Modified transfer RNAS and methods of use

Abstract

The disclosure relates generally to suppressor tRNAs which are engineered to enhance suppression of premature termination in a gene comprising a premature stop codon. The disclosure also generally relates to the use of exogenous tRNA introns to increase production yield of viral vectors encoding suppressor tRNAs.

Description

Attorney Docket No. TVD-012WO MODIFIED TRANSFER RNAS AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The invention claims the benefit of and priority to U.S. Provisional Patent Application No.63 / 602,138, filed November 22, 2023, and U.S. Provisional Patent Application No.63 / 617,968, filed January 5, 2024, the disclosure of each of which is hereby incorporated by reference in its entirety for all purposes. SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on November 21, 2024, is named TVD-012WO_SL.xml and is 1,030,690 bytes in size. FIELD

[0003] The disclosure relates generally to suppressor tRNAs which are engineered to enhance suppression of premature termination. The disclosure also relates generally to methods and compositions for increasing production yield of viral vectors encoding suppressor tRNAs. BACKGROUND

[0004] 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 (PTC), 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.

[0005] 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),Attorney Docket No. TVD-012WO 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, Benign Familial Infantile Seizures (BFIS), Danon disease, Ehlers-Danlos syndrome, Limb-girdle Muscular dystrophies, LAMA2-related muscular dystrophy, and neuronal ceroid liopfuscinoses (NCLs; also collectively referred to as Batten diseases).

[0006] 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.

[0007] 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.

[0008] A number of other disorders are also associated with, or caused by, nonsense mutations, e.g., Duchenne muscular dystrophy, cystic fibrosis, and β-thalassemia. By way of example, dilated cardiomyopathy (DCM) is a disease in which the heart becomes enlarged (dilated) and cannot pump blood effectively. DCM is one of the main causes of heart failure and heart transplant worldwide. Truncating variants in the TTN gene (TTN), which encodes the giant protein titin, represent the most common cause of inherited DCM (Tharp et al. (2019) FRONT. PHYSIOL.10:1436).Attorney Docket No. TVD-012WO

[0009] 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 and DCM. SUMMARY

[0010] Although approaches have been developed for treating certain genetic disorders, such as PTC-mediated disorders, there is an ongoing need for the development of novel suppressor tRNAs that permit an amino acid to be efficiently incorporated into a gene product encoded by a gene in a mammalian cell at a position that that would otherwise result in premature termination and the formation of a truncated protein. The disclosure is based, in part, upon the discovery that modifying a suppressor tRNA to contain an engineered T-stem sequence can increase the ability of the tRNA to suppress termination at a stop codon. The disclosure is also based, in part, upon the discovery that viral vectors encoding suppressor tRNAs have a reduced production yield, and that the production yield of a viral vector encoding a suppressor tRNA can be increased by modifying the nucleic acid encoding the suppressor tRNA to comprise an exogenous intron.

[0011] Accordingly, in one aspect, the disclosure provides a nucleic acid encoding a suppressor tRNA and an exogenous intron located between nucleotides corresponding to positions 37 and 38 of the tRNA, wherein the presence of the intron increases production yield of an AAV or lentiviral vector comprising the nucleic acid relative to an AAV or lentiviral vector comprising the same nucleic acid but that lacks the exogenous intron. For example, in some embodiments, the exogenous intron is a synthetic intron. In other embodiments, the exogenous intron is derived from an intron-containing mammalian tRNA gene (e.g., an intron-containing human tRNA gene). In some embodiments, the intron comprises a nucleic acid sequence selected from SEQ ID NOs: 932-939.

[0012] In some embodiments, the tRNA comprises a tri-nucleotide anticodon, wherein the anticodon is 5′-UCA-3′ and recognizes UGA stop codons, the anticodon is 5ʹ-UUA-3ʹ and recognizes UAA stop codons, or the anticodon is 5ʹ-CUA-3ʹ and recognizes UAG stop codons. In some embodiments, the tRNA is operably linked to arginine, glutamine, or serine. In some embodiments, the tRNA is operably linked to arginine. In some embodiments, the nucleic acid comprises a nucleic acid sequence selected from SEQ ID NOs: 940-955, 957- 965, 967, and 969-1179. In some embodiments, the nucleic acid comprises a nucleic acid sequence selected from SEQ ID NOs: 1049, 1052, 1060, 1079, and 1080, e.g., SEQ ID NO: 1060. In some embodiments, the suppressor tRNA comprises a T-arm having a T-stem and aAttorney Docket No. TVD-012WO T-loop, wherein the T-arm is encoded by a DNA comprising the nucleic acid sequence of SEQ ID NO: 902 or SEQ ID NO: 903, wherein Ns in SEQ ID NO: 902 and SEQ ID NO: 903 correspond to a DNA encoding the T-loop.

[0013] In some embodiments, the nucleic acid comprises a 5′ flanking sequence, a 3′ flanking sequence, or both 5′ and 3′ flanking sequences. In some embodiments, the 5′ flanking sequence or the 3′ flanking sequence comprises a regulatory element. For example, in some embodiments, the 5′ flanking sequence comprises a leader sequence, a promoter element, or a secondary structure (e.g., a hairpin element). In some embodiments, the 3′ flanking sequence comprises a terminator element or a poly-T element, optionally wherein the poly-T element is less than 50, 40, 30, 20, or 10 nucleotides in length. In some embodiments, the 5′ flanking region and / or the 3′ flanking region comprises a nucleotide sequence set forth in TABLE 8. In some embodiments, the nucleic acid comprises an internal tRNA promoter.

[0014] In another aspect, the disclosure provides a vector comprising the nucleic acid disclosed above. The vector can be a viral vector, e.g., a DNA virus vector. In some embodiments, the viral vector is an AAV (e.g., a single-stranded AAV (ssAAV) or a self- complementary AAV (scAAV)) vector or a lentiviral vector.

[0015] In another aspect, the disclosure provides a suppressor tRNA operably linked to an arginine comprising a T-arm having a T-stem and a T-loop, wherein the T-stem comprises an engineered nucleic acid sequence that increases the ability of the suppressor tRNA to suppress termination at a stop codon relative to a similar suppressor tRNA without the engineered nucleic acid sequence. In some embodiments, the suppressor tRNA does not comprise the nucleic acid sequence of SEQ ID NO: 915 or 916, wherein each thymine is replaced by a uracil. In some embodiments of any of the foregoing methods, the T-arm comprises the nucleic acid sequence of SEQ ID NO: 901 or SEQ ID NO: 903, wherein Ns in SEQ ID NO: 901 and SEQ ID NO: 903 correspond to the T-loop.

[0016] In some embodiments, the tRNA comprises a naturally occurring nucleotide modification, for example, one or more nucleotide modifications selected from 5-methyl uridine, 5-carbamoylmethyluridine, 5-carbamoylmethyl-2-O-methyluridine, 5-methoxy- carbonylmethyluridine, 5-methoxycarbonylmethyl-2-thiouridine, pseudouridine, dihydrouridine, 1-methyladenosine, and inosine.

[0017] In some embodiments, the tRNA comprises a tri-nucleotide anticodon, wherein the anticodon is 5′-UCA-3′ and recognizes a UGA stop codon.Attorney Docket No. TVD-012WO

[0018] In some embodiments, the suppressor tRNA is encoded by a DNA comprising a nucleic acid sequence selected from SEQ ID NOs: 912-914, 917, 919, 1020-1083, and 1186. In some embodiments, the suppressor tRNA is encoded by a DNA comprising a nucleic acid sequence selected from SEQ ID NOs: 913, 917, 919, 1049, 1052, 1060, 1079, 1080, and 1186. In some embodiments, the suppressor tRNA is encoded by a DNA comprising the nucleic acid sequence of SEQ ID NO: 917. In some embodiments, the suppressor tRNA is encoded by a DNA comprising the nucleic acid sequence of SEQ ID NO: 1060.

[0019] In some embodiments, the suppressor tRNA comprises a nucleic acid sequence selected from SEQ ID NOs: 912-914, 917, 919, 1020-1083, and 1186, wherein each thymine is replaced by a uracil. In some embodiments, the suppressor tRNA comprises a nucleic acid sequence selected from SEQ ID NOs: 913, 917, 919, 1049, 1052, 1060, 1079, 1080, and 1186, wherein each thymine is replaced by a uracil. In some embodiments, the suppressor tRNA comprises a nucleic acid sequence selected from SEQ ID NOs: 912-914, 917, 919, and 1186. In some embodiments, the suppressor tRNA comprises the nucleic acid sequence of SEQ ID NO: 917. In some embodiments, the suppressor tRNA comprises the nucleic acid sequence of SEQ ID NO: 1060.

[0020] In another aspect, the disclosure provides a nucleic acid encoding one or more copy numbers of the suppressor tRNA of any of the foregoing embodiments. The nucleic acid can, in some embodiments, comprise a 5′ flanking sequence, a 3′ flanking sequence, or both 5′ and 3′ flanking sequences. In some embodiments, the 5′ flanking sequence or the 3′ flanking sequence comprises a regulatory element. For example, in some embodiments, the 5′ flanking sequence comprises a leader sequence, a promoter element, or a secondary structure (e.g., a hairpin element). In some embodiments, the 3′ flanking sequence comprises a terminator element or a poly-T element, optionally wherein the poly-T element is less than 50, 40, 30, 20, or 10 nucleotides in length. In some embodiments, the 5′ flanking region and / or the 3′ flanking region comprises a nucleotide sequence set forth in TABLE 8. In some embodiments, the nucleic acid comprises an internal tRNA promoter.

[0021] In some embodiments, the nucleic acid comprises an intron located between nucleotides corresponding to positions 37 and 38 of the tRNA. In some embodiments, the intron is an exogenous intron. For example, in some embodiments, the exogenous intron is a synthetic intron. In other embodiments, the exogenous intron is derived from an intron- containing mammalian tRNA gene (e.g., an intron-containing human tRNA gene). In someAttorney Docket No. TVD-012WO embodiments, the intron comprises a nucleic acid sequence selected from SEQ ID NOs: 932- 939.

[0022] In another aspect, the disclosure provides a vector comprising the nucleic acid disclosed above. The vector can be a viral vector, e.g., a DNA virus vector.

[0023] In some embodiments, the viral vector is an adeno-associated virus (AAV) vector (e.g., an ssAAV or an scAAV) or a lentiviral vector. In some embodiments, the nucleic acid comprises an exogenous intron between nucleotides corresponding to positions 37 and 38 of the tRNA, wherein the presence of the exogenous intron increases production yield of the AAV or lentiviral vector comprising the nucleic acid relative to an AAV or lentiviral vector comprising the same nucleic acid but that lacks the exogenous intron.

[0024] In another aspect, the disclosure provides a pharmaceutical composition comprising a tRNA disclosed herein, a nucleic acid disclosed herein, or a vector disclosed herein, and a pharmaceutically acceptable excipient.

[0025] In another aspect, the disclosure provides a method of producing a tRNA of interest in a mammalian cell, the method comprising contacting the cell with a nucleic acid disclosed herein, a vector disclosed herein, or a pharmaceutical composition disclosed herein.

[0026] In another aspect, the disclosure provides a method of increasing production in a mammalian cell of a full-length protein encoded by a gene containing a premature termination codon (PTC), the method comprising contacting the cell with a nucleic acid disclosed herein or a vector disclosed herein, and permitting the nucleic acid or vector to be internalized by the cell, whereupon production of the suppressor tRNA permits readthrough of the PTC and the production of the full length protein.

[0027] In another aspect, the disclosure provides a method of increasing the production of a protein of interest in a mammalian cell, the method comprising contacting the cell with a nucleic acid disclosed herein or a vector disclosed herein, and permitting the nucleic acid or vector to be internalized by the cell, whereupon production of the tRNA permits increased translation of the protein relative to a cell not contacted with the nucleic acid or vector.

[0028] In another aspect, the disclosure provides a method of treating a disorder associated with a protein encoded by a gene including a premature termination codon (PTC) in a subject in need thereof, the method comprising administering to the subject an effective amount of a tRNA disclosed herein, a nucleic acid disclosed herein, a vector disclosed herein, or a pharmaceutical composition disclosed herein, thereby to treat the disorder.Attorney Docket No. TVD-012WO

[0029] In another aspect, the disclosure provides a method of treating a haploinsufficiency disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of a tRNA disclosed herein, a nucleic acid disclosed herein, a vector disclosed herein, or a pharmaceutical composition disclosed herein, thereby to treat the disorder. In some embodiments, the haploinsufficiency disorder is Dravet syndrome or dilated cardiomyopathy.

[0030] In another aspect, the disclosure provides a method for generating a recombinant AAV (rAAV) vector, the method comprising: (a) providing a virus-producing cell with a first plasmid comprising a Rep gene and a Cap gene or functional fragments thereof, a transfer plasmid comprising a nucleic acid disclosed herein and flanking inverted terminal repeats (ITR), and a helper plasmid comprising helper genes to mediate rAAV replication; and (b) following step (a), culturing the cell to produce the rAAV vector. In some embodiments, the cell is a human embryonic kidney (HEK) cell. In some embodiments, the presence of the exogenous intron in the nucleic acid increases production yield of the rAAV vector by at least 50%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, or at least 400% relative to an rAAV vector comprising the same nucleic acid but that lacks the exogenous intron.

[0031] In another aspect, the disclosure provides a method of generating a recombinant lentiviral vector, the method comprising: (a) providing a virus-producing cell with one or more plasmids collectively comprising gag, pol, rev, and tat genes or functional fragments thereof; and a transfer plasmid comprising a nucleic acid disclosed herein and flanking long terminal repeats (LTR); and (b) following step (a), culturing the cell to produce the recombinant lentiviral vector. In some embodiments, the cell is a HEK cell. In some embodiments, the presence of the exogenous intron in the nucleic acid increases production yield of the recombinant lentiviral vector by at least 50%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, or at least 400% relative to a recombinant lentiviral vector comprising the same nucleic acid but that lacks the exogenous intron.

[0032] These and other aspects and features of the disclosure are described in the following detailed description and claims.Attorney Docket No. TVD-012WO BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The disclosure can be more completely understood with reference to the following drawings.

[0034] FIGURE 1 is a schematic illustration of a tRNA, numbered according to the “Sprinzl” tRNA numbering system. Circles represent nucleotides which are always present. Ovals represent nucleotides which are not always present in each tRNA structure, including nucleotides before position 1 on the 5′ end, nucleotides before and after the two invariant GMP (2′-O-methylguanosine) residues (positions 18 and 19) in the D-loop, and nucleotides in the variable loop. (Steinberg et al. (1993) NUCLEIC ACIDS RES.21(13): 3011-15; Sprinzl et al. (2005) NUCLEIC ACIDS RES.33: D139-40.)

[0035] FIGURE 2 is a schematic illustration of an exemplary pre-tRNA comprising an intron disposed between positions 37 and 38, and the subsequent removal of the intron to form a mature tRNA.

[0036] FIGURE 3 is a phylogram showing sequence divergence for all human Arg(R)- tRNAs. Branch lengths are proportional to the amount of inferred change between all included human Arg-tRNA isoacceptors. Arg-tRNAs used to generate suppressor tRNAs (with and without modified T-stems and exogenous introns) are indicated with arrows.

[0037] FIGURES 4A-4B are bar graphs depicting the ability of Arg-suppressor tRNAs comprising an endogenous T-stem (hatched bars) or a modified T-stem (TS0036, solid bars) to suppress premature termination of a gene (MeCP2) comprising a nonsense mutation in primary cortical neurons. The indicated tRNAs were packaged into AAV1 and tested at an MOI of 5E+4 (FIGURE 4A) or packaged into AAV1 or AAV9 and tested at a range of MOIs (FIGURE 4B, MOIs: AAV1 = 5E+3, 1.5E+4, 5E+4; AAV9=5E+4, 1.5E+5, 5E+5). Data were quantified based on MeCP2 intensity in NeuN-positive nuclei (FIGURE 4A) or as the number of NeuN-positive cells expressing MECP2 at detectable levels (FIGURE 4B).

[0038] FIGURE 5 is a bar graph depicting the ability of Arg-suppressor tRNAs comprising an endogenous T-stem (tr0115; hatched bars) or comprising the modified T-stem TS0006 (tr0400; solid bars) to suppress premature termination of an EGFP reporter comprising an Arg>TGA nonsense mutation in HEK cells. Nonsense suppression activity was quantified approximately 48 hours post-transfection as percentage of fluorescence activity relative to cells transfected with a wild-type EGFP reporter.

[0039] FIGURE 6 is a phylogram showing sequence divergence for all human and mouse Gln(Q)-tRNAs. Branch lengths are proportional to the amount of inferred change betweenAttorney Docket No. TVD-012WO all included human and mouse Gln-tRNA isoacceptors. Gln-tRNAs used to generate suppressor tRNAs (with and without modified T-stems and exogenous introns) are indicated with arrows.

[0040] FIGURE 7 is a bar graph depicting the ability of Gln-suppressor tRNAs comprising either an endogenous T-stem (hatched bars), the modified T-stem TS0006 (grey bars, “TS06”), or the modified T-stem TS0036 (black bars, “TS36”) to suppress premature termination of a dual-luciferase reporter comprising a Gln>TAA nonsense mutation in HEK cells. Nonsense suppression activity was quantified by normalizing luminescence from the C-terminal NanoLuc luciferase to luminescence from the N-terminal firefly luciferase approximately 24 hours post-transfection.

[0041] FIGURE 8 is a bar graph comparing AAV production yield for vectors encoding a potent suppressor tRNA (vectors A-H, hatched bars) relative to control vectors that do not encode a suppressor tRNA (vectors I-O, solid bars). Vector compositions are summarized in TABLE 18. Vectors that met or exceeded AAV yield expectations in a single production run were defined as being 100% normal yield.

[0042] FIGURES 9A-9B are bar graphs depicting the effect of tRNA introns (INT00##; grey bars) on the ability of Arg-suppressor tRNAs to suppress premature termination. Plasmids encoding a suppressor tRNA without an intron (hatched bars; tr0115 in FIGURE 9A, tr0106 in FIGURE 9B) were modified to comprise one of five tRNA introns (INT0002, INT0003, INT0005, INT0006, INT0011), and the plasmids were transfected into HEK cells in combination with an EGFP reporter comprising an inactivating Arg(R)>TGA nonsense mutation. Nonsense mutation rescue was quantified via EGFP intensity and normalized to cells transfected with a wild-type EGFP plasmid (black bars). All introns were exogenous to the parental suppressor tRNA tested, except where indicated by an asterisk.

[0043] FIGURES 10A-10B are bar graphs depicting the effect of tRNA introns (INT00##; grey bars) on the ability of Gln-suppressor tRNAs to suppress premature termination. Plasmids encoding a suppressor tRNA without an intron (hatched bars; tr0157 in FIGURE 10A, tr0191 in FIGURE 10B) were modified to comprise one of five tRNA introns (INT0002, INT0003, INT0005, INT0006, INT0011), and the plasmids were transfected into HEK cells in combination with of an EGFP reporter comprising an inactivating Gln(Q)>TAA (FIGURE 10A) or Gln(Q)>TAG nonsense mutation (FIGURE 10B). Nonsense mutation rescue was quantified via EGFP intensity and normalized to cellsAttorney Docket No. TVD-012WO transfected with a wild-type EGFP plasmid (black bars). All introns were exogenous to the parental suppressor tRNA tested.

[0044] FIGURE 11 is a bar graph comparing AAV production yield for vectors encoding a potent suppressor tRNA without an intron (hatched bars) with the production yield for vectors encoding a potent suppressor tRNA and comprising one of three exogenous introns (solid bars). Yields for each vector design tested were normalized to the yield obtained for the non-intron-containing version of the suppressor tRNA. Vector compositions are summarized in TABLE 19. Intron 1 was tested in each of vectors A-H, Intron 2 was tested in Vectors E and H, and Intron 3 was tested in Vector E.

[0045] FIGURE 12 is a bar graph comparing AAV production yield for vectors encoding one or three copies of the indicated suppressor tRNA without an intron (hatched bars) or with an exogenous intron (black bars). Yields for each vector tested were normalized to the yield obtained for a vector that did not encode a tRNA (grey bar).

[0046] FIGURE 13 is a graph depicting the percentage of heterozygous Scn1aWT / R613Xmice on a mixed background (50:50) (“HET”), at post-natal day 21 (P21), remaining free of hyperthermia-induced seizures at the indicated temperature as compared to wild-type control mice. At P1, mice received an intracerebroventricular (ICV) injection with a single-stranded (ss) or self-complementary (sc) AAV at a dose of 5E10 viral genomes (VG) per mouse (as determined by qPCR titration) expressing three copies of the suppressor tRNAs tr0115 (SEQ ID NO: 391) or tr0374 (SEQ ID NO: 1060), respectively, as described in TABLE 3 and TABLE 21.

[0047] FIGURES 14A-14G are Kaplan-Meier graphs showing survival of HET mice as compared to wild-type control mice following P1 ICV injection of scAAV vectors at a dose of 5E10 VG / mouse (as determined by qPCR titration) expressing three copies of the suppressor tRNAs tr0104 (SEQ ID NO: 378; FIGURE 14A), tr0115 (SEQ ID NO: 391; FIGURE 14B), tr0119 (SEQ ID NO: 395; FIGURE 14C), tr0315 (SEQ ID NO: 913; FIGURE 14D), tr0374 (SEQ ID NO: 1060; FIGURE 14E), tr0417 (SEQ ID NO: 1079; FIGURE 14F), or tr0418 (SEQ ID NO: 1080; FIGURE 14G), as described in TABLE 3, TABLE 7, and TABLE 21.

[0048] FIGURES 15A-15B are Kaplan-Meier graphs showing survival of HET mice following P1 ICV injection of ssAAV vectors (FIGURE 15A) or scAAV vectors (FIGURE 15B) at a dose of 5E10 VG / mouse (as determined by qPCR titration) expressing one copy of the suppressor tRNA tr0374 (SEQ ID NO: 1060), as described in TABLE 7 and TABLE 21.Attorney Docket No. TVD-012WO

[0049] FIGURE 16 is a bar graph comparing the SCN1A mRNA (dPCR analysis) levels in cortical tissue of P21 HET mice. At P1, mice received ICV injection with an ssAAV at a dose of 1E11 VG / mouse (as determined by ddPCR titration) expressing one copy of the suppressor tRNAs tr0315 (SEQ ID NO: 913), tr0374 (SEQ ID NO: 1060), or tr0590 (SEQ ID NO: 1049), as described in TABLE 3, TABLE 7, and TABLE 21.

[0050] FIGURE 17 is a schematic illustration of an AAV vector encoding three copies of a suppressor tRNA (tr0374) as described in Example 4. Each tRNA copy is functionally linked to a U6 promoter. “ITR” represents the location of inverted terminal repeats.

[0051] FIGURE 18 is a bar graph comparing the TTN mRNA levels in cardiac tissue obtained from TTN+ / R30,277Xheterozygous mice at 6 or 12 weeks post-injection with an AAV encoding three copies of an Arg>TGA suppressor tRNA (tr0374; see TABLE 7). Wild type mice (“WT”) and uninjected heterozygous mice (“Het Untreated”) were used as negative controls.

[0052] FIGURE 19 is a schematic illustration of the TTN R30,277X allele described in Example 4 that was introduced into C57BL / 6J mice. In addition to the premature termination codon (“PTC”) introduced at R30,277 of the TTN gene, a TEV-HaloTag (comprising a TEV protease site and a 912 bp HaloTag) was introduced N-terminal to the PTC. TEV digestion of the prematurely terminated TTN protein yields a 34 kDa cleavage product, whereas TEV digestion of the rescued full-length TTN protein yields a 587 kDa cleavage product.

[0053] FIGURE 20 is a western blot comparing the levels of (TEV-digested) truncated TTN and rescued full-length TTN in cardiac tissue obtained from TTN+ / R30,277Xheterozygous mice (“MyoAAV-2A (3x_U6_tr0374)”) following administration of an AAV encoding a suppressor tRNA. Mice were injected with an AAV encoding three copies of an Arg>TGA suppressor tRNA (tr0374; see TABLE 7), and protein levels in cardiac tissue were measured at 6 or 12 weeks post-injection. Uninjected heterozygous mice (“Untreated”) were used as negative controls. Tissue samples were treated with TEV protease (“+”) to generate the HaloTagged 34 kDa and 587 kDa cleavage products described above. DETAILED DESCRIPTION

[0054] Although approaches have been developed for treating certain genetic disorders, such as PTC-mediated disorders, there is an ongoing need for the development of novelAttorney Docket No. TVD-012WO suppressor tRNAs that permit an amino acid to be efficiently incorporated into a gene product encoded by a gene in a mammalian cell at a position that that would otherwise result in premature termination and the formation of a truncated protein. The disclosure is based, in part, upon the discovery that modifying a suppressor tRNA to contain an engineered T-stem sequence increases the ability of the tRNA to suppress termination at stop codons. The disclosure is also based, in part, upon the discovery that viral vectors encoding potent suppressor tRNAs have a reduced yield, and that the yield of a viral vector encoding a suppressor tRNA can be increased by modifying the suppressor tRNA gene to comprise an exogenous intron. DEFINITIONS

[0055] Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. For example, nomenclatures utilized in connection with, and techniques of, e.g., polypeptide and polynucleotide chemistry and synthesis, molecular and cellular biology, protein biology and biochemistry, immunology, etc. described herein are those well-known and commonly used in the art.

[0056] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless context clearly dictates otherwise. Thus, for example, in some embodiments, reference to, e.g., a nucleic acid encoding a ncRNA includes a single ncRNA, a plurality of ncRNAs, etc.

[0057] As used herein, the expression “and / or” in connection with two or more recited objects includes individually each of the recited objects and the various combinations of two or more of the recited objects, unless otherwise understood from the context and use.

[0058] 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.

[0059] As used herein, unless otherwise indicated, the terms “non-coding gene” and “ncgene” are understood to include a nucleic acid encoding a ncRNA, irrespective of whether the nucleic acid comprises part or all of any 5′ or 3′ regulatory regions that are ordinarily associated with the ncgene in nature. For example, a nucleic acid encoding a tRNA but not a native tRNA promoter is considered to comprise a ncgene. Likewise, a nucleic acidAttorney Docket No. TVD-012WO encoding a tRNA and an ectopic promoter, wherein the tRNA is configured to be expressed by the ectopic promoter, is considered to comprise a ncgene.

[0060] 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 some 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.”

[0061] As used herein, the phrase “percent identity” and “% identity” refers to the extent to which two sequences e.g., two polypeptides or two nucleic acids have the same respective amino acid or nucleotide at the same positions in an alignment. As used herein, “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. Similarly, 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. 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 (Basic Local Alignment Search Tool), BLAST- 2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. For a discussion of basic issues in searching sequence databases see Altschul et al., (1994) NATURE GENETICS 6:119-129, which is fully incorporated by reference herein. 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.

[0062] 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; other elements for expression can be supplied by the host cell or inAttorney Docket No. TVD-012WO an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), retrotransposons (e.g., piggyback or sleeping beauty), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide of interest.

[0063] As used herein, the term “virus” refers to an obligate intracellular parasite having no protein-synthesizing or energy-generating mechanism.

[0064] As used herein, unless otherwise indicated, the term “exogenous intron,” when used in reference to an intron present in a pre-tRNA or a nucleic acid encoding a tRNA, refers to an intron that is not ordinarily present in the gene encoding that tRNA in nature. When used in reference to an intron present in a suppressor pre-tRNA or in a nucleic acid encoding a suppressor tRNA, the term “exogenous intron” is understood to refer to an intron that is not ordinarily present in the wild-type, parental tRNA gene from which the suppressor tRNA is derived. An exogenous intron can be, e.g., an intron derived from another intron- containing tRNA gene from the same species, an intron derived from an intron-containing tRNA gene from a different species, or a synthetic intron which is not present in any naturally occurring tRNA gene.

[0065] As used herein, the phrase “pharmaceutically acceptable” 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.

[0066] As used herein, the phrase “pharmaceutically acceptable carrier” refers to an agent (e.g., excipient, carrier, buffer, etc.) 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 and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Standard pharmaceutical carriers may include, for example 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., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded.2020).Attorney Docket No. TVD-012WO

[0067] As used herein, the phrase “effective amount” refers to the amount of an active agent (e.g., a ncRNA disclosed herein) sufficient to effect beneficial or desired results. An 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.

[0068] The term “therapeutically effective amount” as used herein refers to the amount of an active agent (e.g., a ncRNA according to 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.

[0069] As used herein, “treat,” “treating,” and “treatment” refer to the treatment of a disease, disorder, or symptom or manifestation of such in a subject, e.g., in a human. This includes: (a) preventing a disease or disorder, (b) inhibiting the disease, disorder, etc., i.e., slowing or arresting its progress or development; and (b) relieving the disease, disorder, etc., i.e., causing regression of the disease state. As used herein, “prevent,” “preventing,” and “prevention” refer to causing a disease, disorder, or symptom or manifestation of such not to occur for at least a period of time in at least some subjects.

[0070] 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.

[0071] As used herein, the terms “administering” and “administration” refer to any method of providing an agent to the subject. Such methods are known to those skilled in the art, and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intra-aural administration, intracerebral administration, administration to spinal cord, administration to intracerebral fluid, rectal administration, parenteral administration, intravenous administration, intra-arterial administration, intramuscular administration, intrathecal administration, and subcutaneous administration. Administration can be continuous or intermittent. In some instances a vector described herein can be administered therapeutically or prophylactically, such as administered for prevention of a disease or condition in a subject, or for improvement of one or more functions in a subject.Attorney Docket No. TVD-012WO

[0072] 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.

[0073] 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. Similarly, throughout the description, where compositions are described as consisting essentially of specific components, or where processes and methods are described as consisting essentially of specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist of the recited components, and that there are processes and methods according to the present disclosure that consist of the recited processing steps.

[0074] Throughout the text, 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.

[0075] 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 any invention provided herein. For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of any invention described and depicted herein.Attorney Docket No. TVD-012WO

[0076] 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 any invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of any invention disclosed herein.

[0077] 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.

[0078] 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.

[0079] It should be understood that the order of steps or order for performing certain actions is immaterial so long as disclosed invention(s) remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0080] As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90%-100%, includes 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc. I. TRNAS AND SUPPRESSOR TRNAS a. tRNAs and Suppressor tRNAs

[0081] During protein synthesis, a 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 basesAttorney Docket No. TVD-012WO 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.

[0082] 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; and Agris (1983) “The Modified Nucleosides of Transfer RNA, II,” Alan R. Liss Inc. tRNAs have a conserved general structure comprising an acceptor stem (to which the amino acid is attached), a D-arm, an anticodon arm, a variable loop, and a T arm (also known as a TΨC arm), wherein each arm comprises a double-stranded stem and a single-stranded loop (FIGURE 1). Unless otherwise stated herein, tRNA nucleic acid sequences are numbered and referred to according to the well-known “Sprinzl” tRNA numbering system. See Steinberg et al. (1993) NUCLEIC ACIDS RES.21(13): 3011-15; Sprinzl et al. (2005) NUCLEIC ACIDS RES.33: D139-40. For example, according to the Sprinzl numbering system, the anticodon loop of a tRNA corresponds to positions 32 to 38, and the anticodon corresponds to positions 34 to 36. The Sprinzl numbering system is visually summarized by the schematic in FIGURE 1. A small proportion of tRNA genes contain introns (approximately 7% of human tRNA genes; approximately 5% of mice tRNA genes), typically situated one nucleotide 3′ to the anticodon, immediately 3′ to position 37. Schmidt and Matera (2019), WILEY INTERDISCIP. REV. RNA 11(3): e1583.

[0083] Suppressor tRNAs are modified tRNAs that insert a suitable amino acid at a mutant site, e.g., a PTC, in a protein-encoding gene. The use of the word “suppressor” is based on the fact, that under certain circumstances, the modified tRNA “suppresses” the phenotypic effect of the coding mutation. Suppressor tRNAs typically contain a mutationAttorney Docket No. TVD-012WO (modification) in either the anticodon, changing codon specificity, or at some position that alters the aminoacylation identity of the tRNA.

[0084] In some 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 some 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 some embodiments, the modified anticodon hybridizes with a PTC 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 PTC.

[0085] In some 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 some embodiments, the anticodon hybridizes to a codon selected from UGA to UAA. In some embodiments, the anticodon hybridizes to UGA. In some 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).

[0086] In some 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 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 some embodiments the tRNA is capable of being aminoacylated with serine, leucine, glutamine, or arginine. In some embodiments the tRNA is capable of being aminoacylated with glutamine or arginine. In some embodiments the tRNA is capable of being aminoacylated with arginine.

[0087] In some embodiments, the tRNA (a) comprises an anticodon that hybridizes to a codon as indicated in TABLE 1, and (b) is aminoacylated or is capable of being aminoacylated with an amino acid as indicated in TABLE 1.Attorney Docket No. TVD-012WO TABLE 1

[0088] In some embodiments, a suppressor tRNA is expressed using a single vector. The suppressor tRNA permits an amino acid to be incorporated into a gene product encoded by aAttorney Docket No. TVD-012WO gene at a position that would otherwise result in a truncated gene product caused by a PTC in the target gene, and can be used to treat a disease mediated by a PTC in a gene in a subject.

[0089] In some embodiments, multiple (e.g., two, three, or more) suppressor tRNAs, which can be the same or different, are expressed using a single vector. 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 vector allows for the single 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. Exemplary suppressor tRNAs include, for example, those set forth in International patent application publication numbers WO 2019 / 090154, WO 2020 / 069194, WO 2021 / 087401 and WO 2022 / 235861, as well as those set forth in TABLE 2.

[0090] It is understood that, throughout the description (e.g., TABLES 2-7 and the Sequence Listing), in each instance where a tRNA comprises, consists essentially of, or consists of a nucleotide sequence comprising one or more thymines (T), a uracil (U) may be in place of one or more of the T or a U may be in place of all the T’s. Similarly, in each instance where a tRNA comprises, consists essentially of, or consists of a nucleotide sequence comprising one or more U’s, a T may be in place of one or more of the U’s or a T may be in place of all the U’s. As a result, in TABLES 2-7, each thymine (T) can be replaced by a uracil (U). TABLE 21For some sequence entries in Table 2, the anticodon is denoted via lowercase lettersAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WO

[0091] A suppressor tRNA can be engineered to allow for more efficient suppression of premature termination, e.g., as disclosed in Section Ib herein below. Alternatively or in addition, a nucleic acid encoding a suppressor tRNA can be modified to comprise anAttorney Docket No. TVD-012WO exogenous intron to enhance the yield of a viral vector comprising the nucleic acid, e.g., as described in Section Ic herein below. In some embodiments, a suppressor tRNA can be engineered as described in Section Ib and also be encoded by a nucleic acid comprising an exogenous intron as described in Section Ic.

[0092] In some embodiments, a suppressor 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 some embodiments, the tRNA may comprise 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, e.g., 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2- 6, 2-5, 2-4, 2-3, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-15, 4-14, 4- 13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 8-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-15, 10-14, 10-13, 10-12, 10-11, 11-15, 11-14, 11-13, 11-12, 12-15, 12-14, 12-13, 13-15, 13-14, or 14-15 mutations relative to a reference tRNA sequence (e.g., a tRNA disclosed herein).

[0093] It is contemplated that a suppressor 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; 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.Attorney Docket No. TVD-012WO (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.

[0094] In some embodiments, a suppressor 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 some 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; 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′-Attorney Docket No. TVD-012WO 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.

[0095] In some embodiments, the suppressor 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.

[0096] In some embodiments, multiple (e.g., two or three) suppressor tRNAs are expressed using a vector. Expression of multiple, different types of suppressor tRNAs from a single vector allows for the single 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.

[0097] In eukaryotes, tRNAs are initially produced as precursor tRNAs (pre-tRNAs) that undergo post-transcriptional 5'- and 3'-end processing. CCA-adding enzymes (tRNA nucleotidyltransferases) are specialized polymerases that add a specific invariant sequence (C-C-A) to pre-tRNA 3′-ends without requiring a nucleic acid template. This sequence is a prerequisite for tRNAs to be charged with the corresponding amino acid and to participate in mRNA translation (Neuenfeldt et al. (2008) PROC. NATL. ACAD. SCI.105(23): 7953-7958). Accordingly, in some embodiments of any of the tRNAs disclosed herein, a CCA sequence added to the 5' end of the tRNA. b. Suppressor tRNAs Comprising Engineered T-stemsAttorney Docket No. TVD-012WO

[0098] The disclosure also provides, among other things, suppressor tRNAs engineered to allow for more efficient suppression of premature termination.

[0099] tRNAs have a conserved general structure comprising an acceptor stem (to which the amino acid is attached), a D-arm, an anticodon arm, a variable loop, and a T arm (also known as a TΨC arm), wherein each arm comprises a double-stranded stem and a single- stranded loop. The T-arm, which comprises the T-stem and the T-loop, is understood to facilitate interaction of the tRNA with the ribosome. Each strand of the T-stem is generally 5 nucleotides in length, whereas the T-loop is generally 7 nucleotides in length. In the exemplary tRNA structure depicted in FIGURE 1, the T-stem corresponds to positions 49 to 53 and 61 to 65, and the T-loop corresponds to positions 54 to 60.

[0100] Disclosed herein are suppressor tRNAs comprising a T-arm comprising a T-stem and a T-loop, wherein the T-stem comprises an engineered nucleic acid sequence that increases the ability of the suppressor tRNA to suppress termination at a stop codon, relative to a similar suppressor tRNA without the engineered nucleic acid sequence. In some embodiments, an engineered tRNA of the disclosure comprises a T-arm comprising the nucleotide sequence of GCGGGNNNNNNNCCCGU (SEQ ID NO: 901). In some embodiments, an engineered tRNA of the disclosure comprises a T-arm encoded by a nucleic acid comprising the nucleotide sequence of GCGGGNNNNNNNCCCGT (SEQ ID NO: 902). In SEQ ID NOs: 901 and 902, the N’s in the sequence correspond to the T-loop, and the first five nucleotides and last five nucleotides correspond to the T-stem. The T-stem sequence of SEQ ID NO: 901 (encoded by SEQ ID NO: 902) is referred to herein as TS0036.

[0101] In some embodiments, an engineered tRNA of the disclosure comprises a T-arm comprising the nucleotide sequence of GCGGGNNNNNNNCCCGC (SEQ ID NO: 903) or encoded by the nucleotide sequence of SEQ ID NO: 903. In SEQ ID NO: 903, the N’s in the sequence correspond to the T-loop, and the first five nucleotides and last five nucleotides correspond to the T-stem. The T-stem sequence of SEQ ID NO: 903 is referred to herein as TS0006.

[0102] The T-arm of an engineered tRNA of the disclosure can comprise any appropriate T-loop sequence known in the art. For example, the T-loop can comprise the nucleotide sequence of UUCNANN (SEQ ID NO: 904), wherein N corresponds to any nucleotide. In some embodiments, the T-loop comprises the nucleotide sequence of UUCGAGU (SEQ ID NO: 905), UUCAAAU (SEQ ID NO: 906), or UUCGAAU (SEQ ID NO: 907). In some embodiments, the T-loop is encoded by a nucleic acid comprising the nucleotide sequence ofAttorney Docket No. TVD-012WO TTCNANN (SEQ ID NO: 908), wherein N corresponds to any nucleotide. In some embodiments, the T-loop is encoded by a nucleic acid comprising the nucleotide sequence of TTCGAGT (SEQ ID NO: 909), TTCAAAT (SEQ ID NO: 910), or TTCGAAT (SEQ ID NO: 911).

[0103] In some embodiments, an engineered suppressor tRNA of the disclosure 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 some embodiments, the anticodon hybridizes to a codon selected from UGA and UAA. In some embodiments, the anticodon hybridizes to UGA. In some 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).

[0104] In some embodiments, the engineered suppressor tRNA is aminoacylated or is capable of being aminoacylated with any natural amino acid. For example, a tRNA may be capable of being 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 some embodiments the engineered tRNA is capable of being aminoacylated with serine, leucine, glutamine, or arginine. In some embodiments, the engineered tRNA is capable of being aminoacylated with glutamine or arginine. In some embodiments, the engineered tRNA is capable of being aminoacylated with arginine.

[0105] In some embodiments, the engineered suppressor tRNA (i) comprises an anticodon that hybridizes to a codon as indicated in TABLE 1, and (ii) is aminoacylated or is capable of being aminoacylated with an amino acid as indicated in TABLE 1.

[0106] In some embodiments, the engineered tRNA is capable of being aminoacylated with arginine, and the tRNA comprises an anticodon that hybridizes to UGA. In some embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of a nucleotide sequence shown in TABLE 3. In some embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of a nucleotide sequence having at least 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 3. In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising a nucleotide sequence shown in TABLE 3. In some embodiments, theAttorney Docket No. TVD-012WO engineered suppressor tRNA is encoded by a nucleic acid comprising a nucleotide sequence having at least 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 3. TABLE 3: Exemplary Arg-Suppressor tRNAs Comprising a Modified T-stemAttorney Docket No. TVD-012WO

[0107] In some embodiments, the engineered suppressor tRNA comprises a nucleotide sequence selected from SEQ ID NOs: 912-919, or a nucleotide sequence having at least 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 SEQ ID NOs: 912-919 and 1186. In some embodiments, the engineered suppressor tRNA comprises a nucleotide sequence selected from SEQ ID NOs: 912-914, 917-919, and 1186 or a nucleotide sequence having at least 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 SEQ ID NOs: 912-914, 917-919, and 1186.Attorney Docket No. TVD-012WO

[0108] In some embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 912. In some embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 913. In some embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 914. In some embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 915. In some embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 916. In some embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 917. In some embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 918. In some embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 919. In some embodiments, the tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 1186.

[0109] In some embodiments, the tRNA does not comprise the nucleotide sequence of SEQ ID NO: 915, and / or the tRNA does not comprise the nucleotide sequence of SEQ ID NO: 916.

[0110] In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising a nucleotide sequence selected from SEQ ID NOs: 912-919 and 1186, or a nucleotide sequence having at least 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 SEQ ID NOs: 912-919 and 1186. In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising a nucleotide sequence selected from SEQ ID NOs: 912-914, 917-919, and 1186, or a nucleotide sequence having at least 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 SEQ ID NOs: 912-914, 917-919, and 1186.

[0111] In some embodiments, the tRNA is encoded by a nucleic acid which does not comprise the nucleotide sequence of SEQ ID NO: 915, and / or which does not comprise the nucleotide sequence of SEQ ID NO: 916.

[0112] In some embodiments, the tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 912. In some embodiments, the tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 913. In some embodiments, the tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO:Attorney Docket No. TVD-012WO 914. In some embodiments, the tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 915. In some embodiments, the tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 916. In some embodiments, the tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 917. In some embodiments, the tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 918. In some embodiments, the tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 919. In some embodiments, the tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1186.

[0113] In some embodiments, the tRNA is not encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 915, and / or the tRNA is not encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 916.

[0114] In some embodiments, the engineered tRNA is capable of being aminoacylated with glutamine, and the tRNA comprises an anticodon that hybridizes to UAA or UAG. In some embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of a nucleotide sequence shown in TABLE 4. In some embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of a nucleotide sequence having at least 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 some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising a nucleotide sequence shown in TABLE 4. In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising a nucleotide sequence having at least 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. TABLE 4: Exemplary Gln-Suppressor tRNAs Comprising a Modified T-stemAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WO

[0115] In some embodiments, the engineered suppressor tRNA comprises a nucleotide sequence selected from SEQ ID NOs: 920-931 or 1180-1185, or a nucleotide sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,Attorney Docket No. TVD-012WO 97%, 98%, or 99% sequence identity to a nucleotide sequence selected from SEQ ID NOs: 920-931 or 1180-1185. In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising a nucleotide sequence selected from SEQ ID NOs: 920-931 or 1180-1185, or a nucleotide sequence having at least 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 SEQ ID NOs: 920-931 or 1180-1185.

[0116] In some embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 921. In some embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 922. In some embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 923. In some embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 924. In some embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 925. In some embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 926. In some embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 927. In some embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 928. In some embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 929. In some embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 930. In some embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 931. In some embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 1180. In some embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 1181. In some embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 1182. In some embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 1183. In some embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 1184. In someAttorney Docket No. TVD-012WO embodiments, the engineered suppressor tRNA comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 1185.

[0117] In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 921. In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 922. In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 923. In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 924. In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 925. In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 926. In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 927. In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 928. In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 929. In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 930. In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 931. In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1180. In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1181. In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1182. In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1183. In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1184. In some embodiments, the engineered suppressor tRNA is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1185.

[0118] In some embodiments, the engineered suppressor 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). The one or more mutations may be presentAttorney Docket No. TVD-012WO in the T-arm (e.g., in the T-stem or T-loop) of the engineered tRNA. Additionally or alternatively, the one or more mutations may be present in other parts of the tRNA (e.g., in the acceptor stem, the D-arm, the anticodon arm, and / or the variable loop). In some embodiments, the engineered tRNA may comprise 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, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-15, 3-14, 3-13, 3-12, 3- 11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4- 5, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6- 9, 6-8, 6-7, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8- 9, 8-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-15, 10-14, 10-13, 10-12, 10-11, 11-15, 11-14, 11-13, 11-12, 12-15, 12-14, 12-13, 13-15, 13-14, or 14-15 mutations relative to a reference tRNA sequence (e.g., a tRNA disclosed herein).

[0119] In some embodiments, a nucleic acid encoding an engineered suppressor tRNA of the disclosure further 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).

[0120] In some embodiments, a nucleic acid encoding an engineered suppressor tRNA of the disclosure further comprises a 5′ flanking sequence, a 3′ flanking sequence, or both 5′ and 3′ flanking sequences, e.g., as shown in TABLE 8. Depending upon the circumstances, the 5′ flanking sequence and / or the 3′ flanking sequence comprises a regulatory element. The 5′ flanking sequence can comprise, for example, a leader sequence, a promoter element, or secondary structure (e.g., a hairpin element). The 3′ flanking sequence can comprise, for example, a terminator element or a poly-T element. In some embodiments, the nucleic acid comprises an internal tRNA promoter.

[0121] It is contemplated that an engineered tRNA of the disclosure may comprise one or more modifications, e.g., as described in Section Ia herein above. c. Nucleic Acids Encoding tRNAs and Comprising Exogenous Introns

[0122] The disclosure also provides, among other things, nucleic acids encoding a suppressor tRNA, wherein the nucleic acid comprises an exogenous intron. The exogenous intron can be located between the nucleotides corresponding to positions 37 and 38 of theAttorney Docket No. TVD-012WO suppressor tRNA. In some embodiments, the presence of the exogenous intron in the nucleic acid increases the production yield of a viral vector (e.g., an adeno-associated viral (AAV) vector or a lentiviral vector) comprising the nucleic acid, relative to a viral vector comprising the same nucleic acid but that lacks the exogenous intron. In addition, the disclosure provides a suppressor pre-tRNA which comprises an exogenous intron. In some embodiments the pre-tRNA is an intermediate transcription product, and the exogenous intron will be spliced out of the pre-tRNA by appropriate cellular machinery to generate a mature tRNA.

[0123] It is contemplated that exogenous introns of the disclosure can be derived from a naturally occurring tRNA intron. In some embodiments, the naturally-occurring tRNA intron is derived from, e.g., an intron-containing tRNA gene from a mammal, e.g., a tRNA gene from a mouse, simian, equine, bovine, porcine, canine, feline, or the like. In some embodiments, the exogenous intron is derived from an intron-containing tRNA gene from a mouse. In some embodiments, the exogenous intron is derived from an intron-containing tRNA gene from a human.

[0124] In some embodiments, the exogenous intron is derived from a naturally-occurring tRNA gene, and the exogenous intron is incorporated into a nucleic acid encoding a suppressor tRNA. As described in Section Ia above, the suppressor tRNA can be derived from a naturally-occurring “parental” tRNA. In some embodiments, the exogenous intron is derived from a naturally-occurring tRNA gene from a different species as the parental tRNA. For example, in some embodiments, the exogenous intron is derived from a mouse tRNA gene, and the intron is incorporated into a nucleic acid encoding a human suppressor tRNA. In other embodiments, the exogenous intron is derived from a human tRNA gene, and the intron is incorporated into a nucleic acid encoding a murine suppressor tRNA. Alternatively, the tRNA gene that the exogenous intron is derived from can be from the same species as the parental tRNA gene. For example, in some embodiments, the exogenous intron is derived from a first human tRNA gene, and the intron is incorporated into a nucleic acid encoding a suppressor tRNA derived from a second, distinct human tRNA gene. The first and second tRNA genes may both encode tRNAs that are charged with the same amino acid (e.g., both can be Arg-tRNA genes derived from the same species). Alternatively, the first and second tRNA genes may encode tRNAs that are charged with different amino acids (e.g., the exogenous intron can be derived from a human Ile-tRNA, Leu-tRNA, or Tyr-tRNA gene, and the suppressor tRNA can be derived from a human Arg-tRNA gene).Attorney Docket No. TVD-012WO

[0125] In some embodiments, the exogenous intron is a synthetic intron, i.e., the intron is not found in a naturally-occurring tRNA gene.

[0126] In some embodiments, the exogenous intron is 12 to 24 nucleotides in length. For example, in some embodiments, the exogenous intron is 12 to 24, 13 to 24, 14 to 24, 15 to 24, 16 to 24, 17 to 24, 18 to 24, 19 to 24, 20 to 24, 21 to 24, 22 to 24, 23 to 24, 12 to 23, 13 to 23, 14 to 23, 15 to 23, 16 to 23, 17 to 23, 18 to 23, 19 to 23, 20 to 23, 21 to 23, 22 to 23, 12 to 22, 13 to 22, 14 to 22, 15 to 22, 16 to 22, 17 to 22, 18 to 22, 19 to 22, 20 to 22, 21 to 22, 12 to 21, 13 to 21, 14 to 21, 15 to 21, 16 to 21, 17 to 21, 18 to 21, 19 to 21, 20 to 21, 12 to 20, 13 to 20, 14 to 20, 15 to 20, 16 to 20, 17 to 20, 18 to 20, 19 to 20, 12 to 19, 13 to 19, 14 to 19, 15 to 19, 16 to 19, 17 to 19, 18 to 19, 12 to 18, 13 to 18, 14 to 18, 15 to 18, 16 to 18, 17 to 18, 12 to 17, 13 to 17, 14 to 17, 15 to 17, 16 to 17, 12 to 16, 13 to 16, 14 to 16, 15 to 16, 12 to 15, 13 to 15, 14 to 15, 12 to 14, 13 to 14, or 12 to 13 nucleotides in length. In some embodiments, the exogenous intron is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides in length.

[0127] Particular nucleotide positions within the sequences of tRNA introns may be referred to herein using the terms “Int-5′ +X” and / or “Int-3′ -Y”. The former term (Int-5′ +X) identifies a nucleotide in the intron according to its position relative to the 5′ splice site. For example, Int-5′ +1, Int-5′ +2, and Int-5′ +3 identify the nucleotides in the intron that are, respectively, the first, second, and third closest nucleotides to the 5′ splice site. Similarly, the term “Int-3′ -Y” identifies a nucleotide in the intron according to its position relative to the 3′-splice site, wherein Int-3′ -1, Int-3′ -2, and Int-3′ -3 identify the nucleotides in the intron that are, respectively, the first, second, and third closest nucleotides to the 3′ splice site. For example, in a 12-nucleotide tRNA intron represented by the sequence N1N2N3N4N5N6N7N8N9N10N11N12, the nucleotide N9 may equivalently be referred to as being in position Int-5′ +9, or as being in position Int-3′ -4. This intron numbering scheme is independent of the Sprinzl numbering system used herein. In general, when an exogenous tRNA intron is incorporated into a nucleic acid encoding a tRNA (or incorporated into a pre- tRNA), the nucleotide immediately 5′ to Int-5′ +1 corresponds to tRNA position 37, and the nucleotide immediately 3′ to Int-3′ -1 corresponds to tRNA position 38 (FIGURE 2).

[0128] In some embodiments, an exogenous intron of the disclosure comprises a guanine (G) at position Int-3′ -3.

[0129] In some embodiments, a nucleic acid of the disclosure encodes a tRNA (e.g., a suppressor tRNA) and comprises a thymine (T) at tRNA position 34, and the nucleic acidAttorney Docket No. TVD-012WO comprises an exogenous intron comprising an adenine (A) at position Int-3′ -4. In some embodiments, a pre-tRNA of the disclosure comprises a uracil (U) at tRNA position 34, and the pre-tRNA comprises an exogenous intron comprising an adenine (A) at position Int-3′ -4.

[0130] In some embodiments, a nucleic acid of the disclosure encodes a tRNA (e.g., a suppressor tRNA) and comprises a thymine (T) at tRNA position 35, and the nucleic acid comprises an exogenous intron comprising an adenine (A), thymine (T), guanine (G), or cytosine (C) at position Int-3′ -5. In some embodiments, a pre-tRNA of the disclosure comprises a uracil (U) at tRNA position 35, and the pre-tRNA comprises an exogenous intron comprising an adenine (A), uracil (U), guanine (G), or cytosine (C) at position Int-3′ - 5.

[0131] In some embodiments, a nucleic acid of the disclosure encodes a tRNA (e.g., suppressor tRNA) and comprises a cytosine (C) at tRNA position 35, and the nucleic acid comprises an exogenous intron comprising an adenine (A), thymine (T), guanine (G), or cytosine (C) at position Int-3′ -5. In some embodiments, a pre-tRNA of the disclosure comprises a cytosine (C) at tRNA position 35, and the pre-tRNA comprises an exogenous intron comprising an adenine (A), uracil (U), guanine (G), or cytosine (C) at position Int-3′ - 5.

[0132] In some embodiments, a nucleic acid of the disclosure encodes a tRNA (e.g., a suppressor tRNA) and comprises a thymine (T) at tRNA position 36, and the nucleic acid comprises an exogenous intron comprising an adenine (A) at position Int-3′ -5. In some embodiments, a pre-tRNA of the disclosure comprises a uracil (U) at tRNA position 36, and the pre-tRNA comprises an exogenous intron comprising an adenine (A) at position Int-3′ -5.

[0133] In some embodiments, a nucleic acid of the disclosure encodes a tRNA (e.g., a suppressor tRNA) and comprises an adenine (A) at tRNA position 36, and the nucleic acid comprises an exogenous intron comprising a guanine (G) at position Int-3′ -5. In some embodiments, a pre-tRNA of the disclosure comprises an adenine (A) at tRNA position 36, and the pre-tRNA comprises an exogenous intron comprising a guanine (G) at position Int-3′ -5.

[0134] In some embodiments, a nucleic acid of the disclosure encodes a tRNA (e.g., a suppressor tRNA) and comprises a thymine (T) at position 34, and the nucleic acid comprises an exogenous intron comprising an adenine (A) at position Int-3′ -4 and a guanine (G) at position Int-3′ -3. In some embodiments, a pre-tRNA of the disclosure comprises a uracilAttorney Docket No. TVD-012WO (U) at tRNA position 34, and the pre-tRNA comprises an exogenous intron comprising an adenine (A) at position Int-3′ -4 and a guanine (G) at position Int-3′ -3.

[0135] In some embodiments, a nucleic acid of the disclosure encodes a tRNA (e.g., a suppressor tRNA) and comprises a thymine (T) at tRNA position 35, and the nucleic acid comprises an exogenous intron comprising an adenine (A), thymine (T), guanine (G), or cytosine (C) at position Int-3′ -5 and a guanine (G) at position Int-3′ -3. In some embodiments, a pre-tRNA of the disclosure comprises a uracil (U) at tRNA position 35, and the pre-tRNA comprises an exogenous intron comprising an adenine (A), uracil (U), guanine (G), or cytosine (C) at position Int-3′ -5 and a guanine (G) at position Int-3′ -3.

[0136] In some embodiments, a nucleic acid of the disclosure encodes a tRNA (e.g., suppressor tRNA) and comprises a cytosine (C) at tRNA position 35, and the nucleic acid comprises an exogenous intron comprising an adenine (A), thymine (T), guanine (G), or cytosine (C) at position Int-3′ -5 and a guanine (G) at position Int-3′ -3. In some embodiments, a pre-tRNA of the disclosure comprises a cytosine (C) at tRNA position 35, and the pre-tRNA comprises an exogenous intron comprising an adenine (A), uracil (U), guanine (G), or cytosine (C) at position Int-3′ -5 and a guanine (G) at position Int-3′ -3.

[0137] In some embodiments, a nucleic acid of the disclosure encodes a tRNA (e.g., a suppressor tRNA) and comprises a thymine (T) at tRNA position 36, and the nucleic acid comprises an exogenous intron comprising an adenine (A) at position Int-3′ -5. In some embodiments, a pre-tRNA of the disclosure comprises a uracil (U) at tRNA position 36, and the pre-tRNA comprises an exogenous intron comprising an adenine (A) at position Int-3′ -5.

[0138] In some embodiments, a nucleic acid of the disclosure encodes a tRNA (e.g., a suppressor tRNA) and comprises an adenine (A) at tRNA position 36, and the nucleic acid comprises an exogenous intron comprising a guanine (G) at position Int-3′ -5. In some embodiments, a pre-tRNA of the disclosure comprises an adenine (A) at tRNA position 36, and the pre-tRNA comprises an exogenous intron comprising a guanine (G) at position Int-3′ -5.

[0139] In some embodiments, an exogenous intron of the disclosure is encoded by a nucleic acid sequence set forth in TABLE 5. In some embodiments, an exogenous intron of the disclosure comprises a nucleic acid sequence set forth in TABLE 5, optionally wherein each thymine (T) is replaced with a uracil (U).Attorney Docket No. TVD-012WO TABLE 5: Exemplary tRNA Introns

[0140] In some embodiments, an exogenous intron of the disclosure comprises a nucleic acid sequence selected from SEQ ID NOs: 932-939, optionally wherein each thymine (T) is replaced with a uracil (U). In some embodiments, the exogenous intron comprises the nucleic acid sequence of SEQ ID NO: 932, optionally wherein each thymine (T) is replaced with a uracil (U). In some embodiments, the exogenous intron comprises the nucleic acid sequence of SEQ ID NO: 933, optionally wherein each thymine (T) is replaced with a uracil (U). In some embodiments, the exogenous intron comprises the nucleic acid sequence of SEQ ID NO: 934, optionally wherein each thymine (T) is replaced with a uracil (U). In some embodiments, the exogenous intron comprises the nucleic acid sequence of SEQ ID NO: 935, optionally wherein each thymine (T) is replaced with a uracil (U). In some embodiments, the exogenous intron comprises the nucleic acid sequence of SEQ ID NO: 936, optionally wherein each thymine (T) is replaced with a uracil (U). In some embodiments, the exogenous intron comprises the nucleic acid sequence of SEQ ID NO: 937, optionally wherein each thymine (T) is replaced with a uracil (U). In some embodiments, the exogenous intron comprises the nucleic acid sequence of SEQ ID NO: 938, optionally wherein each thymine (T) is replaced with a uracil (U). In some embodiments, the exogenous intron comprises the nucleic acid sequence of SEQ ID NO: 939, optionally wherein each thymine (T) is replaced with a uracil (U).

[0141] In some embodiments, a nucleic acid of the disclosure encodes a suppressor tRNA and comprises an exogenous intron, and the suppressor tRNA (i) comprises an anticodon that hybridizes to a codon as indicated in TABLE 1, and (ii) is aminoacylated or is capable of being aminoacylated with an amino acid as indicated in TABLE 1. For example, in some embodiments, the tRNA is aminoacylated or is capable of being aminoacylated with arginine. In some embodiments, the tRNA is aminoacylated or is capable of being aminoacylated withAttorney Docket No. TVD-012WO glutamine. In some embodiments, the tRNA is aminoacylated or is capable of being aminoacylated with serine.

[0142] In some embodiments, a nucleic acid of the disclosure encodes a suppressor tRNA and comprises an exogenous intron, and the nucleic acid comprises a sequence set forth in TABLE 6. In some embodiments, the nucleic acid comprises the sequence of any of SEQ ID NOs: 940-1019. In some embodiments, the a pre-tRNA of the disclosure comprising an exogenous intron, and the pre-tRNA comprises a nucleic acid sequence set forth in TABLE 6, wherein each thymine (T) is substituted for a uracil (U). In some embodiments, the pre- tRNA comprises the sequence of any SEQ ID NOs: 940-1019, wherein each thymine (T) is replaced with a uracil (U). TABLE 6: Nucleic Acids Encoding Exemplary Suppressor tRNAs and Comprising an Exogenous IntronAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOContains an intron that is naturally associated with the corresponding wild-type Arg-tRNA.Attorney Docket No. TVD-012WOContains an intron that is naturally associated with the corresponding wild-type Arg-tRNA. Contains an intron that is naturally associated with the corresponding wild-type Arg-tRNA.Attorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WO

[0143] In some embodiments, a nucleic acid of the disclosure encodes a suppressor tRNA and comprises an exogenous intron, and the nucleic acid comprises a nucleotide sequence selected from SEQ ID NOs: 940-1019, or a nucleotide sequence having at least 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 SEQ ID NOs: 940-1019. In some embodiments, a nucleic acid of the disclosure encodes a suppressor tRNA and comprises an exogenous intron having a nucleotide sequence selected from SEQ ID NOs: 932-939, and the nucleic acid comprises a nucleotide sequence selected from SEQ ID NOs: 940-1019, or a nucleotide sequence having at least 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 SEQ ID NOs: 940-1019.

[0144] In some embodiments, a pre-tRNA of the disclosure comprises an exogenous intron, and the pre-tRNA comprises a nucleotide sequence selected from SEQ ID NOs: 940-Attorney Docket No. TVD-012WO 1019, or a nucleotide sequence having at least 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 SEQ ID NOs: 940-1019, wherein each thymine (T) is substituted for uracil (U). In some embodiments, a pre-tRNA of the disclosure comprises an exogenous intron having a nucleotide sequence selected from SEQ ID NOs: 932-939, and the nucleic acid comprises a nucleotide sequence selected from SEQ ID NOs: 940-1019, or a nucleotide sequence having at least 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 SEQ ID NOs: 940- 1019, wherein each thymine (T) is substituted for uracil (U).

[0145] In some embodiments, the suppressor tRNA-encoding nucleic acid or the pre- tRNA may comprise one or more mutations (e.g., nucleotide substitutions, deletions, or insertions) relative to a reference nucleic acid or tRNA sequence (e.g., relative to a tRNA sequence set forth in TABLE 6). The one or more mutations can, for example, be present in a region corresponding to the T-arm (e.g., in the T-stem or T-loop) of the tRNA. Additionally or alternatively, the one or more mutations may be present in other parts of the tRNA (e.g., in the acceptor stem, the D-arm, the anticodon arm, and / or the variable loop). In some embodiments, the tRNA-encoding nucleic acid or pre-tRNA may comprise 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 nucleic acid or pre-tRNA may comprise 1-15, 1-14, 1- 13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3- 4, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-15, 5-14, 5-13, 5-12, 5-11, 5- 10, 5-9, 5-8, 5-7, 5-6, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 8-15, 9-14, 9-13, 9-12, 9-11, 9- 10, 10-15, 10-14, 10-13, 10-12, 10-11, 11-15, 11-14, 11-13, 11-12, 12-15, 12-14, 12-13, 13- 15, 13-14, or 14-15 mutations relative to a reference nucleic acid or tRNA sequence (e.g., relative to a tRNA sequence set forth in TABLE 6).

[0146] In some embodiments, a nucleic acid of the disclosure encodes a suppressor tRNA and comprises an exogenous intron, and the suppressor tRNA comprises an engineered T- stem sequence (e.g., the T-stem sequence of SEQ ID NO: 902 or 903). In some embodiments, the nucleic acid comprises a nucleotide sequence set forth in TABLE 7.

[0147] In some embodiments, a pre-tRNA of the disclosure comprises an exogenous intron and an engineered T-stem sequence (e.g., the T-stem sequence of SEQ ID NO: 901 orAttorney Docket No. TVD-012WO 903). In some embodiments, the pre-tRNA comprises a nucleotide sequence set forth in TABLE 7, wherein each thymine (T) is replaced by a uracil (U). TABLE 7: Nucleic Acids Encoding Exemplary Engineered Suppressor tRNAs and Comprising an Exogenous IntronAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WO

[0148] In some embodiments, a nucleic acid of the disclosure encodes a suppressor tRNA and comprises an exogenous intron, wherein the suppressor tRNA comprises the engineeredAttorney Docket No. TVD-012WO T-stem sequence of SEQ ID NO: 902 or 903, and the exogenous intron comprises the nucleotide sequence of one of SEQ ID NOs: 932-939. In some embodiments, the nucleic acid comprises a nucleotide sequence selected from SEQ ID NOs: 1020-1179, or a nucleotide sequence having at least 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 SEQ ID NOs: 1020-1179.

[0149] In some embodiments, a pre-tRNA of the disclosure comprises an engineered T- stem and an exogenous intron, wherein the pre-tRNA comprises the engineered T-stem sequence of SEQ ID NO: 902 or 903, and the exogenous intron comprises the nucleotide sequence of one of SEQ ID NOs: 932-939, wherein each thymine (T) is replaced with uracil (U). In some embodiments, the pre-tRNA comprises a nucleotide sequence selected from SEQ ID NOs: 1020-1179, or a nucleotide sequence having at least 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 SEQ ID NOs: 1020-1179, wherein each thymine (T) is replaced with uracil (U).

[0150] In some embodiments, a nucleic acid of the disclosure encodes a suppressor tRNA set forth in TABLE 2 and an exogenous intron between suppressor tRNA positions 37 and 38. For example, for suppressor tRNA sequences in TABLE 2 which do not comprise an intron, the nucleic acid encoding the tRNA can be modified to comprise an intron (e.g., an intron selected from SEQ ID NOs: 932-939) between tRNA positions 37 and 38. For suppressor tRNA sequences in TABLE 2 which do comprise an intron, the intron can be replaced with an exogenous intron (e.g., an intron selected from SEQ ID NOs: 932-939).

[0151] In some embodiments, a nucleic acid encoding a suppressor tRNA of the disclosure and comprising an exogenous intron further 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).

[0152] In some embodiments, a nucleic acid encoding an suppressor tRNA and comprising an intron further comprises a 5′ flanking sequence, a 3′ flanking sequence, or both 5′ and 3′ flanking sequences, e.g., as shown in TABLE 8. Depending upon the circumstances, the 5′ flanking sequence and / or the 3′ flanking sequence comprises a regulatory element. The 5′ flanking sequence can comprise, for example, a leader sequence,Attorney Docket No. TVD-012WO a promoter element, or secondary structure (e.g., a hairpin element). The 3′ flanking sequence can comprise, for example, a terminator element or a poly-T element. In some embodiments, the nucleic acid comprises an internal tRNA promoter.

[0153] It is contemplated that a tRNA or pre-tRNA encoded by a nucleic acid of the disclosure may comprise one or more modifications, e.g., as described in Section Ia herein above. d. Methods of Making Engineered Suppressor tRNAs

[0154] It is contemplated that the suppressor tRNAs disclosed herein can be produced by methods known in the art, including extracellular production by synthetic chemical methods and intracellular production by recombinant DNA methods.

[0155] For example, DNA molecules encoding a tRNA of the disclosure can be synthesized chemically or by recombinant DNA methodologies. For example, the sequences of the engineered tRNAs can be synthesized or cloned from libraries by conventional hybridization techniques or polymerase chain reaction (PCR) techniques, using the appropriate synthetic nucleic acid primers. The resulting DNA molecules encoding the tRNAs can be ligated to other appropriate nucleotide sequences, including, for example, expression control sequences to produce conventional gene expression constructs (i.e., expression vectors) encoding the engineered tRNAs. Production of defined gene constructs is within routine skill in the art. Nucleic acids encoding desired tRNAs can be incorporated (ligated) into expression vectors, such as the expression vectors described in the following section, which can be introduced into host cells through conventional transfection or transformation techniques. Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells. Transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the tRNAs. Specific expression and purification conditions will vary depending upon the expression system employed.

[0156] Alternatively, the tRNAs can be chemically synthesized by methods known in art. When a tRNA is aminoacylated prior to introduction into the cell or administration to the subject, the tRNA may be aminoacylated with a desired amino acid by any method known in the art, including chemical or enzymatic aminoacylation.Attorney Docket No. TVD-012WO e. Expression Vectors

[0157] The suppressor tRNAs of the disclosure may be expressed in a cell of interest by incorporating a gene or nucleic acid encoding the tRNA of interest into an appropriate expression vector.

[0158] In some embodiments, the expression vector comprises a regulatory sequence or promoter operably linked to the nucleotide sequence encoding the engineered tRNA. 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.

[0159] In some embodiments, in addition to a tRNA gene or 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).

[0160] In some embodiments, the vector further comprises a 5′ flanking sequence, a 3′ flanking sequence, or both 5′ and 3′ flanking sequences, e.g., as shown in TABLE 8. Depending upon the circumstances, the 5′ flanking sequence and / or the 3′ flanking sequence comprises a regulatory element. The 5′ flanking sequence can comprise, for example, a leader sequence, a promoter element, or secondary structure (e.g., a hairpin element). The 3′ flanking sequence can comprise, for example, a terminator element or a poly-T element. In some embodiments, the nucleic acid comprises an internal tRNA promoter.Attorney Docket No. TVD-012WO TABLE 8Attorney Docket No. TVD-012WOAttorney Docket No. TVD-012WO

[0161] An expression vector of the disclosure can comprise a nucleotide sequence encoding a tRNA set forth in any of TABLES 2-4 and 6-7. In some embodiments, the expression vector comprises a nucleotide sequence encoding a tRNA set forth in any of TABLES 2-4 and 6-7, and further comprises a nucleotide sequence set forth in TABLE 8. In some embodiments, the expression vector comprises a nucleotide sequence selected from SEQ ID NOs: 912-931, 940-1179, and 1180-1185. In some embodiments, the expression vector comprises a nucleotide sequence selected from SEQ ID NOs: 912-919. In some embodiments, the expression vector comprises a nucleotide sequence selected from SEQ ID NOs: 940-1019. In some embodiments, the expression vector comprises a nucleotide sequence selected from SEQ ID NOs: 1020-1179.

[0162] In some embodiments, a gene encoding an engineered tRNAs is operably linked to a strong promoter that is active in a variety of cell types. The promoters for eukaryotic tRNA genes typically are present within the structural sequences encoding the tRNA molecule itself. Although there are elements which regulate transcriptional activity within the 5′ upstream region, the length of an active transcriptional unit may be considerably less than 500 base pairs.

[0163] Additional exemplary promoters which may be employed include, but are not limited to, the retroviral LTR, the SV40 promoter, the human cytomegalovirus (CMV) promoter, the U6 promoter, or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and β-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirusAttorney Docket No. TVD-012WO promoters, TK promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.

[0164] In some embodiments, the expression vector is 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. 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 some 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)), papillomaviruses (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 some embodiments, the viral vector is a 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-I (HIV-1)) and toroviruses.

[0165] 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.

[0166] In some 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 gradientAttorney Docket No. TVD-012WO centrifugation, clarification, enzymatic treatment, e.g., benzonase or protease treatment, chromatographic steps, e.g., ion exchange chromatography or filtration steps. i. Retroviral Vectors Including Lentivirus Vectors

[0167] In some embodiments, the viral vector can be a retroviral vector. Examples of retroviral vectors 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.

[0168] In some 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 immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV), and caprine arthritis encephalitis virus (CAEV).

[0169] 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.Attorney Docket No. TVD-012WO

[0170] 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.

[0171] 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 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 some 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.

[0172] In some 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′Attorney Docket No. TVD-012WO LTR, or both 3′ and 5′ LTRs, are also contemplated to be useful in the practice of the disclosure.

[0173] In some 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.

[0174] 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, 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.

[0175] In some 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.

[0176] In some embodiments, the retroviral vector (e.g., lentiviral vector) further comprises an export element. In one embodiment, retroviral vectors comprise one or moreAttorney Docket No. TVD-012WO 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.

[0177] In some 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′ portion comprises the heterologous nucleic acid coding sequences and whose 3′ portion comprises the posttranscriptional regulatory element sequence.

[0178] 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 some 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 and 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.

[0179] Non-limiting examples of lentiviral vectors include pLVX-EF1alpha-AcGFP1-C1 (Clontech Catalog #631984), pLVX-EF1alpha-IRES-mCherry (Clontech Catalog #631987),Attorney Docket No. TVD-012WO pLVX-Puro (Clontech Catalog #632159), pLVX-IRES-Puro (Clontech Catalog #632186), pLenti6 / V5-DESTTM (Thermo Fisher), pLenti6.2 / V5-DESTTM (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), and 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, 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. WO 2017 / 091786. ii. Adenoviral Vectors

[0180] In some 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.

[0181] 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,Attorney Docket No. TVD-012WO 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. WO 1997 / 012986 and WO 1998 / 053087.

[0182] 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. WO 2013 / 052799, WO 2013 / 052811, and WO 2013 / 052832. The adenoviral vector can also comprise a combination of subtypes and thereby be a “chimeric” adenoviral vector.

[0183] 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.

[0184] Preferably, the adenoviral vector is replication-deficient, such that the replication- deficient adenoviral vector requires complementation of at least one replication-essentialAttorney Docket No. TVD-012WO 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. WO 1994 / 028152, WO 1995 / 002697, WO 1995 / 016772, WO 1995 / 034671, WO 1996 / 022378, WO 1997 / 012986, WO 1997 / 021826, and WO 2003 / 022311.

[0185] 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. WO 1997 / 000326, and U.S. Patent Nos.5,994,128 and 6,033,908), and 293-ORF6 cells (described in, e.g., PCT Publication No. WO 1995 / 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. WO 2003 / 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. WO 2000 / 034444.

[0186] 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.Attorney Docket No. TVD-012WO

[0187] 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. iii. Use of Exogenous tRNA Introns to Increase Yield of Viral Vectors Encoding Suppressor tRNAs

[0188] As described herein above, the present disclosure is based, in part, on the discovery that the incorporation of suppressor tRNA genes into viral vectors (e.g., AAV vectors and lentiviral vectors) can result in a decrease in production yield. This decrease in production can be substantial for viral vectors encoding potent suppressor tRNAs. However, it has also been discovered that incorporating exogenous tRNA introns into suppressor tRNAs alleviates this decrease, thereby restoring production yield of viral vectors encoding the suppressor tRNAs. Surprisingly, it has also been found that incorporating exogenous introns into suppressor tRNAs does not negatively impact the efficiency of suppression of premature termination.

[0189] Accordingly, disclosed herein is a method of increasing production yield of a viral vector encoding a suppressor tRNA, the method comprising modifying the nucleic acid encoding the tRNA to comprise an exogenous tRNA intron described herein. The intron is generally introduced between tRNA nucleotides 37 and 38. In some embodiments, the viral vector is an AAV vector. In some embodiments, the viral vector is a lentiviral vector.

[0190] The disclosure also provides a method for generating a recombinant AAV (rAAV) vector, the method comprising: (a) providing a virus-producing cell with (1) a first plasmid comprising a Rep gene and a Cap gene or functional fragments thereof; (2) a transfer plasmid comprising a nucleic acid comprising a suppressor tRNA and an exogenous intron of the disclosure, and flanking inverted terminal repeats (ITR); and (3) a helper plasmid comprising helper genes to mediate rAAV replication. In some embodiments, after step (a), the method further comprises culturing the cell to produce the rAAV vector.

[0191] The disclosure also provides a method for generating a recombinant lentiviral vector, the method comprising: (a) providing a virus-producing cell with (1) one or more plasmids (e.g., one or more, two or more, or three or more plasmids) collectively comprising gag, pol, rev, and tat genes or functional fragments thereof; and (2) providing a transfer plasmid comprising a nucleic acid comprising a suppressor tRNA and an exogenous intron ofAttorney Docket No. TVD-012WO the disclosure, and flanking long terminal repeats (LTR). In some embodiments, after step (a), the method further comprises culturing the cell to produce the recombinant lentiviral vector.

[0192] In some embodiments, the presence of the exogenous intron in the nucleic acid increases production yield of the viral vector (e.g., the AAV or lentiviral vector), relative to a viral vector comprising the same nucleic acid but that lacks the exogenous intron. In some embodiments, production yield of the viral vector comprising the nucleic acid with the exogenous intron is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, or 2000% relative to the production yield of a viral vector comprising the same nucleic acid but that lacks the exogenous intron. In some embodiments, production yield of the viral vector comprising the nucleic acid with the exogenous intron is increased by 10%-2000%, 10%-1000%, 10%-500%, 10-200%, 10%- 100%, 10%-50%, 10-25%, 25%-2000%, 25%-1000%, 25%-500%, 25%-200%, 25%-100%, 25%-50%, 50%-2000%, 50%-1000%, 50%-500%, 50%-200%, 50%-100%, 100%-2000%, 100%-1000%, 100%-500%, 100%-200%, 200%-2000%, 200%-1000%, 200%-500%, 500%- 2000%, 500%-1000%, or 1000%-2000% relative to the production yield of a viral vector comprising the same nucleic acid but that lacks the exogenous intron.

[0193] In some embodiments, the presence of the exogenous intron in the nucleic acid encoding the suppressor tRNA causes little or no observable reduction in the efficiency of suppression of premature termination. For example, in some embodiments, the presence of an exogenous intron in a nucleic acid encoding a suppressor tRNA results in less than a 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 12.5%, 10%, 9%, 8%, 7%, 6%, or 5% change in the efficiency of suppression of premature termination, relative to the efficiency of termination of a tRNA encoded by and transcribed from the same nucleic acid but lacking the exogenous intron. II. PHARMACEUTICAL COMPOSITIONS, MEDICAMENTS, AND ROUTES OF ADMINISTRATION

[0194] For therapeutic use a tRNA and / or an expression vector preferably is combined with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include 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 problems orAttorney Docket No. TVD-012WO complications, 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., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23d ed.2020). 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.

[0195] In some 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;Attorney Docket No. TVD-012WO excipients; and / or pharmaceutical adjuvants (see, REMINGTON’S PHARMACEUTICAL SCIENCES, 18thed. (Mack Publishing Company, 1990).

[0196] In some 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).

[0197] In some 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, poly (2-hydroxyethyl-inethacrylate), 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.

[0198] Pharmaceutical compositions containing a tRNA and / or 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 some embodiments a tRNA and / or an expression vector disclosed herein is administered by IV infusion. In some embodiments, a tRNA and / or an expression vector disclosed herein is administered intrathecally. Useful formulations can be prepared by methods known in the pharmaceutical art. For example, see Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23d ed.2020). 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.

[0199] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered salineAttorney Docket No. TVD-012WO (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 polyethylene glycol), and suitable mixtures thereof.

[0200] In general, any method of delivering a nucleic acid molecule can be adapted for use with a tRNA (see e.g., Akhtar et al. (1992) TRENDS CELL. BIOL.2(5):139-144 and PCT Publication No. WO 1994 / 02595). The tRNA can be modified or alternatively delivered using a drug delivery system to prevent the rapid degradation of the tRNA by endo- and exo- nucleases in vivo. tRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. tRNA molecules can also be conjugated to or otherwise associated with an aptamer. A tRNA can also be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, a liposome, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of a tRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of a tRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to the RNA, e.g., tRNA, or induced to form a vesicle or micelle (see e.g., Kim et al. (2008) JOURNAL OF CONTROLLED RELEASE 129(2):107-116) that encases the RNA. Methods for making and administering cationic-RNA complexes are well within the abilities of one skilled in the art (see, e.g., Sorensen et al. (2003) J. MOL. BIOL 327:761-766; Verma et al. (2003) CLIN. CANCER RES. 9:1291-1300; Arnold et al. (2007) J. HYPERTENS.25:197-205). Some non-limiting examples of drug delivery systems useful for systemic delivery of RNAs, e.g., tRNAs include DOTAP (Sorensen et al. (2003) supra; Verma et al. (2003), supra), oligofectamine, solid nucleic acid lipid particles (Zimmermann et al. (2006) NATURE 441:111-114), cardiolipin (Chien et al. (2005) CANCER GENE THER.12:321-328; Pal et al. (2005) INT J. ONCOL.26:1087-1091), polyethyleneimine (Bonnet et al. (2008) PHARM. RES.25(12):2972-82; Aigner (2006) J. BIOMED. BIOTECHNOL.71659), Arg-Gly-Asp (RGD) peptides (Liu (2006) MOL. PHARM. 3:472-487), and polyamidoamines (Tomalia et al. (2007) BIOCHEM. SOC. TRANS.35:61-67; Yoo et al. (1999) PHARM. RES.16:1799-1804). In some embodiments, a tRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of RNAs and cyclodextrins can be found in U.S. Patent No. 7,427,605.Attorney Docket No. TVD-012WO

[0201] 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.

[0202] The compositions described herein may be administered locally or systemically. In some embodiments, the pharmaceutical composition is administered parenterally. In some embodiments, the pharmaceutical composition is administered subcutaneously. In further embodiments, the pharmaceutical composition is administered intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.

[0203] Generally, a therapeutically effective amount of active component, for example, a tRNA or 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. 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 active component, 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. 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, and the disease being treated. Exemplary dosing frequencies are once per day, once per week, and once every two weeks. In some embodiments, a polypeptide and / or construct is lyophilized, and then reconstituted in buffered saline, at the time of administration.

[0204] It is to be understood that the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. III. THERAPEUTIC USES

[0205] The disclosure provides a method of expressing in a mammalian cell a functional gene product encoded by a gene containing a PTC, the method comprising contacting or exposing the cell with an effective amount (e.g., a therapeutically effective amount) of any of the foregoing tRNAs, nucleic acids, 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 PTC.Attorney Docket No. TVD-012WO

[0206] The present disclosure also provides a method of treating a disease or disorder associated with a protein encoded by a gene including a PTC in a subject in need thereof (e.g., a human). In some embodiments, the method comprises administering a therapeutically effective amount of any of the foregoing tRNAs, nucleic acids, expression vectors, or pharmaceutical compositions disclosed herein to the subject in need thereof.

[0207] The present disclosure also provides a method of treating a haploinsufficiency disorder in a subject in need thereof. In some embodiments, the method comprises administering a therapeutically effective amount of any of the foregoing tRNAs, nucleic acids, expression vectors, or pharmaceutical compositions disclosed herein to the subject in need thereof.

[0208] The present disclosure also provides a method of increasing production in a mammalian cell of a full length protein encoded by a gene containing a PTC. In some embodiments, the method comprises administering a composition disclosed herein (e.g., a tRNA, nucleic acid, expression vector, or pharmaceutical composition) to a cell, and permitting the complex, nucleic acid, or vector to be internalized by the cell, whereupon production of the suppressor tRNA permits readthrough of the PTC and the production of the full length protein.

[0209] The present disclosure also provides a method of increasing the production of a protein of interest in a mammalian cell. In some embodiments, the method comprises contacting the cell with a composition disclosed herein (e.g., a tRNA, a nucleic acid, an expression vector, or a pharmaceutical composition) and permitting the complex, nucleic acid, or vector to be internalized by the cell, whereupon production of the tRNA permits increased translation of the protein relative to a cell not contacted with the composition.

[0210] In some embodiments of any of the foregoing methods, the cell contains less truncated gene product than a cell without the tRNA (e.g., the tRNA expressed from a nucleic acid or expression vector disclosed herein). For example, in some 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 some 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%Attorney Docket No. TVD-012WO of the truncated gene product relative to a cell without the tRNA. In some 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.

[0211] In some embodiments, the cell contains a greater amount of functional gene product than a cell without the tRNA expressed from a nucleic acid or expression vector disclosed herein. For example, in some 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 some 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%, 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.

[0212] In some 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 positionAttorney Docket No. TVD-012WO corresponding to a native stop codon (i.e., the tRNA does not permit read-through of a native stop codon). For example, in some 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.

[0213] In some embodiments, the diseases or disorder associated with a protein encoded by a gene including a PTC to be treated by a method of the disclosure is a disorder listed in TABLE 9 below, and / or the gene with a premature termination codon is a gene listed in the corresponding row of TABLE 9 below. TABLE 9Attorney Docket No. TVD-012WO

[0214] In some embodiments, the premature termination codon-mediated disorder to be treated by a method of the disclosure is a disorder listed in TABLE 10 below, and / or the gene with a premature termination codon is a gene listed in the corresponding row of TABLE 10 below. TABLE 10Attorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAttorney Docket No. TVD-012WO

[0215] In some 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 some 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 some 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-012WO

[0216] In some embodiments, the PTC-mediated disorder is Dravet syndrome and / or the gene with a premature termination codon is SCN1A. In some 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_ 4130delins ATAATCATACTGAT TGCCTAAAACTAAT, c.3690_3693del, c.3338_3339del, c.1247_1248insGTAGA, c.825_826insGTATA, and c.278_279dup.

[0217] In some 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 some 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,Attorney Docket No. TVD-012WO c.4573C>T, c.5656C>T, and c.5734C>T. In some embodiments, a premature termination codon in the SCN1A gene is caused by a mutation selected from c.1738C>T and c.3985C>T.

[0218] In some embodiments, a premature termination codon in the SCN1A gene is caused by a mutation set forth in TABLE 11, or a combination of mutations set forth in TABLE 11. TABLE 11Attorney Docket No. TVD-012WOAttorney Docket No. TVD-012WO

[0219] 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.31(2):114-30, the SCN1A Registry (available on the world wide web at scn1a.net / scn1a-registry / ), the SCN1A Mutation Database (available on the 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).

[0220] 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 of TABLE 11, the method comprising administering to the subject an effective amount of a suppressor tRNA disclosed herein; or a nucleic acid or expression vector disclosed herein encoding the same, wherein the suppressor tRNA is of the suppressor class indicated in the same row of TABLE 11 as the mutation. “Suppressor Class” as used in TABLE 11 (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 SEQ ID NOs: 2, 19-21, 375, 378-381, 384, 388, 390-391, 393, 395, 397, 912- 919, 940-971, 1020-1083, and 1186. Exemplary Gln>TAA suppressor tRNAs include tRNAs comprising a nucleotide sequence selected from SEQ ID NOs: 37, 39, 40, 44, 442, 449-450, 456-458, and 972-995. Exemplary Gln>TAG suppressor tRNAs include tRNAs comprising a nucleotide sequence selected from SEQ ID NOs: 179, 181, 182, 186, 411, 425- 427, and 996-1019.

[0221] For example, in some 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 SEQ ID NOs: 2, 19- 21, 375, 378-381, 384, 388, 390-391, 393, 395, 397, 912-919, 940-971, 1020-1083, and 1186, or a nucleic acid or expression vector encoding the same. In some 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 theAttorney Docket No. TVD-012WO subject an effective amount of a suppressor tRNA comprising a nucleotide sequence selected from SEQ ID NOs: 37, 39, 40, 44, 442, 456, 458, and 972-995, or a nucleic acid or expression vector encoding the same. In some 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 SEQ ID NOs: 179, 181, 182, 186, 411, 425, 427 and 996-1019, or a nucleic acid or expression vector encoding the same.

[0222] In some embodiments, wherein the gene is a SCN1A gene, the SCN1A gene product produced with the tRNA is a functional SCN1A gene product. In some embodiments, the functional SCN1A gene product has greater activity than the truncated SCN1A gene product, e.g., greater voltage-gated sodium channel activity. In some 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 some 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 aboutAttorney Docket No. TVD-012WO 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.

[0223] In some embodiments, the functional SCN1A gene product is the Nav1.1 protein. In some 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): MEQTVLVPPGPDSFNFFTRESLAAIERRIAEEKAKNPKPDKKDDDENGPKPNSDLEAGKNLP FIYGDIPPEMVSEPLEDLDPYYINKKTFIVLNKGKAIFRFSATSALYILTPFNPLRKIAIKI LVHSLFSMLIMCTILTNCVFMTMSNPPDWTKNVEYTFTGIYTFESLIKIIARGFCLEDFTFL RDPWNWLDFTVITFAYVTEFVDLGNVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLS DVMILTVFCLSVFALIGLQLFMGNLRNKCIQWPPTNASLEEHSIEKNITVNYNGTLINETVF EFDWKSYIQDSRYHYFLEGFLDALLCGNSSDAGQCPEGYMCVKAGRNPNYGYTSFDTFSWAF LSLFRLMTQDFWENLYQLTLRAAGKTYMIFFVLVIFLGSFYLINLILAVVAMAYEEQNQATL EEAEQKEAEFQQMIEQLKKQQEAAQQAATATASEHSREPSAAGRLSDSSSEASKLSSKSAKE RRNRRKKRKQKEQSGGEEKDEDEFQKSESEDSIRRKGFRFSIEGNRLTYEKRYSSPHQSLLS IRGSLFSPRRNSRTSLFSFRGRAKDVGSENDFADDEHSTFEDNESRRDSLFVPRRHGERRNS NLSQTSRSSRMLAVFPANGKMHSTVDCNGVVSLVGGPSVPTSPVGQLLPEVIIDKPATDDNG TTTETEMRKRRSSSFHVSMDFLEDPSQRQRAMSIASILTNTVEELEESRQKCPPCWYKFSNI FLIWDCSPYWLKVKHVVNLVVMDPFVDLAITICIVLNTLFMAMEHYPMTDHFNNVLTVGNLV FTGIFTAEMFLKIIAMDPYYYFQEGWNIFDGFIVTLSLVELGLANVEGLSVLRSFRLLRVFK LAKSWPTLNMLIKIIGNSVGALGNLTLVLAIIVFIFAVVGMQLFGKSYKDCVCKIASDCQLP RWHMNDFFHSFLIVFRVLCGEWIETMWDCMEVAGQAMCLTVFMMVMVIGNLVVLNLFLALLL SSFSADNLAATDDDNEMNNLQIAVDRMHKGVAYVKRKIYEFIQQSFIRKQKILDEIKPLDDL NNKKDSCMSNHTAEIGKDLDYLKDVNGTTSGIGTGSSVEKYIIDESDYMSFINNPSLTVTVP IAVGESDFENLNTEDFSSESDLEESKEKLNESSSSSEGSTVDIGAPVEEQPVVEPEETLEPE ACFTEGCVQRFKCCQINVEEGRGKQWWNLRRTCFRIVEHNWFETFIVFMILLSSGALAFEDI YIDQRKTIKTMLEYADKVFTYIFILEMLLKWVAYGYQTYFTNAWCWLDFLIVDVSLVSLTAN ALGYSELGAIKSLRTLRALRPLRALSRFEGMRVVVNALLGAIPSIMNVLLVCLIFWLIFSIM GVNLFAGKFYHCINTTTGDRFDIEDVNNHTDCLKLIERNETARWKNVKVNFDNVGFGYLSLL QVATFKGWMDIMYAAVDSRNVELQPKYEESLYMYLYFVIFIIFGSFFTLNLFIGVIIDNFNQAttorney Docket No. TVD-012WO QKKKFGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNKFQGMVFDFVTRQVFDISIMIL ICLNMVTMMVETDDQSEYVTTILSRINLVFIVLFTGECVLKLISLRHYYFTIGWNIFDFVVV ILSIVGMFLAELIEKYFVSPTLFRVIRLARIGRILRLIKGAKGIRTLLFALMMSLPALFNIG LLLFLVMFIYAIFGMSNFAYVKREVGIDDMFNFETFGNSMICLFQITTSAGWDGLLAPILNS KPPDCDPNKVNPGSSVKGDCGNPSVGIFFFVSYIIISFLVVVNMYIAVILENFSVATEESAE PLSEDDFEMFYEVWEKFDPDATQFMEFEKLSQFAAALEPPLNLPQPNKLQLIAMDLPMVSGD RIHCLDILFAFTKRVLGESGEMDALRIQMEERFMASNPSKVSYQPITTTLKRKQEEVSAVII QRAYRRHLLKRTVKQASFTYNKNKIKGGANLLIKEDMIIDRINENSITEKTDLTMSTAACPP SYDRVTKPIVEKHEQEGKDEKAKGK (SEQ ID NO: 863); MEQTVLVPPGPDSFNFFTRESLAAIERRIAEEKAKNPKPDKKDDDENGPKPNSDLEAGKNLP FIYGDIPPEMVSEPLEDLDPYYINKKTFIVLNKGKAIFRFSATSALYILTPFNPLRKIAIKI LVHSLFSMLIMCTILTNCVFMTMSNPPDWTKNVEYTFTGIYTFESLIKIIARGFCLEDFTFL RDPWNWLDFTVITFAYVTEFVDLGNVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLS DVMILTVFCLSVFALIGLQLFMGNLRNKCIQWPPTNASLEEHSIEKNITVNYNGTLINETVF EFDWKSYIQDSRYHYFLEGFLDALLCGNSSDAGQCPEGYMCVKAGRNPNYGYTSFDTFSWAF LSLFRLMTQDFWENLYQLTLRAAGKTYMIFFVLVIFLGSFYLINLILAVVAMAYEEQNQATL EEAEQKEAEFQQMIEQLKKQQEAAQQAATATASEHSREPSAAGRLSDSSSEASKLSSKSAKE RRNRRKKRKQKEQSGGEEKDEDEFQKSESEDSIRRKGFRFSIEGNRLTYEKRYSSPHQSLLS IRGSLFSPRRNSRTSLFSFRGRAKDVGSENDFADDEHSTFEDNESRRDSLFVPRRHGERRNS NLSQTSRSSRMLAVFPANGKMHSTVDCNGVVSLVGGPSVPTSPVGQLLPEGTTTETEMRKRR SSSFHVSMDFLEDPSQRQRAMSIASILTNTVEELEESRQKCPPCWYKFSNIFLIWDCSPYWL KVKHVVNLVVMDPFVDLAITICIVLNTLFMAMEHYPMTDHFNNVLTVGNLVFTGIFTAEMFL KIIAMDPYYYFQEGWNIFDGFIVTLSLVELGLANVEGLSVLRSFRLLRVFKLAKSWPTLNML IKIIGNSVGALGNLTLVLAIIVFIFAVVGMQLFGKSYKDCVCKIASDCQLPRWHMNDFFHSF LIVFRVLCGEWIETMWDCMEVAGQAMCLTVFMMVMVIGNLVVLNLFLALLLSSFSADNLAAT DDDNEMNNLQIAVDRMHKGVAYVKRKIYEFIQQSFIRKQKILDEIKPLDDLNNKKDSCMSNH TAEIGKDLDYLKDVNGTTSGIGTGSSVEKYIIDESDYMSFINNPSLTVTVPIAVGESDFENL NTEDFSSESDLEESKEKLNESSSSSEGSTVDIGAPVEEQPVVEPEETLEPEACFTEGCVQRF KCCQINVEEGRGKQWWNLRRTCFRIVEHNWFETFIVFMILLSSGALAFEDIYIDQRKTIKTM LEYADKVFTYIFILEMLLKWVAYGYQTYFTNAWCWLDFLIVDVSLVSLTANALGYSELGAIK SLRTLRALRPLRALSRFEGMRVVVNALLGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFYH CINTTTGDRFDIEDVNNHTDCLKLIERNETARWKNVKVNFDNVGFGYLSLLQVATFKGWMDI MYAAVDSRNVELQPKYEESLYMYLYFVIFIIFGSFFTLNLFIGVIIDNFNQQKKKFGGQDIFAttorney Docket No. TVD-012WO MTEEQKKYYNAMKKLGSKKPQKPIPRPGNKFQGMVFDFVTRQVFDISIMILICLNMVTMMVE TDDQSEYVTTILSRINLVFIVLFTGECVLKLISLRHYYFTIGWNIFDFVVVILSIVGMFLAE LIEKYFVSPTLFRVIRLARIGRILRLIKGAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYA IFGMSNFAYVKREVGIDDMFNFETFGNSMICLFQITTSAGWDGLLAPILNSKPPDCDPNKVN PGSSVKGDCGNPSVGIFFFVSYIIISFLVVVNMYIAVILENFSVATEESAEPLSEDDFEMFY EVWEKFDPDATQFMEFEKLSQFAAALEPPLNLPQPNKLQLIAMDLPMVSGDRIHCLDILFAF TKRVLGESGEMDALRIQMEERFMASNPSKVSYQPITTTLKRKQEEVSAVIIQRAYRRHLLKR TVKQASFTYNKNKIKGGANLLIKEDMIIDRINENSITEKTDLTMSTAACPPSYDRVTKPIVE KHEQEGKDEKAKGK (SEQ ID NO: 864); MEQTVLVPPGPDSFNFFTRESLAAIERRIAEEKAKNPKPDKKDDDENGPKPNSDLEAGKNLP FIYGDIPPEMVSEPLEDLDPYYINKKTFIVLNKGKAIFRFSATSALYILTPFNPLRKIAIKI LVHSLFSMLIMCTILTNCVFMTMSNPPDWTKNVEYTFTGIYTFESLIKIIARGFCLEDFTFL RDPWNWLDFTVITFAYVTEFVDLGNVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLS DVMILTVFCLSVFALIGLQLFMGNLRNKCIQWPPTNASLEEHSIEKNITVNYNGTLINETVF EFDWKSYIQDSRYHYFLEGFLDALLCGNSSDAGQCPEGYMCVKAGRNPNYGYTSFDTFSWAF LSLFRLMTQDFWENLYQLTLRAAGKTYMIFFVLVIFLGSFYLINLILAVVAMAYEEQNQATL EEAEQKEAEFQQMIEQLKKQQEAAQQAATATASEHSREPSAAGRLSDSSSEASKLSSKSAKE RRNRRKKRKQKEQSGGEEKDEDEFQKSESEDSIRRKGFRFSIEGNRLTYEKRYSSPHQSLLS IRGSLFSPRRNSRTSLFSFRGRAKDVGSENDFADDEHSTFEDNESRRDSLFVPRRHGERRNS NLSQTSRSSRMLAVFPANGKMHSTVDCNGVVSLGTTTETEMRKRRSSSFHVSMDFLEDPSQR QRAMSIASILTNTVEELEESRQKCPPCWYKFSNIFLIWDCSPYWLKVKHVVNLVVMDPFVDL AITICIVLNTLFMAMEHYPMTDHFNNVLTVGNLVFTGIFTAEMFLKIIAMDPYYYFQEGWNI FDGFIVTLSLVELGLANVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLV LAIIVFIFAVVGMQLFGKSYKDCVCKIASDCQLPRWHMNDFFHSFLIVFRVLCGEWIETMWD CMEVAGQAMCLTVFMMVMVIGNLVVLNLFLALLLSSFSADNLAATDDDNEMNNLQIAVDRMH KGVAYVKRKIYEFIQQSFIRKQKILDEIKPLDDLNNKKDSCMSNHTAEIGKDLDYLKDVNGT TSGIGTGSSVEKYIIDESDYMSFINNPSLTVTVPIAVGESDFENLNTEDFSSESDLEESKEK LNESSSSSEGSTVDIGAPVEEQPVVEPEETLEPEACFTEGCVQRFKCCQINVEEGRGKQWWN LRRTCFRIVEHNWFETFIVFMILLSSGALAFEDIYIDQRKTIKTMLEYADKVFTYIFILEML LKWVAYGYQTYFTNAWCWLDFLIVDVSLVSLTANALGYSELGAIKSLRTLRALRPLRALSRF EGMRVVVNALLGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFYHCINTTTGDRFDIEDVNN HTDCLKLIERNETARWKNVKVNFDNVGFGYLSLLQVATFKGWMDIMYAAVDSRNVELQPKYE ESLYMYLYFVIFIIFGSFFTLNLFIGVIIDNFNQQKKKFGGQDIFMTEEQKKYYNAMKKLGSAttorney Docket No. TVD-012WO KKPQKPIPRPGNKFQGMVFDFVTRQVFDISIMILICLNMVTMMVETDDQSEYVTTILSRINL VFIVLFTGECVLKLISLRHYYFTIGWNIFDFVVVILSIVGMFLAELIEKYFVSPTLFRVIRL ARIGRILRLIKGAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIFGMSNFAYVKREVGID DMFNFETFGNSMICLFQITTSAGWDGLLAPILNSKPPDCDPNKVNPGSSVKGDCGNPSVGIF FFVSYIIISFLVVVNMYIAVILENFSVATEESAEPLSEDDFEMFYEVWEKFDPDATQFMEFE KLSQFAAALEPPLNLPQPNKLQLIAMDLPMVSGDRIHCLDILFAFTKRVLGESGEMDALRIQ MEERFMASNPSKVSYQPITTTLKRKQEEVSAVIIQRAYRRHLLKRTVKQASFTYNKNKIKGG ANLLIKEDMIIDRINENSITEKTDLTMSTAACPPSYDRVTKPIVEKHEQEGKDEKAKGK (SEQ ID NO: 865); MEQTVLVPPGPDSFNFFTRESLAAIERRIAEEKAKNPKPDKKDDDENGPKPNSDLEAGKNLP FIYGDIPPEMVSEPLEDLDPYYINKKTFIVLNKGKAIFRFSATSALYILTPFNPLRKIAIKI LVHSLFSMLIMCTILTNCVFMTMSNPPDWTKNVEYTFTGIYTFESLIKIIARGFCLEDFTFL RDPWNWLDFTVITFAYVTEFVDLGNVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLS DVMILTVFCLSVFALIGLQLFMGNLRNKCIQWPPTNASLEEHSIEKNITVNYNGTLINETVF EFDWKSYIQDSRYHYFLEGFLDALLCGNSSDAGQCPEGYMCVKAGRNPNYGYTSFDTFSWAF LSLFRLMTQDFWENLYQLTLRAAGKTYMIFFVLVIFLGSFYLINLILAVVAMAYEEQNQATL EEAEQKEAEFQQMIEQLKKQQEAAQAATATASEHSREPSAAGRLSDSSSEASKLSSKSAKER RNRRKKRKQKEQSGGEEKDEDEFQKSESEDSIRRKGFRFSIEGNRLTYEKRYSSPHQSLLSI RGSLFSPRRNSRTSLFSFRGRAKDVGSENDFADDEHSTFEDNESRRDSLFVPRRHGERRNSN LSQTSRSSRMLAVFPANGKMHSTVDCNGVVSLVGGPSVPTSPVGQLLPEGTTTETEMRKRRS SSFHVSMDFLEDPSQRQRAMSIASILTNTVEELEESRQKCPPCWYKFSNIFLIWDCSPYWLK VKHVVNLVVMDPFVDLAITICIVLNTLFMAMEHYPMTDHFNNVLTVGNLVFTGIFTAEMFLK IIAMDPYYYFQEGWNIFDGFIVTLSLVELGLANVEGLSVLRSFRLLRVFKLAKSWPTLNMLI KIIGNSVGALGNLTLVLAIIVFIFAVVGMQLFGKSYKDCVCKIASDCQLPRWHMNDFFHSFL IVFRVLCGEWIETMWDCMEVAGQAMCLTVFMMVMVIGNLVVLNLFLALLLSSFSADNLAATD DDNEMNNLQIAVDRMHKGVAYVKRKIYEFIQQSFIRKQKILDEIKPLDDLNNKKDSCMSNHT AEIGKDLDYLKDVNGTTSGIGTGSSVEKYIIDESDYMSFINNPSLTVTVPIAVGESDFENLN TEDFSSESDLEESKEKLNESSSSSEGSTVDIGAPVEEQPVVEPEETLEPEACFTEGCVQRFK CCQINVEEGRGKQWWNLRRTCFRIVEHNWFETFIVFMILLSSGALAFEDIYIDQRKTIKTML EYADKVFTYIFILEMLLKWVAYGYQTYFTNAWCWLDFLIVDVSLVSLTANALGYSELGAIKS LRTLRALRPLRALSRFEGMRVVVNALLGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFYHC INTTTGDRFDIEDVNNHTDCLKLIERNETARWKNVKVNFDNVGFGYLSLLQVATFKGWMDIM YAAVDSRNVELQPKYEESLYMYLYFVIFIIFGSFFTLNLFIGVIIDNFNQQKKKFGGQDIFMAttorney Docket No. TVD-012WO TEEQKKYYNAMKKLGSKKPQKPIPRPGNKFQGMVFDFVTRQVFDISIMILICLNMVTMMVET DDQSEYVTTILSRINLVFIVLFTGECVLKLISLRHYYFTIGWNIFDFVVVILSIVGMFLAEL IEKYFVSPTLFRVIRLARIGRILRLIKGAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAI FGMSNFAYVKREVGIDDMFNFETFGNSMICLFQITTSAGWDGLLAPILNSKPPDCDPNKVNP GSSVKGDCGNPSVGIFFFVSYIIISFLVVVNMYIAVILENFSVATEESAEPLSEDDFEMFYE VWEKFDPDATQFMEFEKLSQFAAALEPPLNLPQPNKLQLIAMDLPMVSGDRIHCLDILFAFT KRVLGESGEMDALRIQMEERFMASNPSKVSYQPITTTLKRKQEEVSAVIIQRAYRRHLLKRT VKQASFTYNKNKIKGGANLLIKEDMIIDRINENSITEKTDLTMSTAACPPSYDRVTKPIVEK HEQEGKDEKAKGK (SEQ ID NO: 866); MEQTVLVPPGPDSFNFFTRESLAAIERRIAEEKAKNPKPDKKDDDENGPKPNSDLEAGKNLP FIYGDIPPEMVSEPLEDLDPYYINKKTFIVLNKGKAIFRFSATSALYILTPFNPLRKIAIKI LVHSLFSMLIMCTILTNCVFMTMSNPPDWTKNVEYTFTGIYTFESLIKIIARGFCLEDFTFL RDPWNWLDFTVITFAYVTEFVDLGNVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLS DVMILTVFCLSVFALIGLQLFMGNLRNKCIQWPPTNASLEEHSIEKNITVNYNGTLINETVF EFDWKSYIQDSRYHYFLEGFLDALLCGNSSDAGQCPEGYMCVKAGRNPNYGYTSFDTFSWAF LSLFRLMTQDFWENLYQLTLRAAGKTYMIFFVLVIFLGSFYLINLILAVVAMAYEEQNQATL EEAEQKEAEFQQMIEQLKKQQEAAQAATATASEHSREPSAAGRLSDSSSEASKLSSKSAKER RNRRKKRKQKEQSGGEEKDEDEFQKSESEDSIRRKGFRFSIEGNRLTYEKRYSSPHQSLLSI RGSLFSPRRNSRTSLFSFRGRAKDVGSENDFADDEHSTFEDNESRRDSLFVPRRHGERRNSN LSQTSRSSRMLAVFPANGKMHSTVDCNGVVSLGTTTETEMRKRRSSSFHVSMDFLEDPSQRQ RAMSIASILTNTVEELEESRQKCPPCWYKFSNIFLIWDCSPYWLKVKHVVNLVVMDPFVDLA ITICIVLNTLFMAMEHYPMTDHFNNVLTVGNLVFTGIFTAEMFLKIIAMDPYYYFQEGWNIF DGFIVTLSLVELGLANVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLVL AIIVFIFAVVGMQLFGKSYKDCVCKIASDCQLPRWHMNDFFHSFLIVFRVLCGEWIETMWDC MEVAGQAMCLTVFMMVMVIGNLVVLNLFLALLLSSFSADNLAATDDDNEMNNLQIAVDRMHK GVAYVKRKIYEFIQQSFIRKQKILDEIKPLDDLNNKKDSCMSNHTAEIGKDLDYLKDVNGTT SGIGTGSSVEKYIIDESDYMSFINNPSLTVTVPIAVGESDFENLNTEDFSSESDLEESKEKL NESSSSSEGSTVDIGAPVEEQPVVEPEETLEPEACFTEGCVQRFKCCQINVEEGRGKQWWNL RRTCFRIVEHNWFETFIVFMILLSSGALAFEDIYIDQRKTIKTMLEYADKVFTYIFILEMLL KWVAYGYQTYFTNAWCWLDFLIVDVSLVSLTANALGYSELGAIKSLRTLRALRPLRALSRFE GMRVVVNALLGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFYHCINTTTGDRFDIEDVNNH TDCLKLIERNETARWKNVKVNFDNVGFGYLSLLQVATFKGWMDIMYAAVDSRNVELQPKYEE SLYMYLYFVIFIIFGSFFTLNLFIGVIIDNFNQQKKKFGGQDIFMTEEQKKYYNAMKKLGSKAttorney Docket No. TVD-012WO KPQKPIPRPGNKFQGMVFDFVTRQVFDISIMILICLNMVTMMVETDDQSEYVTTILSRINLV FIVLFTGECVLKLISLRHYYFTIGWNIFDFVVVILSIVGMFLAELIEKYFVSPTLFRVIRLA RIGRILRLIKGAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIFGMSNFAYVKREVGIDD MFNFETFGNSMICLFQITTSAGWDGLLAPILNSKPPDCDPNKVNPGSSVKGDCGNPSVGIFF FVSYIIISFLVVVNMYIAVILENFSVATEESAEPLSEDDFEMFYEVWEKFDPDATQFMEFEK LSQFAAALEPPLNLPQPNKLQLIAMDLPMVSGDRIHCLDILFAFTKRVLGESGEMDALRIQM EERFMASNPSKVSYQPITTTLKRKQEEVSAVIIQRAYRRHLLKRTVKQASFTYNKNKIKGGA NLLIKEDMIIDRINENSITEKTDLTMSTAACPPSYDRVTKPIVEKHEQEGKDEKAKGK (SEQ ID NO: 867); or MFLKIIAMDPYYYFQEGWNIFDGFIVTLSLVELGLANVEGLSVLRSFRLLRVFKLAKSWPTL NMLIKIIGNSVGALGNLTLVLAIIVFIFAVVGMQLFGKSYKDCVCKIASDCQLPRWHMNDFF HSFLIVFRVLCGEWIETMWDCMEVAGQAMCLTVFMMVMVIGNLVVLNLFLALLLSSFSADNL AATDDDNEMNNLQIAVDRMHKGVAYVKRKIYEFIQQSFIRKQKILDEIKPLDDLNNKKDSCM SNHTAEIGKDLDYLKDVNGTTSGIGTGSSVEKYIIDESDYMSFINNPSLTVTVPIAVGESDF ENLNTEDFSSESDLEESKEKLNESSSSSEGSTVDIGAPVEEQPVVEPEETLEPEACFTEGCV QRFKCCQINVEEGRGKQWWNLRRTCFRIVEHNWFETFIVFMILLSSGALAFEDIYIDQRKTI KTMLEYADKVFTYIFILEMLLKWVAYGYQTYFTNAWCWLDFLIVDVSLVSLTANALGYSELG AIKSLRTLRALRPLRALSRFEGMRVVVNALLGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGK FYHCINTTTGDRFDIEDVNNHTDCLKLIERNETARWKNVKVNFDNVGFGYLSLLQVATFKGW MDIMYAAVDSRNVELQPKYEESLYMYLYFVIFIIFGSFFTLNLFIGVIIDNFNQQKKKFGGQ DIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNKFQGMVFDFVTRQVFDISIMILICLNMVTM MVETDDQSEYVTTILSRINLVFIVLFTGECVLKLISLRHYYFTIGWNIFDFVVVILSIVGMF LAELIEKYFVSPTLFRVIRLARIGRILRLIKGAKGIRTLLFALMMSLPALFNIGLLLFLVMF IYAIFGMSNFAYVKREVGIDDMFNFETFGNSMICLFQITTSAGWDGLLAPILNSKPPDCDPN KVNPGSSVKGDCGNPSVGIFFFVSYIIISFLVVVNMYIAVILENFSVATEESAEPLSEDDFE MFYEVWEKFDPDATQFMEFEKLSQFAAALEPPLNLPQPNKLQLIAMDLPMVSGDRIHCLDIL FAFTKRVLGESGEMDALRIQMEERFMASNPSKVSYQPITTTLKRKQEEVSAVIIQRAYRRHL LKRTVKQASFTYNKNKIKGGANLLIKEDMIIDRINENSITEKTDLTMSTAACPPSYDRVTKP IVEKHEQEGKDEKAKGK (SEQ ID NO: 868).

[0224] The disclosure provides a method of treating dilated cardiomyopathy (DCM) in a subject in need thereof, wherein the subject has a TTN gene with a premature termination codon. In some embodiments, the method comprises administering to the subject an effective amount of a suppressor tRNA disclosed herein (e.g., an Arg-suppressor tRNA, e.g.,Attorney Docket No. TVD-012WO an Arg>TGA suppressor tRNA, such as tr0321 or tr0374), or a nucleic acid or expression vector disclosed herein encoding the same. Exemplary Arg>TGA suppressor tRNAs include tRNAs comprising a nucleotide sequence selected from SEQ ID NOs: 2, 19-21, 375, 378- 381, 384, 388, 390-391, 393, 395, 397, 912-919, 940-971, 1020-1083, and 1186. In some embodiments, the suppressor tRNA is tr0321 (SEQ ID NO: 917) or tr0374 (SEQ ID NO: 1060).

[0225] For example, in some embodiments, the subject has a TTN gene with a premature termination codon R30,227X, and the method comprises administering to the subject an effective amount of a suppressor tRNA comprising a nucleotide sequence selected from SEQ ID NOs: 2, 19-21, 375, 378-381, 384, 388, 390-391, 393, 395, 397, 912-919, 940-971, 1020- 1083, and 1186, or a nucleic acid or expression vector encoding the same. In some embodiments, the suppressor tRNA is tr0321 (SEQ ID NO: 917) or tr0374 (SEQ ID NO: 1060).

[0226] In some embodiments, the functional TTN gene product produced with the tRNA comprises, consists essentially of, or consists of the amino acid sequence of any one of the amino acid sequences set forth in UniProt entry #A2ASS6, which sequences are hereby incorporated by reference in their entirety. In some embodiments, the functional TTN gene product is the TTN A2ASS6-1 isoform (as set forth in UniProt #A2ASS6), 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 the A2ASS6-1 isoform.

[0227] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or modalities, e.g., in the treatment of a disease or disorder, such as a PTC-associated disorder or a haploinsufficiency disorder. In some embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments, 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 the absence of the first treatment, or the analogous situation is seen with the first treatment. In some 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, whollyAttorney Docket No. TVD-012WO 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.

[0228] In some 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.

[0229] The disclosure also provides a method of producing a tRNA of interest in a mammalian cell. In some embodiments, the method comprises contacting the cell with an effective amount of any of the foregoing compositions, tRNAs, nucleic acids, expression vectors, or pharmaceutical compositions disclosed herein. EXAMPLES

[0230] The following examples are offered for illustrative purposes only and are not intended to limit the scope or content of inventions disclosed herein, in any way. EXAMPLE 1

[0231] This Example describes using engineered T-stems in suppressor tRNAs to enhance suppression of premature termination of a gene comprising a premature stop codon. Materials and Methods AAV Production

[0232] Recombinant AAV (rAAV) particles comprising a viral vector encoding wild-type or suppressor tRNAs were generated using standard methods known in the art. To construct transfer plasmids (pAAVs; also known as vector genome plasmids), elements of the transfer plasmids (including nucleic acids encoding tRNAs, U6 promoter sequences, and miscellaneous vector sequences) were synthesized or excised from commercially available plasmids. The transfer plasmid elements were assembled into pAAV plasmid backbonesAttorney Docket No. TVD-012WO using restriction enzymes or non-restriction-enzyme-based DNA assembly methods, as appropriate.

[0233] Triple-plasmid transfection was conducted using polyethylenimine (PEI, Polyscience) and optionally including other transfection reagents to produce rAAV particles. The transfected plasmids included: (1) a single-stranded (ssAAV) or self-complementary (scAAV) transfer plasmid encoding a tRNA (e.g., a wild-type tRNA or a suppressor tRNA of the disclosure) located between two inverted terminal repeats (ITRs); (2) a pRep2CapX (“RepCap”) plasmid, encoding AAV2 replication (Rep2) protein plus a defined capsid (Cap) protein for the desired AAV serotype (CapX); (3) a “pHelper” plasmid, encoding additional AAV proteins required for producing functional AAV particles.

[0234] The three plasmids were co-transfected into HEK293T cells. Briefly, 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 1:1:1 molar ratio of the pHelper, RepCap, and 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.

[0235] 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 1x PBS + 0.001% pluronic F-68.

[0236] The viral genomic titers were determined by a SYBR Green quantitative polymerase chain reaction (qPCR) (Bio-Rad, USA) assay and / or a droplet digital polymeraseAttorney Docket No. TVD-012WO chain reaction (ddPCR; Bio-Rad, USA). Polymerase chain reaction (PCR) primers were designed using SnapGene for each AAV. MECP2 Primary Neuron Assay Experimental Design

[0237] Cortical cultures were made from MECP2 hemizygous male mouse pups with an inactivating Arg(R)>TGA mutation at R255 (Jackson Laboratories Strain #:012602). Primary neurons were obtained at embryonic day (E)14-E16. 100,000 cells were seeded per well in Poly-D-Lysine (PDL)-coated 48-well plates or 40,000 cells in Poly-D-Lysine (PDL)- coated 96-well plates on in vitro day (DIV)0. At DIV4, cells were transduced with either rAAV1 at 5 x 103, 1.5 x 104, or 5 x 104viral genomes per cell (vg / cell), or with rAAV9 at 1.5 x 104, 5 x 104or 1.5 x 105vg / cell. Each dose was tested in 2-5 wells with two un-dosed wells and / or an AAV with an inert payload serving as controls. On DIV5, half of the medium was replaced with fresh culture medium. On DIV11, cells were fixed with 4% paraformaldehyde (PFA), stained using immunohistochemistry (IHC), imaged, and analyzed as described below. Primary Cortical Culture Preparation

[0238] Dissection of embryos: 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.

[0239] Dissociation and Plating: Digestion media, dissociation media, and plating media with or without fetal bovine serum (see recipes in TABLES 12-15, below) were freshly prepared and all media were warmed to 37 °C before usage. 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 corticalAttorney Docket No. TVD-012WO 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 105cells per well; 500 µL / well (48 well plates, Corning, Cat 356509) or 4 x 104cells per well; 200 µL / well (96 well plates, Corning, Cat 354461). TABLE 12: Digestion MediaTABLE 13: Dissociation MediaTABLE 14: Plating Media with 5% FBSTABLE 15: Plating Media without FBSMaintenance

[0240] Media was replaced at DIV5. Half of the media was removed and replaced with the addition of 5% (vol / vol) of distilled water and AraC (Cytosine β-D-arabinofuranosideAttorney Docket No. TVD-012WO 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 2-3% (vol / vol) of distilled water and AraC (C6645, Sigma), as above. Immunohistochemistry

[0241] Immunohistochemistry was performed using standard techniques 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). Images and Analysis

[0242] Images were taken on an Evos7000 microscope with a 20x objective. Five images per well were acquired from all the wells of the 48 or 96 well plates. Image analysis was performed using Cellprofiler. DAPI / NeuN-positive nuclei were identified, and MECP2 intensity was measured in DAPI / NeuN-positive nuclei. MECP2 signal from a non- transduced well was taken for background normalization. MECP2 intensity was measured and the number of neurons and MECP2-positive neurons were counted. HEK293 Cell Culture and Transient Transfection

[0243] Low passage HEK293 cells were maintained in high glucose Dulbecco’s Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% L-glutamine. Cells were incubated in a 5% CO2humidified incubator at 37 °C. Transient transfections for EGFP experiments used Lipofectamine 3000 according to manufacturer’s instructions. Equal amounts of plasmids expressing (1) an EGFP reporter with an inactivating Arg(R)>TGA nonsense mutation (EGFP-PTC) and (2) a suppressor tRNA (50 ng each) were mixed in Lipofectamine reagent. For conditions without suppressor tRNA, a filler plasmid was used to maintain an equal amount of plasmid DNA. The resulting mixture was incubated for 15 minutes at room temperature and added dropwise to 60% confluent HEK293 cells cultured in 96-well tissue culture plates. Media was changed at 6 hours post-transfection and maintained in supplemented DMEM for 48 hours. EGFP fluorescence was measured using flow cytometry. Analysis was done using FlowJo software. A gate was used to identify single live cells and EGFP positive cells wereAttorney Docket No. TVD-012WO identified using a second gate calibrated on non-transfected cells. EGFP intensity was measured using the geometric mean of EGFP positive viable cells and nonsense mutation rescue was normalized to cells transfected with a wild-type EGFP plasmid.

[0244] Lipofectamine 3000 reagent was also used for NanoLuc luciferase rescue experiments. Equal amounts of plasmids expressing (1) a dual-luciferase reporter (comprising an N-terminal firefly luciferase and a C-terminal NanoLuc luciferase with an inactivating Gln>TAA nonsense mutation; luciferase domains separated by a P2A self- cleaving peptide) and (2) a suppressor tRNA (10 ng each) were mixed in Lipofectamine reagent. For conditions without suppressor tRNA, a filler plasmid was used to maintain an equal amount of plasmid DNA. The resulting mixture was incubated for 15 minutes at room temperature and added dropwise to 60% confluent HEK293 cells cultured in 96-well tissue culture plates. Media was changed 6 hours post-transfection and maintained in supplemented DMEM for 24 hours. The activities of firefly and NanoLuc luciferases were measured sequentially from samples using the Promega Nano-Glo Dual Reporter Assay. Multiple Sequence Alignment and Phylogram of Human Arg-tRNAs and Gln-tRNAs

[0245] Arg-tRNA and Gln-tRNA sequences for alignment were obtained from the GtRNAdb database of transfer RNA genes detected in genomic sequence (http: / / gtrnadb.ucsc.edu / index.html) using the Homo sapiens (GRCh38 / hg38) genome and the Mus musculus (GRCm39 / mm39) genome. Only tRNAs from the High Confidence tRNA Gene Set were included in the analysis. In cases where multiple tRNAs at unique genomic loci produce identical mature tRNAs, only a single copy of the sequence was included in the alignment. Multiple sequence alignment was performed using Clustal Omega from EMBL- EBI's search and sequence analysis tools services. Default parameters were used for the “ClustalW with character counts” output format. Results of the sequence alignments were visualized using a phylograms with branch lengths proportional to the amount of inferred change between all included human Arg-tRNA isoacceptors. Results TS0036 T-stem motif enhances Arg(R)-suppressor tRNA-mediated suppression of premature termination

[0246] FIGURE 3 is a phylogram showing sequence divergence for all endogenous human Arg(R)-tRNAs. The Arg(R)-tRNAs indicated by an arrow in FIGURE 3 wereAttorney Docket No. TVD-012WO converted into suppressor tRNAs (“tr0104,” “tr0106,” “tr0114,” “tr0115,” and “tr0119”) by altering the anticodon to recognize the TGA stop codon. These parental suppressor tRNAs were then further modified to comprise the engineered T-stem sequence GCGGG_NNNNNNN_CCCGT (SEQ ID NO: 902), referred to herein as TS0036 (wherein the first five nucleotides correspond to the T-stem at nucleotide positions 49-53, the N’s correspond to the T-loop at nucleotide positions 54-60, and the last five nucleotides correspond to the T-stem at nucleotide positions 61-65). The modified suppressor tRNAs are summarized in TABLE 16. No endogenous Arg-tRNAs in humans contain the TS0036 T- stem motif. In mammals, the TS0036 motif is almost exclusively associated with Ile-tRNAs and Thr-tRNAs, which comprise 516 of the 520 TS0036-containing tRNAs identified in 46 different species. Only a single mammalian Arg-tRNA, found in the platypus (Ornithorhynchus anatinus), contains the TS0036 T-stem. TABLE 16: Arg(R)-Suppressor tRNAs used in Example 1

[0247] The parental tRNA suppressors and the modified tRNA suppressors were compared for their ability to suppress premature termination in primary mouse cortical neuron cultures (FIGURES 4A-4B). Briefly, primary cortical neurons were obtained from 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 were transduced with rAAV1 or rAAV9 at varying doses (for rAAV1, 5E3, 1.5E4, or 5E4 vg / cell; for rAAV9, 5E4, 1.5E5, or 5E5 vg / cell). Each AAV expression construct comprised three copies of a suppressor5Corresponds to tr0115 with an intron.Attorney Docket No. TVD-012WO tRNA gene comprising either an endogenous T-stem (hatched bars), or an engineered T-stem modified to match the TS0036 T-stem sequence motif (solid black bars). Suppressor tRNAs were paired with upstream RNA polymerase III promoters from either U6 or H1, as indicated. On DIV11, cells were fixed and stained via IHC for NeuN (to identify neurons) and for full-length MECP2. Results were quantified as the number of NeuN-positive cells expressing MECP2 at detectable levels (FIGURE 4B). For the rAAV1 treatment at 5E4 vg / cell, nonsense suppression activity was also quantified based on MeCP2 intensity in NeuN-positive nuclei (FIGURE 4A). MECP2 signal from a non-transduced well was used for background normalization.

[0248] As summarized in FIGURES 4A-4B, Arg(R)>TGA suppressor tRNAs exhibited enhanced rescue of a nonsense mutation in MeCP2 when their endogenous T-stem was modified to match the T-stem sequence motif TS0036. TS0006 T-stem motif enhances Arg(R)-suppressor tRNA-mediated suppression of premature termination

[0249] The tr0115 Arg(R)>TGA suppressor tRNA was also modified to comprise the engineered T-stem motif GCGGG_NNNNNNN_CCCGC (SEQ ID NO: 903), referred to herein as TS0006 (wherein the first five nucleotides correspond to the T-stem at nucleotide positions 49-53, the N’s correspond to the T-loop at nucleotide positions 54-60, and the last five nucleotides correspond to the T-stem at nucleotide positions 61-65). This modified suppressor tRNA is summarized in TABLE 16. No endogenous Arg-tRNAs in humans contain the TS0006 T-stem motif. In mammals, the TS0006 motif is primarily associated with Ala-tRNAs, which comprise 246 of the 324 TS0006-containing tRNAs identified in 46 different species. The TS0006 motif is also found in some mammalian Ile, Lys, Gly, and Arg-tRNAs.

[0250] The parental suppressor tRNA (tr0115) and the modified suppressor tRNA (tr0400) were compared for their ability to suppress premature termination (FIGURE 5). Briefly, HEK293 cells were simultaneously transfected with plasmids encoding (1) an expression construct containing an EGFP-PTC reporter with an inactivating Arg(R)>TGA nonsense mutation and (2) the parental suppressor tRNA (tr0115; hatched bar) or the modified suppressor tRNA (tr0400; grey bar). Suppressor tRNA plasmid expression constructs comprised a single copy of the indicated suppressor tRNA paired with an upstream RNA polymerase III promoter from U6. Nonsense suppression activity wasAttorney Docket No. TVD-012WO quantified approximately 48 hours post-transfection as a percentage of fluorescence activity relative to cells transfected with a wild-type EGFP reporter.

[0251] As summarized in FIGURE 5, the Arg(R)>TGA suppressor tRNA exhibited enhanced rescue of a nonsense mutation when the endogenous T-stem was modified to match the T-stem sequence motif TS0006. TS0006 and TS0036 T-stem motifs negatively impact the activity of Gln(Q)>TAA suppressor tRNAs

[0252] FIGURE 6 is a phylogram showing sequence divergence for all endogenous human (“h”) and mouse (“m”) Gln(Q)-tRNAs. The Gln(Q)-tRNAs indicated by an arrow in FIGURE 6 were converted into suppressor tRNAs (“tr0157,” “tr0162,” “tr0521,” and “tr0522”) by altering the anticodon to recognize the TAA stop codon. These parental suppressor tRNAs were then further modified to comprise the engineered T-stem motif of TS0036 or TS0006. The modified Gln suppressor tRNAs are summarized in TABLE 17. TABLE 17: Gln(Q)-Suppressor tRNAs used in Example 1

[0253] The parental Gln>TAA tRNA suppressors and the modified Gln>TAA tRNA suppressors were compared for their ability to suppress premature termination (FIGURE 7). Briefly, HEK293 cells were simultaneously transfected with plasmids encoding: (1) a dual- luciferase reporter comprising an N-terminal firefly luciferase and a C-terminal NanoLuc luciferase with an inactivating Gln>TAA nonsense mutation, wherein the luciferase domains were separated by a P2A self-cleaving peptide; and (2) either a parental Gln>TAA suppressor tRNA (hatched bars), a modified suppressor tRNA with the TS0006 T-stem (grey bars; “+TS6”), or a modified suppressor tRNA with the TS0036 T-stem (black bars; “+TS36”). Suppressor tRNA plasmid expression constructs comprised a single copy of theAttorney Docket No. TVD-012WO indicated suppressor tRNA paired with an upstream RNA polymerase III promoter from U6. Nonsense suppression activity was measured approximately 24 hours post-transfection. Nonsense suppression activity was quantified by normalizing luminescence from the C- terminal NanoLuc luciferase to luminescence from the N-terminal firefly luciferase. Normalized NanoLuc levels were expressed as a percentage of NanoLuc rescue in the most active parental Gln>TAA suppressor.

[0254] As summarized in FIGURE 7, the Gln(Q)>TAA suppressor tRNAs exhibited a reduced ability to suppress premature termination when the endogenous T-stem was modified to match the T-stem sequence motif TS0006 or TS0036. EXAMPLE 2

[0255] This Example describes using exogenous introns to increase the yield of AAVs encoding potent suppressor tRNAs. AAV vectors encoding potent suppressor tRNAs have reduced AAV yields

[0256] Genes encoding potent suppressor tRNAs were incorporated into AAV vectors, and the effect of the suppressor tRNA genes on AAV yield was investigated. Briefly, single- stranded AAV (ssAAV) transfer plasmids and self-complementary AAV (“Self-Comp” or scAAV) transfer plasmids encoding (1) potent Arg(R)>TGA or Gln(Q)>TAA suppressor tRNAs, and / or (2) non-suppressor tRNA sequences were prepared. The non-suppressor sequences included genes encoding a wild-type tRNA, EGFP, an EGFP-PTC reporter with an inactivating nonsense mutation, or randomized “stuffer” DNA. The composition and elements (“payload”) of each AAV construct are summarized in TABLE 18.

[0257] AAV packaging, purification, and quantification of viral genome copies were carried out by PackGene Biotech, LLC. AAV yield was calculated based on the final yield (divided by number of batches, if multiple production runs were carried out) relative to the expected normal yield for the AAV production scale. Transfer plasmids that met or exceeded yield expectations in a single production run were defined as being 100% normal yield.Attorney Docket No. TVD-012WO TABLE 18: AAV Transfer Plasmids Encoding Potent Suppressor tRNAs and / or Control Sequences

[0258] As summarized in TABLE 18 and in FIGURE 8, vectors encoding potent suppressor tRNAs (vectors A-H; hatched bars in FIGURE 8) had a reduced AAV yield as compared to control vectors encoding EGFP or EGFP-PTC (vectors I, K-N), a vector encoding a wild-type tRNA (vector O), and a vector comprising stuffer DNA (vector J). Incorporating exogenous introns into suppressor tRNAs does not disrupt suppression activity.

[0259] Nucleic acids encoding Arg-suppressor tRNAs described in Example 1 were modified to comprise exogenous tRNA introns, and the intron-containing Arg-suppressor tRNA constructs were compared with the parental constructs for their ability to suppress premature termination. Briefly, the Arg(R)>TGA suppressor tRNAs tr0115 (derived fromAttorney Docket No. TVD-012WO human tRNA-Arg-TCT-1-1) and tr0106 (derived from human tRNA-Arg-CCT-2-1) were modified to comprise one of five introns between nucleotides corresponding to positions 37 and 38 of the tRNA (using the Sprinzl numbering scheme). Four of the introns were exogenous to all tested suppressor tRNAs (INT0003, INT0005, INT0006, and INT011), and one of the introns was derived from human tRNA-Arg-TCT-1-1 (INT0002; endogenous to tr0115 and exogenous to tr0106).

[0260] HEK293 cells were simultaneously transfected with plasmids encoding (1) an expression construct containing an EGFP-PTC reporter with an inactivating Arg(R)>TGA nonsense mutation, and (2) the indicated Arg(R)>TGA suppressor tRNA. All suppressor tRNAs were paired with an upstream RNA polymerase III promoter from U6. Approximately 48 hours post-transfection, readthrough activity was assessed for the suppressor variant without an intron sequence (FIGURES 9A-9B, hatched bars) and for the five otherwise identical constructs in which the indicated intron sequences were included in the suppressor tRNA gene (FIGURES 9A-9B, gray bars). Nonsense mutation rescue was normalized to cells transfected with a wild-type EGFP plasmid (FIGURES 9A-9B, black bars).

[0261] As summarized in FIGURE 9A, the inclusion of any of the four exogenous introns into tr0115 did not disrupt suppression activity. As summarized in FIGURE 9B, the inclusion of any of the five exogenous tRNA introns into tr0106 did not disrupt suppression activity.

[0262] Additionally, nucleic acids encoding Gln-suppressor tRNAs described in Example 1 were modified to comprise exogenous tRNA introns, and the intron-containing Gln- suppressor tRNA constructs were compared with the parental constructs for their ability to suppress premature termination. Briefly, the Gln(Q)>TAA and Gln(Q)>TAG suppressor tRNAs tr0157 and tr0191 (each derived from mouse tRNA-Gln-CTG-3-1, -2, -3) were modified to comprise one of five introns between nucleotides corresponding to positions 37 and 38 of the tRNA (using the Sprinzl numbering scheme). No Gln tRNAs found in humans contain introns, and all of the Gln suppressor tRNA intron variants utilized exogenous introns that are not natively associated with human or mouse tRNA-Gln-CTG-3-1, -2, -3.

[0263] HEK293 cells were simultaneously transfected with plasmids encoding (1) an expression construct containing an EGFP-PTC reporter with an inactivating Gln(Q)>TAA or Gln(Q)>TAG nonsense mutation, and (2) the indicated Gln(Q)>TAA suppressor tRNA (FIGURE 10A) or Gln(Q)>TAG suppressor tRNA (FIGURE 10B), as appropriate. AllAttorney Docket No. TVD-012WO suppressor tRNAs were paired with an upstream RNA polymerase III promoter from U6. Approximately 48 hours post-transfection, readthrough activity was assessed for the suppressor variant without an intron sequence (hatched bars) and for the five otherwise identical suppressor tRNA constructs in which the indicated intron sequences were included in the suppressor tRNA gene (gray bars). Nonsense mutation rescue was normalized to cells transfected with a wild-type EGFP plasmid (black bars).

[0264] As summarized in FIGURES 10A-10B, the inclusion of any of the five exogenous introns into the Gln(Q)>TAA suppressor tRNA (tr0157; FIGURE 10A) or the Gln(Q)>TAG suppressor tRNA (tr0191; FIGURE 10B) did not disrupt suppression activity. Introns increase the yield of AAV vectors encoding potent suppressor tRNAs

[0265] AAV transfer plasmids encoding Arg(R)>TGA or Gln(Q)>TAA suppressor tRNAs (Vectors A-H in TABLE 18) were modified to include a tRNA intron in order to investigate the effect of the introns on AAV yield. The composition of the modified, intron- containing transfer plasmids is summarized in TABLE 19. Vectors E and H (encoding Arg(R)-suppressor tRNAs tr0318 and tr0416) were tested using several different introns. Suppressor tRNAs were paired with either an upstream RNA polymerase III promoter from U6 (“U6”) or an upstream flanking sequence derived from an endogenous human Tyr-tRNA (“Tyr”). TABLE 19: Composition of Intron-Containing AAV Transfer PlasmidsAttorney Docket No. TVD-012WO

[0266] AAV packaging, purification, and quantification of viral genome copies were carried out as described above. AAV yield was calculated based on the final yield (divided by number of batches, if multiple production runs were carried out) relative to the expected normal yield for the AAV production scale. Yields for each vector design tested were then normalized to the yield obtained for the non-intron-containing version of the suppressor tRNA.

[0267] As summarized in TABLE 20 and in FIGURE 11, the presence of tRNA introns improved AAV yield of vectors comprising potent suppressor tRNA genes. The improvement in AAV yield was observed in all tested vectors, irrespective of which intron was tested and irrespective of which suppressor tRNA was encoded by the vector. TABLE 20: Relative AAV Yields of Intron-Containing AAV Transfer PlasmidsAttorney Docket No. TVD-012WO

[0268] To further test the effect of an exogenous intron, AAV transfer plasmids were generated which comprised one or three copies of a potent Arg(R)>TGA suppressor tRNA (tr0115), a potent Gln(Q)>TAA suppressor tRNA (tr0157), or a less potent Arg(R)>TGA tRNA (tr0104). Each suppressor tRNA was paired with either an upstream RNA polymerase III promoter from U6 or an upstream flanking sequence derived from an endogenous human Tyr-tRNA. Each transfer plasmid was modified to include the tRNA intron INT0002, and AAV yield was measured as described above.

[0269] As summarized in FIGURE 12, the presence of the tRNA intron INT0002 improved AAV yield of vectors comprising potent suppressor tRNA genes (tr0115 and tr0157). The inclusion of INT0002 did not substantially improve the already high AAV yield of vectors comprising the less potent suppressor tRNA gene tr0104. The improvement in AAV yield was observed in all tested vectors comprising tr0115 and tr0157, irrespective of suppressor tRNA copy number and irrespective of whether the suppressor tRNA was paired with an upstream RNA polymerase III promoter from U6 or an upstream flanking sequence derived from an endogenous human Tyr-tRNA. EXAMPLE 3

[0270] This Example describes the use of engineered T-stems and exogenous introns in suppressor tRNAs to increase viability and survival of mice, due, at least in part, to readthrough of premature termination codons by the suppressor tRNAs. Materials and Methods

[0271] The Arg suppressor tRNAs are summarized in TABLE 21.Attorney Docket No. TVD-012WOAttorney Docket No. TVD-012WOAAV Production

[0272] Recombinant AAV (rAAV) particles comprising a viral vector encoding suppressor tRNAs were generated using standard methods known in the art. To construct transfer plasmids (pAAVs; also known as vector genome plasmids), elements of the transfer plasmids (including nucleic acids encoding tRNAs, U6 promoter sequences, and miscellaneous vector sequences) were synthesized or excised from commercially available plasmids. The transfer plasmid elements were assembled into pAAV plasmid backbones using restriction enzymes or non-restriction-enzyme-based DNA assembly methods, as appropriate.

[0273] Triple-plasmid transfection was conducted using polyethylenimine (PEI, Polyscience) and optionally including other transfection reagents to produce rAAV particles. The transfected plasmids included: (1) a single-stranded (ssAAV) or self-complementary (scAAV) transfer plasmid encoding a tRNA (e.g., a suppressor tRNA of the disclosure) located between two inverted terminal repeats (ITRs); (2) a pRep2Cap9 (‘RepCap’) plasmid, encoding AAV2 replication (Rep2) protein and a defined capsid (Cap) protein for the AAV9 serotype (Cap9); and (3) a ‘pHelper’ plasmid, encoding additional AAV proteins required for producing functional AAV particles.Attorney Docket No. TVD-012WO

[0274] AAVs were administered to mice by intracerebroventricular (ICV) injection following standard protocols. For ICV injection in mice, P1 pups were used. After injection, mice were monitored following approved protocols and allowed to develop normally until tissue collection.

[0275] The three plasmids were co-transfected into HEK293T cells. Briefly, 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 1:1:1 molar ratio of the pHelper, RepCap, and 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.

[0276] 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 at 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 1x PBS + 0.001% pluronic F-68.

[0277] 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).

[0278] For the hyperthermia-induced seizure (HIS) protocol described below and summarized in FIGURE 13, an scAAV encoding three copes of tr0374 under the control of a U6 promoter was used (5E10 VG / mouse, as determined by qPCR titration). An ssAAV encoding three copies of tr0115 with a 5′ tRNA-Tyr-GTA-5-1 regulatory sequence was used as a control (5E10 VG / mouse, as determined by qPCR titration).Attorney Docket No. TVD-012WO

[0279] For the studies assessing survival of Dravet Syndrome (DS) mice following administration of AAVs encoding a suppressor tRNA (described below and summarized in FIGURES 14A-14G), scAAVs encoding three copies of a suppressor tRNA (tr0104, tr0115 tr0119, tr0315, tr0374, tr0417, tr0418) under the control of a U6 promoter (all except tr0115) or a 5′ tRNA-Tyr-GTA-5-1 (+5) regulatory sequence (tr0115) were used (5E10 VG / mouse, as determined by qPCR titration).

[0280] For the studies assessing survival of DS mice following administration of an AAV encoding the suppressor tRNA tr0374 (described below and summarized in FIGURES 15A- 15B), either an ssAAV or an scAAV encoding one copy of tr0374 under the control of a U6 promoter was used (5E10 VG / mouse, as determined by qPCR titration).

[0281] For the studies assessing the level of SCN1A expression in DS mice following administration of an AAV encoding a suppressor tRNA (described below and summarized in FIGURE 16), an ssAAV encoding one copy of a suppressor tRNA (either tr0374, tr0315, or tr0590) under the control of a U6 promoter was used (1E11 VG / mouse, as determined by ddPCR titration). Animals

[0282] To produce DS mice, male Scn1aWT / R613Xmice with an inactivating arginine (R) 613 to a STOP (X) premature termination codon (PTC) on a 129S1 / SvImJ genetic background (see Mavashov et al. (2023) FRONTIERS IN CELLULAR NEUROSCIENCE 17: 1149391); The Jackson Laboratory, stock no.034129) 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. Genotyping

[0283] PCR was performed using the primers and protocol described by the Jackson Laboratory (129S1 / SvImJ-Scn1aem1Dsf / J, stock no.034129). Intracerebroventricular Injection of AAVs

[0284] For intracerebroventricular (ICV) injections in mice, P1 pups were cryoanesthetized on ice for 6 to 8 minutes following standard protocols, as known in the art. Cryoanesthetized pups were injected with the indicated dose(s) of AAV9 using 10 µlAttorney Docket No. TVD-012WO Hamilton syringes in the right hemisphere, halfway between the coronal and occipital sutures, close to the midline. After injection, pups were allowed to completely recover under a heat lamp and then returned to the home cage. Hyperthermia-induced Seizure (HIS) Protocol

[0285] Thermal induction of seizures was performed on P21 mice. Mice were placed in a temperature controlled module and allowed to acclimate for 5 minutes, followed by insertion of a rectal temperature probe and an additional 2 minute acclimation period. Body temperature was then increased by 0.5 °C every 2 minutes with a heat lamp until a generalized tonic-clonic seizure was provoked. Body temperature was not increased above 43 °C. If no tonic-clonic seizures were observed, mice were maintained at 43 °C for 3 minutes, at which point the experiment was terminated and the mice were euthanized. For analysis, time-to-first tonic-clonic seizure (seconds), as well as the body temperature at the onset of seizure (°C), was used. These measurements were graphed as a survival curve. dPCR for allele specific quantification of WT and R613X SCN1A mRNA Allele-specific digital PCR (dPCR) assays were performed on cortical tissue isolated from WT and Scn1aWT / R613Xmice on a mixed 50:50 C57BL / 6J:129S1 / SvImJ background. After euthanasia, cortical tissue was extracted, flash-frozen in liquid nitrogen, and stored at −70 °C until processing. Total RNA was extracted from tissues using TRI Reagent (Sigma-Aldrich), purified using a miRNeasy Mini Kit (QIAGEN), and reverse transcribed with the SuperScript IV First-Strand Synthesis System (Thermo Fisher, Waltham, MA). For allele-specific detection of Scn1a transcripts (WT vs. R613X mRNA), custom-designed Affinity Plus PCR Probes (IDT, Coralville, IA, USA) were used to enable greater SNP target specificity as previously described (see Mavashov et al. (2023) FRONTIERS IN CELLULAR NEUROSCIENCE 17: 1149391). dPCR for absolute quantification of gene targets was performed using naica System dPCR from Stilla Technologies (Villejuif, France). Results

[0286] WT or heterozygous Scn1aWT / R613Xmice on a mixed background (50:50) (“HET”) were injected with single-stranded (“ss”) or self-complementary (“sc”) AAVs encoding the tRNA suppressors tr0115 or tr0374, respectively, at P1 via intracerebroventricular (ICV) injection at a dose of 5E10 viral genomes (VG) per mouse (as determined by qPCR titration)Attorney Docket No. TVD-012WO and compared to un-injected (“Uninj”) controls for their ability to suppress seizures using the hyperthermia-induced Seizure (HIS) model. WT mice did not exhibit hyperthermia-induced seizures while untreated HET mice developed tonic-clonic seizures between 40 °C and 41 °C. HET mice dosed with AAV9 vectors expressing tr0115 and tr0374 (TABLE 7 and TABLE 21) showed a 1.7 °C and 2.1 °C increase, respectively, in median seizure temperature threshold relative to untreated HET mice (FIGURE 13).

[0287] The survival of un-injected HET control mice was also compared to HET mice injected with scAAVs encoding the tRNA suppressors tr0104, tr0115, tr0119, tr0315, tr0374, tr0417, or tr0418 (TABLE 3, TABLE 7, and TABLE 21), which demonstrated that untreated HET mice exhibited premature mortality relative to WT mice on the same background beginning at about P20. HET mice dosed with AAV9 vectors expressing tr0104, tr0115, tr0119, tr0315, tr0374, tr0417, and tr0418 showed a reduction in premature mortality that persisted through P100 (FIGURES 14A-14G). A similar set of experiments was conducted in HET mice injected with ss or scAAVs expressing the tRNA suppressor tr0374, respectively, both of which showed a reduction in premature mortality in dosed mice that persisted through P100 as compared to HET un-injected controls (FIGURES 15A-15B).

[0288] FIGURE 16 depicts levels of mRNA encoding the mutant (R613X) allele of SCN1A relative to levels of mRNA encoding the WT allele in HET mice dosed with AAV9 vectors expressing suppressor tRNAs tr0315, tr0374, and tr0590 (TABLE 3, TABLE 7, and TABLE 21). Levels of both mutant and WT transcripts were quantified by dPCR using allele-specific PCR probes. As demonstrated by the data, transcripts from the mutant allele were subject to nonsense-mediated mRNA decay (NMD), a translation-coupled mechanism that eliminates mRNAs containing premature translation-termination codons (PTCs), resulting in reduced levels that were about 25% of the WT transcript in untreated HET mice. Functional suppressor tRNAs enabled translation of transcripts encoding the mutant allele, resulting in reduced NMD. Furthermore, it was observed that HET mice dosed with the AAV9 vectors expressing suppressor tRNAs tr0315, tr0374, and tr0590 showed increased levels of the mutant transcript, reflecting a reduction in NMD.

[0289] Taken together, the results of this Example demonstrate that mice with a heterozygous loss-of-function mutation in the SCN1A gene (encoding the voltage-gated sodium channel NaV 1.1) that converts arginine 613 to a TGA STOP codon (R613X), exhibit core aspects of the Dravet syndrome phenotype. This includes susceptibility to heat-induced seizures, premature mortality, and reduced SCN1A expression. In addition, the steady-stateAttorney Docket No. TVD-012WO level of the mutant R613X transcript was significantly less than the wild-type allele, demonstrating that the mutant mRNA underwent strong nonsense-mediated decay (NMD). The data demonstrated that Arg>TGA suppressor tRNAs delivered to HET mice via AAVs rescued both heat-induced seizures (FIGURE 13) and premature mortality (FIGURES 14A- 14G and FIGURES 15A-15B). In addition, the data demonstrated that rescue of the Dravet phenotype in HET mice was due to decreased NMD of the mutant R613X SCN1A transcript (FIGURE 16, left panel (mRNA)). The data further demonstrated that rescue of the Dravet phenotype in HET mice was achieved using suppressor tRNAs wherein the T-stem comprised an engineered nucleic acid sequence that increased the ability of the suppressor tRNA to suppress nonsense mutations (FIGURE 16, see e.g., tr0315, tr0374, and tr0590) and / or using suppressor tRNAs that were encoded by a nucleic acid comprising an exogenous intron (FIGURE 16, see, e.g., tr0590). EXAMPLE 4

[0290] This Example describes the use of engineered T-stems and exogenous introns in a suppressor tRNA to increase TTN mRNA and protein expression. The example demonstrates that the increase in expression is due, at least in part, to readthrough of a premature termination codon by the engineered suppressor tRNA. Materials and Methods AAV Production

[0291] Recombinant AAV (rAAV) particles comprising a viral vector encoding suppressor tRNAs were generated using standard methods known in the art. To construct transfer plasmids (pAAVs; also known as vector genome plasmids), elements of the transfer plasmids (including nucleic acids encoding tRNAs, U6 promoter sequences, and miscellaneous vector sequences) were synthesized or excised from commercially available plasmids. The transfer plasmid elements were assembled into pAAV plasmid backbones using restriction enzymes or non-restriction-enzyme-based DNA assembly methods, as appropriate. The transfer plasmid used in the Titin (TTN) mouse model of dilated cardiomyopathy (DCM) described in this Example comprises a single-stranded (ssAAV) transfer plasmid encoding three copies of a suppressor tRNA of the disclosure, functionally linked to U6 promoters located between two inverted terminal repeats (ITRs) (FIGURE 17).Attorney Docket No. TVD-012WO

[0292] Triple-plasmid transfection was conducted using polyethylenimine (PEI, Polyscience) and optionally including other transfection reagents to produce rAAV particles. The transfected plasmids included: (1) a single-stranded (ssAAV) transfer plasmid encoding a tRNA (e.g., a suppressor tRNA of the disclosure) located between two inverted terminal repeats (ITRs); (2) a pRep2-MyoAAV2A (“RepCap”) plasmid, encoding AAV2 replication (Rep2) protein plus a defined capsid (Cap) protein for the MyoAAV-2A serotype; and (3) a “pHelper” plasmid, encoding additional AAV proteins required for producing functional AAV particles.

[0293] The three plasmids were co-transfected into HEK293T cells. Briefly, 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 1:1:1 molar ratio of the pHelper, RepCap, and 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.

[0294] 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 -70°C until usage. The final formulation buffer consisted of 1x PBS + 0.001% pluronic F-68.

[0295] The viral genomic titers were determined by a droplet digital polymerase chain reaction (ddPCR; Bio-Rad, USA). AnimalsAttorney Docket No. TVD-012WO

[0296] TTN+ / R30,277Xheterozygous (het) mice in a C57BL / 6J background were used for the study. This mouse model of dilated cardiomyopathy (DCM) contains an inactivating arginine (R) to a STOP (X) nonsense mutation at amino acid position 30,277 of the TTN gene (R30,277X). In addition to the nonsense mutation, a 912 bp TEV-HaloTag (TEV-HT) sequence was inserted just upstream of the nonsense mutation (FIGURE 19). The HaloTag is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands and serves as a unique epitope label to identify TTN protein that has been translated specifically from the mRNA containing the nonsense mutation. The TEV site is recognition site of the TEV protease, a highly sequence-specific cysteine protease from Tobacco Etch Virus (TEV). The glycine-serine-glycine (GSG) spacer increases cleavage efficiency at the TEV site. The TEV-HT insert enables analysis of TTN protein translated from the mutant allele independently from protein translated from the wild-type allele.

[0297] Both male and female mice were used in the study. Mice were housed in a standard animal facility (Jackson laboratories) at a constant temperature, on a 12-hour light / dark cycle, with ad libitum access to food and water. Retro-Orbital Injection of AAVs

[0298] AAV virus stocks were provided by the manufacturer in PBS (phosphate buffered saline) containing 0.001% pluronic F-68 and stored at -70°C and thawed on wet ice prior to usage. Thawed stock vector aliquots were diluted to a working concentration in the same buffer and finally to the concentration necessary to inject the required dose. Uninjected WT and HET mice were used as negative controls. The vector was administered by retro-orbital injection at P40 at doses of 1E14 or 2E14 viral genomes (VG) / kg. Different cohorts of mice were sacrificed at 6- or 12-weeks after treatment. Mice were euthanized by inhalation of 5% isoflurane and decapitation prior to tissue collection. Left ventricle (LV), gluteus muscle, and liver punches were collected according to standard methods. Tissues were flash frozen in liquid nitrogen and stored at -70°C. TEV protease digestion, protein extraction and quantification

[0299] Cardiac tissue samples were permeabilized in Triton X-100 solution at room temperature. After washing at 4 °C the samples were incubated with TEV protease for 6 hours at room temperature. Tissue was then homogenized manually in Urea:Thiourea:SDS solution with a protease inhibitor cocktail in a mortar and pestle at 60 °C. Total proteinAttorney Docket No. TVD-012WO concentration was determined using the Pierce BCA assay and stored at -70 °C. Aliquots of the soluble phase were then analyzed by vertical agarose gel electrophoresis (VAGE). RNA processing and quantification

[0300] RNA was extracted using the Direct-zol RNA kit (Zymo) according to manufacturer’s protocol. RNA concentration and quality was measured by photo spectrometry. Samples were stored at -70 °C until use. SuperScript IV Reverse Transcriptase (ThermoFisher) was used according to manufacturer’s protocol to synthesize cDNA for quantification of TTN mRNA. Residual RNA was degraded with E. coli RNase H and the cDNA was stored at -20 °C until use. A protocol for Quant-M tRNA-Seq (Pinkard et al. (2020) NAT. COMMUN.11:4104) was adapted to synthesize cDNA for quantification of tRNAs. Briefly, extracted RNA was deacylated and ligated with an adaptor that binds to the CCA overhang in mature tRNAs. Using a reverse transcription (RT) primer binding sequence on the adaptor, tRNAs were then reverse transcribed into cDNA using SuperScript IV Reverse Transcriptase (ThermoFisher). Residual RNA was degraded by incubation with NaOH and the cDNA stored at -20 °C until use.

[0301] TTN mRNA was quantified by droplet digital PCR (ddPCR). PCR for TTN was performed using primer sets that specifically recognize transcripts from the wild-type (WT) or mutant (R30,277X) TTN alleles using naica System dPCR from Stilla Technologies. All values were normalized to Actin Alpha Cardiac Muscle 1 (ACTC1) using a commercially available primer and probe mix (IDT). For each sample, the number of mutant transcripts was normalized to the number of WT transcripts.

[0302] Suppressor tRNA levels were quantified by ddPCR. PCR utilized a reverse primer that binds to a common target sequence that was added to all cDNA products by the adaptor and RT primer used for cDNA synthesis. Forward primers are specific to the tRNA being quantified and bind to a region on the anticodon arm of the tRNA that distinguishes it from other similar tRNA sequences. Forward primers were validated prior to use to ensure they do not cross-amplify endogenous tRNAs. The naica System from Stilla Technologies was used for the dPCR reaction, imaging, and quantification. For each sample, the suppressor tRNA levels were normalized to those of an endogenous tRNA (Gly-TCC-2).

[0303] Statistical comparisons were performed utilizing One-Way ANOVA followed by Dunnett’s multiple comparison test (mean ±SD). Statistical significance was set at p-value ≤ 0.05 (∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001).Attorney Docket No. TVD-012WO Results

[0304] TTN+ / R30,277Xheterozygous mice were retro-orbitally injected with an AAV encoding three copies of the Arg>TGA tRNA suppressor tr0374, which comprises a modified T-stem and is encoded by a nucleic acid comprising an exogenous intron (see TABLE 7 and TABLE 21). TTN mRNA and protein levels were measured in cardiac tissue, using uninjected wild-type (“WT”) and heterozygous (“het”) mice as negative controls. AAV-delivered Arg>TGA suppressor tRNA increases TTN mRNA levels in mouse cardiac tissue

[0305] In TTN+ / R30,277Xhet mice, mRNA transcribed from the mutant TTN allele was detected at only 30.8% ± 1.4% of mRNA transcribed from the wild-type WT allele (FIGURE 18, “Het Untreated”). This reduction in the PTC-containing TTN transcript is consistent with the process of nonsense-mediated mRNA decay (NMD), which has been described for many genes that contain nonsense mutations. Treatment of the heterozygous animals with the suppressor tRNA-expressing AAV led to restoration of mutant transcript levels to near normal (90.9% ± 6.5% of wild-type level) (FIGURE 18, “Het + 3x-tr0374”). Suppression of the nonsense mutation to allow complete translation of the mutant mRNA is the only mechanism known to enable restoration of transcript levels. AAV-delivered Arg>TGA suppressor tRNA rescues full-length TTN protein in mouse cardiac tissue

[0306] To determine directly whether suppressor tRNA treatment leads to translation of full-length TTN protein from the mutant transcript, proteins from untreated and suppressor tRNA-treated left ventricle (LV) tissue were analyzed. The large size of the TTN protein – 363 exons encoding 38,138 amino acid residues (4200 kDa) – combined with the presence of multiple splice isoforms – ranging in size from ~27,000 to ~35,000 amino acids – complicates detection of the protein when using standard western blotting protocols. In order to clearly discriminate between full-length and truncated TTN protein transcribed from the mutant allele, cardiac tissue was first treated with TEV protease. Proteolytic cleavage of truncated TTN protein will result in a 34 kDa peptide comprising just the HaloTag, while cleavage of full-length rescued TTN will result in a 587 kDa C-terminal fragment (FIGURE 19).Attorney Docket No. TVD-012WO

[0307] Following TEV protease digestion, protein was separated by vertical agarose gel electrophoresis (VAGE) on 1% agarose SDS gels, transferred to PVDF membranes, and probed with a monoclonal antibody directed against the HaloTag epitope that was knocked in to the mutant allele upstream of the PTC (FIGURE 19). A small amount of residual undigested high molecular weight HaloTagged TTN was observed in TEV protease-treated samples due to incomplete digestion. A faint protein band corresponding to the 34 kDa HaloTag peptide – indicative of PTC-truncated TTN protein – was detected in left ventricle tissue from untreated het mice following TEV protease digestion (FIGURE 20, “untreated”). No larger 587 kDa band corresponding to full-length rescued TTN was observed in this cohort. In treated animals, there was an increase in signal from the 34 kDa HaloTag band (FIGURE 20, “MyoAAV-2A (3x_U6_tr0374)”). This presumably arises from suppressor- mediated rescue of NMD mutant at the level of the mutant mRNA, which occasionally still undergoes premature termination during translation. Importantly, the dominant TTN band detected in left ventricle tissue from treated mice was the 587 kDa C-terminal fragment. This fragment could only arise from successful readthrough of the PTC by the suppressor tRNA and therefore confirms rescue of full-length TTN protein from the mutant allele. Quantification of truncated (34 kDa) and full-length (587 kDa) band intensities indicated that 79.0% ± 5.2% of TTN protein transcribed from the mutant allele was full-length.

[0308] Taken together, the results of this Example demonstrate that mice with a heterozygous loss-of-function R30,277X mutation in the TTN gene results in a decrease in TTN transcript levels, demonstrating that the mutant mRNA underwent strong nonsense- mediated decay (NMD). The data demonstrate that AAV-mediated delivery of an Arg>TGA suppressor tRNA with a modified T-stem (TS0036) and encoded by a nucleic acid comprising an exogenous intron (INT0002) significantly increased TTN transcript levels (FIGURE 18) and full-length TTN protein expression (FIGURE 20). INCORPORATION BY REFERENCE

[0309] All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety for all purposes. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.Attorney Docket No. TVD-012WO EQUIVALENTS

[0310] Inventions of 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 any invention disclosed herein. Scope of any claimed inventions is determined 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-012WO CLAIMS What is claimed is:

1. A nucleic acid encoding a suppressor tRNA and an exogenous intron located between nucleotides corresponding to positions 37 and 38 of the tRNA, wherein the presence of the intron increases production yield of an AAV or lentiviral vector comprising the nucleic acid relative to an AAV or lentiviral vector comprising the same nucleic acid but that lacks the exogenous intron.

2. The nucleic acid of claim 1, wherein the exogenous intron is a synthetic intron.

3. The nucleic acid of claim 1, wherein the exogenous intron is derived from an intron- containing mammalian tRNA gene.

4. The nucleic acid of claim 3, wherein the exogenous intron is derived from an intron- containing human tRNA gene.

5. The nucleic acid of any one of claims 1-4, wherein the intron comprises a nucleic acid sequence selected from SEQ ID NOs: 932-939.

6. The nucleic acid of any one of claims 1-5, wherein the tRNA comprises a tri- nucleotide anticodon, wherein the anticodon is 5′-UCA-3′ and recognizes a UGA stop codon.

7. The nucleic acid of any one of claims 1-5, wherein the tRNA comprises a tri- nucleotide anticodon, wherein the anticodon is 5ʹ-UUA-3ʹ and recognizes UAA stop codons.

8. The nucleic acid of any one of claims 1-5, wherein the tRNA comprises a tri- nucleotide anticodon, wherein the anticodon is 5ʹ-CUA-3ʹ and recognizes UAG stop codons.

9. The nucleic acid of any one of claims 1-8, wherein the tRNA is operably linked to arginine, glutamine, or serine.

10. The nucleic acid of claim 9, wherein the tRNA is operably linked to arginine.

11. The nucleic acid of any one of claims 1-10, wherein the nucleic acid comprises a nucleic acid sequence selected from SEQ ID NOs: 940-955, 957-965, 967, and 969-1179.Attorney Docket No. TVD-012WO 12. The nucleic acid of any one of claims 1-11, wherein the nucleic acid comprises a nucleic acid sequence selected from SEQ ID NOs: 1049, 1052, 1060, 1079, and 1080.

13. The nucleic acid sequence of any one of claims 1-12, wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 1060.

14. The nucleic acid of any one of claims 1-13, wherein the suppressor tRNA comprises a T-arm having a T-stem and a T-loop, wherein the T-arm is encoded by a DNA comprising the nucleic acid sequence of SEQ ID NO: 902 or SEQ ID NO: 903, wherein Ns in SEQ ID NO: 902 and SEQ ID NO: 903 correspond to a DNA encoding the T-loop.

15. The nucleic acid of any one of claims 1-14, wherein the nucleic acid comprises a 5′ flanking sequence, a 3′ flanking sequence, or both 5′ and 3′ flanking sequences.

16. The nucleic acid of claim 15, wherein the 5′ flanking sequence or the 3′ flanking sequence comprises a regulatory element.

17. The nucleic acid of claim 15 or 16, wherein the 5′ flanking sequence comprises a leader sequence, a promoter element, or a secondary structure.

18. The nucleic acid of claim 17, wherein the secondary structure is a hairpin element.

19. The nucleic acid of any one of claims 15-18, wherein the 3′ flanking sequence comprises a terminator element or a poly-T element.

20. The nucleic acid of claim 19, wherein the poly-T element is less than 50, 40, 30, 20, or 10 nucleotides in length.

21. The nucleic acid of any one of claims 15-20, wherein the 5′ flanking region comprises a nucleotide sequence set forth in TABLE 8.

22. The nucleic acid of any one of claims 15-21, wherein the 3′ flanking region comprises a nucleotide sequence set forth in TABLE 8.

23. The nucleic acid of any one of claims 1-22, wherein the nucleic acid comprises an internal tRNA promoter.Attorney Docket No. TVD-012WO 24. A vector comprising the nucleic acid of any one of claims 1-23.

25. The vector of claim 24, wherein the vector is a viral vector.

26. The vector of claim 25, wherein the viral vector is a DNA virus vector.

27. The vector of claim 25 or 26, wherein the viral vector is an AAV vector or a lentiviral vector.

28. The vector of claim 27, wherein the viral vector is an AAV.

29. The vector of claim 28, wherein the AAV is a single-stranded AAV or a self- complementary AAV.

30. A suppressor tRNA operably linked to an arginine comprising a T-arm having a T- stem and a T-loop, wherein the T-stem comprises an engineered nucleic acid sequence that increases the ability of the suppressor tRNA to suppress termination at a stop codon relative to a similar suppressor tRNA without the engineered nucleic acid sequence, and wherein the suppressor tRNA does not comprise the nucleic acid sequence of SEQ ID NO: 915 or 916, wherein each thymine is replaced by a uracil.

31. The suppressor tRNA of claim 30, wherein the T-arm comprises the nucleic acid sequence of SEQ ID NO: 901 or SEQ ID NO: 903, wherein Ns in SEQ ID NO: 901 and SEQ ID NO: 903 correspond to the T-loop.

32. The suppressor tRNA of claim 30 or 31, wherein the tRNA comprises a naturally occurring nucleotide modification.

33. The suppressor tRNA of any one of claims 30-32, wherein the suppressor tRNA comprises one or more nucleotide modifications selected from 5-methyl uridine, 5- carbamoylmethyluridine, 5-carbamoylmethyl-2-O-methyluridine, 5-methoxy- carbonylmethyluridine, 5-methoxycarbonylmethyl-2-thiouridine, pseudouridine, dihydrouridine, 1-methyladenosine, and inosine.

34. The suppressor tRNA of any one of claims 30-33, wherein the tRNA comprises a tri- nucleotide anticodon, wherein the anticodon is 5′-UCA-3′ and recognizes a UGA stop codon.Attorney Docket No. TVD-012WO 35. The suppressor tRNA of any one of claims 30-34, wherein the suppressor tRNA is encoded by a DNA comprising a nucleic acid sequence selected from SEQ ID NOs: 912-914, 917, 919, 1020-1083, and 1186.

36. The suppressor tRNA of any one of claims 30-35, wherein the suppressor tRNA is encoded by a DNA comprising a nucleic acid sequence selected from SEQ ID NOs: 913, 917, 919, 1049, 1052, 1060, 1079, 1080, and 1186.

37. The suppressor tRNA of any one of claims 30-36, wherein the suppressor tRNA is encoded by a DNA comprising the nucleic acid sequence of SEQ ID NO:

917.

38. The suppressor tRNA of any one of claims 30-36, wherein the suppressor tRNA is encoded by a DNA comprising the nucleic acid sequence of SEQ ID NO: 1060.

39. The suppressor tRNA of any one of claims 30-38, wherein the suppressor tRNA comprises a nucleic acid sequence selected from SEQ ID NOs: 912-914, 917, 919, 1020- 1083, and 1186, wherein each thymine is replaced by a uracil.

40. The suppressor tRNA of any one of claims 30-39, wherein the suppressor tRNA comprises a nucleic acid sequence selected from SEQ ID NOs: 913, 917, 919, 1049, 1052, 1060, 1079, 1080, and 1186, wherein each thymine is replaced by a uracil.

41. The suppressor tRNA of any one of claims 30-39, wherein the suppressor tRNA comprises a nucleic acid sequence selected from SEQ ID NOs: 912-914, 917, 919, and 1186.

42. The suppressor tRNA of any one of claims 30-39, wherein the suppressor tRNA comprises the nucleic acid sequence of SEQ ID NO:

917.

43. The suppressor tRNA of any one of claims 30-39, wherein the suppressor tRNA comprises the nucleic acid sequence of SEQ ID NO: 1060.

44. A nucleic acid encoding one or more copy numbers of the suppressor tRNA of any one of claims 30-43.

45. The nucleic acid of claim 44, wherein the nucleic acid comprises a 5′ flanking sequence, a 3′ flanking sequence, or both 5′ and 3′ flanking sequences.Attorney Docket No. TVD-012WO 46. The nucleic acid of claim 45, wherein the 5′ flanking sequence or the 3′ flanking sequence comprises a regulatory element.

47. The nucleic acid of claim 45 or 46, wherein the 5′ flanking sequence comprises a leader sequence, a promoter element, or a secondary structure.

48. The nucleic acid of claim 47, wherein the secondary structure is a hairpin element.

49. The nucleic acid of any one of claims 45-48, wherein the 3′ flanking sequence comprises a terminator element or a poly-T element.

50. The nucleic acid of claim 49, wherein the poly-T element is less than 50, 40, 30, 20, or 10 nucleotides in length.

51. The nucleic acid of any one of claims 45-50, wherein the 5′ flanking region comprises a nucleotide sequence set forth in TABLE 8.

52. The nucleic acid of any one of claims 45-51, wherein the 3′ flanking region comprises a nucleotide sequence set forth in TABLE 8.

53. The nucleic acid of any one of claims 44-52, wherein the nucleic acid comprises an internal tRNA promoter.

54. The nucleic acid of any one of claims 44-53, wherein the nucleic acid comprises an intron located between nucleotides corresponding to positions 37 and 38 of the tRNA.

55. The nucleic acid of claim 54, wherein the intron is an exogenous intron.

56. The nucleic acid of claim 55, wherein the exogenous intron is a synthetic intron.

57. The nucleic acid of claim 55, wherein the wherein the exogenous intron is derived from an intron-containing mammalian tRNA gene.

58. The nucleic acid of claim 55, wherein the exogenous intron is derived from an intron- containing human tRNA gene.Attorney Docket No. TVD-012WO 59. The nucleic acid of any one of claims 54-58, wherein the intron comprises a nucleic acid sequence selected from SEQ ID NOs: 932-939.

60. A vector comprising the nucleic acid of any one of claims 44-59.

61. The vector of claim 60, wherein the vector is a viral vector.

62. The vector of claim 61, wherein the viral vector is a DNA virus vector.

63. The vector of claim 61 or 62, wherein the viral vector is an adeno-associated virus (AAV) vector or a lentiviral vector.

64. The vector of claim 63, wherein the viral vector is an AAV.

65. The vector of claim 64, wherein the AAV is a single-stranded AAV or a self- complementary AAV.

66. The vector of any one of claims 63-65, wherein the nucleic acid comprises an exogenous intron between nucleotides corresponding to positions 37 and 38 of the tRNA, and wherein the presence of the exogenous intron increases production yield of the AAV or lentiviral vector comprising the nucleic acid relative to an AAV or lentiviral vector comprising the same nucleic acid but that lacks the exogenous intron.

67. A pharmaceutical composition comprising the nucleic acid of any one of claims 1-23 and 44-59, the vector of any one of claims 24-29 and 60-66, or the tRNA of any one of claims 30-43, and a pharmaceutically acceptable excipient.

68. A method of producing a tRNA of interest in a mammalian cell, the method comprising contacting the cell with the nucleic acid of any one of claims 1-23 and 44-59, the vector of any one of claims 24-29 and 60-66, or the pharmaceutical composition of claim 67.

69. A method of increasing production in a mammalian cell of a full-length protein encoded by a gene containing a premature termination codon (PTC), the method comprising contacting the cell with the nucleic acid of any one of claims 1-23 and 44-59 or with the vector of any one of claims 24-29 and 60-66, and permitting the nucleic acid or vector to beAttorney Docket No. TVD-012WO internalized by the cell, whereupon production of the suppressor tRNA permits readthrough of the PTC and the production of the full length protein.

70. A method of increasing the production of a protein of interest in a mammalian cell, the method comprising contacting the cell with the nucleic acid of any one of claims 1-23 and 44-59, or with the vector of any one of claims 24-29 and 60-66, and permitting the nucleic acid or vector to be internalized by the cell, whereupon production of the tRNA permits increased translation of the protein relative to a cell not contacted with the nucleic acid or vector.

71. A method of treating a disorder associated with a protein encoded by a gene including a premature termination codon (PTC) in a subject in need thereof, the method comprising administering to the subject an effective amount of the nucleic acid of any one of claims 1-23 and 44-59, the vector of any one of claims 24-29 and 60-66, the tRNA of any one of claims 30-43, or the pharmaceutical composition of claim 67, thereby to treat the disorder.

72. A method of treating a haploinsufficiency disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the nucleic acid of any one of claims 1-23 and 44-59, the vector of any one of claims 24-29 and 60-66, the tRNA of any one of claims 30-43, or the pharmaceutical composition of claim 67, thereby to treat the disorder.

73. The method of claim 72, wherein the haploinsufficiency disorder is Dravet syndrome or dilated cardiomyopathy.

74. A method for generating a recombinant AAV (rAAV) vector, the method comprising: (a) providing a virus-producing cell with i. a first plasmid comprising a Rep gene and a Cap gene or functional fragments thereof; ii. a transfer plasmid comprising the nucleic acid of any one of claims 1-23 and 44-59 and flanking inverted terminal repeats (ITR); and iii. a helper plasmid comprising helper genes to mediate rAAV replication; and (b) following step (a), culturing the cell to produce the rAAV vector.Attorney Docket No. TVD-012WO 75. A method of generating a recombinant lentiviral vector, the method comprising: (a) providing a virus-producing cell with i. one or more plasmids collectively comprising gag, pol, rev, and tat genes or functional fragments thereof; and ii. a transfer plasmid comprising the nucleic acid of any one of claims 1-23 and 44-59 and flanking long terminal repeats (LTR); and (b) following step (a), culturing the cell to produce the recombinant lentiviral vector.

76. The method of claim 74 or 75, wherein the cell is a human embryonic kidney (HEK) cell.

77. The method of any one of claims 74-76, wherein the presence of the exogenous intron in the nucleic acid increases production yield of the rAAV or recombinant lentiviral vector by at least 50%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, or at least 400% relative to an rAAV or recombinant lentiviral vector comprising the same nucleic acid but that lacks the exogenous intron.

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