is transcriptionally operative in a first direction to transcribe the antisense strand of the gene of interest and produce the ncRNA. The second promoter is transcriptionally operative in a second direction opposite to the first direction of the first promoter to transcribe the sense strand of the gene of interest. The transcriptional activity of the second promoter can be regulated to interfere with transcriptional activity of the first promoter and reduce production of the ncRNA. In this system, the two promoters are transcriptionally operative in opposite directions, such that transcription from the first promoter interferes with transcription from the second promoter. Such transcriptional interference permits conditional expression of a gene of interest, e.g., a non- coding RNA (ncRNA), e.g., a suppressor tRNA. [0052] In another aspect, the expression vector comprises (a) a first promoter; (b) a second promoter; and (c) a gene of interest comprising an antisense strand encoding a non-coding RNA (ncRNA) and a complementary sense strand. The complementary strand encodes a complementary non-functional antisense ncRNA. The first promoter is transcriptionally operative in a first direction to transcribe the antisense strand of the gene of interest and produce the ncRNA. The second promoter is transcriptionally operative in a second direction opposite to the first direction of the first promoter to transcribe the sense strand of the gene of interest. The second promoter is a tissue- or cell type-specific promoter, a stress-responsive promoter, and/or a human promoter. For example, the second promoter can be a human promoter, for example, a human tissue- or cell type-specific promoter. Alternatively, the second promoter can be a human stress-responsive promoter. The transcriptional activity of the second promoter can be regulated to interfere with transcriptional activity of the first promoter and reduce production of the ncRNA. Attorney Docket No.: TVD-009WO [0053] When the expression vector is to be incorporated into a viral particle, the expression vector may also include additional elements to facilitate the integration of the expression vector into the viral particle. For example, when the expression vector is incorporated in an AAV, the expression vector can further comprise AAV inverted terminal repeats (ITRs) flanking the first promoter and the second promoter. [0054] In another aspect, the disclosure provides an expression vector, e.g., a viral expression vector, such as an AAV vector that comprises: (a) a first promoter; (b) a second promoter; and (c) a gene of interest (e.g., a gene encoding a ncRNA) comprising an antisense strand and a complementary sense strand where the antisense strand encodes or otherwise serves as a template for the ncRNA. The complementary strand encodes a complementary non-functional antisense ncRNA. The first promoter is transcriptionally operative in a first direction to transcribe the antisense strand of the gene of interest (e.g., ncRNA). The second promoter is transcriptionally operative in a second direction opposite to the first direction of the first promoter to transcribe the sense strand of the gene of interest. The transcriptional activity of the second promoter can be regulated to interfere with transcriptional activity of the first promoter and reduce production of the gene of interest (e.g., ncRNA). When the expression vector is an AAV vector, the vector further comprises AAV ITRs flanking the first promoter and the second promoter. [0055] In certain embodiments, the expression vector, e.g., an AAV vector, further comprises a third promoter disposed upstream of the first promoter. The third promoter is transcriptionally operative in the first direction opposite to the second direction of the second promoter. The third promoter, in some embodiments, can be an RNA Polymerase III promoter, such as a constitutively active promoter, such as the U6 promoter. It is contemplated that other constitutive promoters can be used instead of the U6 promoter. [0056] For example and without limitation, when the gene of interest is a suppressor tRNA, suitable tissue-specific promoters can be used and can include, for example, one or more of the liver-specific, heart-specific, muscle-specific, retinal-specific, inner ear-specific, spinal cord- specific, and dorsal root ganglion-specific promoters discussed below. [0057] For example and without limitation, exemplary tissue-specific promoters of the disclosure include the tissue-specific promoters set forth in TABLE 1. TABLE 1

Attorney Docket No.: TVD-009WO

Attorney Docket No.: TVD-009WO

[0058] It is contemplated that other tissue- or cell type-specific promoters can be used in the expression systems disclosed herein. [0059] Depending upon the circumstances, the expression vector can contain a plurality of tissue-specific promoters, e.g., 2 or more, 3 or more, 4 or more, or 5 or more tissue-specific promoters. In certain embodiments, a first tissue-specific promoter is used in tandem with a second tissue-specific promoter. It is contemplated that the first tissue specific promoter can be used in tandem with multiple tissue specific promoters (e.g., 2, 3, 4, 5 or more tissue specific promoters). For example, a first tissue-specific promoter is used in tandem with a second tissue- specific promoter and a third tissue-specific promoter. Alternatively, a first tissue-specific promoter is used in tandem with a second tissue-specific promoter, a third tissue-specific promoter, and a fourth tissue-specific promoter. Alternatively, a first tissue-specific promoter is used in tandem with a second tissue-specific promoter, a third tissue-specific promoter, a fourth tissue-specific promoter, and a fifth tissue-specific promoter. [0060] Alternatively, in some embodiments, the second promoter is a stress-responsive promoter. As used herein, the term “stress-responsive promoter” refers to a promoter that can be regulated based upon an external stress condition and/or an intrinsic stress condition. Examples of stress stimuli that can trigger stress-response signaling include, for example, cell-extrinsic factors such as hypoxia, amino acid deprivation, glucose deprivation, or viral infection and cell- intrinsic stresses such as endoplasmic reticulum (ER) stress, caused by the accumulation of unfolded proteins in the ER. Both cell-extrinsic and cell-intrinsic stimuli can activate a common adaptive pathway, termed the integrated stress response (ISR), to restore cellular homeostasis. It is believed that the common point of convergence for all the stress stimuli that activate ISR is phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α). eIF2α phosphorylation causes a reduction in global protein synthesis while allowing the translation of selected genes including activating transcription factor 4 (ATF4), aiding cell survival and recovery (Pakos-Zebrucka et al. (2016) EMBO REP., 17(10):1374-95). [0061] An exemplary stress-responsive promoter can contain one or more tandemly-repeated (e.g., up to 10 tandem repeats) stress-responsive response elements operably linked to a promoter, such as a minimal promoter. As used herein, the term “responsive element” refers to a nucleic acid sequence that can be disposed within a gene promoter or enhancer region that is able to bind specific transcription factors and regulate transcription of genes. For example, Attorney Docket No.: TVD-009WO under conditions of stress, a transcription activator protein can bind to a stress-responsive element and stimulates transcription. As used herein, a “minimal promoter” refers to a nucleic acid sequence that allows for the formation of the RNA polymerase initiation complex at a transcription start site. Exemplary minimal promoters include the CMV minimal promoter and the MinP promoter (Promega, Madison WI). [0062] For example and without limitation, exemplary stress-responsive response elements include the tissue-specific response elements ERSE1, ERSE2, AARE, ATF4RE, ATF6RE, and CARE set forth in TABLE 2. TABLE 2

[0063] An exemplary ERSE1 stress-responsive response element comprises the nucleic acid sequence of: CCTTCACCAATCGGCGGCCTCCACGACGG (SEQ ID NO 889). An exemplary ERSE2 stress-responsive response element comprises the nucleic acid sequence of: GGACGCCGATTGGGCCACGTTGGGAGAGTGCCT (SEQ ID NO 890). An exemplary AARE stress-responsive response element comprises the nucleic acid sequence of: AACATTGCATCATCCCCGC (SEQ ID NO 891). An exemplary ATF4RE stress-responsive response element comprises the nucleic acid sequence of:GTTTCATCA (SEQ ID NO 892). An Attorney Docket No.: TVD-009WO exemplary ATF6RE stress-responsive response element comprises the nucleic acid sequence of: ATCGAGACAGGTGCTGACGTGGCATTC (SEQ ID NO 893). An exemplary CARE stress- responsive response element comprises the nucleic acid sequence of: GCAGGCATGATGAAACTTC (SEQ ID NO 894). It is contemplated that other stress-responsive response elements can be used in the expression systems described herein. [0064] Alternatively or in addition, in some embodiments, the second promoter is a human promoter, such as, for example, an endogenous human promoter. Depending upon the circumstances, the second promoter can comprise an RNA Polymerase II promoter. [0065] In certain embodiments, the first promoter comprises an RNA Polymerase III promoter. The RNA Polymerase III promoter can be a gene-internal type 1 RNA Polymerase III promoter, a gene-internal type 2 RNA Polymerase III promoter, or a gene-external type 3 RNA Polymerase III promoter. Alternatively or in addition, the RNA Polymerase III promoter can be a synthetic hybrid promoter. As used herein, a “synthetic hybrid promoter” refers to a non-naturally occurring promoter comprising, for example, an enhancer operatively joined (fused) to a promoter. Any suitable enhancer may be used in a synthetic hybrid promoter, for example and without limitation, any suitable enhancer derived from an RNA Polymerase II promoter-based system. Any suitable promoter may be used in a synthetic hybrid promoter, for example and without limitation, any suitable RNA Polymerase III promoter. One example of a synthetic hybrid promoter comprises a CMV-derived enhancer and an RNA Polymerase III promoter. [0066] Expression vectors disclosed herein include, for example, the nucleic acid molecules depicted in FIG.13. For example, a nucleic acid molecule of the disclosure can include one or more tissue-specific RNA polymerase II promoters convergently-aligned to one or more type 2 RNA polymerase III promoters that regulate the transcription of one or more tRNAs, respectively. For example, a nucleic acid molecule of the disclosure can include a tissue-specific RNA polymerase II promoter convergently-aligned to a type 2 RNA polymerase III promoter that regulates the transcription of a tRNA (FIG.13, top panel). Alternatively, a nucleic acid construct of the disclosure can be similar to that as shown in the bottom panel of FIG.13, with the construct containing two tandem tissue-specific RNA polymerase II promoters convergently- aligned with RNA polymerase III promoter(s) that control the transcription of one or more tRNAs. Alternatively, for example, the tissue-specific RNA polymerase II promoters of FIG. 13, could be replaced with cell type-specific RNA polymerase II promoters. Alternatively, for example, the tissue-specific RNA polymerase II promoters of FIG.13, could be replaced with a human RNA polymerase II promoter. As depicted in FIGs.1 and 2, respectively, the various Attorney Docket No.: TVD-009WO types of RNA polymerase III promoters recruit different proteins to initiate transcription (FIG. 1) and when two or more promoters are aligned convergently, the transcription of a gene of interest (e.g., a tRNA) can be controlled by promoter collision (see, FIG.2B). [0067] Additionally, expression vectors disclosed herein include, for example, the nucleic acid molecules depicted in FIG.14. For example, a nucleic acid molecule of the disclosure can include a stress-responsive promoter (e.g., a RNA polymerase II promoter) convergently-aligned to one or more type 2 RNA polymerase III promoters that regulate the transcription of one or more tRNAs, respectively. [0068] Additionally, expression vectors disclosed herein include, for example, the nucleic acid molecules depicted in FIG.3. For example, a nucleic acid molecule of the disclosure can include an RNA polymerase II promoter convergently-aligned to a type 2 RNA polymerase III promoter that regulates the transcription of a tRNA. The RNA polymerase II promoter may be a cell- or tissue-specific promoter or a conditional promoter, such as a tetracycline (Tet)-Off or Tet-On promoter (FIG.3A). Alternatively, for example, a nucleic acid molecule of the disclosure can include an RNA polymerase II promoter convergently-aligned to a type 2 RNA polymerase III promoter that regulates the transcription of a tRNA, and further includes a type 3 RNA polymerase III promoter upstream of the type 2 RNA polymerase III promoter (FIG.3B). Alternatively, for example, a nucleic acid molecule of the disclosure can include an RNA polymerase II promoter convergently-aligned to a type 2 RNA polymerase III promoter that regulates the transcription of a first gene of interest (e.g., a tRNA), and further includes a type 3 RNA polymerase III promoter upstream of the type 2 RNA polymerase III promoter. The nucleic acid molecule further includes a second type 3 RNA polymerase III promoter upstream of a second type 2 RNA polymerase III promoter that regulates the transcription of a second gene of interest (e.g., a tRNA) (FIG.3C). Alternatively, a nucleic acid construct of the disclosure can be similar to that as shown in FIG.3C, except that the construct contains two adjacent RNA polymerase II promoters convergently-aligned with promoters that control the transcription of the two genes of interest (FIG.3D). [0069] FIG.3E depicts an exemplary system for expressing multiple genes of interest (e.g., tRNAs) in a host cell (e.g., a HEK293 cell line). The expression vector comprises: (a) a first promoter (e.g., a U6 RNA polymerase III promoter); (b) a second, regulatable promoter (e.g., a Tet induced CMV RNA polymerase II promoter); and (c) multiple genes of interest (tRNAs denoted as tr0115) separated by stuffer sequences, where the genes of interest are under the transcriptional control of the first promoter. The antisense strand of each gene of interest Attorney Docket No.: TVD-009WO encodes a tRNA gene. The first promoter is transcriptionally operative in a first direction (from right to left), and when operative transcribes the antisense strand of the genes of interest and produce the ncRNAs, namely the tRNAs. The second promoter is transcriptionally operative in a second direction (from left to right) and, when operative transcribes the sense strand of the gene of interest. As shown, the transcriptional activity of the second promoter is regulated via Tet induced CMV RNA polymerase II promoter. When the inducer (e.g., doxycycline) is present, the CMV RNA polymerase II promoter is active (in a left to right direction) and interferes with the transcriptional activity of the U6 RNA polymerase III promoter (in a right to left direction) and reduces expression of the tRNAs. [0070] Depending upon the circumstances, when the gene of interest is a tRNA (see, FIG.4), the intragenic A-box and B-box sequences of a tRNA enable its transcription by the molecular machinery that facilitates transcription from a type 2 RNA polymerase III promoter. Type 2 RNA polymerase III promoters typically comprise an A-box and a B-box that are recognized by the TFIIIC transcription factor which functions in RNA polymerase III transcription. The A-box and a B-box are generally internal to the genes being transcribed, for example, as is the case for tRNAs (as demonstrated in FIG.4). [0071] By way of example, FIG.10 depicts a schematic representation of an exemplary expression system of the disclosure. The expression system includes an antisense strand of a gene of interest, exemplified in the figure as three tRNAs (“TCA-115”) each under control of a tRNA-gene internal RNA Polymerase III promoter. Depending on the circumstances, the tRNAs may be linked to one or more gene-external promoter(s) (such as “TRE3GV promoter” in FIG. 10). Convergently-aligned is a second promoter, exemplified as a Tet-Off 3G tTA element upstream of a CMV promoter, which encodes the complementary, non-functional sense strand of the tRNA. Due to such convergent-alignment of the RNA Polymerase III/TRE3GV and CMV promoters, the transcriptional activity of the CMV promoter, in this example, interferes with the transcriptional activity of the promoter encoding the tRNA. [0072] In certain embodiments, the first promoter comprises an RNA Polymerase II promoter. Alternatively, or in addition, the second promoter comprises a conditional RNA Polymerase II promoter. As used herein, the term “conditional promoter” refers to a promoter that is modulated (e.g. activated) in response to a signal, such as a tissue-specific transcription factor, an exogenous ligand, or a factor present in the integrated stress response pathway. Exemplary conditional promoters include tissue-specific promoters (e.g., a liver-specific promoter, a muscle-specific promoter, a heart-specific promoter, or an oligodendrocyte-specific promoter), Attorney Docket No.: TVD-009WO inducible promoters (e.g., a tetracycline-inducible promoter), and stress-responsive promoters (e.g, an ERSE1 stress-responsive response element operably linked to a minimal promoter). [0073] Depending upon the circumstances, the second promoter can comprise a conditional promoter. For example, depending upon the circumstances, the conditional promoter comprises an inducible promoter. As used herein, the term “inducible promoter” refers to a type of conditional promoter that is activated in response to an exogenous signal, such as a ligand. For example an inducible promoter can be selected from a tetracycline-inducible promoter (e.g., Tet- On and Tet-Off promoters and variants thereof), a Lac-inducible promoter (e.g., IPTG-inducible LacO promoters and variants thereof), a Bad-inducible promoter (e.g., arabinose-inducible BAD promoters and variants thereof), a temperature-inducible promoter (e.g., human HSP17 derived promoters, HSP family protein-derived promoters, and variants thereof), a light-inducible promoter (e.g., rhodopsin-based and or Split-dCas protein-based promoters, and variants thereof), and a CRISPR/Cas-based promoter (e.g., CRISPRa-, CRISPRi-, and CRISPR- VP16/VP64 fusion protein-based promoters and variants thereof). In certain embodiments, the conditional promoter comprises a tetracycline-inducible promoter (e.g., a tetracycline-on (Tet- On) promoter or a tetracycline-off (Tet-Off) promoter). Alternatively or in addition, the conditional promoter exhibits tissue or cell-type specific activities. For example, the condition promoter comprises one or more tissue-specific promoters (e.g., a liver-specific promoter (e.g., a human AAT promoter and human TBG promoter), a heart-specific promoter (e.g., a MLC2v promoter and TNNT2 promoter), a muscle-specific promoter (e.g., a muscle-hybrid promoter (see Piekarowicz et al. (2019) MOL. THER. METHODS CLIN. DEV.12(15): 157-169) and tMCK promoter), a retinal-specific promoter (e.g., a human L-opsin promoter and Nefh promoter), an inner ear-specific promoter (e.g., an Atoh1 promoter), a spinal cord-specific promoter (e.g., an Hb9 promoter), or a dorsal root ganglion-specific promoter). [0074] A conditional promoter comprises a regulatable element (e.g., for a tetracycline-inducible promoter, the regulatable element is the Tet-operon), and a minimal promoter. Suitable minimal promoters include, for example and without limitation, a minimal CMV promoter, a minimal SV40 promoter, a minimal Beta Globin promoter, and a minimal EF1 alpha promoter. Depending on the circumstances, a minimal promoter may be replaced with a different minimal promoter, for example, depending on properties of the promoter such as strength or leakiness. Such promoters with replaced “minimal promoters” relative to the endogenous promoter (e.g., a Tet-On promoter) are described herein as “variants thereof” (e.g., a variant of the Tet-On promoter). Attorney Docket No.: TVD-009WO Vector Backbone [0075] It is contemplated that the convergent promoters and the gene or genes of interest can be integrated into a the backbone of a viral vector. Exemplary viral vectors include retroviral vectors (e.g., lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpesviruses vectors, epstein-barr virus (EBV) vectors, polyomavirus vectors (e.g., simian vacuolating virus 40 (SV40) vectors), poxvirus vectors, and pseudotype virus vectors. [0076] The virus may be an RNA virus (having a genome that is composed of RNA) or a DNA virus (having a genome composed of DNA). In certain embodiments, the viral vector is a DNA virus vector. Exemplary DNA viruses include parvoviruses (e.g., adeno-associated viruses), adenoviruses, asfarviruses, herpesviruses (e.g., herpes simplex virus 1 and 2 (HSV-1 and HSV- 2), epstein-barr virus (EBV), cytomegalovirus (CMV)), papillomoviruses (e.g., HPV), polyomaviruses (e.g., simian vacuolating virus 40 (SV40)), and poxviruses (e.g., vaccinia virus, cowpox virus, smallpox virus, fowlpox virus, sheeppox virus, myxoma virus). In certain embodiments, the viral vector is an RNA virus vector. Exemplary RNA viruses include bunyaviruses (e.g., hantavirus), coronaviruses, flaviviruses (e.g., yellow fever virus, west nile virus, dengue virus), hepatitis viruses (e.g., hepatitis A virus, hepatitis C virus, hepatitis E virus), influenza viruses (e.g., influenza virus type A, influenza virus type B, influenza virus type C), measles virus, mumps virus, noroviruses (e.g., Norwalk virus), poliovirus, respiratory syncytial virus (RSV), retroviruses (e.g., human immunodeficiency virus-1 (HIV-1)) and toroviruses. Adeno-Associated virus (AAV) Vectors [0077] In certain embodiments, the backbone of the expression vector is an AAV vector. AAV is a small, nonenveloped icosahedral virus of the genus Dependoparvovirus and family Parvovirus. AAV has a single-stranded linear DNA genome of approximately 4.7 kb. AAV is capable of infecting both dividing and quiescent cells of several tissue types, with different AAV serotypes exhibiting different tissue tropism. [0078] AAV includes numerous serologically distinguishable types including serotypes AAV-1 to AAV-12, as well as more than 100 serotypes from nonhuman primates (See, e.g., Srivastava (2008) J. CELL BIOCHEM., 105(1): 17–24, and Gao et al. (2004) J. VIROL., 78(12), 6381–6388). The serotype of the AAV vector used in the present disclosure can be selected by a skilled person in the art based on the efficiency of delivery, tissue tropism, and immunogenicity. For example, AAV-1, AAV-2, AAV-4, AAV-5, AAV-8, and AAV-9 can be used for delivery to the central nervous system; AAV-1, AAV-8, and AAV-9 can be used for delivery to the heart; AAV-2 can be used for delivery to the kidney; AAV-7, AAV-8, and AAV-9 can be used for Attorney Docket No.: TVD-009WO delivery to the liver; AAV-4, AAV-5, AAV-6, AAV-9 can be used for delivery to the lung, AAV-8 can be used for delivery to the pancreas, AAV-2, AAV-5, and AAV-8 can be used for delivery to the photoreceptor cells; AAV-1, AAV-2, AAV-4, AAV-5, and AAV-8 can be used for delivery to the retinal pigment epithelium; AAV-1, AAV-6, AAV-7, AAV-8, and AAV-9 can be used for delivery to the skeletal muscle. In certain embodiments, the AAV capsid protein comprises a sequence as disclosed in U.S. Patent No.7,198,951, such as, but not limited to, AAV-9 (SEQ ID NOs: 1-3 of U.S. Patent No.7,198,951), AAV-2 (SEQ ID NO: 4 of U.S. Patent No.7,198,951), AAV-1 (SEQ ID NO: 5 of U.S. Patent No.7,198,951), AAV-3 (SEQ ID NO: 6 of U.S. Patent No.7,198,951), and AAV-8 (SEQ ID NO: 7 of U.S. Patent No.7,198,951). AAV serotypes identified from rhesus monkeys, e.g., rh.8, rh.10, rh.39, rh.43, and rh.74, are also contemplated in the instant disclosure. Besides the natural AAV serotypes, modified AAV capsids have been developed for improving efficiency of delivery, tissue tropism, and immunogenicity. Exemplary natural and modified AAV capsids are disclosed in U.S. Patent Nos.7,906,111, 9,493,788, and 7,198,951, and PCT Publication No. WO2017189964A2. [0079] The wild-type AAV genome contains two 145 nucleotide ITRs, which contain signal sequences directing AAV replication, genome encapsidation and integration. In addition to the ITRs, three AAV promoters, p5, p19, and p40, drive expression of two open reading frames encoding rep and cap genes. Two rep promoters, coupled with differential splicing of the single AAV intron, result in the production of four rep proteins (Rep 78, Rep 68, Rep 52, and Rep 40) from the rep gene. Rep proteins are responsible for genomic replication. The Cap gene is expressed from the p40 promoter, and encodes three capsid proteins (VP1, VP2, and VP3) which are splice variants of the cap gene. These proteins form the capsid of the AAV particle. [0080] Because the cis-acting signals for replication, encapsidation, and integration are contained within the ITRs, some or all of the 4.3 kb internal genome may be replaced with foreign DNA, for example, an expression cassette for an exogenous gene of interest. Accordingly, in certain embodiments, the AAV vector comprises a genome comprising an expression cassette for an exogenous gene flanked by a 5′ ITR and a 3′ ITR. The ITRs may be derived from the same serotype as the capsid or a derivative thereof. Alternatively, the ITRs may be of a different serotype from the capsid, thereby generating a pseudotyped AAV. In certain embodiments, the ITRs are derived from AAV-2. In certain embodiments, the ITRs are derived from AAV-5. At least one of the ITRs may be modified to mutate or delete the terminal resolution site, thereby allowing production of a self-complementary AAV vector. Attorney Docket No.: TVD-009WO [0081] The rep and cap proteins can be provided in trans, for example, on a plasmid, to produce an AAV vector. A host cell line permissive of AAV replication must express the rep and cap genes, the ITR-flanked expression cassette, and helper functions provided by a helper virus, for example adenoviral genes E1a, E1b55K, E2a, E4orf6, and VA (Weitzman et al., Adeno- associated virus biology. Adeno-Associated Virus: Methods and Protocols, pp.1–23, 2011). Methods for generating and purifying AAV vectors have been described in detail (see e.g., Mueller et al., (2012) CURRENT PROTOCOLS IN MICROBIOLOGY, 14D.1.1-14D.1.21, Production and Discovery of Novel Recombinant Adeno-Associated Viral Vectors). Numerous cell types are suitable for producing AAV vectors, including HEK293 cells, COS cells, HeLa cells, BHK cells, Vero cells, as well as insect cells such as SF9 cells (See, e.g. U.S. Patent Nos.6,156,303, 5,387,484, 5,741,683, 5,691,176, 5,688,676, and 8,163,543, U.S. Patent Publication No. 20020081721, and PCT Publication Nos. WO00/47757, WO00/24916, and WO96/17947). AAV vectors are typically produced in these cell types by one plasmid containing the ITR-flanked expression cassette, and one or more additional plasmids providing the additional AAV and helper virus genes. [0082] It is contemplated that any AAV serotype can be used with the convergent promoter system described herein. Similarly, it is contemplated that any adenoviral type may be used, and a person of skill in the art will be able to identify AAV and adenoviral types suitable for the production of their desired recombinant AAV vector (rAAV). AAV particles may be purified, for example by affinity chromatography, iodixonal gradient, or CsCl gradient. [0083] AAV vectors may have single-stranded genomes that are 4.7 kb in size, or are larger or smaller than 4.7 kb, including oversized genomes that are as large as 5.2 kb, or as small as 3.0 kb. Thus, where the exogenous gene of interest to be expressed from the AAV vector is small, the AAV genome may comprise a stuffer sequence. Further, vector genomes may be substantially self-complementary thereby allowing for rapid expression in the cell. In certain embodiments, the genome of a self-complementary AAV vector comprises from 5′ to 3′: a 5′ ITR; a first nucleic acid sequence comprising a promoter and/or enhancer operably linked to a coding sequence of a gene of interest; a modified ITR that does not have a functional terminal resolution site; a second nucleic acid sequence complementary or substantially complementary to the first nucleic acid sequence; and a 3′ ITR. AAV vectors containing genomes of all types are suitable for use in the method of the present disclosure. [0084] Non-limiting examples of AAV vectors include pAAV-MCS (Agilent Technologies), pAAVK-EF1α-MCS (System Bio Catalog # AAV502A-1), pAAVK-EF1α-MCS1-CMV-MCS2 Attorney Docket No.: TVD-009WO (System Bio Catalog # AAV503A-1), pAAV-ZsGreen1 (Clontech Catalog #6231), pAAV- MCS2 (Addgene Plasmid #46954), AAV-Stuffer (Addgene Plasmid #106248), pAAVscCBPIGpluc (Addgene Plasmid #35645), AAVS1_Puro_PGK1_3xFLAG_Twin_Strep (Addgene Plasmid #68375), pAAV-RAM-d2TTA::TRE-MCS-WPRE-pA (Addgene Plasmid #63931), pAAV-UbC (Addgene Plasmid #62806), pAAVS1-P-MCS (Addgene Plasmid #80488), pAAV-Gateway (Addgene Plasmid #32671), pAAV-Puro_siKD (Addgene Plasmid #86695), pAAVS1-Nst-MCS (Addgene Plasmid #80487), pAAVS1-Nst-CAG-DEST (Addgene Plasmid #80489), pAAVS1-P-CAG-DEST (Addgene Plasmid #80490), pAAVf-EnhCB-lacZnls (Addgene Plasmid #35642), and pAAVS1-shRNA (Addgene Plasmid #82697). These vectors can be modified to be suitable for therapeutic use. For example, an exogenous gene of interest can be inserted in a multiple cloning site, and a selection marker (e.g., puro or a gene encoding a fluorescent protein) can be deleted or replaced with another (same or different) exogenous gene of interest. Further examples of AAV vectors are disclosed in U.S. Patent Nos.5,871,982, 6,270,996, 7,238,526, 6,943,019, 6,953,690, 9,150,882, and 8,298,818, U.S. Patent Publication No.2009/0087413, and PCT Publication Nos. WO2017075335A1, WO2017075338A2, and WO2017201258A1. [0085] In certain embodiments, the expression vector is an AAV vector capable of targeting the nervous system, e.g., the central nervous system, in a subject, e.g., a human subject. Exemplary AAV vectors that can target the nervous system include the AAV9 variants AAV-PHP.B (See, e.g., Deverman et al. (2016) NAT. BIOTECHNOL.34(2):204–209), AAV-AS (See, e.g., Choudhury et al. (2016) MOL. THER.24:726–35), and AAV-PHP.eB (See, e.g., Chan et al. (2017) NAT. NEUROSCI.20:1172–79). Additional exemplary AAV-based strategies for targeting the nervous system are described in Bedrook et al. (2018) ANNU REV NEUROSCI.41:323-348. In certain embodiments, the AAV vector is an AAV-PHP.eB vector. Manufacturing of AAV [0086] The virus-producing cells can be selected for productivity by infecting each clonal population with a recombinant virus expression vector (e.g., baculovirus expression vector (BEV)) void of any AAV elements. Clones can be screened in antibiotic and/or serum-free conditions. Infection can initiate expression of genomically-integrated AAV Rep and AAV Cap genes, which can express the AAV genomes and facilitate their packaging into assembled AAV capsids. Following the production phase, AAV can then be harvested from cell monolayers by freeze-thaw and nuclease treatment and the resulting AAV content produced by each clonal population can be quantified by PCR. Attorney Docket No.: TVD-009WO [0087] In some embodiments, the clones are then expanded to a production bioreactor to produce a recombinant viral vector. In some embodiments, the recombinant viral vector (e.g., rAAV) is subsequently harvested, adenovirus is inactivated (e.g., by heat) and/or removed, and the viral particles are purified. In some embodiments, recombinant viral vectors are purified and formulated. [0088] In some embodiments, suitable media is used for the production of recombinant vectors. These media comprise, without limitation, media appropriate for cell type (e.g., mammalian, insect, etc.), such as, for example, media produced by Hyclone Laboratories and JRH comprising Modified Eagle Medium (MEM), Roswell Park Memorial Institute (RPMI) 1640, Eagle’s Minimal Essential Medium (EMEM), Dulbecco’s Modified Eagle Medium (DMEM), ExpiSf- CD media (Thermo Fisher Scientific), Sf-900 II (Thermo Fisher Scientific), Sf-900 III (Thermo Fisher Scientific), ESF-AF (Expression Systems), IS Sf Insect ACF (FUJIFILM Irvine Scientific), 4 Cell Insect Media (Sartorius), Hyclone SFX (Cytiva Life Sciences), EX-Cell (Sigma Aldrich), and/or custom formulations, particularly with respect to custom media formulations for use in production of recombinant vectors. [0089] In some embodiments, suitable production culture media of the present disclosure is supplemented with serum or serum-derived recombinant proteins at a level of 0.5 -20 (v/v or w/v). In some embodiments, vectors are produced in serum-free conditions which are also referred to as media with no animal-derived products. In some embodiments, commercial or custom media is designed to support production of vectors, comprising supplementation of without limitation glucose, vitamins, amino acids, and or growth factors, in order to increase the titer and/or yield of vector in production cultures. [0090] Vector production cultures comprise a variety of conditions (over a wide temperature range, for varying lengths of time, and the like) suitable to the particular host cell being utilized. Vector production cultures comprise attachment-dependent cultures which are cultured in suitable attachment-dependent vessels such as, for example, plates, flasks, cell stacks, roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors. In some embodiments, vector production cultures comprise suspension-adapted host cells such as HeLa, SF-9, HEK-293, HEK-293T, and HEK293F cells, and other suspension HEK293-derived cell lines for AAV particle production which are cultured in a variety of ways comprising, for example, spinner flasks, stirred tank bioreactors, single use bioreactors such as Cytiva Xcellerex and Sartorius, and disposable systems such as the Wave bag system. Attorney Docket No.: TVD-009WO [0091] In some embodiments, viral particles of the disclosure are harvested from vector production cultures by lysis of the host cells of the production culture or by harvest of the spent media from the production culture, provided the cells are cultured under conditions to cause release of viral particles into the media from intact cells. Suitable methods of lysing cells comprise for example multiple freeze/thaw cycles, sonication, microfluidization, and treatment with chemicals, such as detergents and/or proteases. [0092] The resulting viral particles can then be purified. The term “purified” as used herein comprises a preparation of viral particles devoid of at least some of the other components that are present where the viral particles naturally occur or are initially prepared from. Thus, for example, in some embodiments, isolated viral particles are prepared using a purification technique to enrich it from a source mixture, such as a culture lysate or production culture supernatant. In some embodiments, enrichment is measured in a variety of ways, such as, for example, by the proportion of DNase-resistant particles (DRPs) or genome copies (gc) present in a solution, or by infectivity, or measured in relation to a second, potentially interfering substance present in the source mixture, such as contaminants, comprising production culture contaminants or in-process contaminants, comprising helper virus, media components, and the like. [0093] In some embodiments, the vector production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtration through a series of depth filters comprising, for example, a grade DOHC Millipore Millistak+ HC Pod Filter, a grade A1HC Millipore Millistak+ HC Pod Filter, and a 0.2 μm Filter Opticap XL 10 Millipore Express SHC Hydrophilic Membrane filter. Clarification can also be achieved by a variety of other standard techniques, such as, centrifugation or filtration through any cellulose acetate filter of 0.2 μm or greater pore size. [0094] The vector production culture harvest can be further treated with Benzonase® to digest any high molecular weight DNA present in the production culture. In some embodiments, the Benzonase® digestion is performed under standard conditions comprising, for example, a final concentration of 1-2.5 units/ml of Benzonase® at a temperature ranging from ambient to 37 °C for a period of 30 minutes to several hours. [0095] Depending upon the circumstances, the viral particles are isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) for concentrating the viral particles; vector capture by apatite chromatography; heat inactivation of helper virus; vector capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography Attorney Docket No.: TVD-009WO (SEC); nanofiltration; and vector capture by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. It is contemplated that these steps can be used alone, in various combinations, or in different orders. [0096] In some embodiments, methods for generating a recombinant vector (e.g., rAAV) comprise providing stably-integrated virus-producing cells with a helper plasmid. The cells can be transfected with a helper plasmid that provides helper functions to the AAV. In some embodiments, the helper plasmid provides adenovirus functions including, but not limited to, E1A, E1B, E4, and E2A. In some embodiments, the helper plasmid provides other virus functions including, but not limited to, VA RNA, Gag, Pol, Tat, Rev, Env, and VSV-G. The sequences of adenovirus gene providing these functions, in some embodiments, are obtained from any known adenovirus serotype, such as serotypes 2, 3, 4, 7, 12 and 40, and further comprising any of the presently identified human types. In some embodiments, the methods involve transfecting the cell with vectors expressing one or more genes necessary for AAV replication, AAV gene transcription, and/or AAV packaging. [0097] Methods disclosed herein result in improvements in recombinant viral vector manufacturing, including improved quantity of recombinant virus, more efficient and faster production time, and greater reproducibility and scalability without any decrease in efficacy of recombinant viral produce produced. [0098] Other details for making and using recombinant viruses containing a gene of interest, including AAV, can be found, for example, in PCT Publications Nos. WO2010114948 and WO2017181162. Retroviral Vectors Including Lentivirus Vectors [0099] In certain embodiments, the viral vector can be a retroviral vector. Examples of retroviral vectbacors include moloney murine leukemia virus vectors, spleen necrosis virus vectors, and vectors derived from retroviruses such as rous sarcoma virus, harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus. Retroviral vectors are useful as agents to mediate retroviral-mediated gene transfer into eukaryotic cells. [00100] In certain embodiments, the retroviral vector is a lentiviral vector. Exemplary lentiviral vectors include vectors derived from human immunodeficiency virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), feline Attorney Docket No.: TVD-009WO immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV), and caprine arthritis encephalitis virus (CAEV). [00101] Retroviral vectors typically are constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene(s) of interest. Often, the structural genes (i.e., gag, pol, and env), are removed from the retroviral backbone using genetic engineering techniques known in the art. Accordingly, a minimum retroviral vector comprises from 5′ to 3′: a 5′ long terminal repeat (LTR), a packaging signal, an optional exogenous promoter and/or enhancer, an exogenous gene of interest, and a 3′ LTR. If no exogenous promoter is provided, gene expression is driven by the 5′ LTR, which is a weak promoter and requires the presence of Tat to activate expression. The structural genes can be provided in separate vectors for manufacture of the lentivirus, rendering the produced virions replication-defective. Specifically, with respect to lentivirus, the packaging system may comprise a single packaging vector encoding the Gag, Pol, Rev, and Tat genes, and a third, separate vector encoding the envelope protein Env (usually VSV‐G due to its wide infectivity). To improve the safety of the packaging system, the packaging vector can be split, expressing Rev from one vector, Gag and Pol from another vector. Tat can also be eliminated from the packaging system by using a retroviral vector comprising a chimeric 5′ LTR, wherein the U3 region of the 5′ LTR is replaced with a heterologous regulatory element. [00102] The genes can be incorporated into the proviral backbone in several general ways. Potentially straightforward constructions are ones in which the structural genes of the retrovirus are replaced by a single gene that is transcribed under the control of the viral regulatory sequences within the LTR. Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter. [00103] Typically, the new gene(s) are flanked by 5′ and 3′ LTRs, which serve to promote transcription and polyadenylation of the virion RNAs, respectively. The term “long terminal repeat” or “LTR” refers to domains of base pairs located at the ends of retroviral DNAs which, in their natural sequence context, are direct repeats and contain U3, R and U5 regions. LTRs generally provide functions fundamental to the expression of retroviral genes (e.g., promotion, initiation and polyadenylation of gene transcripts) and to viral replication. The LTR contains numerous regulatory signals including transcriptional control elements, polyadenylation signals, and sequences needed for replication and integration of the viral genome. The U3 region Attorney Docket No.: TVD-009WO contains the enhancer and promoter elements. The U5 region is the sequence between the primer binding site and the R region and contains the polyadenylation sequence. The R (repeat) region is flanked by the U3 and U5 regions. In certain embodiments, the R region comprises a trans-activation response (TAR) genetic element, which interacts with the trans-activator (tat) genetic element to enhance viral replication. This element is not required in embodiments wherein the U3 region of the 5′ LTR is replaced by a heterologous promoter. [00104] In certain embodiments, the retroviral vector comprises a modified 5′ LTR and/or 3′ LTR. Modifications of the 3′ LTR are often made to improve the safety of lentiviral or retroviral systems by rendering viruses replication-defective. In specific embodiments, the retroviral vector is a self-inactivating (SIN) vector. As used herein, a SIN retroviral vector refers to a replication-defective retroviral vector in which the 3′ LTR U3 region has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is because the 3′ LTR U3 region is used as a template for the 5′ LTR U3 region during viral replication and, thus, the viral transcript cannot be made without the U3 enhancer- promoter. In a further embodiment, the 3′ LTR is modified such that the U5 region is replaced, for example, with an ideal polyadenylation sequence. It should be noted that modifications to the LTRs such as modifications to the 3′ LTR, the 5′ LTR, or both 3′ and 5′ LTRs, are also contemplated to be useful in the practice of the disclosure. [00105] In certain embodiments, the U3 region of the 5′ LTR is replaced with a heterologous promoter to drive transcription of the viral genome during production of viral particles. Examples of heterologous promoters which can be used include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. Typical promoters are able to drive high levels of transcription in a Tat-independent manner. This replacement reduces the possibility of recombination to generate replication-competent virus, because there is no complete U3 sequence in the virus production system. [00106] Adjacent the 5′ LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient packaging of viral RNA into particles (the Psi site). As used herein, the term “packaging signal” or “packaging sequence” refers to sequences located within the retroviral genome which are required for encapsidation of retroviral RNA strands during viral particle formation (see e.g., Clever et al., 1995 J. VIROLOGY, Attorney Docket No.: TVD-009WO 69(4):2101-09). The packaging signal may be a minimal packaging signal (also referred to as the psi [Ψ] sequence) needed for encapsidation of the viral genome. [00107] In certain embodiments, the retroviral vector (e.g., lentiviral vector) further comprises a FLAP. As used herein, the term “FLAP” refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and in Zennou et al. (2000) CELL, 101:173. During reverse transcription, central initiation of the plus-strand DNA at the cPPT and central termination at the CTS lead to the formation of a three-stranded DNA structure: a central DNA flap. While not wishing to be bound by any theory, the DNA flap may act as a cis-active determinant of lentiviral genome nuclear import and/or may increase the titer of the virus. In particular embodiments, the retroviral vector backbones comprise one or more FLAP elements upstream or downstream of the heterologous genes of interest in the vectors. For example, in particular embodiments, a transfer plasmid includes a FLAP element. In one embodiment, a vector of the disclosure comprises a FLAP element isolated from HIV-1. [00108] In certain embodiments, the retroviral vector (e.g., lentiviral vector) further comprises an export element. In one embodiment, retroviral vectors comprise one or more export elements. The term “export element” refers to a cis-acting post-transcriptional regulatory element which regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) RRE (see e.g., Cullen et al., (1991) J. VIROL.65: 1053; and Cullen et al., (1991) CELL 58: 423) and the hepatitis B virus post-transcriptional regulatory element (HPRE). Generally, the RNA export element is placed within the 3′ UTR of a gene, and can be inserted as one or multiple copies. [00109] In certain embodiments, the retroviral vector (e.g., lentiviral vector) further comprises a posttranscriptional regulatory element. A variety of posttranscriptional regulatory elements can increase expression of a heterologous nucleic acid, e.g., woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; see Zufferey et al., (1999) J. VIROL., 73:2886); the posttranscriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al., MOL. CELL. BIOL., 5:3864); and the like (Liu et al., (1995), GENES DEV., 9:1766). The posttranscriptional regulatory element is generally positioned at the 3′ end the heterologous nucleic acid sequence. This configuration results in synthesis of an mRNA transcript whose 5′ Attorney Docket No.: TVD-009WO portion comprises the heterologous nucleic acid coding sequences and whose 3′ portion comprises the posttranscriptional regulatory element sequence. [00110] Elements directing the efficient termination and polyadenylation of the heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal. Accordingly, in certain embodiments, the retroviral vector (e.g., lentiviral vector) further comprises a polyadenylation signal. The term “polyadenylation signal” or “polyadenylation sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase H. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a polyadenylation signal are unstable and are rapidly degraded. Illustrative examples of polyadenylation signals that can be used in a vector of the disclosure, includes an ideal polyadenylation sequence (e.g., AATAAA, ATTAAA AGTAAA), a bovine growth hormone polyadenylation sequence (BGHpA), a rabbit β-globin polyadenylation sequence (rβgpA), or another suitable heterologous or endogenous polyadenylation sequence known in the art. [00111] Non-limiting examples of lentiviral vectors include pLVX-EF1alpha-AcGFP1-C1 (Clontech Catalog #631984), pLVX-EF1alpha-IRES-mCherry (Clontech Catalog #631987), pLVX-Puro (Clontech Catalog #632159), pLVX-IRES-Puro (Clontech Catalog #632186), pLenti6/V5-DEST^TM (Thermo Fisher), pLenti6.2/V5-DEST^TM (Thermo Fisher), pLKO.1 (Plasmid #10878 at Addgene), pLKO.3G (Plasmid #14748 at Addgene), pSico (Plasmid #11578 at Addgene), pLJM1-EGFP (Plasmid #19319 at Addgene), FUGW (Plasmid #14883 at Addgene), pLVTHM (Plasmid #12247 at Addgene), pLVUT-tTR-KRAB (Plasmid #11651 at Addgene), pLL3.7 (Plasmid #11795 at Addgene), pLB (Plasmid #11619 at Addgene), pWPXL (Plasmid #12257 at Addgene), pWPI (Plasmid #12254 at Addgene), EF.CMV.RFP (Plasmid #17619 at Addgene), pLenti CMV Puro DEST (Plasmid #17452 at Addgene), pLenti-puro (Plasmid #39481 at Addgene), pULTRA (Plasmid #24129 at Addgene), pLX301 (Plasmid #25895 at Addgene), pHIV-EGFP (Plasmid #21373 at Addgene), pLV-mCherry (Plasmid #36084 at Addgene), pLionII (Plasmid #1730 at Addgene), pInducer10-mir-RUP-PheS (Plasmid #44011 at Addgene). These vectors can be modified to be suitable for therapeutic use. For example, a selection marker (e.g., puromycin, EGFP, or mCherry) can be deleted or replaced with a second exogenous gene of interest. Further examples of lentiviral vectors are disclosed in U.S. Patent Nos.7,629,153, 7,198,950, 8,329,462, 6,863,884, 6,682,907, 7,745,179, 7,250,299, Attorney Docket No.: TVD-009WO 5,994,136, 6,287,814, 6,013,516, 6,797,512, 6,544,771, 5,834,256, 6,958,226, 6,207,455, 6,531,123, and 6,352,694, and PCT Publication No. WO2017/091786. Adenoviral Vectors [00112] In certain embodiments, the viral vector can be an adenoviral vector. Adenoviruses are medium-sized (90-100 nm), non-enveloped (naked), icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome. The term “adenovirus” refers to any virus in the genus Adenoviridiae including, but not limited to, human, bovine, ovine, equine, canine, porcine, murine, and simian adenovirus subgenera. Typically, an adenoviral vector is generated by introducing one or more mutations (e.g., a deletion, insertion, or substitution) into the adenoviral genome of the adenovirus so as to accommodate the insertion of a non-native nucleic acid sequence, for example, for gene transfer, into the adenovirus. [00113] A human adenovirus can be used as the source of the adenoviral genome for the adenoviral vector. For instance, an adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31 ), subgroup B (e.g., serotypes 3, 7, 11 , 14, 16, 21 , 34, 35, and 50), subgroup C (e.g., serotypes 1 , 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-48), subgroup E (e.g., serotype 4), subgroup F (e.g., serotypes 40 and 41 ), an unclassified serogroup (e.g., serotypes 49 and 51), or any other adenoviral serogroup or serotype. Adenoviral serotypes 1 through 51 are available from the American Type Culture Collection (ATCC, Manassas, Virginia). Non-group C adenoviral vectors, methods of producing non-group C adenoviral vectors, and methods of using non- group C adenoviral vectors are disclosed in, for example, U.S. Patent Nos.5,801 ,030, 5,837,511, and 5,849,561, and PCT Publication Nos. WO1997/012986 and WO1998/053087. [00114] Non-human adenovirus (e.g., ape, simian, avian, canine, ovine, or bovine adenoviruses) can be used to generate the adenoviral vector (i.e., as a source of the adenoviral genome for the adenoviral vector). For example, the adenoviral vector can be based on a simian adenovirus, including both new world and old world monkeys (see, e.g., Virus Taxonomy: VHIth Report of the International Committee on Taxonomy of Viruses (2005)). A phylogeny analysis of adenoviruses that infect primates is disclosed in, e.g., Roy et al. (2009) PLOS PATHOG.5(7):e1000503. A gorilla adenovirus can be used as the source of the adenoviral genome for the adenoviral vector. Gorilla adenoviruses and adenoviral vectors are described in, e.g., PCT Publication Nos.WO2013/052799, WO2013/052811, and WO2013/052832. The adenoviral vector can also comprise a combination of subtypes and thereby be a “chimeric” adenoviral vector. Attorney Docket No.: TVD-009WO [00115] The adenoviral vector can be replication-competent, conditionally replication- competent, or replication-deficient. A replication-competent adenoviral vector can replicate in typical host cells, i.e., cells typically capable of being infected by an adenovirus. A conditionally-replicating adenoviral vector is an adenoviral vector that has been engineered to replicate under pre-determined conditions. For example, replication-essential gene functions, e.g., gene functions encoded by the adenoviral early regions, can be operably linked to an inducible, repressible, or tissue-specific transcription control sequence, e.g., a promoter. Conditionally-replicating adenoviral vectors are further described in U.S. Patent No.5,998,205. A replication-deficient adenoviral vector is an adenoviral vector that requires complementation of one or more gene functions or regions of the adenoviral genome that are required for replication, as a result of, for example, a deficiency in one or more replication-essential gene function or regions, such that the adenoviral vector does not replicate in typical host cells, especially those in a human to be infected by the adenoviral vector. [00116] Preferably, the adenoviral vector is replication-deficient, such that the replication- deficient adenoviral vector requires complementation of at least one replication-essential gene function of one or more regions of the adenoviral genome for propagation (e.g., to form adenoviral vector particles). The adenoviral vector can be deficient in one or more replication- essential gene functions of only the early regions (i.e., E1-E4 regions) of the adenoviral genome, only the late regions (i.e., L1-L5 regions) of the adenoviral genome, both the early and late regions of the adenoviral genome, or all adenoviral genes (i.e., a high capacity adenovector (HC- Ad)). See, e.g., Morsy et al. (1998) PROC. NATL. ACAD. SCI. USA 95: 965-976, Chen et al. (1997) PROC. NATL. ACAD. SCI. USA 94: 1645-1650, and Kochanek et al. (1999) HUM. GENE THER.10(15):2451-9. Examples of replication-deficient adenoviral vectors are disclosed in U.S. Patent Nos.5,837,511, 5,851,806, 5,994,106, 6,127,175, 6,482,616, and 7,195,896, and PCT Publication Nos. WO1994/028152, WO1995/002697, WO1995/016772, WO1995/034671, WO1996/022378, WO1997/012986, WO1997/021826, and WO2003/022311. [00117] The replication-deficient adenoviral vector of the disclosure can be produced in complementing cell lines that provide gene functions not present in the replication-deficient adenoviral vector, but required for viral propagation, at appropriate levels in order to generate high titers of viral vector stock. Such complementing cell lines are known and include, but are not limited to, 293 cells (described in, e.g., Graham et al. (1977) J. GEN. VIROL.36: 59-72), PER.C6 cells (described in, e.g., PCT Publication No. WO1997/000326, and U.S. Patent Nos. 5,994,128 and 6,033,908), and 293-ORF6 cells (described in, e.g., PCT Publication No. Attorney Docket No.: TVD-009WO WO1995/034671 and Brough et al. (1997) J. VIROL.71: 9206-9213). Other suitable complementing cell lines to produce the replication-deficient adenoviral vector of the disclosure include complementing cells that have been generated to propagate adenoviral vectors encoding transgenes whose expression inhibits viral growth in host cells (see, e.g., U.S. Patent Publication No.2008/0233650). Additional suitable complementing cells are described in, for example, U.S. Patent Nos.6,677,156 and 6,682,929, and PCT Publication No. WO2003/020879. Formulations for adenoviral vector-containing compositions are further described in, for example, U.S. Patent Nos.6,225,289, and 6,514,943, and PCT Publication No. WO2000/034444. [00118] Additional exemplary adenoviral vectors, and/or methods for making or propagating adenoviral vectors are described in U.S. Patent Nos.5,559,099, 5,837,511, 5,846,782, 5,851,806, 5,994,106, 5,994,128, 5,965,541, 5,981,225, 6,040,174, 6,020,191, 6,083,716, 6,113,913, 6,303,362, 7,067,310, and 9,073,980. [00119] Commercially available adenoviral vector systems include the ViraPower™ Adenoviral Expression System available from Thermo Fisher Scientific, the AdEasy™ adenoviral vector system available from Agilent Technologies, and the Adeno-X™ Expression System 3 available from Takara Bio USA, Inc. Viral Vector Production [00120] Methods for producing viral vectors are known in the art. Typically, a virus of interest is produced in a suitable host cell line using conventional techniques including culturing a transfected or infected host cell under suitable conditions so as to allow the production of infectious viral particles. Nucleic acids encoding viral genes and/or tRNAs can be incorporated into plasmids and introduced into host cells through conventional transfection or transformation techniques. Exemplary suitable host cells for production of disclosed viruses include human cell lines such as HeLa, Hela-S3, HEK293, 911, A549, HER96, or PER-C6 cells. Specific production and purification conditions will vary depending upon the virus and the production system employed. [00121] In certain embodiments, producer cells may be directly administered to a subject, however, in other embodiments, following production, infectious viral particles are recovered from the culture and optionally purified. Typical purification steps may include plaque purification, centrifugation, e.g., ultra-centrifugation or cesium chloride gradient centrifugation, clarification, enzymatic treatment, e.g., benzonase or protease treatment, chromatographic steps, e.g., ion exchange chromatography or filtration steps. Attorney Docket No.: TVD-009WO II. Genes of Interest [00122] A non-coding gene of interest can be expressed using an expression vector disclosed herein. By way of example, the expression vector comprises: (a) a first promoter; (b) a second, regulatable promoter; and (c) a gene of interest comprising an antisense strand encoding non-coding RNA (ncRNA) and a complementary sense strand. The first promoter is transcriptionally operative in a first direction to transcribe the antisense strand of the gene of interest and produce the ncRNA. The second promoter is transcriptionally operative in a second direction opposite to the first direction of the first promoter to transcribe the sense strand of the gene of interest. The transcriptional activity of the second promoter can be regulated to interfere with transcriptional activity of the first promoter and reduce production of the ncRNA. [00123] In certain embodiments, the ncRNA is selected from the group consisting of a transfer RNA (tRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), single guide RNA (sgRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), and long noncoding RNA (lncRNA). In certain embodiments, the ncRNA is a suppressor tRNA. Suppressor tRNAs [00124] During protein synthesis, a transfer RNA (tRNA) delivers an amino acid to a ribosome for incorporation into a growing protein (polypeptide) chain. tRNAs typically are about 70 to 100 nucleotides in length, and active tRNAs contain a 3′ CCA sequence that may be transcribed into the tRNA during its synthesis or may be added later during post-transcriptional processing. During aminoacylation, the amino acid that is attached to a given tRNA molecule is covalently attached to the 2’ or 3′ hydroxyl group of the 3′-terminal ribose to form an aminoacyl- tRNA (aa-tRNA). It is understood that an amino acid can spontaneously migrate from the 2’- hydroxyl group to the 3′-hydroxyl group and vice versa, but it is incorporated into a growing protein chain at the ribosome from the 3′-OH position. A loop at the other end of the folded aa- tRNA molecule contains a sequence of three bases known as the anticodon. When this anticodon sequence hybridizes or base-pairs with a complementary three-base codon sequence in a ribosome-bound messenger RNA (mRNA), the aa-tRNA binds to the ribosome and its amino acid is incorporated into the polypeptide chain being synthesized by the ribosome. Because all tRNAs that base-pair with a specific codon are aminoacylated with a single specific amino acid, the translation of the genetic code is effected by tRNAs. Each of the 61 non-termination codons in an mRNA directs the binding of its cognate aa-tRNA and the addition of a single specific amino acid to the growing polypeptide chain being synthesized by the ribosome. Attorney Docket No.: TVD-009WO [00125] tRNAs are generally highly conserved and are often functional across species. Accordingly, a tRNA derived from a bacterial tRNA, a non-mammalian eukaryotic tRNA, or a mammalian (e.g., human) tRNA may be useful in the practice of the disclosure. Nucleotide sequences encoding naturally occurring human tRNAs are known and generally available to those of skill in the art through sources such as Genbank. See also Sprinzl et al. (2005) NUCLEIC ACIDS RES.33: D139-40; Buckland et al. (1996) GENOMICS 35(1):164-71; Schimmel et al. (Eds.) (1979) “Transfer-RNA: Structure, Properties, and Recognition,” Cold Spring Harbor Laboratory; Agris (1983) “The Modified Nucleosides of Transfer RNA, II,” Alan R. Liss Inc. tRNAs are generally highly conserved and are often functional across species. [00126] Suppressor tRNAs are modified tRNAs that insert a suitable amino acid at a mutant site, e.g., a PTC, in protein encoding gene. The use of the word in suppressor is based on the fact, that under certain circumstance, the modified tRNA “suppresses” the phenotypic effect of the coding mutation. Suppressor tRNAs typically contain a mutation (modification) in either the anticodon, changing codon specificity, or at some position that alters the aminoacylation identity of the tRNA. [00127] In certain embodiments, a tRNA (e.g., a suppressor tRNA) contains a modified anticodon region, such that the modified anticodon hybridizes with a different codon than the corresponding naturally occurring anticodon. In certain embodiments, the modified anticodon hybridizes with a termination codon, e.g., a PTC, and as a result, the tRNA incorporates an amino acid into a gene product rather than terminating protein synthesis. In certain embodiments, the modified anticodon hybridizes with a premature termination codon and, and as a result, the tRNA incorporates an amino acid into a gene product at a position that would otherwise result in a truncated gene product caused by the premature termination codon. [00128] In certain embodiments, a tRNA comprises an anticodon that hybridizes to a codon selected from UAG (i.e., an “amber” termination codon), UGA (i.e., an “opal” termination codon), and UAA (i.e., an “ochre” termination codon). In certain embodiments, the anticodon hybridizes to a codon selected from UGA to UAA. In certain embodiments, the anticodon hybridizes to UGA. In certain embodiments, a tRNA comprises an anticodon that hybridizes to a non-standard termination codon, e.g., a 4-nucleotide codon (See, for example, Moore et al. (2000) J. MOL. BIOL.298:195, and Hohsaka et al. (1999) J. AM. CHEM. SOC. 121:12194). [00129] In certain embodiments, the tRNA is aminoacylated or is capable of being aminoacylated with any natural amino acid. For example, a tRNA may be capable of being Attorney Docket No.: TVD-009WO aminoacylated with alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In certain embodiments the tRNA is capable of being aminoacylated with serine, leucine, glutamine, or arginine. In certain embodiments the tRNA is capable of being aminoacylated with glutamine or arginine. In certain embodiments the tRNA is capable of being aminoacylated with arginine. [00130] In certain embodiments, the tRNA (i) comprises an anticodon that hybridizes to a codon as indicated in TABLE 3, and (ii) is aminoacylated or is capable of being aminoacylated with an amino acid as indicated in TABLE 3. TABLE 3

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[00131] In certain embodiments, a suppressor tRNA is expressed using a single expression vector. The suppressor tRNA permits an amino acid to be incorporated into a gene product encoded by a gene at a position that would otherwise result in a truncated gene product caused by a premature termination codon (PTC) in the target gene, and can be used to treat a disease mediated by a PTC in a gene in a subject. [00132] In certain embodiments, multiple (e.g., two or three) suppressor tRNAs, which can be the same or different, are expressed using a single expression vector (see, FIG.3E). Each suppressor tRNA permits an amino acid to be incorporated into a gene product encoded by a gene in a mammalian cell at a position that would otherwise result in a truncated gene product caused by a PTC in the target gene. Expression of multiple suppressor tRNAs from a single expression vector allows for the single expression vector to treat a disease mediated by multiple, different PTCs in the same subject and/or treat a disease mediated by multiple, different PTCs in multiple, different subjects. [00133] Exemplary expression vectors can comprise a tRNA sequence set forth in International patent applications WO2019/090154, WO2020/069194, WO2021/087401 and WO2022/235861. Furthermore, exemplary expression vectors can comprise a tRNA comprising a nucleotide sequence set forth in TABLE 4, wherein the expression vector optionally can comprise 1, 2, 3, 4, or more than 4 copy numbers of the nucleotide sequence encoding the tRNA. In certain embodiments, the tRNA comprises, consists essentially of, or consists of a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to a nucleotide sequence shown in TABLE 4. In certain embodiments, the tRNA comprises, consists essentially of, or consists of a nucleotide sequence selected from any one of SEQ ID NOs: 19-21, 37, 39, 40, 44, 179, 181, 182, and 186, or a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 19-21, 37, 39, 40, 44, 179, 181, 182, and 186. Attorney Docket No.: TVD-009WO [00134] As used herein, percent “identity” between a nucleic acid sequence and a reference sequence is defined as the percentage of nucleotides in the nucleic acid sequence that are identical to the nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Similarly, “percent identity” between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity (e.g., nucleic acid sequence identity or amino acid sequence identity) can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. [00135] It is understood that, throughout the description (e.g., TABLES 4 and 5, and the Sequence Listing), in each instance where a tRNA comprises, consists essentially of, or consists of a nucleotide sequence including one or more thymines (T), a tRNA is also contemplated that comprises, consists essentially of, or consists of the same nucleotide sequence including a uracil (U) in place of one or more of the thymines (T), or a uracil (U) in place of all the thymines (T). Similarly, in each instance where a tRNA comprises, consists essentially of, or consists of a nucleotide sequence including one or more uracils (U), a tRNA is also contemplated that comprises, consists essentially of, or consists of a nucleotide sequence including a thymine (T) in place of the one or more of the uracils (U), or a thymine (T) in place of all the uracils (U). As a result, in TABLES 4 and 5, each thymine (T) can be replaced by a uracil (U). TABLE 4

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[00136] Similarly, exemplary expression vectors can comprise a tRNA comprising a nucleotide sequence encoding a tRNA set forth in TABLE 5, wherein the expression vector optionally can comprise 1, 2, 3, 4, or more than 4 copy numbers of the nucleotide sequence encoding the tRNA. In certain embodiments, the tRNA comprises, consists essentially of, or consists of a nucleotide sequence shown in TABLE 5. In certain embodiments, the tRNA comprises, consists essentially of, or consists of a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence shown in TABLE 5. Attorney Docket No.: TVD-009WO TABLE 5

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[00137] In certain embodiments, the tRNA comprises, consists essentially of, or consists of a nucleotide sequence selected from any one of SEQ ID NOs: 6-9, 11, 16-18, 22, 35, 36, 38, 45, 178, 180, and 187, or a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 6-9, 11, 16-18, 22, 35, 36, 38, 45, 178, 180, and 187. In certain embodiments, the tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 6-9, 11, 16-18, 22, 35, 36, 38, 45, 178, 180, and 187. In certain embodiments, the tRNA comprises, consists essentially of, or consists of a nucleotide sequence selected from any one of SEQ ID NOs: 6, 8, 17, 18, 22, 36, 39, 178, and 181, or a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 6, 8, 17, 18, 22, 36, 39, 178, and 181. In certain embodiments, the tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 6, 8, 17, 18, 22, 36, 39, 178, and 181. [00138] In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 6. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 8. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 9. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 11. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 16. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 17. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 18. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 19. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 20. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 21. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 22. In certain embodiments, the tRNA comprises, consists essentially Attorney Docket No.: TVD-009WO of, or consists of the nucleotide sequence of SEQ ID NO: 35. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 36. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 37. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 38. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 39. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 40. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 44. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 45. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 178. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 179. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 180. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 181. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 182. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 186. In certain embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 187. [00139] In certain embodiments, the tRNA may comprise one or more mutations (e.g., nucleotide substitutions, deletions, or insertions) relative to a reference tRNA sequence (e.g., a tRNA disclosed herein). In certain embodiments, the tRNA may comprise, consist, or consist essentially of, a single mutation, or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more than 15 mutations. It is contemplated that the tRNA may comprise, consist, or consist essentially 1-15, 1-10, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-15, 2-10, 2-7, 2-6, 2-5, 2-4, 2-3, 3-15, 3-10, 3-7, 3-6, 3-5, or 3-4 mutations. [00140] In certain embodiments, in addition to a tRNA coding sequence, the expression vector comprises a nucleotide sequence corresponding to a genomic DNA sequence flanking a wild-type tRNA gene (i.e., a DNA sequence from the same genome as a wild-type tRNA gene and which is 5′ or 3′ to the wild-type tRNA gene in the genome, e.g., immediately 5′ or 3′ to the wild-type tRNA gene in the genome). Attorney Docket No.: TVD-009WO [00141] In certain embodiments, the expression vector further comprises a nucleotide sequence shown in TABLE 6. TABLE 6

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[00142] In certain embodiments, the expression vector comprises a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence shown in TABLE 6. In certain embodiments, in the expression vector, the nucleotide sequence set forth in TABLE 6 is operably linked to the nucleotide sequence encoding the tRNA. In certain embodiments, in the expression vector, the nucleotide sequence set forth in TABLE 6 is 5′ or 3′ (e.g., immediately 5′ or immediately 3) to the nucleotide sequence encoding the tRNA. In certain embodiments, the expression vector comprises a nucleotide sequence selected from any one of SEQ ID NOs: 869-888, or a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from any one of SEQ ID NOs: 869-888. In certain embodiments, the nucleotide sequence set forth in TABLE 6 is selected from any one of SEQ ID NOs: 869-888. In certain embodiments, the nucleotide sequence set forth in TABLE 6 is operably linked to the nucleotide sequence encoding the tRNA. In certain embodiments, in the expression vector, the nucleotide sequence set forth in TABLE 6 is 5′ to the nucleotide sequence encoding the tRNA. In certain embodiments, in the expression vector, the nucleotide sequence set forth in TABLE 6 is immediately 5′ to (i.e., adjacent) the nucleotide sequence encoding the tRNA. [00143] It is contemplated that an expression vector can comprise a nucleotide sequence encoding a tRNA set forth in TABLE 5, wherein the expression vector further comprises a nucleotide sequence set forth in TABLE 6. In certain embodiments, the tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 6-9, 11, 16-18, 22, 35, 36, 38, 45, 178, 180, and 187. In certain embodiments, the tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 6, 8, 17, 18, 22, 36, 39, 178, and 181. In certain embodiments, the nucleotide sequence set forth in TABLE 6 is selected from any one of SEQ ID NOs: 869- 888. The nucleotide sequence set forth in TABLE 6 is operably linked to the nucleotide Attorney Docket No.: TVD-009WO sequence encoding the tRNA. In the expression vector, the nucleotide sequence set forth in TABLE 6 is 5′ to the nucleotide sequence encoding the tRNA. In certain embodiments, in the expression vector, the nucleotide sequence set forth in TABLE 6 is immediately 5′ to (i.e., adjacent) the nucleotide sequence encoding the tRNA. [00144] It is contemplated that a tRNA may comprise one or more modifications. Exemplary modified tRNAs include: acylated tRNA; alkylated tRNA; a tRNA containing one or more bases other than adenine, cytosine, guanine, or uracil; a tRNA covalently modified by the attachment of a specific ligand or antigenic, fluorescent, affinity, reactive, spectral, or other probe moiety; a tRNA containing one or more ribose moieties that are methylated or otherwise modified; aa-tRNAs that are aminoacylated with an amino acid other than the 20 natural amino acids, including non-natural amino acids that function as a carrier for reagents, specific ligands, or as an antigenic, fluorescent, reactive, affinity, spectral, or other probe; or any combination of these compositions. Exemplary modified tRNA molecules are described in Soll et al. (1995) “tRNA: Structure, Biosynthesis, and Function,” ASM Press; El Yacoubi et al. (2012) ANNU. REV. GENET.46:69-95; Grosjean et al. (1998) “Modification and Editing of RNA.” ASM Press; Hendrickson et al. (2004) ANNU. REV. BIOCHEM.73:147-176, 2004; Ibba et al. (2000) ANNU. REV. BIOCHEM.69:617-650; Johnson et al. (1995) COLD SPRING HARBOR SYMP. QUANT. BIOL. 60:71-82; Johnson et al. (1982) J. MOL. BIOL.156:113-140; Crowley et al. (1994) CELL 78:61- 71; Beier et al. (2001) NUCLEIC ACIDS RES.29:4767-4782; Torres et al. (2014) TRENDS MOL. MED.20:306-314; Bjork et al. (1987) ANNU. REV. BIOCHEM.56:263-287; Schaffrath et al. (2017) RNA BIOL.14(9):1209-1222; and Johansson et al. (2008) MOL. CELL. BIOL. 28(10):3301-12. [00145] In certain embodiments, a tRNA comprises a naturally occurring nucleotide modification. Naturally occurring tRNAs contain a wide variety of post-transcriptionally modified nucleotides, which are described, for example, in Machnicka et al. (2014) RNA BIOLOGY 11(12): 1619-1629. In certain embodiments, the tRNA comprises one or more of the residues selected from the group consisting of: 2’-O-methylguanosine or G at position 0; pseudouridine or U at position 1; 2’-O-methyladenosine, A, 2’-O-methyluridine, U, 2’-O- methylcytidine, C, 2’-O-methylguanosine, or G at position 4; N2-methylguanosine or G at position 6; N2-methylguanosine or G at position 7; 1-methyladenosine, A, 1-methylguanosine, G, or a modified G at position 9; N2-methylguanosine or G at position 10; N4-acetylcytidine or C at position 12; pseudouridine, U, 2’-O-methylcytidine, or C at position 13; 1-methyladenosine, A, or a modified A at position 14; dihydrouridine (D) or U at position 16; D or U at position 17; Attorney Docket No.: TVD-009WO 2’-O-methylguanosine or G at position 18; 3-(3-amino-3-carboxypropyl)uridine, D, or U at position 20; 3-(3-amino-3-carboxypropyl)uridine, D, pseudouridine, U, or a modified U at position 20a; D, pseudouridine, or U at position 20b; pseudouridine or U at position 25; pseudouridine, U, N2,N2-dimethylguanosine, N2-methylguanosine, G, or a modified G at position 26; pseudouridine, U, N2,N2-dimethylguanosine, or G at position 27; pseudouridine or U at position 28; pseudouridine or U at position 30; pseudouridine or U at position 31; 2′-O- methylpseudouridine, 2′-O-methyluridine, pseudouridine, U, 2′-O-methylcytidine, 3- methylcytidine, C, or a modified C at position 32; inosine, A, 2-thiouridine, 2′-O-methyluridine, 5-(carboxyhydroxymethyl)uridine methyl ester, 5-carbamoylmethyluridine, 5- carboxymethylaminomethyl-2′-O-methyluridine, 5-methoxycarbonylmethyl-2-thiouridine, 5- methoxycarbonylmethyluridine, pseudouridine, U, a modified U, 2′-O-methylcytidine, 5-formyl- 2′-O-methylcytidine, 5-methylcytidine, C, a modified C, queuosine, mannosyl-queuosine, galactosyl-queuosine, 2′-O-methylguanosine, or G at position 34; pseudouridine or U at position 35; pseudouridine, U, or a modified U at position 36; 1-methylinosine, 2-methylthio-N6- threonylcarbamoyladenosine, N6-isopentenyladenosine, N6-methyl-N6- threonylcarbamoyladenosine, N6-threonylcarbamoyladenosine, A, a modified A, 1- methylguanosine, peroxywybutosine, wybutosine, G, or a modified G at position 37; pseudouridine, U, 5-methylcytidine, C, or a modified C at position 38; 1-methylpseudouridine, 2′-O-methylpseudouridine, 2′-O-methyluridine, pseudouridine, U, 2′-O-methylguanosine, or G at position 39; pseudouridine, U, 5-methylcytidine, or C at position 40; 2′-O-methyluridine, U, or a modified U at position 44; pseudouridine or U at position e11; pseudouridine or U at position e12; pseudouridine or U at position e14; 3-methylcytidine or C at position e2; 7- methylguanosine or G at position 46; D, U, or a modified U at position 47; D, U, 5- methylcytidine, C, or a modified C at position 48; A, a modified A, 5-methylcytidine, C, or a modified C at position 49; pseudouridine, U, 5-methylcytidine, or C at position 50; 5,2′-O- dimethyluridine, 5-methyluridine, pseudouridine, or U at position 54; pseudouridine or U at position 55; 1-methyladenosine, A, or a modified A at position 58; 2′-O-ribosyladenosine (phosphate), A, 2′-O-ribosylguanosine (phosphate), G, or a modified G at position 64; pseudouridine or U at position 65; pseudouridine, U, N2-methylguanosine, or G at position 67; pseudouridine or U at position 68; and, pseudouridine, U, 5-methylcytidine, or C at position 72. A, C, G, and U, refer to unmodified adenine, cytosine, guanine, and uracil, respectively. The numbering of the residues is based on the tRNA numbering system described in Steinberg et al., (1993) NUCLEIC ACIDS RES.21:3011-15. Attorney Docket No.: TVD-009WO [00146] In certain embodiments, the tRNA comprises one or more nucleotide modifications selected from 5-methyl uridine, 5-carbamoylmethyluridine, 5-carbamoyl-methyl-2-O- methyluridine, 5-methoxy-carbonylmethyluridine, 5-methoxycarbonylmethyl-2-thiouridine, pseudouridine, dihydrouridine, 1-methyladenosine, and inosine. [00147] In certain embodiments, multiple (e.g., two or three) suppressor tRNAs are expressed using a single expression vector. Expression of multiple, different types of suppressor tRNAs from a single expression vector allows for the single expression vector to treat a disease mediated by multiple, different premature termination codons (PTCs) in the same subject and/or treat a disease mediated by multiple, different PTCs in multiple, different subjects. [00148] Accordingly, it is contemplated that an expression vector disclosed herein can comprise:(a) a first nucleotide sequence encoding a first suppressor tRNA that comprises an anticodon that hybridizes to a first premature stop codon (e.g., TGA), and is capable of being aminoacylated with a first amino acid; (b) a second nucleotide sequence encoding a second suppressor tRNA that comprises an anticodon that hybridizes to a second premature stop codon (e.g., TAG), and is capable of being aminoacylated with a second amino acid; and optionally, (c) a third nucleotide sequence encoding a third suppressor tRNA that comprises an anticodon that hybridizes to a third premature stop codon (e.g., TAA), and is capable of being aminoacylated with a third amino acid. [00149] It is contemplated that the first amino acid can be selected from arginine, tryptophan, cysteine, serine, glycine, and leucine (e.g., the first amino acid is arginine). Alternatively or in addition, it is contemplated that the second amino acid can be selected from glutamine, glutamic acid, tyrosine, tryptophan, lysine, serine, and leucine (e.g., the second amino acid is glutamine). Alternatively or in addition, it is contemplated that the third amino acid can be selected from glutamine, glutamic acid, tyrosine, lysine, serine, and leucine. In certain embodiments, the second and third amino acid are the same, for example, the second and third amino acid are selected from glutamine, glutamic acid, tyrosine, lysine, serine, and leucine. [00150] In certain embodiments: (i) the first amino acid is arginine, the second amino acid is glutamine, and the third amino acid is lysine; (ii) the first amino acid is arginine, the second amino acid is glutamine, and the third amino acid is glutamic acid; (iii) the first amino acid is arginine, the second amino acid is glutamine, and the third amino acid is tyrosine; (iv) the first amino acid is arginine, the second amino acid is glutamine, and the third amino acid is leucine; (v) the first amino acid is arginine, the second amino acid is tryptophan, and the third amino acid is glutamic acid; or (vi) the first amino acid is arginine, the second amino acid is tyrosine, and Attorney Docket No.: TVD-009WO the third amino acid is glutamic acid. [00151] In certain embodiments: (i) the first amino acid is arginine, the second amino acid is glutamine, and the third amino acid is glutamine; (ii) the first amino acid is arginine, the second amino acid is glutamine, and the third amino acid is glutamic acid; (iii) the first amino acid is arginine, the second amino acid is glutamine, and the third amino acid is lysine; (iv) the first amino acid is arginine, the second amino acid is tryptophan, and the third amino acid is glutamine; or (v) the first amino acid is arginine, the second amino acid is glutamic acid, and the third amino acid is glutamine. [00152] In certain embodiments: (i) the first amino acid is arginine, the second amino acid is glutamine, and the third amino acid is glutamine; (ii) the first amino acid is tryptophan, the second amino acid is glutamic acid, and the third amino acid is glutamic acid; (iii) the first amino acid is cysteine, the second amino acid is tyrosine, and the third amino acid is tyrosine; (iv) the first amino acid is serine, the second amino acid is lysine, and the third amino acid is lysine; (v) the first amino acid is glycine, the second amino acid is serine, and the third amino acid is serine; or (vi) the first amino acid is leucine, the second amino acid is leucine, and the third amino acid is leucine. [00153] In certain embodiments, the expression vector comprises, in order (e.g., in a 5′ to 3′ orientation): (i) the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence; (ii) the first nucleotide sequence, the third nucleotide sequence, and the second nucleotide sequence; (iii) the second nucleotide sequence, the first nucleotide sequence, and the third nucleotide sequence; (iv) the second nucleotide sequence, the third nucleotide sequence, and the first nucleotide sequence; (v) the third nucleotide sequence, the first nucleotide sequence, and the second nucleotide sequence; or (vi) the third nucleotide sequence, the second nucleotide sequence, and the first nucleotide sequence. III. Pharmaceutical Compositions [00154] For therapeutic use, an expression vector or viral particle comprising an expression vector preferably is combined with a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” as used herein refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Attorney Docket No.: TVD-009WO [00155] The term “pharmaceutically acceptable carrier” as used herein refers to buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, e.g., Martin, Remington’s Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA [1975]. Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art. [00156] In certain embodiments, a pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta- cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt- forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or Attorney Docket No.: TVD-009WO pharmaceutical adjuvants (See Remington’s Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). [00157] In certain embodiments, a pharmaceutical composition may contain nanoparticles, e.g., polymeric nanoparticles, liposomes, or micelles (See Anselmo et al. (2016) BIOENG. TRANSL. MED.1: 10-29). In certain embodiments, the composition does not comprise (or is substantially free of, for example, the composition comprises less than 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of) a nanoparticle or an aminolipid delivery compound, e.g., as described in U.S. Patent Publication No.2017/0354672. In certain embodiments, the tRNA or expression vector introduced into the cell or administered to the subject is not conjugated to or associated with another moiety, e.g., a carrier particle, e.g., an aminolipid particle. As used herein, the term “conjugated,” when used with respect to two or more moieties, means that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which structure is used, e.g., physiological conditions. Typically the moieties are attached either by one or more covalent bonds or by a mechanism that involves specific binding. Alternately, a sufficient number of weaker interactions can provide sufficient stability for moieties to remain physically associated. [00158] In certain embodiments, a pharmaceutical composition may contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Sustained-release preparations may include, e.g., porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, ethylene vinyl acetate, or poly-D(−)-3-hydroxybutyric acid. Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art. [00159] Pharmaceutical compositions containing an expression vector and/or viral particle containing an expression vector disclosed herein can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, intrathecal and rectal administration. In certain embodiments, an expression vector and/or viral particle containing an expression vector disclosed herein is administered intrathecally. In certain Attorney Docket No.: TVD-009WO embodiments, expression vector and/or viral particle containing an expression vector disclosed herein is administered by injection. Useful formulations can be prepared by methods known in the pharmaceutical art. For example, see Remington’s Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. [00160] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol), and suitable mixtures thereof. [00161] Pharmaceutical formulations preferably are sterile. Sterilization can be accomplished by any suitable method, e.g., filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution. [00162] The compositions described herein may be administered locally or systemically. Administration will generally be parenteral administration. In a preferred embodiment, the pharmaceutical composition is administered subcutaneously and in an even more preferred embodiment intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. [00163] Generally, a therapeutically effective amount of active component, for example, an expression vector, is in the range of 0.1 mg/kg to 100 mg/kg, e.g., 1 mg/kg to 100 mg/kg, 1 mg/kg to 10 mg/kg. In certain embodiments, a therapeutically effective amount of a viral particles containing an expression vector is in the range of 10² to 10¹⁵ plaque forming units (pfus), e.g., 10² to 10¹⁰, 10² to 10⁵, 10⁵ to 10¹⁵, 10⁵ to 10¹⁰, or 10¹⁰ to 10¹⁵ plaque forming units. The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the antibody, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood-level or tissue-level. Attorney Docket No.: TVD-009WO Alternatively, the initial dosage can be smaller than the optimum, and the daily dosage may be progressively increased during the course of treatment. Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study designed to run from 0.5 mg/kg to 20 mg/kg. Dosing frequency can vary, depending on factors such as route of administration, dosage amount, serum half-life, and the disease being treated. Exemplary dosing frequencies are once per day, once per week and once every two weeks. A preferred route of administration is parenteral, e.g., intravenous infusion. In certain embodiments, a polypeptide and/or multimeric protein is lyophilized, and then reconstituted in buffered saline, at the time of administration. [00164] In certain embodiments, the expression vector is not conjugated to or associated with another moiety, e.g., a carrier particle, e.g., an aminolipid particle. In certain embodiments, the expression vector is introduced into the cell or administered to subject in a dosage form lacking a nanoparticle. In certain embodiments, the expression vector is introduced into the cell or administered to subject in a dosage form lacking an aminolipid delivery compound, e.g., as described in U.S. Patent Publication No.2017/0354672. [00165] In certain circumstances, the pharmaceutical composition comprises an expression vector or viral particle (e.g., AAV particle) including an expression vector, where the expression vector encodes (a) a first suppressor tRNA that comprises an anticodon that hybridizes to a first premature stop codon (e.g., TGA), and is capable of being aminoacylated with a first amino acid; alternatively or in addition, (b) a second suppressor tRNA that comprises an anticodon that hybridizes to a second premature stop codon (e.g., TAG), and is capable of being aminoacylated with a second amino acid; and alternatively or in addition, (c) a third suppressor tRNA that comprises an anticodon that hybridizes to a third premature stop codon (e.g., TAA), and is capable of being aminoacylated with a third amino acid. IV. Uses of the Expression Vectors and Viral Particles Containing the Expression Vectors [00166] The expression vectors and viral particles disclosed herein have a number of uses including the production of viral particles for the delivery of one or more genes or interest, for inducing tissue specific expression of one or more genes of interest, or reducing off-target toxicity in a tissue by one or more genes of interest. Manufacturing Uses [00167] It is understood the expression vectors disclosed herein can be used in the production of viral particles (e.g., for use in therapy), wherein premature expression of the genes of interest in a host cells used to produce the viral particles (producer cells) can be toxic to the Attorney Docket No.: TVD-009WO host cells or can result in suboptimal production conditions for the producer cells. As a result the expression vectors disclosed herein can be employed to prevent or reduce the premature expression of the gene or genes of interest in the producer cells. The expression vectors can be used in the production of any of the viral particles and delivery systems (e.g., adeno-associated virus particles, adenoviral particles, and retroviral particles (e.g., lentiviral particles). [00168] In one embodiment, the expression vectors described herein can be used in producing AAV particles from a producer cell. The method comprises contacting (e.g., transfecting) a producer cell with an effective amount of the expression vector of any one of the above embodiments, thereby to produce the AAV. The AAV can be a high titer AAV, such as, for example, between about 1x10¹² vg/L and about 1x10¹⁶ vg/L. A variety of producer cells can be used including for example, human embryonic kidney (HEK) cells or SF9 insect cells as disclosed in section I. Depending upon the production conditions, the second, regulatable promoter is transcriptionally active in the producer cell to interfere with expression of the gene or genes of interest under the control of the first promoter. Therapeutic Uses [00169] The disclosure provides a method of expressing in a mammalian cell a functional gene product encoded by a gene containing a premature termination codon, the method comprising contacting or exposing the cell with an effective amount (e.g., a therapeutically effective amount) of any of the foregoing expression vectors or pharmaceutical compositions, thereby permitting an amino acid to be incorporated into the gene product at a position that would otherwise result in a truncated gene product caused by the premature termination codon. The gene of interest can be, for example, SCN1A or dystrophin. Under certain circumstances, the tRNA becomes aminoacylated in the cell. [00170] The term “effective amount” as used herein refers to the amount of an active agent (e.g., expression vector according to the present disclosure) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Depending upon the circumstances, e.g., during the treatment of a subject, an effective amount also includes a therapeutically effective amount. [00171] In addition, the disclosure provides a method of expressing in a mammalian cell a functional gene product encoded by a gene containing a first, second, and/or third premature termination codon, the method comprising contacting the cell with effective amount of an expression vector comprising: (a) a nucleotide sequence encoding a first suppressor tRNA that comprises an anticodon that hybridizes to a first premature stop codon (e.g., TGA), and is Attorney Docket No.: TVD-009WO capable of being aminoacylated with a first amino acid; (b) a nucleotide sequence encoding a second suppressor tRNA that comprises an anticodon that hybridizes to a second premature stop codon (e.g., TAG), and is capable of being aminoacylated with a second amino acid; and optionally, (c) a nucleotide sequence encoding a third suppressor tRNA that comprises an anticodon that hybridizes to a third premature stop codon (e.g., TAA), and is capable of being aminoacylated with a third amino acid; thereby permitting an amino acid to be incorporated into the gene product at a position that would otherwise result in a truncated gene product caused by the premature termination codon. [00172] In certain embodiments of any of the foregoing methods, the cell contains less truncated gene product than a cell without the tRNA expressed from a vector disclosed herein. For example, in certain embodiments, the cell contains less than about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the truncated gene product relative to a cell without the tRNA. In certain embodiments, the cell contains from about 5% to about 80%, about 5% to about 60%, about 5% to about 40%, about 5% to about 20%, about 5% to about 10%, about 10% to about 80%, about 10% to about 60%, about 10% to about 40%, about 10% to about 20%, about 20% to about 80%, about 20% to about 60%, about 20% to about 40%, about 40% to about 80%, about 40% to about 60%, or about 60% to about 80% of the truncated gene product relative to a cell without the tRNA. In certain embodiments, there is no detectable truncated gene product in the cell. Truncated gene product amount or expression may be measured by any method known in the art, for example, Western blot or ELISA. [00173] In certain embodiments, the cell contains a greater amount of functional gene product than a cell without the tRNA expressed from a vector disclosed herein. For example, in certain embodiments, the method increases the amount of functional gene product in a cell, tissue, or subject by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, or about 500% relative to a cell, tissue, or subject without the tRNA. In certain embodiments, the method increases the amount of functional gene product in a cell, tissue, or subject, by from about 20% to about 200%, about 20% to about 180%, about 20% to about 160%, about 20% to about 140%, about 20% to about 120%, about 20% to about 100%, about 20% to about 80%, about 20% to about 60%, about 20% to about 40%, about 40% to about 200%, about 40% to about 180%, about 40% to about 160%, Attorney Docket No.: TVD-009WO about 40% to about 140%, about 40% to about 120%, about 40% to about 100%, about 40% to about 80%, about 40% to about 60%, about 60% to about 200%, about 60% to about 180%, about 60% to about 160%, about 60% to about 140%, about 60% to about 120%, about 60% to about 100%, about 60% to about 80%, about 80% to about 200%, about 80% to about 180%, about 80% to about 160%, about 80% to about 140%, about 80% to about 120%, about 80% to about 100%, about 100% to about 200%, about 100% to about 180%, about 100% to about 160%, about 100% to about 140%, about 100% to about 120%, about 120% to about 200%, about 120% to about 180%, about 120% to about 160%, about 120% to about 140%, about 140% to about 200%, about 140% to about 180%, about 140% to about 160%, about 160% to about 200%, about 160% to about 180%, or about 180% to about 200% relative to a cell, tissue, or subject without the tRNA. Functional gene product amount or expression may be measured by any method known in the art, for example, Western blot or ELISA. [00174] In certain embodiments, the tRNA permits an amino acid to be incorporated into the gene product at a position corresponding to a premature termination codon (i.e., the tRNA permits read-through of the premature termination codon), but the tRNA does not permit a substantial amount of amino acid to be incorporated into a gene product at a position corresponding to a native stop codon (i.e., the tRNA does not permit read-through of a native stop codon). For example, in certain embodiments, a disclosed tRNA does not increase read- through of a native stop codon (or all native stop codons) in a cell, tissue, or subject, or increases read-through by less than about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, or about 50%, relative to a cell, tissue, or subject that has not been contacted with the tRNA. Read-through of a native stop codon may be measured by any method known in the art, for example, ribosome profiling. [00175] The compositions and methods disclosed herein can be used to treat a premature termination codon (PTC)-mediated disorder in a subject. As used herein, the term “PTC- mediated disorder” refers to a disorder that is mediated, enhanced, exacerbated, or otherwise facilitated by or associated with a PTC in a gene. Accordingly, provided herein is a method of treating a PTC-mediated disorder in a subject (e.g., human) in need thereof wherein the subject has a gene with a premature termination codon, the method comprising contacting the cell with an effective amount of the expression vector, the virus, or the pharmaceutical composition of any one of the above embodiments, thereby to treat the disorder in the subject. [00176] The term “therapeutically effective amount” as used herein refers to the amount of an active agent (e.g., expression vector, viral particle encoding such expression vector according to Attorney Docket No.: TVD-009WO the present disclosure or a secondary active agent in a combination therapy) sufficient to effect beneficial or desired results in a subject. A therapeutically effective amount can be an amount of an active agent to treat a disorder in a subject in need thereof. A therapeutically effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route. [00177] As used herein, “treat”, “treating” and “treatment” mean the treatment of a disease in a subject, e.g., in a human. This includes: (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease, i.e., causing regression of the disease state. As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably includes humans. [00178] In certain embodiments, the premature termination codon-mediated disorder is a disorder listed in TABLE 7 below, and/or the gene with a premature termination codon is a gene listed in the corresponding row of TABLE 7 below. TABLE 7

Attorney Docket No.: TVD-009WO

[00179] In certain embodiments, the premature termination codon-mediated disorder is a disorder listed in TABLE 8 below, and/or the gene with a premature termination codon is a gene listed in the corresponding row of TABLE 8 below. TABLE 8

Attorney Docket No.: TVD-009WO

[00180] In certain embodiments, the PTC-mediated disorder is an epilepsy (e.g., Dravet syndrome), wherein the method reduces seizure frequency, seizure severity, and/or cognitive impairment in the subject. For example, in certain embodiments, the method reduces seizure frequency in the subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% over the period of, e.g., a day, a week, or a month. In certain embodiments, the method reduces seizure frequency by 50% over the period of, e.g., a day, a week, or a month. Attorney Docket No.: TVD-009WO [00181] In certain embodiments, the PTC-mediated disorder is Dravet and/or the gene with a premature termination codon is SCN1A. In certain embodiments, a premature termination codon in the SCN1A gene is caused by a mutation, or a combination of mutations, selected from c.5745C>G, c.5713G>T, c.5701C>T, c.5677C>T, c.5641C>T, c.5629C>T, c.5623C>T, c.5503A>T, c.5473G>T, c.5437G>T, c.5428C>T, c.5403G>A, c.5402G>A, c.5383G>T, c.5371G>T, c.5049T>G, c.4921G>T, c.4900C>T, c.4873C>T, c.4779del, c.4778G>A, c.4774G>T, c.4761T>G, c.4648G>T, c.4540C>T, c.4516A>T, c.4514C>A, c.4508T>G, c.4488C>G, c.4471G>T, c.4300A>T, c.4269G>A, c.4268G>A, c.4233T>A, c.4222G>T, c.4191G>A, c.4190G>A, c.4186C>T, c.4159A>T, c.4155C>A, c.3964del, c.3952C>T, c.3825G>A, c.3824G>A, c.3819G>A, c.3818G>A, c.3795T>A, c.3789T>G, c.3779G>A, c.3750C>G, c.3724G>T, c.3700C>T, c.3697C>T, c.3657dup, c.3624G>A, c.3604C>T, c.3582G>A, c.3578G>A, c.3574C>T, c.3463C>T, c.3454del, c.3424G>T, c.3422C>A, c.3406G>T, c.3328G>T, c.3273C>A, c.3262G>T, c.3073C>T, c.3060T>A, c.2844T>A, c.2749C>T, c.2695C>T, c.2645T>A, c.2560C>T, c.2551C>T, c.2546C>A, c.2462G>A, c.2298del, c.2228G>A, c.2181G>A, c.2180G>A, c.2101C>T, c.2038A>T, c.1958T>A, c.1837C>T, c.1834C>T, c.1804G>T, c.1795G>T, c.1738C>T, c.1702C>T, c.1660C>T, c.1624C>T, c.1516C>T, c.1378C>T, c.1363C>T, c.1354A>T, c.1348C>T, c.1345G>T, c.1344dup, c.1306G>T, c.1278C>A, c.1278C>G, c.1151G>A, c.1129C>T, c.1118T>A, c.942del, c.751del, c.644T>A, c.327C>G, c.249C>A, c.121A>T, c.4846_4850dup, c.4787_4788del, c.4578_4612dup, c.4211_4212del, c.4125_ 4130delinsATAATCATACTGAT TGCCTAAAACTAAT, c.3690_3693del, c.3338_3339del, c.1247_1248insGTAGA, c.825_826insGTATA, and c.278_279dup. In certain embodiments, a premature termination codon in the SCN1A gene is caused by a mutation, or a combination of mutations, selected from c.58G>T, c.575G>A, c.664C>T, c.962C>G, c.1095dupT, c.1129C>T, c.1315C>T, c.1348C>T, c.1366G>T, c.1492A>T, c.1537G>T, c.1624C>T, c.1738C>T, c.1804G>T, c.1837C>T, c.2134C>T, c.2370T>A, c.2495G>A, c.2593C>T, c.2635delC, c.2904C>A, c.3295G>T, c.3311C>A, c.3452C>G, c.3637C>T, c.3656G>A, c.3733C>T, c.3783C>A, c.3829C>T, c.3985C>T, c.4359T>G, c.4547C>A, c.4573C>T, c.4721C>G, c.4954G>T, c.5641G>T, c.5656C>T, and c.5734C>T. In certain embodiments, a premature termination codon in the SCN1A gene is caused by a mutation selected from c.664C>T, c.1129C>T, c.1492A>T, c.1624C>T, c.1738C>T, c.1837C>T, c.2134C>T, c.2593C>T, c.3637C>T, c.3733C>T, c.3985C>T, c.4573C>T, c.5656C>T, and c.5734C>T. In certain embodiments, a premature termination codon in the SCN1A gene is caused by a mutation selected from c.1738C>T and c.3985C>T. Attorney Docket No.: TVD-009WO [00182] In certain embodiments, a premature termination codon in the SCN1A gene is caused by a mutation set forth in TABLE 9, or a combination of mutations set forth in TABLE 9. TABLE 9

Attorney Docket No.: TVD-009WO

[00183] Additional exemplary mutations, including exemplary mutations causing a premature termination codon in a gene, e.g., the SCN1A gene, can be found in ClinVar (available on the world wide web at ncbi.nlm.nih.gov/clinvar/), “A catalog of SCN1A variants” Lossin et al. (2009) BRAIN DEV.200931(2):114-30, the SCN1A Registry (available on the world wide web at scn1a.net/scn1a-registry/), the SCN1A Mutation Database (available on the Attorney Docket No.: TVD-009WO world wide web at gzneurosci.com/scn1adatabase), and the Leiden Open Variation Database (LOVD v.3.0; available on the world wide web at databases.lovd.nl/shared/genes/SCN1A). Unless indicated otherwise, any SCN1A mutations described herein are relative to SCN1a isoform 1 (NCBI reference sequence NM_001165963, SEQ ID NO: 863). [00184] The disclosure provides a method of treating Dravet syndrome in a subject in need thereof wherein the subject has a SCN1A gene with a mutation set forth in a row TABLE 9, the method comprising administering to the subject an effective amount of an expression vector disclosed herein encoding a suppressor tRNA of the suppressor class indicated in the same row of TABLE 9 as the mutation. “Suppressor Class” as used in TABLE 9 (e.g., Arg>TGA) refers to the endogenous tRNA type from which the suppressor tRNA is derived (e.g., an arginine tRNA) and the termination codon recognized by the suppressor tRNA (e.g., TGA). Exemplary Arg>TGA suppressor tRNAs include tRNAs comprising a nucleotide sequence selected from any one of SEQ ID NOs: 6-9, 11, 16-18, 19-22, and 35. Exemplary Gln>TAA suppressor tRNAs include tRNAs comprising a nucleotide sequence selected from any one of SEQ ID NOs: 36-40, 44, and 45. Exemplary Gln>TAG suppressor tRNAs include tRNAs comprising a nucleotide sequence selected from any one of SEQ ID NOs: 178-182, 186, and 187. [00185] For example, in certain embodiments, the subject has a SCN1A gene with a premature termination codon selected from c.664C>T, c.3637C>T, c.3733C>T, c.2134C>T, and c.1837C>T, and the method comprises administering to the subject an effective amount of a suppressor tRNA comprising a nucleotide sequence selected from any one of SEQ ID NOs: 6-9, 11, 16-18, 19-22, and 35. In certain embodiments, the subject has a SCN1A gene with a premature termination codon selected from c.3607C>T, c.2782C>T, c.3829C>T, and c.2893C>T, and the method comprises administering to the subject an effective amount of a suppressor tRNA comprising a nucleotide sequence selected from any one of SEQ ID NOs: 36- 40, 44, and 45. In certain embodiments, the subject has a SCN1A gene with a premature termination codon selected from c.3106C>T, c.3496C>T, c.5662C>T, c.5461C>T, and c.3730C>T, and the method comprises administering to the subject an effective amount of a suppressor tRNA comprising a nucleotide sequence selected from any one of SEQ ID NOs: 178- 182, 186, and 187. [00186] In certain embodiments, wherein the gene is a SCN1A gene, the SCN1A gene product produced with the tRNA is a functional SCN1A gene product. In certain embodiments, the functional SCN1A gene product has greater activity than the truncated SCN1A gene product, Attorney Docket No.: TVD-009WO e.g., greater voltage-gated sodium channel activity. In certain embodiments, the method increases voltage-gated sodium channel activity in a cell, tissue, or subject by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000% relative to a cell, tissue, or subject without the tRNA. In certain embodiments, the method increases voltage-gated sodium channel activity in a cell, tissue, or subject by from about 20% to about 200%, about 20% to about 180%, about 20% to about 160%, about 20% to about 140%, about 20% to about 120%, about 20% to about 100%, about 20% to about 80%, about 20% to about 60%, about 20% to about 40%, about 40% to about 200%, about 40% to about 180%, about 40% to about 160%, about 40% to about 140%, about 40% to about 120%, about 40% to about 100%, about 40% to about 80%, about 40% to about 60%, about 60% to about 200%, about 60% to about 180%, about 60% to about 160%, about 60% to about 140%, about 60% to about 120%, about 60% to about 100%, about 60% to about 80%, about 80% to about 200%, about 80% to about 180%, about 80% to about 160%, about 80% to about 140%, about 80% to about 120%, about 80% to about 100%, about 100% to about 200%, about 100% to about 180%, about 100% to about 160%, about 100% to about 140%, about 100% to about 120%, about 120% to about 200%, about 120% to about 180%, about 120% to about 160%, about 120% to about 140%, about 140% to about 200%, about 140% to about 180%, about 140% to about 160%, about 160% to about 200%, about 160% to about 180%, or about 180% to about 200% relative to a cell, tissue, or subject without the tRNA. Voltage-gated sodium channel activity may be measured by any method known in the art, for example, as described in Kalume et al. (2007) J. NEUROSCI.27(41):11065-74, Yu et al. (2007) NAT. NEUROSCI.9(9): 1142-9, and Han et al. (2012) NATURE 489(7416): 385–390. [00187] In certain embodiments, the functional SCN1A gene product is the Nav1.1 protein. In certain embodiments, the functional SCN1A gene product comprises, consists essentially of, or consists of the amino acid sequence of any one of the following amino acid sequences, or an amino acid sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the following amino acid sequences (each corresponding to different isoforms of SCN1A): MEQTVLVPPGPDSFNFFTRESLAAIERRIAEEKAKNPKPDKKDDDENGPKPNSDLEAGKNLPFI YGDIPPEMVSEPLEDLDPYYINKKTFIVLNKGKAIFRFSATSALYILTPFNPLRKIAIKILVHS Attorney Docket No.: TVD-009WO LFSMLIMCTILTNCVFMTMSNPPDWTKNVEYTFTGIYTFESLIKIIARGFCLEDFTFLRDPWNW LDFTVITFAYVTEFVDLGNVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF CLSVFALIGLQLFMGNLRNKCIQWPPTNASLEEHSIEKNITVNYNGTLINETVFEFDWKSYIQD SRYHYFLEGFLDALLCGNSSDAGQCPEGYMCVKAGRNPNYGYTSFDTFSWAFLSLFRLMTQDFW ENLYQLTLRAAGKTYMIFFVLVIFLGSFYLINLILAVVAMAYEEQNQATLEEAEQKEAEFQQMI EQLKKQQEAAQQAATATASEHSREPSAAGRLSDSSSEASKLSSKSAKERRNRRKKRKQKEQSGG EEKDEDEFQKSESEDSIRRKGFRFSIEGNRLTYEKRYSSPHQSLLSIRGSLFSPRRNSRTSLFS FRGRAKDVGSENDFADDEHSTFEDNESRRDSLFVPRRHGERRNSNLSQTSRSSRMLAVFPANGK MHSTVDCNGVVSLVGGPSVPTSPVGQLLPEVIIDKPATDDNGTTTETEMRKRRSSSFHVSMDFL EDPSQRQRAMSIASILTNTVEELEESRQKCPPCWYKFSNIFLIWDCSPYWLKVKHVVNLVVMDP FVDLAITICIVLNTLFMAMEHYPMTDHFNNVLTVGNLVFTGIFTAEMFLKIIAMDPYYYFQEGW NIFDGFIVTLSLVELGLANVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLV LAIIVFIFAVVGMQLFGKSYKDCVCKIASDCQLPRWHMNDFFHSFLIVFRVLCGEWIETMWDCM EVAGQAMCLTVFMMVMVIGNLVVLNLFLALLLSSFSADNLAATDDDNEMNNLQIAVDRMHKGVA YVKRKIYEFIQQSFIRKQKILDEIKPLDDLNNKKDSCMSNHTAEIGKDLDYLKDVNGTTSGIGT GSSVEKYIIDESDYMSFINNPSLTVTVPIAVGESDFENLNTEDFSSESDLEESKEKLNESSSSS EGSTVDIGAPVEEQPVVEPEETLEPEACFTEGCVQRFKCCQINVEEGRGKQWWNLRRTCFRIVE HNWFETFIVFMILLSSGALAFEDIYIDQRKTIKTMLEYADKVFTYIFILEMLLKWVAYGYQTYF TNAWCWLDFLIVDVSLVSLTANALGYSELGAIKSLRTLRALRPLRALSRFEGMRVVVNALLGAI PSIMNVLLVCLIFWLIFSIMGVNLFAGKFYHCINTTTGDRFDIEDVNNHTDCLKLIERNETARW KNVKVNFDNVGFGYLSLLQVATFKGWMDIMYAAVDSRNVELQPKYEESLYMYLYFVIFIIFGSF FTLNLFIGVIIDNFNQQKKKFGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNKFQGMVFD FVTRQVFDISIMILICLNMVTMMVETDDQSEYVTTILSRINLVFIVLFTGECVLKLISLRHYYF TIGWNIFDFVVVILSIVGMFLAELIEKYFVSPTLFRVIRLARIGRILRLIKGAKGIRTLLFALM MSLPALFNIGLLLFLVMFIYAIFGMSNFAYVKREVGIDDMFNFETFGNSMICLFQITTSAGWDG LLAPILNSKPPDCDPNKVNPGSSVKGDCGNPSVGIFFFVSYIIISFLVVVNMYIAVILENFSVA TEESAEPLSEDDFEMFYEVWEKFDPDATQFMEFEKLSQFAAALEPPLNLPQPNKLQLIAMDLPM VSGDRIHCLDILFAFTKRVLGESGEMDALRIQMEERFMASNPSKVSYQPITTTLKRKQEEVSAV IIQRAYRRHLLKRTVKQASFTYNKNKIKGGANLLIKEDMIIDRINENSITEKTDLTMSTAACPP SYDRVTKPIVEKHEQEGKDEKAKGK (SEQ ID NO: 863); MEQTVLVPPGPDSFNFFTRESLAAIERRIAEEKAKNPKPDKKDDDENGPKPNSDLEAGKNLPFI YGDIPPEMVSEPLEDLDPYYINKKTFIVLNKGKAIFRFSATSALYILTPFNPLRKIAIKILVHS LFSMLIMCTILTNCVFMTMSNPPDWTKNVEYTFTGIYTFESLIKIIARGFCLEDFTFLRDPWNW LDFTVITFAYVTEFVDLGNVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF Attorney Docket No.: TVD-009WO CLSVFALIGLQLFMGNLRNKCIQWPPTNASLEEHSIEKNITVNYNGTLINETVFEFDWKSYIQD SRYHYFLEGFLDALLCGNSSDAGQCPEGYMCVKAGRNPNYGYTSFDTFSWAFLSLFRLMTQDFW ENLYQLTLRAAGKTYMIFFVLVIFLGSFYLINLILAVVAMAYEEQNQATLEEAEQKEAEFQQMI EQLKKQQEAAQQAATATASEHSREPSAAGRLSDSSSEASKLSSKSAKERRNRRKKRKQKEQSGG EEKDEDEFQKSESEDSIRRKGFRFSIEGNRLTYEKRYSSPHQSLLSIRGSLFSPRRNSRTSLFS FRGRAKDVGSENDFADDEHSTFEDNESRRDSLFVPRRHGERRNSNLSQTSRSSRMLAVFPANGK MHSTVDCNGVVSLVGGPSVPTSPVGQLLPEGTTTETEMRKRRSSSFHVSMDFLEDPSQRQRAMS IASILTNTVEELEESRQKCPPCWYKFSNIFLIWDCSPYWLKVKHVVNLVVMDPFVDLAITICIV LNTLFMAMEHYPMTDHFNNVLTVGNLVFTGIFTAEMFLKIIAMDPYYYFQEGWNIFDGFIVTLS LVELGLANVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLVLAIIVFIFAVV GMQLFGKSYKDCVCKIASDCQLPRWHMNDFFHSFLIVFRVLCGEWIETMWDCMEVAGQAMCLTV FMMVMVIGNLVVLNLFLALLLSSFSADNLAATDDDNEMNNLQIAVDRMHKGVAYVKRKIYEFIQ QSFIRKQKILDEIKPLDDLNNKKDSCMSNHTAEIGKDLDYLKDVNGTTSGIGTGSSVEKYIIDE SDYMSFINNPSLTVTVPIAVGESDFENLNTEDFSSESDLEESKEKLNESSSSSEGSTVDIGAPV EEQPVVEPEETLEPEACFTEGCVQRFKCCQINVEEGRGKQWWNLRRTCFRIVEHNWFETFIVFM ILLSSGALAFEDIYIDQRKTIKTMLEYADKVFTYIFILEMLLKWVAYGYQTYFTNAWCWLDFLI VDVSLVSLTANALGYSELGAIKSLRTLRALRPLRALSRFEGMRVVVNALLGAIPSIMNVLLVCL IFWLIFSIMGVNLFAGKFYHCINTTTGDRFDIEDVNNHTDCLKLIERNETARWKNVKVNFDNVG FGYLSLLQVATFKGWMDIMYAAVDSRNVELQPKYEESLYMYLYFVIFIIFGSFFTLNLFIGVII DNFNQQKKKFGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNKFQGMVFDFVTRQVFDISI MILICLNMVTMMVETDDQSEYVTTILSRINLVFIVLFTGECVLKLISLRHYYFTIGWNIFDFVV VILSIVGMFLAELIEKYFVSPTLFRVIRLARIGRILRLIKGAKGIRTLLFALMMSLPALFNIGL LLFLVMFIYAIFGMSNFAYVKREVGIDDMFNFETFGNSMICLFQITTSAGWDGLLAPILNSKPP DCDPNKVNPGSSVKGDCGNPSVGIFFFVSYIIISFLVVVNMYIAVILENFSVATEESAEPLSED DFEMFYEVWEKFDPDATQFMEFEKLSQFAAALEPPLNLPQPNKLQLIAMDLPMVSGDRIHCLDI LFAFTKRVLGESGEMDALRIQMEERFMASNPSKVSYQPITTTLKRKQEEVSAVIIQRAYRRHLL KRTVKQASFTYNKNKIKGGANLLIKEDMIIDRINENSITEKTDLTMSTAACPPSYDRVTKPIVE KHEQEGKDEKAKGK (SEQ ID NO: 864); MEQTVLVPPGPDSFNFFTRESLAAIERRIAEEKAKNPKPDKKDDDENGPKPNSDLEAGKNLPFI YGDIPPEMVSEPLEDLDPYYINKKTFIVLNKGKAIFRFSATSALYILTPFNPLRKIAIKILVHS LFSMLIMCTILTNCVFMTMSNPPDWTKNVEYTFTGIYTFESLIKIIARGFCLEDFTFLRDPWNW LDFTVITFAYVTEFVDLGNVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF CLSVFALIGLQLFMGNLRNKCIQWPPTNASLEEHSIEKNITVNYNGTLINETVFEFDWKSYIQD SRYHYFLEGFLDALLCGNSSDAGQCPEGYMCVKAGRNPNYGYTSFDTFSWAFLSLFRLMTQDFW Attorney Docket No.: TVD-009WO ENLYQLTLRAAGKTYMIFFVLVIFLGSFYLINLILAVVAMAYEEQNQATLEEAEQKEAEFQQMI EQLKKQQEAAQQAATATASEHSREPSAAGRLSDSSSEASKLSSKSAKERRNRRKKRKQKEQSGG EEKDEDEFQKSESEDSIRRKGFRFSIEGNRLTYEKRYSSPHQSLLSIRGSLFSPRRNSRTSLFS FRGRAKDVGSENDFADDEHSTFEDNESRRDSLFVPRRHGERRNSNLSQTSRSSRMLAVFPANGK MHSTVDCNGVVSLGTTTETEMRKRRSSSFHVSMDFLEDPSQRQRAMSIASILTNTVEELEESRQ KCPPCWYKFSNIFLIWDCSPYWLKVKHVVNLVVMDPFVDLAITICIVLNTLFMAMEHYPMTDHF NNVLTVGNLVFTGIFTAEMFLKIIAMDPYYYFQEGWNIFDGFIVTLSLVELGLANVEGLSVLRS FRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLVLAIIVFIFAVVGMQLFGKSYKDCVCKIA SDCQLPRWHMNDFFHSFLIVFRVLCGEWIETMWDCMEVAGQAMCLTVFMMVMVIGNLVVLNLFL ALLLSSFSADNLAATDDDNEMNNLQIAVDRMHKGVAYVKRKIYEFIQQSFIRKQKILDEIKPLD DLNNKKDSCMSNHTAEIGKDLDYLKDVNGTTSGIGTGSSVEKYIIDESDYMSFINNPSLTVTVP IAVGESDFENLNTEDFSSESDLEESKEKLNESSSSSEGSTVDIGAPVEEQPVVEPEETLEPEAC FTEGCVQRFKCCQINVEEGRGKQWWNLRRTCFRIVEHNWFETFIVFMILLSSGALAFEDIYIDQ RKTIKTMLEYADKVFTYIFILEMLLKWVAYGYQTYFTNAWCWLDFLIVDVSLVSLTANALGYSE LGAIKSLRTLRALRPLRALSRFEGMRVVVNALLGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGK FYHCINTTTGDRFDIEDVNNHTDCLKLIERNETARWKNVKVNFDNVGFGYLSLLQVATFKGWMD IMYAAVDSRNVELQPKYEESLYMYLYFVIFIIFGSFFTLNLFIGVIIDNFNQQKKKFGGQDIFM TEEQKKYYNAMKKLGSKKPQKPIPRPGNKFQGMVFDFVTRQVFDISIMILICLNMVTMMVETDD QSEYVTTILSRINLVFIVLFTGECVLKLISLRHYYFTIGWNIFDFVVVILSIVGMFLAELIEKY FVSPTLFRVIRLARIGRILRLIKGAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIFGMSNF AYVKREVGIDDMFNFETFGNSMICLFQITTSAGWDGLLAPILNSKPPDCDPNKVNPGSSVKGDC GNPSVGIFFFVSYIIISFLVVVNMYIAVILENFSVATEESAEPLSEDDFEMFYEVWEKFDPDAT QFMEFEKLSQFAAALEPPLNLPQPNKLQLIAMDLPMVSGDRIHCLDILFAFTKRVLGESGEMDA LRIQMEERFMASNPSKVSYQPITTTLKRKQEEVSAVIIQRAYRRHLLKRTVKQASFTYNKNKIK GGANLLIKEDMIIDRINENSITEKTDLTMSTAACPPSYDRVTKPIVEKHEQEGKDEKAKGK (SEQ ID NO: 865); MEQTVLVPPGPDSFNFFTRESLAAIERRIAEEKAKNPKPDKKDDDENGPKPNSDLEAGKNLPFI YGDIPPEMVSEPLEDLDPYYINKKTFIVLNKGKAIFRFSATSALYILTPFNPLRKIAIKILVHS LFSMLIMCTILTNCVFMTMSNPPDWTKNVEYTFTGIYTFESLIKIIARGFCLEDFTFLRDPWNW LDFTVITFAYVTEFVDLGNVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF CLSVFALIGLQLFMGNLRNKCIQWPPTNASLEEHSIEKNITVNYNGTLINETVFEFDWKSYIQD SRYHYFLEGFLDALLCGNSSDAGQCPEGYMCVKAGRNPNYGYTSFDTFSWAFLSLFRLMTQDFW ENLYQLTLRAAGKTYMIFFVLVIFLGSFYLINLILAVVAMAYEEQNQATLEEAEQKEAEFQQMI EQLKKQQEAAQAATATASEHSREPSAAGRLSDSSSEASKLSSKSAKERRNRRKKRKQKEQSGGE Attorney Docket No.: TVD-009WO EKDEDEFQKSESEDSIRRKGFRFSIEGNRLTYEKRYSSPHQSLLSIRGSLFSPRRNSRTSLFSF RGRAKDVGSENDFADDEHSTFEDNESRRDSLFVPRRHGERRNSNLSQTSRSSRMLAVFPANGKM HSTVDCNGVVSLVGGPSVPTSPVGQLLPEGTTTETEMRKRRSSSFHVSMDFLEDPSQRQRAMSI ASILTNTVEELEESRQKCPPCWYKFSNIFLIWDCSPYWLKVKHVVNLVVMDPFVDLAITICIVL NTLFMAMEHYPMTDHFNNVLTVGNLVFTGIFTAEMFLKIIAMDPYYYFQEGWNIFDGFIVTLSL VELGLANVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLVLAIIVFIFAVVG MQLFGKSYKDCVCKIASDCQLPRWHMNDFFHSFLIVFRVLCGEWIETMWDCMEVAGQAMCLTVF MMVMVIGNLVVLNLFLALLLSSFSADNLAATDDDNEMNNLQIAVDRMHKGVAYVKRKIYEFIQQ SFIRKQKILDEIKPLDDLNNKKDSCMSNHTAEIGKDLDYLKDVNGTTSGIGTGSSVEKYIIDES DYMSFINNPSLTVTVPIAVGESDFENLNTEDFSSESDLEESKEKLNESSSSSEGSTVDIGAPVE EQPVVEPEETLEPEACFTEGCVQRFKCCQINVEEGRGKQWWNLRRTCFRIVEHNWFETFIVFMI LLSSGALAFEDIYIDQRKTIKTMLEYADKVFTYIFILEMLLKWVAYGYQTYFTNAWCWLDFLIV DVSLVSLTANALGYSELGAIKSLRTLRALRPLRALSRFEGMRVVVNALLGAIPSIMNVLLVCLI FWLIFSIMGVNLFAGKFYHCINTTTGDRFDIEDVNNHTDCLKLIERNETARWKNVKVNFDNVGF GYLSLLQVATFKGWMDIMYAAVDSRNVELQPKYEESLYMYLYFVIFIIFGSFFTLNLFIGVIID NFNQQKKKFGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNKFQGMVFDFVTRQVFDISIM ILICLNMVTMMVETDDQSEYVTTILSRINLVFIVLFTGECVLKLISLRHYYFTIGWNIFDFVVV ILSIVGMFLAELIEKYFVSPTLFRVIRLARIGRILRLIKGAKGIRTLLFALMMSLPALFNIGLL LFLVMFIYAIFGMSNFAYVKREVGIDDMFNFETFGNSMICLFQITTSAGWDGLLAPILNSKPPD CDPNKVNPGSSVKGDCGNPSVGIFFFVSYIIISFLVVVNMYIAVILENFSVATEESAEPLSEDD FEMFYEVWEKFDPDATQFMEFEKLSQFAAALEPPLNLPQPNKLQLIAMDLPMVSGDRIHCLDIL FAFTKRVLGESGEMDALRIQMEERFMASNPSKVSYQPITTTLKRKQEEVSAVIIQRAYRRHLLK RTVKQASFTYNKNKIKGGANLLIKEDMIIDRINENSITEKTDLTMSTAACPPSYDRVTKPIVEK HEQEGKDEKAKGK (SEQ ID NO: 866); MEQTVLVPPGPDSFNFFTRESLAAIERRIAEEKAKNPKPDKKDDDENGPKPNSDLEAGKNLPFI YGDIPPEMVSEPLEDLDPYYINKKTFIVLNKGKAIFRFSATSALYILTPFNPLRKIAIKILVHS LFSMLIMCTILTNCVFMTMSNPPDWTKNVEYTFTGIYTFESLIKIIARGFCLEDFTFLRDPWNW LDFTVITFAYVTEFVDLGNVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF CLSVFALIGLQLFMGNLRNKCIQWPPTNASLEEHSIEKNITVNYNGTLINETVFEFDWKSYIQD SRYHYFLEGFLDALLCGNSSDAGQCPEGYMCVKAGRNPNYGYTSFDTFSWAFLSLFRLMTQDFW ENLYQLTLRAAGKTYMIFFVLVIFLGSFYLINLILAVVAMAYEEQNQATLEEAEQKEAEFQQMI EQLKKQQEAAQAATATASEHSREPSAAGRLSDSSSEASKLSSKSAKERRNRRKKRKQKEQSGGE EKDEDEFQKSESEDSIRRKGFRFSIEGNRLTYEKRYSSPHQSLLSIRGSLFSPRRNSRTSLFSF RGRAKDVGSENDFADDEHSTFEDNESRRDSLFVPRRHGERRNSNLSQTSRSSRMLAVFPANGKM Attorney Docket No.: TVD-009WO HSTVDCNGVVSLGTTTETEMRKRRSSSFHVSMDFLEDPSQRQRAMSIASILTNTVEELEESRQK CPPCWYKFSNIFLIWDCSPYWLKVKHVVNLVVMDPFVDLAITICIVLNTLFMAMEHYPMTDHFN NVLTVGNLVFTGIFTAEMFLKIIAMDPYYYFQEGWNIFDGFIVTLSLVELGLANVEGLSVLRSF RLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLVLAIIVFIFAVVGMQLFGKSYKDCVCKIAS DCQLPRWHMNDFFHSFLIVFRVLCGEWIETMWDCMEVAGQAMCLTVFMMVMVIGNLVVLNLFLA LLLSSFSADNLAATDDDNEMNNLQIAVDRMHKGVAYVKRKIYEFIQQSFIRKQKILDEIKPLDD LNNKKDSCMSNHTAEIGKDLDYLKDVNGTTSGIGTGSSVEKYIIDESDYMSFINNPSLTVTVPI AVGESDFENLNTEDFSSESDLEESKEKLNESSSSSEGSTVDIGAPVEEQPVVEPEETLEPEACF TEGCVQRFKCCQINVEEGRGKQWWNLRRTCFRIVEHNWFETFIVFMILLSSGALAFEDIYIDQR KTIKTMLEYADKVFTYIFILEMLLKWVAYGYQTYFTNAWCWLDFLIVDVSLVSLTANALGYSEL GAIKSLRTLRALRPLRALSRFEGMRVVVNALLGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKF YHCINTTTGDRFDIEDVNNHTDCLKLIERNETARWKNVKVNFDNVGFGYLSLLQVATFKGWMDI MYAAVDSRNVELQPKYEESLYMYLYFVIFIIFGSFFTLNLFIGVIIDNFNQQKKKFGGQDIFMT EEQKKYYNAMKKLGSKKPQKPIPRPGNKFQGMVFDFVTRQVFDISIMILICLNMVTMMVETDDQ SEYVTTILSRINLVFIVLFTGECVLKLISLRHYYFTIGWNIFDFVVVILSIVGMFLAELIEKYF VSPTLFRVIRLARIGRILRLIKGAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIFGMSNFA YVKREVGIDDMFNFETFGNSMICLFQITTSAGWDGLLAPILNSKPPDCDPNKVNPGSSVKGDCG NPSVGIFFFVSYIIISFLVVVNMYIAVILENFSVATEESAEPLSEDDFEMFYEVWEKFDPDATQ FMEFEKLSQFAAALEPPLNLPQPNKLQLIAMDLPMVSGDRIHCLDILFAFTKRVLGESGEMDAL RIQMEERFMASNPSKVSYQPITTTLKRKQEEVSAVIIQRAYRRHLLKRTVKQASFTYNKNKIKG GANLLIKEDMIIDRINENSITEKTDLTMSTAACPPSYDRVTKPIVEKHEQEGKDEKAKGK (SEQ ID NO: 867); or MFLKIIAMDPYYYFQEGWNIFDGFIVTLSLVELGLANVEGLSVLRSFRLLRVFKLAKSWPTLNM LIKIIGNSVGALGNLTLVLAIIVFIFAVVGMQLFGKSYKDCVCKIASDCQLPRWHMNDFFHSFL IVFRVLCGEWIETMWDCMEVAGQAMCLTVFMMVMVIGNLVVLNLFLALLLSSFSADNLAATDDD NEMNNLQIAVDRMHKGVAYVKRKIYEFIQQSFIRKQKILDEIKPLDDLNNKKDSCMSNHTAEIG KDLDYLKDVNGTTSGIGTGSSVEKYIIDESDYMSFINNPSLTVTVPIAVGESDFENLNTEDFSS ESDLEESKEKLNESSSSSEGSTVDIGAPVEEQPVVEPEETLEPEACFTEGCVQRFKCCQINVEE GRGKQWWNLRRTCFRIVEHNWFETFIVFMILLSSGALAFEDIYIDQRKTIKTMLEYADKVFTYI FILEMLLKWVAYGYQTYFTNAWCWLDFLIVDVSLVSLTANALGYSELGAIKSLRTLRALRPLRA LSRFEGMRVVVNALLGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFYHCINTTTGDRFDIEDV NNHTDCLKLIERNETARWKNVKVNFDNVGFGYLSLLQVATFKGWMDIMYAAVDSRNVELQPKYE ESLYMYLYFVIFIIFGSFFTLNLFIGVIIDNFNQQKKKFGGQDIFMTEEQKKYYNAMKKLGSKK PQKPIPRPGNKFQGMVFDFVTRQVFDISIMILICLNMVTMMVETDDQSEYVTTILSRINLVFIV Attorney Docket No.: TVD-009WO LFTGECVLKLISLRHYYFTIGWNIFDFVVVILSIVGMFLAELIEKYFVSPTLFRVIRLARIGRI LRLIKGAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIFGMSNFAYVKREVGIDDMFNFETF GNSMICLFQITTSAGWDGLLAPILNSKPPDCDPNKVNPGSSVKGDCGNPSVGIFFFVSYIIISF LVVVNMYIAVILENFSVATEESAEPLSEDDFEMFYEVWEKFDPDATQFMEFEKLSQFAAALEPP LNLPQPNKLQLIAMDLPMVSGDRIHCLDILFAFTKRVLGESGEMDALRIQMEERFMASNPSKVS YQPITTTLKRKQEEVSAVIIQRAYRRHLLKRTVKQASFTYNKNKIKGGANLLIKEDMIIDRINE NSITEKTDLTMSTAACPPSYDRVTKPIVEKHEQEGKDEKAKGK (SEQ ID NO: 868). [00188] In addition, the expression vectors described herein can be used to reduce off-target toxicity in a subject, for example, in a tissue of the subject. The method comprises administered to the subject an effective amount of an expression vector, virus encoding an expression vector, or a pharmaceutical composition containing such an expression vector or virus, thereby to reduce off-target toxicity in the subject. Similarly, the expression vectors can be used to reduce expression of a gene of interest in a tissue of a subject. The method comprises administering to the subject an effective amount of an expression vector, virus encoding an expression vector, or a pharmaceutical composition containing such an expression vector or virus thereby to reduce the expression of the gene of interest in the tissue of the subject. In certain embodiments of each method, the tissue (e.g., human tissue) is liver, heart, muscle, retina, inner-ear, spinal cord, or dorsal root ganglion. In certain embodiments of each method, the second promoter comprises a liver-specific promoter, heart-specific promoter, muscle-specific promoter, retinal-specific promoter, inner ear-specific promoter, spinal cord-specific promoter, or dorsal root ganglion- specific promoter. [00189] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and/or modalities. The term administered “in combination,” as used herein, is understood to mean that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, such that the effects of the treatments on the patient overlap at a point in time. In certain embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in Attorney Docket No.: TVD-009WO the absence of the first treatment, or the analogous situation is seen with the first treatment. In certain embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered. [00190] In certain embodiments, a method or composition described herein is administered in combination with one or more additional therapeutic agents, e.g., DIACOMIT^® (stiripentol), EPIODOLEX^® (cannabidiol), a ketogenic diet, ONFI^® (clobazam), TOPAMAX^® (topiramate), fenfluramine, or valproic acid. For example, during the treatment of Dravet Syndrome, a method or composition described herein is administered in combination with one or more additional therapeutic agents, e.g., DIACOMIT^® (stiripentol), EPIODOLEX^® (cannabidiol), a ketogenic diet, ONFI^® (clobazam), TOPAMAX^® (topiramate), fenfluramine, or valproic acid. [00191] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps. [00192] In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components. [00193] Further, it should be understood that elements and/or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present disclosure, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present disclosure and/or in methods of the present disclosure, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and disclosure(s). For Attorney Docket No.: TVD-009WO example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the disclosure(s) described and depicted herein. [00194] It should be understood that the expression “at least one of” includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and/or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context. [00195] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context. [00196] Where the use of the term “about” is before a quantitative value, the present disclosure also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred. [00197] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present disclosure remain operable. Moreover, two or more steps or actions may be conducted simultaneously. [00198] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present disclosure and does not pose a limitation on the scope of the disclosure unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present disclosure. EXAMPLES [00199] The following Examples are merely illustrative and are not intended to limit the scope or content of the disclosure in any way. Example 1: Design and Characterization of Convergent Promoters that Transcribe tRNAs for Rescuing Premature Stop Codons [00200] This Example describes the design and characterization of an anticodon-edited tRNA for the rescue of MECP2, which is a gene that often has point mutations that introduce a premature termination codon (PTC) leading to Rett syndrome. Further, the Example describes Attorney Docket No.: TVD-009WO the design of a recombinant adeno-associated virus (rAAV) encoding a nucleic acid molecule of the disclosure which includes convergently-aligned promoters. Such convergent promoters allow for conditional expression of the gene of interest, such as a tRNA, which requires regulation because overexpression of anticodon-edited tRNAs can elicit toxicity. Materials and Methods Plasmids and Cloning [00201] All plasmids were cloned using DNA synthesis or restriction enzymes, or a combination of both methods. [00202] Plasmid maps were designed in silico using SnapGene and subsequently synthesized with the required restriction enzyme cutting sites and DNA fragments were combined using DNA recombination or ligation. The final plasmid sequences were confirmed using restriction enzymes and DNA sequencing. Construction of Expression Vector and Expression System [00203] Elements of the plasmids, including Tet-Off 3G and/or Tet-On 3G promoter sequences, as well as a tTA coding sequence, were synthesized using DNA thesis; U6 promoter- driven tRNA constructs were synthesized and subsequently cloned into the Tet-promoter containing plasmids using restriction enzymes to generate the expression vector for use. The vector includes a ssAAV.TRE3G.MCS.BGHpA.CMV.Tet3G.WPRE.SV40pA plasmid backbone, with a Tet-On TRE3GV promoter driving expression of the tRNA, and a Tet-Off Tet3G tTA element. A map of this expression vector is provided in FIG.10. AAV Production (PackGene) [00204] Triple-plasmid transfection was conducted using polyethylenimine (PEI, Polyscience) and optionally including other transfection reagents to produce rAAV particles. Transfected plasmids included single-stranded (ssAAV) or self-complementary (scAAV) transfer plasmids (“Transfer”) including a phospho-AAV (pAAV) backbone containing a nucleic acid molecule of the disclosure encoding a gene of interest (GOI) located between two ITRs. [00205] Helper plasmids used included the plasmid pRep2CapX (“RepCap”), which encodes one or more AAV serotype 2 (in this example, AAV2) (Rep) proteins, and a defined capsid (Cap) protein for the desired serotype (CapX), or a mixture of different Cap variants, such as engineered capsid libraries. The additional helper plasmid was the “pHelper” plasmid, Attorney Docket No.: TVD-009WO which encodes the other additional AAV proteins required for producing functional AAV particles. [00206] The three helper plasmids were co-transfected into HEK293T cells. In this approach, the HEK2293T cells were cultured in Dulbecco’s modified essential medium (DMEM; Invitrogen, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% streptomycin and penicillin (S/P) antibiotics (Gibco, USA) at 37 °C. When the cells reached 80% confluence, they were transfected with a molar ratio of 1:1:1 of pHelper:RepCap:Transfer plasmids. At 72 hours post-transfection, cells were harvested by 4,000 g centrifugation at 4 °C for 30 minutes. The pellet was collected and re-suspended in buffer containing 10 mM Tris- HCl, pH 8.0. The suspension was subjected to four freeze-thaw cycles by dry ice/ethanol and a 37 °C water bath. The cell debris was sonicated and then digested with DNase I (200 units in 1.5 mL) for 1 hour at 37 °C. Following centrifugation at 10,000 g for 10 mins at 4 °C, the supernatant was collected as AAV crude lysate. [00207] The crude lysate was diluted with 10 mM Tris-HCl, pH 8.0 to a final volume of 10 mL and then bottom-loaded to a discontinuous gradient of 15%, 25%, 40%, and 60% iodixanol in a 39 mL ultracentrifuge tube (QuickSeal, 342414). After ultracentrifugation at 350,000 g and 18 °C for 1 hour, 3 mL fractions of a lower layer (the lower 40% of the total volume) and 0.5 mL of an upper layer (the upper 60% of the total volume) were collected. Ultracentrifugation was then repeated at 350,000 g at 18 °C for 1 hour, and the fractions were de-salted using a 100 kDa Cutoff Ultrafiltration tube (15 ml; Millipore, USA). The purified AAVs were stored at -80 °C until usage. The final formulation buffer consisted of: AAV2 including 1x Tris + 0.001% pluronic F-68. AAV Particle Genome Titration [00208] The viral genomic titers were determined by a SYBR Green quantitative polymerase chain reaction (qPCR) (Bio-Rad, USA) assay and/or a droplet digital polymerase chain reaction (ddPCR; Bio-Rad, USA). Polymerase chain reaction (PCR) primers were designed using SnapGene for each AAV. The viral infectious titers and genome copies (GC; see FIG.5; and relative production scale (FIG.6) were measured by transducing primary neurons. The composition of each vector is summarized in TABLE 10. TABLE 10

Attorney Docket No.: TVD-009WO

MECP2 Primary Neuron Assay Experimental Design [00209] Cortical cultures were made from MECP2 hemizygous mouse pups at embryonic day (E)14-E16. 100,000 cells were seeded per well in Poly-D-Lysine (PDL)-coated 48-well plates on in vitro day (DIV)0. At DIV4, cells were transduced with rAAV at 7.5 x 10³ or 2.5 x 10⁴ viral genomes per cell (vg/cell). Each dose was tested in two wells with two un-dosed wells serving as controls. On DIV5, half of the medium was replaced with fresh culture medium. At that time, one of two wells for each dose also received freshly prepared Doxycycline hyclade (50 ng/mL final concentration). Doxycycline hyclade was supplemented every 2 days with subsequent media changes. On DIV11, cells were fixed with 4% paraformaldehyde (PFA), tissues were collected and stained using immunohistochemistry (IHC), imaged, and analyzed, as described below. Primary Cortical Culture Preparation Dissection of embryos [00210] Embryos were dissected and the respective cortices were collected. The olfactory bulb and meninges were removed. The two brain hemispheres were placed into an Eppendorf tube filled with cold nutrient broth (NB) media, while minimizing the transfer of Hanks’ Balanced Salt Solution (HBSS), which was present in the dish in which the brains were dissected. Brain tissues were kept on ice. Dissociation and Plating [00211] Digestion media, dissociation media, and plating media with or without fetal bovine serum (FBS; see recipes in TABLES 11-14, below) were freshly prepared and all media were warmed to 37 °C before usage. TABLE 11 - Digestion media

Attorney Docket No.: TVD-009WO

TABLE 12 - Dissociation media

TABLE 13 - Plating media with 5% FBS

TABLE 14 - Plating media without FBS

[00212] 3-4 pairs of cortices were added into 2-3 mL of digestion medium and put in a 37 °C water bath for 12 minutes and swirled every 5 minutes. The digestion medium was removed and 1 mL of dissociation medium was added. The tissues were triturated and the supernatant was collected. 0.5 mL of dissociation medium was added and triturated, and supernatant was collected. This was repeated until debris was absent. Cells were filtered and spun at room temperature for 4 minutes at 200 g. The supernatant was aspirated, and then 5 mL of plating medium containing 5% FBS was added and cell pellets were resuspended. After resuspension, 10 µL of the cortical suspension was added to 10 μL of trypan blue and the cells were counted. Plating media with 5% FBS was used to dilute the cells to a seeding density of 1 x 10⁵ cells per well; 500 µL/well (48 well plates, Corning, Cat 356509). Attorney Docket No.: TVD-009WO Maintenance [00213] Media was replaced at DIV5. 250 μL was removed and replaced with the addition of 25 μL of distilled water and AraC (Cytosine β-D-arabinofuranoside hydrochloride , C6645, Sigma) to inhibit the growth of glial cells. The AraC was prepared by adding 2 µM AraC to plating media, making a final concentration of AraC of 1 μM. At DIV8 and at every following 2-3 days from that point on, half of the media was removed and replaced with 25 μL of distilled water and AraC (C6645, Sigma), as above. Immunohistochemistry [00214] Immunohistochemistry was performed, as is known in the art, with primary antibodies including mouse anti-NeuN (Abcam ab104224, 1:250) and rabbit anti-MECP2 (CST D4F33456, 1:200). Secondary antibodies included Alexa 568 goat anti-mouse, A11031 (1:1000) and Alexa 647 goat anti-rabbit, A21245 (1:1000). Imaging and Analysis [00215] Images were taken on an Evos7000 microscope with a 20x objective. Five images per well were acquired from all the wells of the 48 well plate. Image analysis was performed using Cellprofiler. DAPI/NeuN-positive nuclei were identified. Following, MECP2 intensity was measured in DAPI/NeuN-positive nuclei. MECP2 signal from a non-transduced well was taken for background normalization. MECP2 intensity (FIG.7) was measured and the number of neurons (FIG.9) and MECP2-positive neurons (FIG.8) were counted. Results AAV Production [00216] As shown in FIGs.5-6, the inclusion of a gene encoding a suppressor tRNA into an AAV vector decreased AAV production and, consequently, increased the scale of AAV production required to generate at least 2e¹³ AAV genome copies. However, the use of a convergently-aligned promoter system to regulate suppressor tRNA expression resulted in an improved AAV yield, and reduced the need to scale up AAV production to reach a target yield of 2e¹³ genome copies. Rescue of Expression [00217] Rescue of the expression of MECP2 in the MECP2 hemizygous mice transfected with a codon-edited tRNA controlled by a type 2 RNA polymerase III promoter convergently- aligned against a RNA polymerase II promoter (here, a Tet-On promoter as described and Attorney Docket No.: TVD-009WO exemplified above), were assessed. The tRNA permits an amino acid to be incorporated into the gene product at a position corresponding to a premature termination codon (PTC; i.e., the tRNA permits read-through of the PTC), such that the higher MECP2 expression intensity in FIG.7 demonstrates that the tRNA was present and allowed PTC read-through to a greater extent when doxycycline (Dox) was provided. A similar trend was observed when MECP2 rescue was quantified by the percentage of MECP2-positive neurons (FIG.8). Across conditions, there was no reduction in the number of neurons (FIG.9), indicating that there was no notable toxicity. [00218] Taken together, these results demonstrate that a gene of interest, such as a codon- modified tRNA, can be conditionally regulated by a convergently-aligned promoter, such as a convergently-aligned Tet-On promoter. Example 2: Design and Characterization of an Exemplary System for Tissue Specifically Transcribing Suppressor tRNAs for Rescuing Premature Stop Codons [00219] This Example describes the design of a system to tissue-specifically transcribe suppressor tRNAs, which require regulation because, under certain circumstances, overexpression of anticodon-edited suppressor tRNAs can elicit toxicity in particular tissues. Materials and Methods Animals [00220] To produce Dravet Syndrome (DS) mice, male Scn1a^WT/R613X mice on a 129S1/SvImJ genetic background were crossed with wild-type (WT) female mice on a C57BL/6J background (The Jackson Laboratory, stock no.000664), generating F1 mice on a 50:50 genetic background. Both male and female offspring were used for experiments. AAV Viral Vectors [00221] AAV viral vectors were generated comprising a TRE3GV RNA Polymerase II promoter (Tetracycline Response Element) transcriptionally operative in a direction opposite to three copies of an Arg>TGA suppressor tRNA encoded by a nucleic acid sequence of SEQ ID NO: 18. Vectors also contained a Tet-Off tetracycline transactivator (tTA) element capable of binding and initiating transcription from the Tetracycline Response Element. The Tet-Off tTA element was placed under transcriptional control of either the liver tissue-specific human alpha- 1-antitrypsin (hAAT) RNA Polymerase II promoter or the liver tissue-specific thyroxine binding globulin (TBG) RNA Polymerase II promoter (FIGs.11B and 11C, respectively). Activation of the liver specific expression promoter induces collision with the convergent Pol III promoter expressing the suppressor tRNAs. A control vector lacking the Tet-Off tTA element was also Attorney Docket No.: TVD-009WO generated to assess suppressor tRNA expression in the absence of tissue-specific RNA Polymerase II promoter collision regulation (FIG.11A). Recombinant AAV9 (rAAV9) particles comprising these viral vectors were generated using standard methods known in the art, such as those described in Example 1. The viral genomic titers were determined by a SYBR green qPCR (Bio-Rad, USA) assay. IV Facial Vein Injection of AAVs [00222] AAVs were administered to mice by facial vein injection at P1. Pups were dosed with 5E¹² viral genomes (VG) per kilogram. After injection, mice were monitored following approved protocols and allowed to develop normally until tissue collection. Tissue Collection [00223] To assess suppressor tRNA expression in the heart and liver, AAV-dosed mice were euthanized two weeks post-injection. Heart and liver tissue were dissected and flash frozen for subsequent analysis. Quantification of Suppressor tRNA Levels by ddPCR [00224] Total RNA was extracted from dissected liver and heart tissues using an miRNeasy Micro kit (Qiagen). Suppressor tRNA expression was quantified by reverse transcription droplet digital PCR (RT-ddPCR) and normalized relative to the endogenous tRNA- Gly-TCC-2, which was also quantified using RT-ddPCR. Results [00225] As shown in FIG.12, the system described herein enabled tissue-specific expression of suppressor tRNAs, as demonstrated by significantly greater expression in the heart, as compared to the liver. In the control system with no tissue specific Pol II promoter, no difference in the levels of suppressor tRNA were observed between the heart and liver tissue. Example 3: Design and In Vivo Characterization of Convergent Promoters that Transcribe tRNAs in a Tissue-Specific Manner for Rescuing Premature Stop Codons [00226] This Example describes the design of a rAAV encoding a nucleic acid molecule of the disclosure which includes convergently-aligned promoters. Such convergent promoters allow for tissue-specific expression of the gene of interest, such as a suppressor tRNA, which requires regulation because overexpression of anticodon-edited tRNAs can elicit toxicity. [00227] Using the Materials and Methods described in Example 1, an AAV viral vector encoding one or more tissue-specific RNA polymerase II promoters transcriptionally operative Attorney Docket No.: TVD-009WO in a direction opposite to one or more suppressor tRNAs is generated (FIG.13). The tissue- specific RNA polymerase II promoter can be, for example, a tissue-specific promoter described herein, such as a tissue-specific promoter set forth in TABLE 1 or any suitable tissue-specific promoter. The suppressor tRNA(s), for example, can be encoded by a nucleotide sequence set forth in TABLE 4 or TABLE 5. The selected suppressor tRNA(s) can be transcribed from either their endogenous Type 2 RNA Polymerase III promoter and/or an upstream Type 3 RNA polymerase III promoter (see e.g., FIGs.3A-3D). rAAV particles comprising the viral vector can be generated using standard methods known in the art. The viral genomic titers are determined by a SYBR green qPCR (Bio-Rad, USA) assay and/or ddPCR (Bio-Rad, USA). [00228] AAVs are administered to mice, such as transgenic mice having a PTC in a gene of interest, either systemically (e.g., by IV tail or facial vein injection) or by ICV injection following standard protocols, such as those described in Example 2. For ICV injection in mice, P1 pups are used. IV injections are carried out in pups, juveniles, or adult mice. After injection, mice are monitored following approved protocols and allowed to develop normally until tissue collection, which can be performed as e.g., described in Example 2. [00229] The ability of tissue-specific RNA polymerase II promoters to downregulate suppressor tRNA expression in targeted tissues/organs is assessed by quantifying tRNA levels in both targeted and non-targeted tissues/organs using standard methods known in the art. For example, detection methods may include tRNA-seq (e.g., as described in Pinkard et al. (2020) NATURE COMMUNICATIONS, 11.1: 4104), reverse transcription digital PCR, and reverse transcription quantitative PCR. Tissue-specific downregulation is determined relative to control AAV viral vectors that contain equivalent suppressor tRNAs but lack convergently-aligned tissue-specific RNA polymerase II promoters. Example 4: Design and In Vitro Characterization of Convergent Promoters that Transcribe tRNAs in a Stress-Responsive Manner for Rescuing Premature Stop Codons [00230] This Example describes the design of a rAAV encoding a nucleic acid molecule of the disclosure which includes convergently-aligned promoters. Such convergent promoters allow for stress-responsive expression of the gene of interest, such as a suppressor tRNA, where it would be favorable to downregulate expression of anticodon-edited tRNAs in cells exhibiting cellular stress. [00231] Using the Materials and Methods described in Example 1, an AAV viral vector encoding one or more RNA polymerase II stress-responsive promoters transcriptionally operative in a direction opposite to one or more suppressor tRNAs is generated (FIG.14). The Attorney Docket No.: TVD-009WO stress-responsive promoter can be, for example, a stress-responsive promoter described herein, including, for example, a stress-responsive response element set forth in TABLE 2 operably linked to a minimal promoter, or any suitable combination of a stress-responsive response element operably linked to a promoter, such as a minimal promoter. The suppressor tRNA(s), for example, can be encoded by a nucleotide sequence set forth in TABLE 4 or TABLE 5. The selected suppressor tRNA(s) can be transcribed from either their endogenous Type 2 RNA Polymerase III promoter and/or an upstream Type 3 RNA polymerase III promoter (see e.g., FIGs.3A-3D). rAAV particles comprising the viral vector are generated using standard methods known in the art. The viral genomic titers are determined by a SYBR green qPCR (Bio-Rad, USA) assay and/or ddPCR (Bio-Rad, USA). [00232] The ability of RNA polymerase II stress-responsive promoters to reduce the toxicity of AAV viral vectors that express suppressor tRNAs, while still enabling suppression of PTCs, is assessed in primary mouse cortical neuron cultures. Briefly, primary cortical neurons are obtained from either wild-type mouse pups or hemizygous male mouse pups with an inactivating Arg(R) > TGA mutation in the gene encoding MECP2 (R255X). At in vitro day 4 (DIV4), the cells are transduced with rAAV1 or rAAV9 at varying doses (for rAAV1, suitable doses include, for example, 5E3, 1.5E4, and 5E4 vg/cell, while for rAAV9, suitable doses include, for example, 5E4, 1.5E5, and 5E5 vg/cell). Primary cortical neurons from wild-type mice are co-transduced with AAV viral vectors expressing a PTC reporter construct (e.g., EGFP containing an inactivating PTC, luciferase containing an inactivating PTC, etc,) to quantify their ability to suppress nonsense mutations. On DIV11, cells are fixed and stained via immunohistochemistry for (1) NeuN (to identify neurons) and for either (2a) a full-length PTC reporter or (2b) full-length MECP2. Toxicity is assessed based upon neuronal cell survival (e.g., counting NeuN-positive cells) and/or a lactic dehydrogenase (LDH) based in vitro toxicology assay (Sigma-Aldrich). Example 5: Design and In Vivo Characterization of Exemplary Convergent Promoters that Transcribe tRNAs in a Stress-Responsive Manner for Rescuing Premature Stop Codons [00233] This Example describes the design of a rAAV encoding a nucleic acid molecule of the disclosure which includes convergently-aligned promoters. Such convergent promoters allow for stress-responsive expression of the gene of interest, such as a suppressor tRNA, where it would be favorable to downregulate expression of anticodon-edited tRNAs in cells exhibiting cellular stress. [00234] Using the Materials and Methods described in Example 1, an AAV viral vector Attorney Docket No.: TVD-009WO encoding one or more RNA polymerase II stress-responsive promoters transcriptionally operative in a direction opposite to one or more suppressor tRNAs is generated (FIG.14). The stress-responsive promoter can be, for example, a stress-responsive promoter described herein, including, for example, a stress-responsive response element set forth in TABLE 2 operably linked to a minimal promoter, or any suitable combination of a stress-responsive response element operably linked to a promoter, such as a minimal promoter. The suppressor tRNA(s), for example, can be encoded by a nucleotide sequence set forth in TABLE 4 or TABLE 5. The selected suppressor tRNA(s) can be transcribed from either their endogenous Type 2 RNA Polymerase III promoter and/or an upstream Type 3 RNA polymerase III promoter (see e.g., FIGs.3A-3D). rAAV particles comprising the viral vector are generated using standard methods known in the art. The viral genomic titers are determined by a SYBR green qPCR (Bio-Rad, USA) assay and/or ddPCR (Bio-Rad, USA). [00235] AAVs are administered to mice, such as transgenic mice having a PTC in a gene of interest, either systemically (e.g., by IV tail or facial vein injection) or by ICV injection following standard protocols, such as those described in Example 2. For ICV injection in mice, P1 pups are used. IV injections are carried out in pups, juveniles, or adult mice. After injection, mice are monitored following approved protocols and allowed to develop normally until tissue collection, which can be performed as e.g., described in Example 2. [00236] The ability of RNA polymerase II stress-responsive promoters to reduce the in vivo toxicity of AAV viral vectors that express suppressor tRNAs, while still facilitating suppression of PTCs, is assessed by quantifying both adverse events associated with toxicity and readthrough of PTCs in AAV-dosed mice. Adverse events and PTC readthrough are evaluated relative to mice dosed with control AAV viral vectors that contain equivalent suppressor tRNAs but lack convergently-aligned RNA polymerase II stress-responsive promoters. Adverse events are quantified at multiple defined timepoints following AAV administration using one or more of the following metrics: body weight, locomotor activity, fur condition, eye squinting, posture, and/or molecular markers of toxicity. Readthrough of PTCs is quantified using either (1) co- administration of AAV viral vectors expressing a PTC reporter construct (e.g., EGFP containing an inactivating PTC, luciferase containing an inactivating PTC, etc.) or (2) mouse lines with an inactivating mutation in one or more endogenous genes (e.g., Scn1a^WT/R613X mice with an inactivating PTC at R613 (The Jackson Laboratory, stock no.000664) or MECP2 hemizygous male mice with an inactivating PTC at R255 (The Jackson Laboratory, stock no.012602)). Attorney Docket No.: TVD-009WO INCORPORATION BY REFERENCE [00237] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes. EQUIVALENTS [00238] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the disclosure described herein. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

Attorney Docket No.: TVD-009WO WHAT IS CLAIMED IS: 1. An expression vector comprising: (a) a first promoter; (b) a second promoter; and (c) a gene of interest comprising an antisense strand encoding a non-coding RNA (ncRNA) and a complementary sense strand; the first promoter is transcriptionally operative in a first direction to transcribe the antisense strand of the gene of interest and produce the ncRNA; the second promoter is transcriptionally operative in a second direction opposite to the first direction of the first promoter to transcribe the sense strand of the gene of interest, wherein the second promoter is a tissue- or cell type-specific promoter, a stress-responsive promoter, and/or a human promoter; and wherein transcriptional activity of the second promoter can be regulated to interfere with transcriptional activity of the first promoter and reduce production of the ncRNA.

2. The expression vector of claim 1, wherein the second promoter is a tissue or cell type- specific promoter.

3. The expression vector of claim 1 or 2, wherein the second promoter is a stress-responsive promoter.

4. The expression vector of any one of claims 1-3, wherein the second promoter is a human promoter.

5. The expression vector of any one of claims 1-4, wherein the expression vector further comprises adeno-associated virus (AAV) inverted terminal repeats (ITRs) flanking the first promoter and the second promoter.

6. An expression vector comprising: (a) a first promoter; (b) a second promoter; and (c) a gene of interest comprising an antisense strand encoding a non-coding RNA (ncRNA) and a complementary sense strand; the first promoter is transcriptionally operative in a first direction to transcribe the antisense strand of the gene of interest; the second promoter is transcriptionally operative in a second direction opposite to the first direction of the first promoter to transcribe the sense strand of the gene of interest and produce the ncRNA; wherein transcriptional activity of the second promoter can be regulated to interfere with transcriptional activity of the first promoter and reduce production of the ncRNA, wherein the expression vector is an adeno-associated virus (AAV) vector that further comprises AAV inverted terminal repeats (ITRs) flanking the first promoter and the second promoter.

7. The expression vector of any one of claims 4-6, wherein the second promoter comprises a conditional promoter.

8. The expression vector of claim 7, wherein the conditional promoter comprises a tissue- or cell type-specific promoter.

9. The expression vector of claim 2 or 8, wherein the tissue-specific promoter comprises a liver-specific promoter.

10. The expression vector of claim 2 or 8, wherein the tissue-specific promoter comprises a heart-specific promoter.

11. The expression vector of claim 2 or 8, wherein the tissue-specific promoter comprises a muscle-specific promoter.

12. The expression vector of claim 2 or 8, wherein the tissue-specific promoter comprises a retinal-specific promoter.

13. The expression vector of claim 2 or 8, wherein the tissue-specific promoter comprises an inner ear-specific promoter.

14. The expression vector of claim 2 or 8, wherein the tissue-specific promoter comprises a spinal cord-specific promoter.

15. The expression vector of claim 2 or 8, wherein the tissue-specific promoter comprises a dorsal root ganglion-specific promoter.

16. The expression vector of any one of claims 1 or 8-15, wherein the second promoter comprises a plurality of tissue-specific promoters.

17. The expression vector of any one of claims 4-16, wherein the second promoter comprises a stress-responsive promoter.

18. The expression vector of any one of claims 4-17, wherein the second promoter comprises an endogenous human promoter.

19. The expression vector of claim 7, wherein the conditional promoter comprises an inducible promoter.

20. The expression vector of claim 19, wherein the inducible promoter is selected from the group consisting of a tetracycline-inducible promoter, a Lac-inducible promoter, a Bad-inducible promoter, a temperature-inducible promoter, a light-inducible promoter, and a CRISPR/Cas- based promoter.

21. The expression vector of claim 19 or 20, wherein the inducible promoter comprises a tetracycline-inducible promoter.

22. The expression vector of claim 21, wherein the tetracycline-inducible promoter comprises a tetracycline-on (Tet-On) promoter.

23. The expression vector of claim 21, wherein the tetracycline-inducible promoter comprises a tetracycline-off (Tet-Off) promoter.

24. The expression vector of any one of claims 1-23, wherein the second promoter comprises an RNA Polymerase II promoter.

25. The expression vector of any one of claims 1-24, wherein the first promoter comprises an RNA Polymerase III promoter.

26. The expression vector of claim 25, wherein the RNA Polymerase III promoter comprises a gene-internal type 1 RNA Polymerase III promoter or a gene-internal type 2 RNA Polymerase III promoter.

27. The expression vector of claim 25, wherein the RNA Polymerase III promoter comprises a gene-external type 3 RNA Polymerase III promoter.

28. The expression vector of any one of claims 25-27, wherein the RNA Polymerase III promoter comprises a synthetic hybrid promoter.

29. The expression vector of any one of claims 1-28, wherein the expression vector further comprises a third promoter disposed upstream of the first promoter and transcriptionally operative in the first direction opposite to the second direction of the second promoter.

30. The expression vector of claim 29, wherein the third promoter comprises an RNA Polymerase III promoter.

31. The expression vector of any one of claims 1-30, wherein the expression vector further comprises a second gene of interest disposed between the first promoter and the second promoter.

32. The expression vector of any one of claims 1-31, wherein the expression vector further comprises a plurality of genes of interest disposed between the first promoter and the second promoter.

33. The expression vector of claim 31 or 32, wherein each gene of interest is operatively linked to a Polymerase III promoter transcriptionally operative to transcribe the gene of interest.

34. The expression vector of any one of claims 1-33, wherein the ncRNA is selected from the group consisting of a tRNA, an siRNA, an shRNA, an sgRNA, an miRNA, a piRNA, a snoRNA, an snRNA, and a lncRNA.

35. The expression vector of claim 34, wherein the ncRNA is a tRNA.

36. The expression vector of claim 35, wherein the tRNA is a suppressor tRNA.

37. The expression vector of claim 36, wherein the suppressor tRNA comprises a nucleotide sequence set forth in TABLE 4 or TABLE 5.

38. The expression vector of claim 36 or 37, wherein the suppressor tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 6-9, 11, 16-22, and 35.

39. The expression vector of any one of claims 36-38, wherein the suppressor tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 178-182, 186, and 187.

40. The expression vector of any one of claims 36-39, wherein the suppressor tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 36-40, 44, and 45.

41. The expression vector of any one of claims 36-40, wherein the expression vector comprises 1, 2, 3, 4, or more than 4 nucleotide sequences each encoding the same suppressor tRNA.

42. The expression vector of any one of claims 36-41, wherein the expression vector further comprises a nucleotide sequence set forth in TABLE 6 disposed immediately upstream of the suppressor tRNA.

43. The expression vector of claim 42, wherein the expression vector comprises a nucleotide sequence selected from any one of SEQ ID NOs: 869-888.

44. The expression vector of any one of claims 36-43, wherein the suppressor tRNA is flanked by a nucleotide sequence set forth in TABLE 6.

45. The expression vector of any one of claims 36-44, wherein the suppressor tRNA is flanked by a nucleotide sequence selected from any one of SEQ ID NOs: 869-888.

46. The expression vector of any one of claims 1-4, wherein the expression vector is a viral vector.

47. The expression vector of claim 46, wherein the viral vector is a DNA virus vector.

48. The expression vector of claim 46 or 47, wherein the viral vector is an AAV vector.

49. A virus comprising the expression vector of any one of claims 1-48.

50. The virus of claim 49, wherein the virus is an AAV.

51. A system comprising the expression vector of any one of claims 1-48 or the virus of claim 49 or 50.

52. The system of claim 51, further comprising an agent for regulating the second promoter.

53. The system of claim 52, wherein the agent for regulating the second promoter is an activator.

54. The system of claim 53, wherein the activator regulates the second promoter in cis.

55. The system of claim 53, wherein the activator regulates the second promoter in trans.

56. The system of any one of claims 51-55, wherein the system is a cell.

57. The system of claim 56, wherein the cell is a producer cell for AAV production.

58. The system of claim 57, wherein the producer cell is a human embryonic kidney (HEK) cell or SF9 (Spodoptera frugiperda) insect cell.

59. A pharmaceutical composition comprising the expression vector of any one of claims 1- 48 or the virus of claim 49 or 50 and a pharmaceutically acceptable excipient.

60. A method of expressing in a mammalian cell a functional gene product encoded by a gene containing a premature termination codon, the method comprising contacting the cell with an effective amount of the expression vector of any one of claims 1-48, the virus of claim 49 or 50, or the pharmaceutical composition of claim 59, thereby permitting an amino acid to be incorporated into the gene product at a position that would otherwise result in a truncated gene product caused by the premature termination codon.

61. The method of claim 60, wherein the gene is SCN1A or dystrophin.

62. The method of claim 60 or 61, wherein the cell is a human cell.

63. The method of any one of claims 60-62, wherein the tRNA becomes aminoacylated in the cell.

64. A method of treating a premature termination codon-mediated disorder in a subject in need thereof, wherein the subject has a gene with a premature termination codon, the method comprising administering to the subject a therapeutically effective amount of the expression vector of any one of claims 1-48, the virus of claim 49 or 50, or the pharmaceutical composition of claim 59, thereby to treat the disorder in the subject.

65. The method of any claim 64, wherein the disorder is Dravet Syndrome or Duchenne Muscular Dystrophy.

66. The method of claim 64 or 65, wherein the subject is a human.

67. A method of producing AAV particles from a producer cell, the method comprising contacting the producer cell with an effective amount of the expression vector of any one of claims 5-48, thereby to produce the AAV.

68. The method of claim 67, wherein the AAV is a high titer AAV.

69. The method of claim 67 or 68, wherein the producer cell is a HEK or SF9 insect cell.

70. The method of any one of claims 67-69, wherein the contacting comprises transfecting the producer cell with the expression vector.

71. The method of any one of claims 67-70, wherein the second promoter is transcriptionally active in the producer cell.

72. A high-titer AAV produced by the method of any one of claims 67-71.

73. A method of reducing off-target toxicity in a tissue of a subject, the method comprising administering to the subject a therapeutically effective amount of the expression vector of any one of claims 1-48, the virus of claim 49 or 50, or the pharmaceutical composition of claim 59, thereby to reduce the off-target toxicity in the tissue of the subject.

74. A method of reducing expression of a gene of interest in a tissue of a subject, the method comprising administering to the subject a therapeutically effective amount of the expression vector of any one of claims 1-48, the virus of claim 49 or 50, or the pharmaceutical composition of claim 59, thereby to reduce the expression of the gene of interest in the tissue of the subject.

75. The method of claim 73 or 74, wherein the tissue is liver, heart, muscle, retina, inner ear, spinal cord, or dorsal root ganglion.

76. The method of any one of claims 73-75, wherein the tissue is a human tissue.