Engineered transfer RNAS and methods of use

Abstract

The disclosure relates generally to suppressor tRNAs engineered to enhance suppression of premature termination and their use in treatment of disorders associated with a premature terminal codon, such as muscular dystrophy and cardiology disorders.

Description

Attorney Docket No. TVD-013WO2 ENGINEERED TRANSFER RNAS AND METHODS OF USECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U. S. Provisional Application Numbers 63 / 744,496, filed January 13, 2025, 63 / 787,174, filed April 11, 2025, and 63 / 891,628, filed October 1, 2025, the contents of each of which are incorporated by reference herein in their entirety.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 January 13, 2026, is named TVD-013WO_SL.xml and is 585,085 bytes in size.FIELD

[0003] The disclosure relates generally to suppressor tRNAs engineered to enhance suppression of premature termination and their use in treatment of disorders associated with a premature terminal codon, such as muscular dystrophy and cardiology disorders.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 Duchenne muscular dystrophy (DMD), limb girdle syndrome, congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy, McArdle disease, Glycogen storage disease type V, a desminopathy, hypokalemic periodic paralysis, malignant hyperthermia, actin-accumulation myopathy, congenital myopathy, congenital myasthenic syndrome, dystroglycanopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy,Attorney Docket No. TVD-013WO2 Brugada syndrome, long QT syndrome, Friderich’s ataxia, and cholesterol emia.

[0006] 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. Truncated variants in the TTN gene (TTN), which encodes the giant protein titin, represents the most common cause of inherited DCM (Tharp et al. (2019) FRONT. PHYSIOL. 10: 1436).

[0007] Despite the efforts made to date, there is a need in the art for improved compositions and methods for treating disorders mediated by PTCs, such as DCM and DMD.SUMMARY

[0008] Although approaches have been developed for treating certain genetic disorders, such as premature termination codon (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 of novel engineered suppressor tRNAs with the ability to suppress termination at a preselected stop codon. The disclosure is also based, in part, upon the discovery that certain suppressor tRNAs can be used to treat a disease mediated by a PTC in a gene in a subject, such as muscular dystrophy (c.g, Duchenne muscular dystrophy) or a cardiology disorder (c.g, dilated cardiomyopathy).

[0009] Accordingly, in one aspect, the disclosure provides a nucleic acid encoding a suppressor tRNA, wherein the nucleic acid includes a nucleic acid sequence selected from any one of SEQ ID NOs: 254-274, 918, 925, 1149, and 1150. In certain embodiments, the nucleic acid sequence selected from any one of SEQ ID NOs: 254-274, 918, 925, 1149, and 1150 includes Ni, N2, and N3, wherein the combination of Ni, N2, and N3 in a given sequence is one of: (i) thymine (T), thymine (T), and adenosine (A), respectively; (ii) thymine (T), cytosine (C), and adenosine (A), respectively; or (iii) cytosine (C), thymine (T), and adenosine (A), respectively. In certain embodiments, Ni is a thymine (T) or cytosine (C) and N2 is thymine (T) or cytosine (C), provided that when Ni is a cytosine (C) then N2 is a thymine (T).

[0010] In another aspect, the disclosure provides a nucleic acid encoding a suppressor tRNA, wherein the suppressor tRNA includes a nucleic acid sequence selected from any oneAttorney Docket No. TVD-013WO2 of SEQ ID NOs: 175-237, 901-909, 915-916, 921-924, 1000-1119, 1122-1123, and 1125-1131, where each thymine in any one of SEQ ID NOs: 175-237, 901-909, 915-916, 921-924, 1000-1119, 1122-1123, and 1125-1131 is replaced by uracil.

[0011] In certain embodiments, the nucleic acid includes a 5' flanking sequence, a 3' flanking sequence, or both 5' and 3' flanking sequences. Depending upon the circumstances, the 5' flanking sequence and / or the 3' flanking sequence can include a regulatory element, for example, the 5' flanking sequence can include a leader sequence, a promoter element, or a secondary structure (e.g., a hairpin element) and / or the 3' flanking sequence can include a terminator element or a poly-T element. In certain embodiments, the 5' flanking region or 3' flanking region includes a nucleotide sequence set forth in TABLE 16.

[0012] In certain embodiments, the nucleic acid includes an internal tRNA promoter.

[0013] In another aspect, the disclosure provides a nucleic acid including: (a) a U6 promoter including a nucleic acid sequence selected from any one of SEQ ID NOs: 917, 1132-1134, and 1151-1152; and (b) a nucleic acid encoding a suppressor tRNA. In certain embodiments, the U6 promoter includes a U6-1 promoter comprising the nucleic acid sequence of SEQ ID NO: 917. In certain embodiments, the U6 promoter includes a U6-8 promoter comprising the nucleic acid sequence of SEQ ID NO: 1132, SEQ ID NO: 1133, SEQ ID NO: 1151, or SEQ ID NO: 1152. In certain embodiments, the U6 promoter includes a U6-8 promoter comprising the nucleic acid sequence of SEQ ID NO: 1132. In certain embodiments, the U6 promoter includes a U6-9 promoter comprising the nucleic acid sequence of SEQ ID NO: 1133.

[0014] In another aspect, the disclosure provides a nucleic acid including: (a) a 5' flanking sequence (e.g., leader sequence) including a nucleic acid sequence selected from any one of SEQ ID NOs: 1153-1156 and 1159; and (b) a nucleic acid encoding a suppressor tRNA.

[0015] In certain embodiments, the nucleic acid encoding the suppressor tRNA includes a nucleic acid sequence selected from any one of SEQ ID NOs: 254-274, 918, 925, 1149, and 1150, wherein Ni is a thymine (T) or cytosine (C) and N2 is thymine (T) or cytosine (C), provided that when Ni is a cytosine (C) then N2 is a thymine (T). In other embodiments, the suppressor tRNA includes a nucleic acid sequence selected from any one of SEQ ID NOs: 175-237, 901-909, 915-916, 921-924, 1000-1119, 1122-1123, and 1125-1131, wherein each thymine in any one of SEQ ID NOs: 175-237, 901-909, 915-916, 921-924, 1000-1119, 1122-1123, and 1125-1131 is replaced by uracil. In other embodiments, the suppressor tRNAAttorney Docket No. TVD-013WO2 includes a nucleic acid sequence selected from any one of SEQ ID NOs: 238-252, 910-914, 1120, 1121, and 1124 or the nucleic acid encoding the suppressor tRNA includes the nucleic acid sequence of any one of SEQ ID NOs: 238-253, 910-914, 1120, 1121, and 1124.Depending upon the circumstances, the nucleic acid encodes an intron located between nucleotides corresponding to positions 37 and 38 of the tRNA. In certain embodiments, the intron includes the nucleic acid sequence TTGGCTGTGTCCTTAGAC (SEQ ID NO: 1135).

[0016] In another aspect, the disclosure provides a nucleic acid including: (a) a 5' flanking sequence (e.g., leader sequence) including a nucleic acid sequence selected from any one of SEQ ID NO: 1157 and SEQ ID NO: 1158; and (b) a nucleic acid encoding a suppressor tRNA. In certain embodiments, the nucleic acid encoding the suppressor tRNA includes a nucleic acid sequence selected from any one of SEQ ID NOs: 254-274, 918, 925, 1149, and 1150, wherein Ni is a thymine (T) or cytosine (C) and N2 is thymine (T) or cytosine (C), provided that when Ni is a cytosine (C) then N2 is a thymine (T). In other embodiments, the suppressor tRNA includes a nucleic acid sequence selected from any one of SEQ ID NOs: 175-237, 901-909, 915-916, 921-924, 1000-1119, 1122-1123, and 1125-1131, wherein each thymine in any one of SEQ ID NOs: 175-237, 901-909, 915-916, 921-924, 1000-1119, 1122-1123, and 1125-1131 is replaced by uracil. In other embodiments, the suppressor tRNA includes a nucleic acid sequence selected from any one of SEQ ID NOs: 238-252, 910-914, 1120, 1121, and 1124 or the nucleic acid encoding the suppressor tRNA includes the nucleic acid sequence of any one of SEQ ID NOs: 238-253, 910-914, 1120, 1121, and 1124.Depending upon the circumstances, the nucleic acid encodes an intron located between nucleotides corresponding to positions 37 and 38 of the tRNA. In certain embodiments, the intron includes the nucleic acid sequence TTGGCTGTGTCCTTAGAC (SEQ ID NO: 1135).

[0017] In another aspect, the disclosure provides a vector comprising the nucleic acid of any one of the foregoing aspects or embodiments. Depending on the circumstances, the vector is a viral vector (e.g., a DNA virus vector). In certain embodiments, the viral vector is an adeno-associated virus (AAV) vector or a lentiviral vector, such as a single-stranded AAV or a self-complementary AAV. In certain embodiments, the AAV includes an AAV inverted terminal repeat (ITR) derived from an AAV serotype selected from the group consisting of: AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03,Attorney Docket No. TVD-013WO2 AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16, or a derivative thereof.

[0018] Depending upon the circumstances, the vector includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copy numbers of the nucleic acid encoding the suppressor tRNA.

[0019] In another aspect, the disclosure provides a tRNA encoded by the nucleic acid of any one of the foregoing aspects or embodiments. In certain embodiments, the tRNA is aminoacylated with glutamine, serine, tyrosine, or leucine.

[0020] Depending upon the circumstances, the tRNA includes a naturally-occurring nucleotide modification (e.g., 5-methyl uridine, 5-carbamoylmethyluridine, 5-carbamoylmethyl-2-O-m ethyluridine, 5-methoxy-carbonylmethyluridine, 5-methoxycarbonylmethyl-2-thiouridine, pseudouridine, dihydrouridine, 1 -methyladenosine, and inosine).

[0021] The tRNA can include a tri -nucleotide anticodon, wherein the anticodon is 5'-UCA-3' and recognizes an UGA stop codon, the anticodon is 5'-UUA-3' and recognizes an UAA stop codon, or the anticodon is a 5'-CUA-3' and recognizes an UAG stop codon.

[0022] In another aspect, the disclosure provides a pharmaceutical composition comprising the nucleic acid or the vector of any one of the foregoing aspects or embodiments and a pharmaceutically acceptable excipient.

[0023] In another aspect, the disclosure provides a method of producing a tRNA of interest in a mammalian cell. The method includes contacting the cell with the nucleic acid, the vector, or the pharmaceutical composition any one of the foregoing aspects or embodiments.

[0024] In another aspect, the disclosure provides a method of treating a disorder in a subject in need thereof. The method includes administering to the subject an effective amount of the nucleic acid, the vector, or the pharmaceutical composition any one of the foregoing aspects or embodiments, thereby to treat the disorder. In certain embodiments, the disorder is a muscular dystrophy or a cardiology disorder.

[0025] In another aspect, the disclosure provides a method of treating a disorder in a subject in need thereof. The method includes administering to the subject an effectiveAttorney Docket No. TVD-013WO2 amount of a nucleic acid encoding a suppressor tRNA, a vector comprising the nucleic acid, or a pharmaceutical composition comprising the nucleic acid or the vector and a pharmaceutically acceptable excipient, wherein the suppressor tRNA includes a nucleic acid sequence selected from any one of SEQ ID NOs: 238-252, 910-914, 1120, 1121, and 1124, thereby to treat the disorder. In certain embodiments, the nucleic acid encoding the suppressor tRNA includes the nucleic acid sequence of any one of SEQ ID NOs: 238-253, 910-914, 1120, 1121, and 1124. In certain embodiments, the nucleic acid encodes an intron located between nucleotides corresponding to positions 37 and 38 of the tRNA. In certain embodiments, the intron includes the nucleic acid sequence TTGGCTGTGTCCTTAGAC (SEQ ID NO: 1135). In certain embodiments, the disorder is a muscular dystrophy (e.g., a disorder listed in TABLE 17) or a cardiology disorder (e.g., a disorder listed in TABLE 18).

[0026] Exemplary muscular dystrophies include Duchenne muscular dystrophy, limb girdle syndrome, congenital muscular dystrophy, Emery -Dreifuss muscular dystrophy, McArdle disease, Glycogen storage disease type V, a desminopathy, hypokalemic periodic paralysis, malignant hyperthermia, actin-accumulation myopathy, congenital myopathy, congenital myasthenic syndrome, dystroglycanopathy, Muscular dystrophy, limb-girdle, autosomal dominant 4, autosomal recessive limb-girdle muscular dystrophy, autosomal dominant limb-girdle muscular dystrophy type ID, muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A 14, autosomal recessive limb-girdle muscular dystrophy type 2P, autosomal recessive limb-girdle muscular dystrophy type 2N, autosomal recessive limb-girdle muscular dystrophy type 2B, muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type Al, muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 4, emery -Dreifuss muscular dystrophy 3, autosomal recessive, X-linked myopathy with postural muscle atrophy, emery-Dreifuss muscular dystrophy 4, autosomal dominant, X-linked Emery-Dreifuss muscular dystrophy, Emery-Dreifuss muscular dystrophy 5, autosomal dominant, glycogen storage disease, type V, McArdle disease, desmin-related myofibrillar myopathy, congenital myopathy 18, congenital myopathy with fiber type disproportion, actin accumulation myopathy, congenital myopathy 11, myopathy, congenital, with tremor, congenital myopathy with fiber type disproportion, Bailey-Bloch congenital myopathy, myopathy, proximal, and ophthalmoplegia, congenital myasthenic syndrome 5, congenital myasthenic syndrome 10, congenital myasthenic syndrome 4 A, congenital myasthenic syndrome 11, congenital myasthenic syndrome 9, congenital myasthenic syndrome 2 A,Attorney Docket No. TVD-013WO2 congenital myasthenic syndrome 13, muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 7, autosomal recessive limb-girdle muscular dystrophy type 2D, autosomal recessive limb-girdle muscular dystrophy type 2E, autosomal recessive limb-girdle muscular dystrophy type 2F, myofibrillar myopathy 3, autosomal dominant limb-girdle muscular dystrophy type IF, autosomal recessive limb-girdle muscular dystrophy type 2 J, autosomal dominant limb-girdle muscular dystrophy type 1G, autosomal recessive limb-girdle muscular dystrophy type 2Q, autosomal recessive limb-girdle muscular dystrophy type R18, autosomal recessive limb-girdle muscular dystrophy type 2G, autosomal recessive limb-girdle muscular dystrophy type 2C, autosomal recessive limb-girdle muscular dystrophy type 2L, limb-girdle muscular dystrophy due to POMK deficiency, limb-girdle muscular dystrophy type 2A, and merosin deficient congenital muscular dystrophy. Under certain circumstances, the muscular dystrophy is Duchenne muscular dystrophy.

[0027] In certain embodiments, the muscular dystrophy is associated with a PTC in a DMD, CAPN3, FKRP, DNAJB6, GMPPB, DYSF, DAG1, POMT2, POMT1, FKTN, LMNA, FHL1, SYNE1, EMD, SYNE2, PYGM, DES, CACNA1S, RYR1, ACTA1, MYBPC1, TPM3, STAC3, MYH2, COLQ, D0K7, CHRNE, RAPSN, MUSK, CHRNB1, DPAGT1, CRPPA, SGCA, SGCB, SGCD, SGCG, MYOT, TNPO3, HNRNPDL, PLEC, TRAPPCI 1, TCAP, AN05, POMK, LAMA2, CAPN3, FKRP, DNAJB6, GMPPB, DAG1, POMT2, DYSF, POMT1, FKTN, LMNA, FHL1, SYNE1, EMD, SYNE2, PYGM, DES, CACNA1S, RYR1, ACTA1, HDAC1, MYBPC1, TPM3, STAC3, MYH2, COLQ, DOK7, CHRNE, RAPSN, MUSK, CHRNB1, DPAGT1, CRPPA, SGCA, SGCB, SGCD, MYOT, TNPO3, TTN, HNRNPDL, PLEC, TRAPPCI 1, TCAP, SGCG, AN05, POMK, and / or LAMA2 gene.

[0028] In certain embodiments, the muscular dystrophy is Duchenne muscular dystrophy and the muscular dystrophy is associated with a PTC in a DMD gene. For example, in certain embodiments, a DMD gene in the subject includes a PTC, wherein production of the tRNA in the subject increases expression of the DMD gene, and the increased expression ameliorates at least one symptom of DMD. In certain embodiments, the production of the tRNA in the subject increases expression of full-length dystrophin encoded by the DMD gene, and the increased expression ameliorates at least one symptom of DMD.

[0029] Depending upon the circumstances, production of the tRNA in the subject increases expression of the DMD gene to at least 5% of a DMD gene expression level in aAttorney Docket No. TVD-013WO2 subject not having DMD or the subject being treated prior to the start of treatment.Additionally or alternatively, in certain embodiments, production of the tRNA in the subject increases expression of the full-length dystrophin in at least one muscle (e.g., gastrocnemius, quadriceps, diaphragm, triceps, heart, and tibialis anterior) of the subject to at least 3% of the expression level of full-length dystrophin in a subject without DMD or the subject being treated prior to the start of treatment.

[0030] Depending upon the circumstances, production of the tRNA in the subject improves proteomic dysregulation arising from the loss of full-length dystrophin (e.g., as measured by LC-MS / MS). In some embodiments, production of the tRNA in the subject decreases expression of one or more proteins selected from the group consisting of: Utrn, Anxal, Anxa2, Anxa5, Fnl, Tubb2A, Tubb5, Tubb6, TubalC, Vim, Serpinhl, Clicl, Coll4Al, Dtna, Sgcb, Sgcd, Sgcg, Dagl, Sspn, Sgca, Calr, and Cacnbl relative to the expression of the one or more proteins before production of the tRNA. In some embodiments, production of the tRNA in the subject increases expression of one or more proteins selected from the group consisting of: Plec, Synm, SntAl, SntB2, SlOOall, and S100al3 relative to the expression of the one or more proteins before production of the tRNA.

[0031] Depending upon the circumstances, production of the tRNA in the subject improves histopathology of dystrophin (e.g., as measured by immunohistochemistry).

[0032] Exemplary cardiology disorders include titinopathies, dilated cardiomyopathy, hypertrophic cardiomyopathy, Brugada syndrome, long QT syndrome, Friderich’s ataxia, and cholesterolemia. In certain embodiments, the cardiology disorder is associated with a PTC in a TTN, LMNA, FLNC, MYBPC3, BAG3, RBM20, LAMP2, SCN5A, KCNH2, KCNQ1, FXN, and / or LDLR gene.

[0033] In certain embodiments, the cardiology disorder is a titinopathy or a dilated cardiomyopathy, and the cardiology disorder is associated with a PTC in a TTN gene. In certain embodiments, the TTN gene in the subject includes a PTC, wherein production of the tRNA in the subject increases expression of the TTN gene, and the increased expression ameliorates at least one symptom of the cardiology disorder.

[0034] Depending upon the circumstances, production of the tRNA in the subject increases expression of the TTN gene by at least 5%, as compared to expression of the TTN in the subject prior to administration or in the subject being treated prior to the start ofAttorney Docket No. TVD-013WO2 treatment.

[0035] These and other aspects and features of the disclosure are described in the following detailed description and claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The disclosure can be more completely understood with reference to the following drawings.

[0037] FIG. 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.)

[0038] FIG. 2 is a schematic illustration of an adeno-associated virus (AAV) vector encoding three copies of a suppressor tRNA. Each tRNA copy is functionally linked to a U6 promoter, which are all disposed between two inverted terminal repeats (ITRs).

[0039] FIG. 3A is a schematic illustration of three batches of suppressor tRNAs targeting TAA stop codons that were assessed in dystrophin-deficient H2K-mdx cells for suppression of an inactivating glutamine (Q) to a STOP (X) nonsense mutation in the endogenous dystrophin gene. Suppressor tRNAs with solely the anticodon modified (hereinafter “parental suppressor tRNA”) as well as suppressor tRNAs with one or more mutations relative to a parental suppressor tRNA were included in Batch 1 -Batch 3. Batch 1 included 13 Gln(Q)> TAA, Ser(S)> TAA, and Tyr(Y)> TAA suppressor tRNAs (tr0157, tr0523, tr0524, tr0525, tr0903, tr0904, tr0905, tr0906, trO538, trl233, trl344, tr0211, and tr0212). Batch 2 included 11 Gln(Q)> TAA suppressor tRNAs (trl499, trl503, trl505, trl502, tr0524, trl500, trl504, trl506, tr0163, tri 501, and tr0904). Batch 3 included eleven Gln(Q)> TAA suppressor tRNAs (tri 511, tri 513, tri 515, tri 517, tri 519, tri 521, tri 523, tri 525, tri 531, tri 621, and tri 622).

[0040] FIG. 3B is a sequence alignment (Smith-Waterman algorithm) of unique canonical human Gln-tRNAs (“CTG-1,” “CTG-2,” “CTG-3,” “TTG-1,” “TTG-2,” “TTG-3,” etc.) as well as tr0524 (CTG-6) and tr0904 (the top performing suppressor tRNAs from BatchAttorney Docket No. TVD-013WO2 1 in a mouse model of Duchenne Muscular Dystrophy) (SEQ ID NOS 1162-1174, respectively, in order of appearance). The anticodon nucleotides are shown as “NNN”. The alignment shows nucleotide conservation within the body of Gln-tRNAs. The suppressor tRNA tr0524 derives from the endogenous Gln-CTG-6 tRNA without any additional sequence changes outside of the anticodon. The suppressor tRNA tr0904 contains additional second-site changes at Sprinzl positions 31 and 39 in the anticodon stem and therefore does not correspond to any endogenous Gln-tRNA. Nucleotide motifs present in tr0524 and / or tr0904 that deviate from Gln-tRNA consensus features are indicated and include C3 in the acceptor stem, C20a in the D-loop, the T31, A39 nucleotide pair in the anticodon stem, and C73 in the acceptor stem. The lowercase ‘a’ in C20a in FIG. 3B comes from the Sprinzl tRNA numbering system as depicted in FIG. 1. In particular, because some tRNAs have more or less bases than others, in order to develop a universal numbering scheme, bases that are not present in all tRNAs include a lowercase letter (e.g., 20, 20a, 20b, 21, etc).Abbreviations: Adenine (A), Thymine (T), Guanine (G), and Cytosine (C).

[0041] FIG. 3C is a diagram of exemplary Gln(Q)> TAA suppressor tRNAs designed to explore the functional importance of the unique nucleotides identified in tr0524 and / or tr0904 from Batch 1. By way of example, the engineered suppressor tRNA tri 500 was engineered by substitution the cytosine (C) at nucleotide position 3 relative to the nucleic acid sequence of tr0904 with thymine (T). Abbreviations: Adenine (A), Thymine (T), Guanine (G), and Cytosine (C).

[0042] FIG. 4 is a schematic illustration of a study design to assess rescue of full-length dystrophin protein in dystrophin-deficient H2K-mdxamp; cells, which contain an inactivating glutamine (Q) to a STOP (X) nonsense mutation in the endogenous dystrophin gene. H2K-mdx cells were plated on Day 0 and allowed to differentiate into myotubes until Day 3, at which time they were transduced with suppressor tRNAs from Batch 1, Batch 2, and Batch 3 at a multiplicity of infection (MOI) of 3E5 vector genomes (VG). On Day 10, cells were collected and lysed for protein analysis.

[0043] FIG. 5A-5C are a set of bar graphs depicting the ability of Gln(Q)> TAA, Ser(S)> TAA, and Tyr(Y)> TAA suppressor tRNAs to suppress premature termination of a gene (dystrophin; DMD) comprising a nonsense mutation in H2K-mdxamp; cells as described in FIG. 4, with FIG. 5A depicting the results of suppressor tRNAs from Batch 1, FIG. 5B depicting the results of suppressor tRNAs from Batch 2, and FIG. 5C depicting the results ofAttorney Docket No. TVD-013WO2 suppressor tRNAs from Batch 3. Dystrophin levels were measured by normalizing dystrophin to total protein signal intensity. Data plots represent dystrophin expression of treated H2K-mdxamp; cells normalized to H2K wild-type levels average. Plots show individual data and mean ± SD, * P < 0.05, ** P< 0.01, *** P < 0.001, **** P < 0.0001.

[0044] FIG. 6 is a schematic illustration of a study design to assess rescue of full-length dystrophin protein by suppressor tRNAs described herein via intramuscular (IM) injection in a mouse model of Duchenne Muscular Dystrophy. Twelve-week-old D2-mt& mice were dosed via IM administration of the tibialis anterior (TA) on both sides at 1E11 VG / muscle with a MyoAAV-2A vector expressing suppressor tRNAs described herein. Animals were euthanized at 18 weeks of age (6 weeks post-treatment). Both the TA muscles were collected and analyzed.

[0045] FIG. 7 is a bar graph depicting the ability of Gln(Q)> TAA, Ser(S)> TAA, and Tyr(Y)> TAA suppressor tRNAs to suppress premature termination of a dystrophin gene comprising a nonsense mutation in a mouse model of Duchenne Muscular Dystrophy as described in FIG. 6. Dystrophin levels were measured by normalizing dystrophin to total protein signal intensity. Data plots represent dystrophin expression of treated mutant groups (D2-mt&) normalized to D2-wild-type (D2-WT) average levels. Plots show individual data and mean ± SD, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0046] FIGs. 8A-8B shows photomicrographs (FIG. 8A) and quantification thereof (FIG.8B) of dystrophin-positive muscle fibers from TA muscles of untreated D2-WT, untreated D2-mt&, and D2-mt& mice treated with tr0157, tr0524, tr0904, and tr1344 suppressor tRNAs as described in FIG. 6. Muscle fibers were stained for dystrophin (FIG. 8A, bottom row) and for laminin (FIG. 8A, top row; used as a marker of muscle fibers). Data plots represent dystrophin-positive muscle fibers as a fraction of total muscle fibers (FIG. 8B). Each group shows percentage of individual mice from at least 300 fibers per muscle cross-section and mean ± SD, *** P < 0.001, **** P < 0.0001.

[0047] FIG. 9 is a schematic illustration of a study design to assess the rescue of full-length dystrophin protein in a mouse model of Duchenne Muscular Dystrophy via intravascular (IV; tail vein) injection of a MyoAAV-2A vector expressing the Gln(Q)> TAA suppressor tRNA tr0904. Twelve-week-old D2-mt& were dosed via IV administration at 1E14 and 2E14 VG / mouse. Animals were euthanized at 18 weeks of age (6 weeks posttreatment), tissues were extracted and analyzed.Attorney Docket No. TVD-013WO2

[0048] FIGs. 10A-10B show a bar graph (FIG. 10A) and set of Jess Western blots (FIG.10B) depicting the ability of the suppressor tRNA Gln(Q)> TAA tr0904 to suppress premature termination of a dystrophin gene having a nonsense mutation in a mouse model of Duchenne Muscular Dystrophy as described in FIG. 9. Full-length dystrophin protein was detected in all four muscle groups from untreated wild-type mice (D2-WT) and in D2-mt& hemizygous male mice (D2-mt&) dosed with tr0904 (1E14 VG / kg or 2E14 VG / kg), but not in untreated D2-mt& hemizygous male mice. Levels of full-length dystrophin were significantly increased relative to D2-mt& controls in all muscle groups and at both doses, and dystrophin levels increased in a dose-dependent manner in all muscles. Plots show individual data and mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, **** p < 0.0001.

[0049] FIG. 11 is a schematic representation of a study design to assess rescue of full-length dystrophin protein by suppressor tRNAs described herein via IM injection in a mouse model of Duchenne Muscular Dystrophy. Twelve-week-old D2-mt& mice were dosed via IM administration of the TA on both sides at 1E11 VG / muscle with a MyoAAV-2A vector expressing suppressor tRNAs described herein. Animals were euthanized at 18 weeks of age (6 weeks post-treatment).

[0050] FIG. 12 is a box and whiskers plot depicting the ability of Gln(Q)> TAA suppressor tRNAs to suppress premature termination of a dystrophin gene comprising a nonsense mutation in a mouse model of Duchenne Muscular Dystrophy as described in FIG.11. Dystrophin levels were measured by normalizing dystrophin to total protein signal intensity. Data plots represent dystrophin expression of treated mutant groups (D2-mt&) normalized to D2-wild-type (D2-WT) average levels. Boxes extend from the 25th to 75th percentiles; and whiskers going down to the smallest value and up to the largest, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0051] FIG. 13 is a schematic illustration of a study design to assess the rescue of full-length dystrophin protein in a mouse model of Duchenne Muscular Dystrophy via intravenous (IV; tail vein) injection of a MyoAAV-2A vector expressing the Gln(Q)> TAA suppressor tRNA tr0904. Twelve-week-old D2-mt& were dosed via IV administration at 1E14 and 2E14 VG / mouse. At 23 weeks of age (11 weeks post-treatment), motor function was assessed in grip strength and rotarod assays. Animals were euthanized at 24 weeks of age (12 weeks post-treatment) and tissues were extracted and analyzed.Attorney Docket No. TVD-013WO2

[0052] FIG. 14 is a box and whiskers plot depicting the ability of the suppressor tRNA Gln(Q)> TAA tr0904 to restore normal motor function in a mouse model of Duchenne Muscular Dystrophy as described in FIG. 13. All-limb grip strength was measured using a grip-strength apparatus. The mean measurement from six consecutive trials was taken as an index of all-limb grip strength. For each animal, grip strength was normalized to body weight.

[0053] FIG. 15 is a box and whiskers plot depicting the ability of the suppressor tRNA Gln(Q)> TAA tr0904 to restore normal motor function in a mouse model of Duchenne Muscular Dystrophy as described in FIG. 13. Motor function was assessed on a rotarod apparatus. The time to fall from the rod was measured. Each mouse was subjected to five trials and the average latency to fall from the five trials was recorded. Data plots represent performance of treated mutant groups (D2-mt&) normalized to D2-wild-type (D2-WT) average levels. Boxes extend from the 25th to 75th percentiles; and whiskers go down to the smallest value and up to the largest, ** P< 0.01, *** P < 0.001, **** p < 0.0001.

[0054] FIG. 16 is a box and whiskers plot depicting the ability of the suppressor tRNA Gln(Q)> TAA tr0904 to suppress premature termination of a dystrophin gene having a nonsense mutation in a mouse model of Duchenne Muscular Dystrophy as described in FIG.13. Full-length dystrophin protein was detected in hearts from untreated wild-type mice (D2-WT) and in l-mdx hemizygous mice (D2-mt&) dosed with tr0904 (1E14 VG / kg, 2E14, or 2E14 VG / kg), but not in untreated D2-mt& hemizygous mice. Levels of full-length dystrophin were significantly increased relative to D2-mt& controls in a dose-dependent manner in the hearts. Plots show individual data and mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0055] FIG. 17 is a box and whiskers plot depicting the ability of the suppressor tRNA Gln(Q)> TAA tr0904 to suppress premature termination of a dystrophin gene having a nonsense mutation in a mouse model of Duchenne Muscular Dystrophy as described in FIG.13. Full-length dystrophin protein was detected in gastrocnemius from untreated wild-type mice (D2-WT) and in D2-mt& hemizygous mice (D2-mt&) dosed with tr0904 (1E14 VG / kg, 2E14, or 2E14 VG / kg), but not in untreated D2-mt& hemizygous mice. Levels of full-length dystrophin were significantly increased relative to D2-mt& controls in a dose-dependent manner in the gastrocnemius. Plots show individual data and mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.Attorney Docket No. TVD-013WO2

[0056] FIG. 18 is a box and whiskers plot depicting the ability of the suppressor tRNA Gln(Q)> TAA tr0904 to suppress premature termination of a dystrophin gene having a nonsense mutation in a mouse model of Duchenne Muscular Dystrophy as described in FIG.13. Full-length dystrophin protein was detected in diaphrams from untreated wild-type mice (D2-WT) and in D2-mt& hemizygous mice (D2-mt&) dosed with tr0904 (1E14 VG / kg, 2E14 VG / kg, or 2E14 VG / kg), but not in untreated D2-mt& hemizygous mice. Levels of full-length dystrophin were significantly increased relative to D2-mt& controls in a dosedependent manner in the diaphrams. Plots show individual data and mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0057] FIGs. 19A-19B show a reporter gene assay construct (FIG. 19A) and resulting data (FIG. 19B) showing the interchangeability of an anticodon in a tRNA Gln(Q)> TAA suppressor. FIG. 19A is a schematic illustration of a dual luciferase reporter gene assay system designed to detect and quantitate both Firefly luciferase and Nanoluciferase (NanoLuc). The Firefly luciferase and NanoLuc were separated by a linker containing a stop codon such that the level of NanoLuc expression was dependent on the readthrough activity of a suppressor tRNA supplied in trans. FIG. 19B is a graph depicting the ability of otherwise identical Gln(Q)> TAA and Gln(Q)> TAG suppressor tRNAs (the only difference being their anticodon sequences) to suppress premature termination of a TAA or a TAG stop codon respectively in the dual luciferase reporter gene assay system. The correlation plot of stop codon readthrough activity was expressed as the ratio of Firefly luminescence to NanoLuc luminescence for 24 otherwise identical pairs of Gln(Q)> TAA and TAG suppressor tRNAs.

[0058] FIG. 20 is a box and whiskers plot depicting the ability of 24 screened Gln> TAA suppressor tRNAs to suppress premature termination in a dual luciferase assay. n=6 technical replicates per group. Abbreviations: hU6, human U6 promoter; T, RNA polymerase III termination signal; P2A, P2A self-cleaving peptide.

[0059] FIG. 21 is a schematic illustration of single-stranded adeno-associated virus (ssAAV) vectors encoding three copies (“SS 3x_U6_trl622”), two copies (“SS 2x_U6_trl622”), or one copy (“SS lx_U6_trl622”) of suppressor tRNA trl622 or a self-complementary adeno-associated virus (scAAV) vector encoding one copy of trl622 (“SC lx_U6_trl622”).

[0060] FIG. 22 is a schematic illustration of a study design to assess rescue of full-lengthAttorney Docket No. TVD-013WO2 dystrophin protein by suppressor tRNAs described herein via IM injection in a mouse model of Duchenne Muscular Dystrophy. Twelve-week-old D2-mt& mice were dosed via IM administration of the TA on both sides at 1E11 VG / muscle with a MyoAAV-2A vector expressing suppressor tRNAs described herein. Animals were euthanized at 18 weeks of age (6 weeks post-treatment). Both the TA muscles were collected and analyzed.

[0061] FIG. 23 is a box and whiskers plot depicting the ability of the indicated AAV vectors described in FIG. 21 expressing suppressor tRNA Gln(Q)> TAA tr1622 to suppress premature termination of a dystrophin gene having a nonsense mutation in a mouse model of Duchenne Muscular Dystrophy as described in FIG. 22. Box plots show the middle line as the median; upper and lower quartiles as box limits; and whiskers going down to the smallest value and up to the largest. *<0.05, ***<0.0005.

[0062] FIG. 24 is a schematic illustration of self-complementary adeno-associated virus (scAAV) vectors encoding one copy of suppressor tRNA tri 622 functionally linked to a human U6-1 (U6) promoter (“SC lx_U6_trl622”) or one copy of a suppressor tRNA functionally linked to a human U6-8 promoter (“SC lx_U6-8_trl622”) between two inverted terminal repeats (ITRs).

[0063] FIG. 25 is a box and whiskers plot depicting the ability of the indicated AAV vectors described in FIG. 24 expressing suppressor tRNA Gln(Q)> TAA tri 622 to suppress premature termination of a dystrophin gene having a nonsense mutation in a mouse model of Duchenne Muscular Dystrophy as described in FIG. 22. Box plots show middle line as the median; upper and lower quartiles as box limits; and whiskers going down to the smallest value and up to the largest. **<0.005.

[0064] FIG. 26 is a schematic illustration of single-stranded adeno-associated virus (ssAAV) vectors encoding three copies of a suppressor tRNA, with each copy of the suppressor functionally linked to a U6 promoter, which are all disposed between two inverted terminal repeats (ITRs).

[0065] FIG. 27 is a schematic illustration of a study design to assess rescue of full-length dystrophin protein by suppressor tRNAs described herein via IM injection in a mouse model of Duchenne Muscular Dystrophy. Twelve-week-old D2-mt& mice were dosed via IM administration of the TA on both sides at 1E11 VG / muscle with a MyoAAV-2A vector expressing suppressor tRNAs described herein. Animals were euthanized at 18 weeks of age (6 weeks post-treatment). Both the TA muscles were collected and analyzed.Attorney Docket No. TVD-013WO2

[0066] FIG. 28 is a box and whiskers plot depicting the ability of Gln(Q)> TAA suppressor tRNAs to suppress premature termination of a dystrophin gene having a nonsense mutation in a mouse model of Duchenne Muscular Dystrophy as described in FIG. 27. Box plots showing middle line as the median; upper and lower quartiles as box limits; and whiskers going down to the smallest value and up to the largest.

[0067] FIG. 29 is a box and whiskers plot depicting 50 screened Gln> UUA suppressor tRNAs to suppress premature termination (bottom panel) in a dual luciferase assay (top panel). Data were normalized to a control dual luciferase reporter lacking the Q586* nonsense mutation (WT reporter). n=6 technical replicates per group. Abbreviations: hU6, human U6 promoter; T, RNA polymerase III termination signal; P2A, P2A self-cleaving peptide.

[0068] FIG. 30 is a schematic diagram of the suppressor tRNA AAV expression cassette used for in vivo studies (top panel) and quantification of dystrophin expression in TA muscles following IM injection of 1 x 1011vg / muscle of AAV expressing the indicated suppressor tRNAs (bottom panel). Full-length dystrophin was measured by capillary Western blot 6 weeks after treatment. Dystrophin levels were quantified by normalizing dystrophin to total protein signal intensity. Plots show average dystrophin levels in each animal normalized to the average of wild-type mice. Box plots with individual data points showing middle line as the median; upper and lower quartiles as box limits; whiskers go down to the smallest value and up to the largest. ****<0.00005.

[0069] FIG. 31 is a box and whiskers plot depicting the quantification of dystrophin expression in tissues from wild-type, D2-mt&, and AAV-tr0904-treated mice at 1, 2, or 4xl014vg / kg. Full-length dystrophin was measured by capillary Western blot 12 weeks after IV injection. Dystrophin levels were quantified by normalizing dystrophin to total protein signal intensity. Plots show average dystrophin level in each cohort normalized to the average of wild-type mice. Box plots with individual data points showing middle line as the median; upper and lower quartiles as box limits; whiskers go down to the smallest value and up to the largest. *<0.05, **<0.005, ***<0.0005, ****<0.00005.

[0070] FIG. 32 is a set of box and whisker plots depicting the number of peptides (left) and peptide intensity normalized to untreated wild-type (right) detected after the premature stop codon (Q995*) by LC-MS / MS. Statistical analysis was performed using one-way ANOVA test ****<0.00005. Box plots with individual data points showing middle line asAttorney Docket No. TVD-013WO2 the median; upper and lower quartiles as box limits; whiskers go down to the smallest value and up to the largest. ****<0.00005.

[0071] FIG. 33 is a plot depicting all proteins detected by LC-MS / MS in quadriceps muscles of untreated D2-mdx mice. 3'-UTR extension products were only detected from four proteins (Myl 1, Aldoa, Ttn, and Mybpcl; upper dashed line) present at levels more than 2 x 103above the lower limit of detection established by the least abundant identified proteins (lower dashed line).

[0072] FIG. 34 is a set of box and whisker plots depicting raw peptide intensities of detected 3'-UTR extended peptides (3'-UTR peptide) relative to peptide intensities of top tryptic peptides from the coding region of each associated protein (canonical). In this figure, 1 denotes 1 x 1014vg / kg; 2 denotes 2 x 1014; and 4 denotes 4 x 1014vg / kg. Box plots with individual data points showing middle line as the median; upper and lower quartiles as box limits; whiskers go down to the smallest value and up to the largest.

[0073] FIG. 35 is a box and whisker plot depicting the quantification of immunofluorescence staining of dystrophin-positive muscle fibers in the heart from wildtype, D2-mdx, and tr0904-treated mice. Box plots with individual data points showing middle line as the median; upper and lower quartiles as box limits; whiskers go down to the smallest value and up to the largest. **<0.005, ****<0.00005.

[0074] FIG. 36 is a set of box and whisker plots depicting quantification of immunofluorescence staining of dystrophin-positive muscle fibers (left panel) and fibers with central nuclei in TA muscle (right panel) in TA muscle from wild-type, D2-mdx, and tr0904-treated mice (left). Box plots with individual data points showing middle line as the median; upper and lower quartiles as box limits; whiskers go down to the smallest value and up to the largest. **<0.005, ***<0.0005, ****<0.00005.

[0075] FIG. 37 is a set of plots depicting muscle fiber size distribution (pm2) in heart (left panel) and TA (right panel) muscle from wild-type, D2-mdx, and tr0904-treated mice. Plots show mean ± SEM normalized to total numbers of fibers (%). Statistical analysis was performed using one-way ANOVA test comparing each group to untreated mdx group.*<0.05, **<0.005, ***<0.0005, ****<0.00005.

[0076] FIG. 38 is a box and whiskers plot depicting the ability of the suppressor tRNAs Gln(Q)> TAA tri 622 and tri 626 to restore normal motor function in a mouse model of Duchenne Muscular Dystrophy. All-limb grip strength was measured using a grip-strengthAttorney Docket No. TVD-013WO2 apparatus. The mean measurement from six consecutive trials was taken as an index of alllimb grip strength. For each animal, grip strength was normalized to body weight.

[0077] FIG. 39 is a box and whiskers plot depicting the ability of the suppressor tRNAs Gln(Q)> TAA tri 622 and tri 626 to restore normal motor function in a mouse model of Duchenne Muscular Dystrophy. Rotarod performance was measured by latency to fall. The mean measurement from five consecutive trials was taken as an index of Rotarod performance.

[0078] FIG. 40 is a box and whiskers plot depicting the ability of the suppressor tRNAs Gln(Q)> TAA tri 622 and tri 626 to restore normal motor function in a mouse model of Duchenne Muscular Dystrophy. Wire hang performance was measured by latency to fall. The mean measurement from three consecutive trials was taken as an index of wire hang performance.

[0079] FIG. 41 is a box and whiskers plot depicting the ability of Gln(Q)> TAA tri 622 suppressor tRNA, with the suppressor functionally linked to a U6 promoter (U6.1, U6.8, U6.8 AACTT, U6.8 AGTGA, or U6.9), to suppress premature termination of a dystrophin gene having a nonsense mutation in a mouse model of Duchenne Muscular Dystrophy. Box plots showing middle line as the median; upper and lower quartiles as box limits; and whiskers going down to the smallest value and up to the largest.

[0080] FIG. 42 is a box and whiskers plot depicting the ability of Gln(Q)> TAA tri 622 suppressor tRNA, with each copy (one (lx), two (2x), or three (3x) of the suppressor functionally linked to a U6 promoter or L0458 5' flanking (e.g., leader) sequence, to suppress premature termination of a dystrophin gene having a nonsense mutation in a mouse model of Duchenne Muscular Dystrophy. Box plots showing middle line as the median; upper and lower quartiles as box limits; and whiskers going down to the smallest value and up to the largest.

[0081] FIG. 43 is a box and whiskers plot depicting the ability of 33 screened Gln> TAA suppressor tRNAs to suppress premature termination in a dual luciferase assay. n=6 or more technical replicates per group. Abbreviations include: hU6, human U6 promoter; T, RNA polymerase III termination signal; P2A, P2A self-cleaving peptide.Attorney Docket No. TVD-013WO2DETAILED DESCRIPTION

[0082] Although approaches have been developed for treating certain genetic disorders, such as premature termination codon (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 would otherwise result in premature termination and the formation of a truncated protein. The disclosure is based, in part, upon the discovery of novel, engineered suppressor tRNAs with the ability to suppress termination at a stop codon. The disclosure is also based, in part, upon the discovery that certain suppressor tRNAs can be used to treat a disease mediated by a PTC in a gene in a subject, such as muscular dystrophy (e.g., Duchenne muscular dystrophy) or a cardiology disorder (e.g., dilated cardiomyopathy).DEFINITIONS

[0083] 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.

[0084] 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 non-coding RNA (ncRNA) includes a single ncRNA, a plurality of ncRNAs, etc.

[0085] 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.

[0086] As used herein, the term “about” refers to a ± 10% variation from the nominal value unless otherwise indicated or inferred from the context, such as in the context of a given assay. Where the use of the term “about” precedes a quantitative value, the present disclosure also includes the specific quantitative value itself, unless specifically stated otherwise.

[0087] As used herein, unless otherwise indicated, the terms “non-coding gene” andAttorney Docket No. TVD-013WO2 “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 acid 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.

[0088] 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 a 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.”

[0089] 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.Attorney Docket No. TVD-013WO2

[0090] 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 in 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.

[0091] As used herein, the term “virus” refers to an obligate intracellular parasite having no protein-synthesizing or energy-generating mechanism.

[0092] 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 problems or complications, commensurate with a reasonable benefit / risk ratio.

[0093] 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 problems or complications, 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).

[0094] As used herein, the phrase “effective amount” refers to the amount of an active agent (e.g., a ncRNA disclosed herein) sufficient to effectuate 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.

[0095] 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 activeAttorney Docket No. TVD-013WO2 agent in a combination therapy) sufficient to effectuate 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.

[0096] 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.

[0097] 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.

[0098] 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, systemic administration, intramuscular administration, intramyocardial administration, transvascular administration, antegrade intracoronary administration, retrograde administration, 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, 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.

[0099] 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.Attorney Docket No. TVD-013WO2

[0100] 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.

[0101] 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.

[0102] 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.

[0103] The use of any and all examples, or exemplary language herein, including “for example” or “such as” are intended merely to illustrate better the present disclosure and do 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 theAttorney Docket No. TVD-013WO2 practice of any invention disclosed herein.

[0104] 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.

[0105] The use of the terms “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.

[0106] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the disclosed invention(s) remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0107] 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 95%-100%, includes 95%, 96%, 97%, 98%, 99%, and 100%.I. TRNAS AND SUPPRESSOR TRNAStRNAs and Suppressor tRNAs

[0108] 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 terminal 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. An anticodon loop in the folded aa-tRNA molecule contains a sequence of three bases known as the anticodon. When this anticodon sequence hybridizes or base-pairs with a complementary three-base codon sequence in a ribosome-bound messenger RNA (mRNA), the aa-tRNA binds to the ribosome and its amino acid is incorporated into the polypeptideAttorney Docket No. TVD-013WO2 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.

[0109] 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 the California Santa Cruz genomic tRNA database (GtRNAdb) as well 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 T C arm), wherein each arm comprises a double-stranded stem and a single-stranded loop (FIG.1; in the context of the structure of a tRNA, the terms “loop” refers to the unpaired bases, while “stem” refers to the paired bases. The “arm” refers to the combined “loop” and “stem” regions). 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 etal. (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 FIG. 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): el583.

[0110] Suppressor tRNAs are modified tRNAs that insert a suitable amino acid at a mutant site, e.g., a premature termination codon (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 tRNAsAttorney Docket No. TVD-013WO2 typically contain a mutation (modification) in either the anticodon, changing codon specificity, or at some position that alters the aminoacylation identity of the tRNA.

[0111] 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. Similarly, 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.

[0112] In some embodiments, a tRNA comprises an anticodon that hybridizes to a codon selected from UAG (i.e., an “amber” termination codon), UGA ( / .<., an “opal” termination codon), and UAA (i.e., an “ochre” termination codon). In some embodiments, the anticodon hybridizes to a codon selected from UAG, UGA, or UAA. In some embodiments, the anticodon hybridizes to UAG. In some embodiments, the anticodon hybridizes to UGA. In some embodiments, the anticodon hybridizes to UAA. 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 eta!. (1999)1 AM. CHEM. SOC. 121:12194).

[0113] 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, tyrosine, glutamine, or leucine. In some embodiments the tRNA is capable of being aminoacylated with serine. In some embodiments the tRNA is capable of being aminoacylated with tyrosine. In some embodiments the tRNA is capable of being aminoacylated with glutamine. In some embodiments the tRNA is capable of being aminoacylated with leucine.

[0114] 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 a gene at a position that would otherwise result in a truncated gene product caused by a PTC inAttorney Docket No. TVD-013WO2 the target gene, and can be used to treat a disease mediated by a PTC in a gene in a subject.

[0115] In some embodiments, multiple (e.g., two, three, four, five, six, seven, eight, nine, ten, 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 TABLES 1-15.

[0116] It is understood that, throughout the description (e.g., TABLES 1-15), 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 1-15, each thymine (T) can be replaced by a uracil (U).TABLE 1: Gln-TAA Suppressor tRNAsSEQ ID Suppressor Suppressor tRNA Sequence1NO. ID175 tr0903 GGTTCCATGGTGTAATGGTAAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGAGCCT176 tr0904 GGCCCCATGGTGTAATGGTTAGCACTCTGGTCTttaAAA CCAGCGATCCGAGTTCAAATCTCGGTGGGACCT177 tr0905 GGTTCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCGATACGAGTTCAAATCTCGTTGGAACCT178 tr0906 GGTTCCGTGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCGACCCGAGTTCAAATCTCGGCGGGACCT179 trl499 GGTCCCATGGTGTAATGGTCAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCAAATCTCGGTGGGACCC180 tri 500 GGTCCCATGGTGTAATGGTTAGCACTCTGGTCTttaAAACCAGCGATCCGAGTTCAAATCTCGGTGGGACCT1For sequence entries in TABLE 1, the 3-nucleotide anticodon is denoted via lowercase letters.Attorney Docket No. TVD-013WO2 SEQ ID Suppressor Suppressor tRNA Sequence1NO. ID181 trl501 GGCTCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCGATCCGAGTTCAAATCTCGGTGGGACCT182 tri 502 GGCCCCATGGTGTAATGGTCAGCACTCTGGTCTttaAAA CCAGCGATCCGAGTTCAAATCTCGGTGGGACCC183 tri 503 GGCCCCATGGTGTAATGGTCAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCAAATCTCGGTGGGACCT184 tri 504 GGCCCCATGGTGTAATGGTTAGCACTCTGGTCTttaAAA CCAGCGATCCGAGTTCAAATCTCGGTGGGACCC185 tri 505 GGCCCCATGGTGTAATGGTTAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCAAATCTCGGTGGGACCC186 tri 506 GGCCCCATGGTGTAATGGTCAGCACTCTGGTCTttaAAA CCAGCGATCCGAGTTCAAATCTCGGTGGGACCT187 trl511 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGAACCC188 trl513 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCAATCTGAGTTCAAATCTCGGTGGGACTT189 trl515 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCGAGCCTCGGTGGAACCT190 trl517 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTttaAAT CCAGCGACCCGAGTTCGAGTCTCGGTGGAACCT191 trl519 GGTTCCATGGTGTAATGGTAAGCACTCTGGACTttaAAT CCAGCGACCCGAGTTCGAGTCTCGGTGGAACCT192 tri 521 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGTAATCCGAGTTCGAATCTCGGTGGGACCT193 tri 523 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTttaAAT CCAGTGATCCGAGTTCGAGTCTCGGTGGAACCT194 tri 525 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCAACCCGAGTTCGAATCTCGGTGGGACCT195 tri 531 GGTCCCATGGTGTAATGGTAAGCACTCTGGACTttaAAT CCAGCAATCCGAGTTCGAATCTCGGTGGGACCT915 tri 621 GGCCCCATGGTGTAATGGTTAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGGACCT916 tri 622 GGCCCCATGGTGTAATGGTCAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGGACCC1125 tri 626 GGACCCATGGTGTAATGGTGAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGGTCCT1126 tri 627 GGCGCCATGGTGTAATGGTGAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGCGCCT1088 tri 723 GGtCCCATGGTGTAATGGTCAGCACTCTGGACTttaAATC CAGCGATCCGAGTTCGAGTCTCGGTGGGACCC1089 tri 771 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCAACCCGAGTTCGAATCTCGGTGGGACCC1090 tri 772 GGTCCCATGGTGTAATGGTtAGCACTCTGGACTttaAATC CAGCGATCCGAGTTCAAATCTCGGTGGGACCC1091 tri 773 GGTTCCATGGTGTAATGGTAAGCACTCTGGACTttaAATCCAGCGATCCGAGTTCGAGTCTCGGTGGAACCCAttorney Docket No. TVD-013WO2 SEQ ID Suppressor Suppressor tRNA Sequence1NO. ID1092 tri 774 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGTAATCCGAGTTCGAATCTCGGTGGGACCC1093 tri 775 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCAATCTGAGTTCGAGTCTCGGTGGGACTC1094 tri 777 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCAATCCGAGTTCGAATCTCGGTGGGACCC1095 tri 778 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTttaAAT CCAGCGACCCGAGTTCGAGTCTCGGTGGAACCC1096 trl780 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCGACCCGAGTTCGAATCTCGGTGGGACTT1097 tri 781 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCGACCCGAGTTCGAATCTCGGTGGGACCC1098 tri 782 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTttaAAT CCAGCGATACGAGTTCGAGTCTCGTTGGAACCC1099 trl785 GGTCCCATGGTGTAATGGTtAGCACTCTGGACTttaAATC CAGCAATCTGAGTTCAAATCTCGGTGGGACCC1100 trl787 GGCCCCATGGTGTAATGGTTAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGGACCC1101 trl788 GGCCCCATGGTGTAATGGTTAACACTCTGGACTttaAAT CCAGCGCTCCGAGTTCAAATCTCGGTGGGACCC1102 tri 791 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTttaAAT CCAGTGATCCGAGTTCGAGTCTCGGTGGAACCC1103 tri 793 GGCCCCATGGTGTAATGGTCAGCACTCTGGTCTttaAAA CCAGCGATCCGAGTTCGAGTCTCGGTGGGACCT1104 tri 794 GGTCCCATGGTGTAATGGTCAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGGACTC1105 tri 795 GGTTCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCGATCCGAGTTCAAGTCTCGGTGGAACCC1106 tri 806 GGTTCCATGGTGTAATGGTAAGCACTCTGGACTttaAAT CCAGCGACCCGAGTTCGAGTCTCGGTGGAACCC1107 tri 807 AGTTCCATGGTGTAATGGTAAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGAACTC1108 tri 809 GGTTCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCGATCCGAGTTCAAATCTCGGTGGGACCC1109 trl810 GGTTCCGTGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCGACCCGAGTTCGAGTCTCGGCGGGACCT1110 trl814 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCAAATCTCGGTGGGACCC1111 trl815 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CTGCGATCCGAGTTCAAATCTCGGTGGGACCC1112 tri 821 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCGATCCGAGTTCGAGTCTCGGTGGGACCC1113 tri 822 GGTTCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCGACCCGAGTTCGAGTCTCGGTGGGACCC1114 tri 823 GGTTCCATGGTGTAATGGTTAGCACTCTGGACTttaAATCCAGCGATCCGAGTTCGAGTCTCGGTGGAACCCAttorney Docket No. TVD-013WO2 SEQ ID Suppressor Suppressor tRNA Sequence1NO. ID1115 tri 824 GGTTCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCGATCCGAGTTCGAGTCTCGGTGGGACCC1116 tri 825 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGGACCC1117 tri 827 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCAATCTGAGTTCGAGTCTCGGTGGGACCC1118 trl830 GGTTCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CGGTAATCCGAGTTCGAGTCTCGGTGGAACCC1119 tri 831 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCAATCCGAGTTCGAGTCTCGGTGGGACCC1129 trl857 GGACCCATGGTGTAATGGTGAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGGTCCC1130 trl858 GGCGCCATGGTGTAATGGTGAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGCGCCC1000 oT_1526 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCGATCCGAGTTCAAATCTCGGTGGGGTCT1001 oT_1525 GGTACGATGGTGTAATGGTTAGCACTCTGGACTttaAAT CCAGCGACCCGAGTTCAAATCTCGGTCGTACCT1002 oT_1524 GGTTCCATGGTGTAATGGTAAGCACGCTGGACTttaAAT CCAGGCATCTGAGTTCGAGTCTCTGTGGAACCT1003 oT_1523 GGTACCATGGTGTAATGGTTAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCAAGTCTCGGTGGTACCT1004 oT_1522 GGTTCCATGGTGTAATGGGGAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTGAAATCTCGGTGGGACCT1005 oT_1521 GATTCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CGGTAATCCGAGTTCAAATCTCGGTGGGATCT1006 oT_1520 GGTTCCATGGTGTAATGGTTATCACTCTGGACTttaAATC CAGCGCTCCGAGTTCAAGTCTCGGTGGAACCT1007 oT_1519 GGTCCCCTGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCAATCTGAGTTCAAATCTCGGTGGGACCT1008 oT_1518 GGTTCCATTGTGTAATGGTAAGCACTCTGGACTttaAATC CAGCGATCCGAGTTCGAGTCTCGGTGGAACCT1009 oT_1516 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCGATCCGAGTTCAAATCTCGTTGGGACCT1010 oT_1515 GGTTCCATGGTGTAATGGTTATCACTCTGGACTttaAATC CGGTTATCCGAGTTCAAATCTCGGTGGAACCT1011 oT_1514 GGTCGGATGGTGTAATGGTTAGCACACTGGACTttaAGT CCAGCAATCCGAGTTCGAGTCTTGGTTTGACCA1012 oT_1513 GGACCCATGGTGTAATGGCTAGCACTCTGGACTttaAAT CCAGCAATCCAAGTTCAAATCTCGGTGGGACCT1013 oT_1512 GGTCCCATGCTGTAATGGTTAGCACTCTGGACTttaAATC CAGCAACCTGAGTTCAAATCTCGGTGGGACCT1014 OT 1511 GGTTCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CGGTAATACGAGTTCAAATCTCGGTGGAACCT1015 oT_1509 GGTTTCATGGTGTAATGGCTAGCACTCTGGACTttaAATCCAGCGATCCGAGTTCAAATCTCGGTGAAATTTAttorney Docket No. TVD-013WO2 SEQ ID Suppressor Suppressor tRNA Sequence1NO. ID1016 oT_1508 TGTTCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCGACCCGAGTTCAAATCTCGGTGGGACCT1017 oT_1507 GGACCCATGGTGTAATGGTTAGCACTCTGGACTttaAAT CCAGCAATTAAAGTTCAAATCTCCGTGGGACCT1018 oT_1506 GGTTCCATGATGTAATGGTAAGCACTCTGGACTttaAAT CCAGCGGTCCGAGTTCGAGTCTCGGTGGAACCT1019 oT_1505 GGTTGCATGGTGTAATGGTGAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGTAACCT1020 oT_1504 GGTCGCATGGTGTAATGGTTAGCACACTGGACTttaAGT CCAGCAATCCGAGTTCGAGTCTTGGTGAGACCA1021 oT_1503 GGCTCGATGGTGTAATGGTTAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCAAATCTCGGTCGGGCCT1022 oT_1502 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCAATCTGAGTTCAAATCTCGGTGGGCCCT1023 oT_1501 GGTCCCATGTTGTAATGGTTAGCACTCTGGACTttaAATC CAGCAATCCGAGTTCGAATCTCGGTGGGACCT1024 oT_1500 GGTCCCATGGTGTAATGGTTAGCACTCTGCGCTttaAATC CAGCAATCCGAGTTCGAATCTTGGTGGGACCT1025 oT_1499 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCAATCCGAGCTCGTATCTCGGTGGGACCT1026 oT_1498 GGTCCGATGGTGTAATGGTTAGCACTCTGGACTttaAAT CCAGCAATCCGAGTTCGAATCTCGGTCGGGCCT1027 oT_1497 GGTCCCATGGTGTAATGGTTAGCACTCTGGCCTttaAAG CCAGCAATCCGAGTTCGAATCTCGGTGGGACCT1028 oT_1496 GGTTCCATGGTGTAATGGTAAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGAAGCT1029 oT_1495 GGTTCCATGATGTAATGGTGAGCACTCTGGACTttaAAT CCAGCCATCCGAGTTCAAATCTCGGTGGGACCT1030 oT_1494 GGTTCCATGGTGTTATGGTTAGCACTCTGGACTttaAATC CAGCGATCCGAGTTCAAGTCTCGGTGGAACCT1031 oT_1493 GGTTCCATGGTGTAATGGCTAGCACTCTGGACCttaAAT CCAGCGATCCGAGTTCAAATCTCGGTGGGATTT1032 oT_1492 GAACGCATGGTGTAATGGTTAGCACTCTGGACTttaAAT CCAGCAATCCAAGTTCAAATCTCGGTGTGATCT1033 oT_1490 GTTTTCATGGTGTAATGGTAAGCACTCTGGACTttaAATC CAGCGATCCGAGTTCGAGTCTCGGTGGAAACT1034 oT_1489 GGTGCGATGGTGTAATGGTTAGCACTCTGGACTttaAAT CCAGCAATCCGAGTTCGAATCTCGGTCGCACCT1035 oT_1488 GGTTCCATGCTGTAATGGTTAGCACTCTGGACTttaAATC CAGCTACCCGAGTTCAAATCTCGGTGGGACCT1036 oT_1486 GGACCCATGGTGTAATGGTTAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCAAGTCTCGGTGGGACCT1037 oT_1485 GATCTGATGGTGTAATGGTTAGCACACTGGACTttaAGT CCAGCAATCCGAGTTCGAGTCTTGGTCAGATCA1038 oT_1484 GGTTCCATGGTGTAATGGCTAGCACTCTGGCCTttaAAGCCAGCGATCCGAGTTCAAATCTCGGTGGGATTTAttorney Docket No. TVD-013WO2 SEQ ID Suppressor Suppressor tRNA Sequence1NO. ID1039 oT_1483 GGTTCCATGGTGTAATGGTTAGCACTCTGGAATttaAATC CAGCGACCCGAGTTCAAATCTCGGTGGGACCT1040 oT_1482 GGGCCCATGGTGTAATGGTCAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCAAATCTCGGTGGGCTCC1041 oT_1481 GGACCCATGCTGTAATGGTTAGCACTCTGGACTttaAAT CCAGCATTCCAAGTTCAAATCTCGGTGGGACCT1042 oT_1480 GGTTCTATGGTGTAATGGTTAGCACTCTGGACTttaAATC CGGTAATCCGAGTTCAAATCTCGGTAGGACCT1043 oT_1479 GGTCTCATGGTGTAATGGTTAGCACACTGGACTttaAGT CCAGCAATCCGAGTTCGAGTCTTTGTGAGACCA1044 oT_1478 GGACCCATGGTGTAATGGTTAGCACTCTTGACTttaAATC CAGCAATCCAAGTTCAAATCTCGGTGGGACCT1045 oT_1477 GGTCCCATGGTATAATGGTTAGTACTCTGGACTttaAATC CAGCGATCCGAGTTCAAATCTCGGTGGGACCT1046 oT_1476 GGTTCCATGCTGTAATGGCTAGCACTCTGGACTttaAATC CAGCGCTCCGAGTTCAAATCTCGGTGGGATTT1047 oT_1475 GGTGCTATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCGATCCGAGTTCAAATCTCGGTAGTACCT1048 oT_1474 GGGCCGATGGTGTAATGGTTAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCAAATCTCGGTCGGTCCT1049 oT_1473 GCTTTCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCGATCCGAGTTCAAGTCTCGGTGGAAGCT1050 oT_1472 GGTTCCATGGTGTAATGGTAAGCACTCTGGACTttaAAT CCAGCGATCGGAGTTCGAGTCTCATTGGAACCT1051 oT_1471 GGTGTCATGGTGTAATGGCTAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCAAATCTCGGTGACATTT1052 oT_1470 GGTTCCATGGTGTAATGGTTAGCACTCTGGTCTttaAAGC CAGCGACCCGAGTTCAAATCTCGGTGGGACCT1053 oT_1469 GGTTTTATGGTGTAATGGTAAGCACTCTGGACTttaAATC CAGCGATCCGAGTTCGAGTCTCGGTGGAACCT1054 oT_1468 GGATACATGGTGTAATGGTTAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCAAATCTCGGTGTGTCCT1055 oT_1467 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCAATCGGAGTTCAAATCTCGGTGGGACCT1056 oT_1466 GACTCCATGGTGTAATGGTGAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCAAATCTCGGTGGGGTCT1057 oT_1465 GGTCCCATGGTGTAATGGTTAGCACTCTGGGCTttaAATC CAGCAATGTGAGTTCGAATCTTTATGGGACCT1058 oT_1464 GGTTCCATAGTGTAATGGTTAGTACTCTGGACTttaAATC CGGTAATCCGAGTTCAAATCTCGGTGGAACCT1059 oT_1463 GGCCCCATGGTGTAATGGTCACCACTCTGGACTttaAAT CCAGCGTTCCGAGTTCAAATCTCGGTGGGACCC1060 oT_1462 GGTCGTATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCAATCCGAGTTCGAATCTCGGTGTGACCT1061 oT_1461 GGGTCCATGGTGTAATGGTTAGCACTCTGGACTttaAATCCGGTAATCCGAGTTCAAATCTCGGTGGACCCTAttorney Docket No. TVD-013WO2 SEQ ID Suppressor Suppressor tRNA Sequence1NO. ID1062 oT_1460 GTTCTTATGGTGTAATGGTTAGCACACTGGACTttaAGTC CAGCAATCCGAGTTCGAGTCTTGGTGAGAGCA1063 oT_1459 GGTCCAATGGTGTAATGGTTAGCACTCTGGACTttaAAT CCGGTAATCCGAGTTCAAATCTCGGTTGGACCT1064 oT_1458 GGCCCCATGGTGTAATGGTCAGCACCCTGGACTttaAAT CCAGTGATCCGAGTTCAAATCTCGGTGGGACCC1065 oT_1457 GGTCGGATGGTGTAATGGTTAGCACACTGGACTttaAGT CCAGCAATCCGAGTTCGAGTCTTGGTCTGACCA1066 oT_1456 GGTTCCATGGTGTCATGGTGAGCACTCTGGACTttaAATC CAGCGATCCGAGTTCGAGTCTCGGTGGAACCT1067 oT_1455 GGTTGCATGGTGTAATGGCTAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCAAATCTCGGTGTAATTT1068 oT_1454 GGTCCCATGGTGTAATGGTTAACACTCTGGACTttaAATC CAGAGATCCGAGTTCAAATCTCGGTGGGACCT1069 oT_1453 GGTTCCATGGTGTAATGGTTAGCACTCCGGACTttaAATC CGGTAATCCGAGTTCAAATCTCGGTGGAACCT1070 oT_1452 GGACCCATGTTGTAATGGTTAGCACTCTGGACTttaAATC CAGCAACCCAAGTTCAAATCTCGGTGGGACCT1071 oT_1451 GGCCGTATGGTGTAATGGTTAGCACTCTGGACTttaAAT CCAGCGATCCGAGTTCAAATCTCGGTACGACCT1072 oT_1450 GGTTCCATGTTGTAATGGTTAGCACTCTGGACTttaAATC CAGAGATCCGAGTTCAAATCTCGGTGGGACCT1073 oT_1449 GGATCCATGGTGTAATGGTTAGCACTCTGGACTttaAAT CCAGCGACCCGAGTTCAAATCTCGGTGGGTTCT1074 oT_1448 GGCCCCATGGTGTAATGGTTAGCACACTGGACTttaAGT CCAGCAATCCGAGTTCGAGTCTTGGTGGGGCCA1075 oT_1447 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTttaAAT CCAGCGATGCGGGTTCAAATCCCGCTGGGACCT1076 oT_1446 GGTTCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCGATGCGGGTTCAAATCCCGTTGGAACCT1077 oT_1444 GGTTCCATGGTGTAATGGTAAGCACTCTGGACTttaAAT CCAGCCATGCGGGTTCGAGTCCCGCTGGAACCT1078 oT_1442 GGTTCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCGATGCGGGTTCAAGTCCCGTTGGAACCT1079 oT_1440 GGTCCCATGGTGTAATGGTTAGCACTCTGGGCTttaAATC CAGCAATGCGGGTTCGAATCCCGTTGGGACCT1080 oT_1439 GGCCCCATGGTGTAATGGTCAGCACTCTGGACTttaAAT CCAGCGATGCGGGTTCAAATCCCGTTGGGACCC1081 oT_1438 GGCCCCATGGTGTAATGGTCAGCACTCTGGACTttaAAT CCAGCGATGCGGGTTCAAATCCCGCTGGGACCC1082 oT_1436 GGTCTCATGGTGTAATGGTTAGCACACTGGACTttaAGT CCAGCAATGCGGGTTCGAGTCCCGTTGAGACCA1083 oT_1435 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTttaAAT CCAGCGATGCGGGTTCGAGTCCCGCTGGAACCT1084 oT_1433 GGTTCCATGGTGTAATGGTTAGCACTCTGGACTttaAATCCAGCGATGCGGGTTCAAGTCCCGCTGGAACCTAttorney Docket No. TVD-013WO2 SEQ ID Suppressor Suppressor tRNA Sequence1NO. ID1085 oT_1432 GGTTCCATGGTGTAATGGCTAGCACTCTGGACTttaAATC CAGCGATGCGGGTTCAAATCCCGTTGGGATTT1086 oT_1431 GGACCCATGGTGTAATGGTTAGCACTCTGGACTttaAAT CCAGCAATGCGGGTTCAAATCCCGCTGGGACCT1087 oT_1429 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATCCAGCGATGCGGGTTCAAATCCCGTTGGGACCTTABLE 2: Gln-TAG Suppressor tRNAsSEQ ID Suppressor Suppressor tRNA Sequence2NO. ID199 trl496 GGTTCCGTGGTGTAATGGTTAGCACTCTGGACTctaAATC CAGCGACCCGAGTTCAAATCTCGGCGGGACCT197 trl498 GGCCCCATGGTGTAATGGTTAGCACTCTGGTCTctaAAA CCAGCGATCCGAGTTCAAATCTCGGTGGGACCT205 tri 507 GGCCCCATGGTGTAATGGTTAGCACTCTGGTCTctaAAA CCAGCGATCCGAGTTCAAATCTCGGTGGGACCC203 tri 508 GGCCCCATGGTGTAATGGTCAGCACTCTGGTCTctaAAA CCAGCGATCCGAGTTCAAATCTCGGTGGGACCC216 tri 509 GGTCCCATGGTGTAATGGTAAGCACTCTGGACTctaAAT CCAGCAATCCGAGTTCGAATCTCGGTGGGACCT208 trl510 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTctaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGAACCC209 trl512 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTctaAAT CCAGCAATCTGAGTTCAAATCTCGGTGGGACTT210 trl514 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTctaAAT CCAGCGATCCGAGTTCGAGCCTCGGTGGAACCT211 trl516 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTctaAAT CCAGCGACCCGAGTTCGAGTCTCGGTGGAACCT212 trl518 GGTTCCATGGTGTAATGGTAAGCACTCTGGACTctaAAT CCAGCGACCCGAGTTCGAGTCTCGGTGGAACCT213 tri 520 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTctaAAT CCAGTAATCCGAGTTCGAATCTCGGTGGGACCT214 tri 522 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTctaAAT CCAGTGATCCGAGTTCGAGTCTCGGTGGAACCT215 tri 524 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTctaAAT CCAGCAACCCGAGTTCGAATCTCGGTGGGACCT196 trl619 GGTTCCATGGTGTAATGGTAAGCACTCTGGACTctaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGAGCCT1122 tri 624 GGACCCATGGTGTAATGGTGAGCACTCTGGACTctaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGGTCCT1123 tri 625 GGCGCCATGGTGTAATGGTGAGCACTCTGGACTctaAATCCAGCGATCCGAGTTCGAGTCTCGGTGGCGCCTFor sequence entries in TABLE 2, the 3-nucleotide anticodon is denoted via lowercase letters.Attorney Docket No. TVD-013WO2 SEQ ID Suppressor Suppressor tRNA Sequence2NO. ID200 tri 629 GGTCCCATGGTGTAATGGTCAGCACTCTGGACTctaAAT CCAGCGATCCGAGTTCAAATCTCGGTGGGACCC201 tri 628 GGTCCCATGGTGTAATGGTTAGCACTCTGGTCTctaAAA CCAGCGATCCGAGTTCAAATCTCGGTGGGACCT204 trl630 GGCCCCATGGTGTAATGGTCAGCACTCTGGACTctaAAT CCAGCGATCCGAGTTCAAATCTCGGTGGGACCT206 tri 631 GGCCCCATGGTGTAATGGTTAGCACTCTGGACTctaAAT CCAGCGATCCGAGTTCAAATCTCGGTGGGACCC198 trl632 GGTTCCATGGTGTAATGGTTAGCACTCTGGACTctaAATC CAGCGATACGAGTTCAAATCTCGTTGGAACCT202 trl633 GGCTCCATGGTGTAATGGTTAGCACTCTGGACTctaAAT CCAGCGATCCGAGTTCAAATCTCGGTGGGACCT207 trl634 GGCCCCATGGTGTAATGGTCAGCACTCTGGTCTctaAAA CCAGCGATCCGAGTTCAAATCTCGGTGGGACCT921 trl732 GGCCCCATGGTGTAATGGTTAGCACTCTGGACTctaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGGACCT922 trl734 GGCCCCATGGTGTAATGGTcAGCACTCTGGACTctaAATC CAGCGATCCGAGTTCGAGTCTCGGTGGGACCC1131 trl855 GGACCCATGGTGTAATGGTGAGCACTCTGGACTctaAATCCAGCGATCCGAGTTCGAGTCTCGGTGGGTCCCTABLE 3: Gln-TGA Suppressor tRNAsSEQ ID Suppressor Suppressor tRNA Sequence3NO. ID217 tr0453 GGTTCCATGGTGTAATGGTTAGCACTCTGGACTtcaAATC CAGCGATCCGAGTTCAAATCTCGGTGGGACCT218 trl636 GGCCCCATGGTGTAATGGTTAGCACTCTGGTCTtcaAAA CCAGCGATCCGAGTTCAAATCTCGGTGGGACCT219 trl637 GGTCCCATGGTGTAATGGTCAGCACTCTGGACTtcaAAT CCAGCGATCCGAGTTCAAATCTCGGTGGGACCC220 tri 638 GGTCCCATGGTGTAATGGTTAGCACTCTGGTCTtcaAAA CCAGCGATCCGAGTTCAAATCTCGGTGGGACCT221 trl639 GGCTCCATGGTGTAATGGTTAGCACTCTGGACTtcaAAT CCAGCGATCCGAGTTCAAATCTCGGTGGGACCT222 tri 640 GGCCCCATGGTGTAATGGTCAGCACTCTGGTCTtcaAAA CCAGCGATCCGAGTTCAAATCTCGGTGGGACCC223 tri 641 GGCCCCATGGTGTAATGGTCAGCACTCTGGACTtcaAAT CCAGCGATCCGAGTTCAAATCTCGGTGGGACCT224 tri 642 GGCCCCATGGTGTAATGGTTAGCACTCTGGTCTtcaAAA CCAGCGATCCGAGTTCAAATCTCGGTGGGACCC225 tri 643 GGCCCCATGGTGTAATGGTTAGCACTCTGGACTtcaAATCCAGCGATCCGAGTTCAAATCTCGGTGGGACCCFor sequence entries in TABLE 3, the 3-nucleotide anticodon is denoted via lowercase letters.Attorney Docket No. TVD-013WO2 SEQ ID Suppressor Suppressor tRNA Sequence3NO. ID226 tri 644 GGCCCCATGGTGTAATGGTCAGCACTCTGGTCTtcaAAA CCAGCGATCCGAGTTCAAATCTCGGTGGGACCT227 tri 645 GGTCCCATGGTGTAATGGTAAGCACTCTGGACTtcaAAT CCAGCAATCCGAGTTCGAATCTCGGTGGGACCT228 tri 646 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTtcaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGAACCC229 tri 647 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTtcaAAT CCAGCAATCTGAGTTCAAATCTCGGTGGGACTT230 tri 648 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTtcaAAT CCAGCGATCCGAGTTCGAGCCTCGGTGGAACCT231 tri 649 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTtcaAAT CCAGCGACCCGAGTTCGAGTCTCGGTGGAACCT232 trl650 GGTTCCATGGTGTAATGGTAAGCACTCTGGACTtcaAAT CCAGCGACCCGAGTTCGAGTCTCGGTGGAACCT233 tri 651 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTtcaAAT CCAGTAATCCGAGTTCGAATCTCGGTGGGACCT234 trl652 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTtcaAAT CCAGTGATCCGAGTTCGAGTCTCGGTGGAACCT235 trl653 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTtcaAAT CCAGCAACCCGAGTTCGAATCTCGGTGGGACCT923 trl733 GGCCCCATGGTGTAATGGTTAGCACTCTGGACTtcaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGGACCT924 tri 735 GGCCCCATGGTGTAATGGTcAGCACTCTGGACTtcaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGGACCC1127 trl853 GGACCCATGGTGTAATGGTGAGCACTCTGGACTtcaAAT CCAGCGATCCGAGTTCGAGTCTCGGTGGGTCCT1128 tri 854 GGCGCCATGGTGTAATGGTGAGCACTCTGGACTtcaAATCCAGCGATCCGAGTTCGAGTCTCGGTGGCGCCTTABLE 4: Ser-TAA Suppressor tRNASEQ ID Suppressor Suppressor tRNA Sequence4NO. ID236 tr1344 GTAGTCGTGGCCGAGTGGTTAAGGCGGCAGGCTttaAAC CTGTTGGGGTTTCCCCGCACGGGTTCGAATCCCGTCGA CTACG901 trl345 GTAGTCGTGGCCGAGTGGTTAAGGCGATGGTCTttaAAA CCATTGGGGTTTCCCCGCACGGGTTCGAATCCCGTCGA CTACG902 trl346 GTAGTCGTGGCCGAGTGGTTAAGGCGATGGACTttaAATCCATTGGGGTTTCCCCGCACGGGTTCGAATCCCGTCGACTACGFor the sequence entries in TABLE 4, the 3 -nucleotide anticodon is denoted via lowercase letters.Attorney Docket No. TVD-013WO2 903 trl347 GTAGTCGTGGCCGAGTGGTTAAGGCGATGGTCTttaAAACCATTGGGGTTTCCCCGCGCAGGTTCGAATCCTGCCGACTACGTABLE 5: Ser-TGA Suppressor tRNASEQ ID Suppressor Suppressor tRNA Sequence5NO. ID905 tri 529 GTAGTCGTGGCCGAGTGGTTAAGGCGATGGACTtcaAAT CCATTGGGGTTTCCCCGCACGGGTTCGAATCCCGTCGA CTACG906 trl530 GTAGTCGTGGCCGAGTGGTTAAGGCGATGGTCTtcaAAA CCATTGGGGTTTCCCCGCGCAGGTTCGAATCCTGCCGA CTACG904 tr0879 GTAGTCGTGGCCGAGTGGTTAAGGCGATGGTCTtcaAAA CCATTGGGGTTTCCCCGCACGGGTTCGAATCCCGTCGA CTACGTABLE 6: Ser-TAG Suppressor tRNASEQ ID Suppressor Suppressor tRNA Sequence6NO. ID237 trl497 GTAGTCGTGGCCGAGTGGTTAAGGCGGCAGGCTctaAAC CTGTTGGGGTTTCCCCGCACGGGTTCGAATCCCGTCGA CTACG907 tri 526 GTAGTCGTGGCCGAGTGGTTAAGGCGATGGTCTctaAAA CCATTGGGGTTTCCCCGCACGGGTTCGAATCCCGTCGA CTACG908 tri 527 GTAGTCGTGGCCGAGTGGTTAAGGCGATGGACTctaAAT CCATTGGGGTTTCCCCGCACGGGTTCGAATCCCGTCGA CTACG909 tri 528 GTAGTCGTGGCCGAGTGGTTAAGGCGATGGTCTctaAAACCATTGGGGTTTCCCCGCGCAGGTTCGAATCCTGCCGACTACGTABLE 7: Gln-TAA Suppressor tRNAsSEQ ID Suppressor Suppressor tRNA Sequence7NO. ID238 tr0157 GGTTCCATGGTGTAATGGTTAGCACTCTGGACTttaAATCCAGCGATCCGAGTTCAAATCTCGGTGGGACCTFor the sequence entries in TABLE 5, the 3-nucleotide anticodon is denoted via lowercase letters.For the sequence entries in TABLE 6, the 3-nucleotide anticodon is denoted via lowercase letters.For sequence entries in TABLE 7, the 3-nucleotide anticodon is denoted via lowercase letters.Attorney Docket No. TVD-013WO2 SEQ ID Suppressor Suppressor tRNA Sequence7NO. ID239 tr0163 GGCCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCGATCCGAGTTCAAATCTCGGTGGGACCT1124 tr0520 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTttaAATC CAGCGATCCGAGTTCGAGTCTCGGTGGAACCT240 tr0523 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTttaAATC CAGCAATCCGAGTTCGAATCTCGGTGGGACCT241 tr0524 GGCCCCATGGTGTAATGGTCAGCACTCTGGACTttaAATC CAGCGATCCGAGTTCAAATCTCGGTGGGACCC242 tr0525 GGTTCCATGGTGTAATGGTTAGCACTCTGGACTttaAATCCAGCGATCCGAGTTCAAGTCTCGGTGGAACCTTABLE 8: Gln-TAG Suppressor tRNAsSEQ ID Suppressor Suppressor tRNA Sequence8NO. ID243 tr0191 GGTTCCATGGTGTAATGGTTAGCACTCTGGACTctaAATC CAGCGATCCGAGTTCAAATCTCGGTGGGACCT244 tr0198 GGCCCCATGGTGTAATGGTTAGCACTCTGGACTctaAAT CCAGCGATCCGAGTTCAAATCTCGGTGGGACCT246 tr0216 GGCCCCATGGTGTAATGGTCAGCACTCTGGACTctaAAT CCAGCGATCCGAGTTCAAATCTCGGTGGGACCC245 tr0606 GGTCCCATGGTGTAATGGTTAGCACTCTGGACTctaAAT CCAGCAATCCGAGTTCGAATCTCGGTGGGACCT247 tr0869 GGTTCCATGGTGTAATGGTTAGCACTCTGGACTctaAATC CAGCGATCCGAGTTCAAGTCTCGGTGGAACCT1121 tr0870 GGTTCCATGGTGTAATGGTGAGCACTCTGGACTctaAATCCAGCGATCCGAGTTCGAGTCTCGGTGGAACCTTABLE 9: Gln-TGA Suppressor tRNASEQ ID Suppressor Suppressor tRNA Sequence9NO. ID248 tri 635 GGCCCCATGGTGTAATGGTTAGCACTCTGGACTtcaAATCCAGCGATCCGAGTTCAAATCTCGGTGGGACCTTABLE 10: Ser-TAA Suppressor tRNAsSEQ ID Suppressor Suppressor tRNA Sequence10NO. ID250 trO538 GTAGTCGTGGCCGAGTGGTTAAGGCGATGGACTttaAATCCATTGGGGTTTCCCCGCGCAGGTTCGAATCCTGCCGACTACGFor sequence entries in TABLE 8, the 3-nucleotide anticodon is denoted via lowercase lettersFor the sequence entry in TABLE 9, the 3-nucleotide anticodon is denoted via lowercase letters.For sequence entries in TABLE 10, the 3-nucleotide anticodon is denoted via lowercase letters.Attorney Docket No. TVD-013WO2 249 tr!233 GTAGTCGTGGCCGAGTGGTTAAGGCGATGGACTttaAATCCATTGGGGTCTCCCCGCGCAGGTTCGAATCCTGCCGACTACGTABLE 11: Ser-TGA Suppressor tRNASEQ ID Suppressor Suppressor tRNA Sequence11NO. ID910 tr0878 GTAGTCGTGGCCGAGTGGTTAAGGCGGCAGGCTtcaAAC CTGTTGGGGTTTCCCCGCACGGGTTCGAATCCCGTCGA CTACG911 trl232 GTAGTCGTGGCCGAGTGGTTAAGGCGATGGACTtcaAAT CCATTGGGGTTTCCCCGCGCAGGTTCGAATCCTGCCGA CTACG912 trl235 GTAGTCGTGGCCGAGTGGTTAAGGCGATGGACTtcaAATCCATTGGGGTCTCCCCGCGCAGGTTCGAATCCTGCCGACTACGTABLE 12: Ser-TAG Suppressor tRNASEQ ID Suppressor Suppressor tRNA Sequence12NO. ID913 trl231 GTAGTCGTGGCCGAGTGGTTAAGGCGATGGACTctaAAT CCATTGGGGTTTCCCCGCGCAGGTTCGAATCCTGCCGA CTACG914 trl234 GTAGTCGTGGCCGAGTGGTTAAGGCGATGGACTctaAATCCATTGGGGTCTCCCCGCGCAGGTTCGAATCCTGCCGACTACGTABLE 13: Tyr-TAA Suppressor tRNAsSEQ ID Suppressor Suppressor tRNA Sequence13NO. ID251 tr0211 CCTTCGATAGCTCAGCTGGTAGAGCGGAGGACTttaGAT CCTTAGGTCGCTGGTTCGATTCCGGCTCGAAGGA252 tr0212 CCTTCGATAGCTCAGTTGGTAGAGCGGAGGACTttaGAT CCTTAGGTCGCTGGTTCGAATCCGGCTCGAAGGA253 trl348 CCTTCGATAGCTCAGTTGGTAGAGCGGAGGACTttaG / / gg c7g / g / cc7 / r / gacATCCTTAGGTCGCTGGTTCGAATCCGGCTCGAAGGA11For the sequence entry in TABLE 11, the 3-nucleotide anticodon is denoted via lowercase letters.12For the sequence entries in TABLE 12, the 3-nucleotide anticodon is denoted via lowercase letters.13For the sequence entries in TABLE 13, the 3-nucleotide anticodon is denoted via lowercase letters, while an endogenous intron is indicated by lowercase and italics.Attorney Docket No. TVD-013WO2TABLE 14: Leu-TGA Suppressor tRNAsSEQ ID Suppressor Suppressor tRNA Sequence14NO. ID1120 tri 090 ACCAGAATGGCCGAGTGGTTAAGGCGTTGGACTtcaGATCCAATGGATTCATATCCGCGTGGGTTCGAACCCCACTTCTGGTATABLE 15: Gin Suppressor Consensus Sequences SEQ Corollary Suppressor tRNA Consensus Sequence15ID SuppressorNO: IDs254 tr903 and GGTTCCATGGTGTAATGGTAAGCACTCTGGACTN1N2N3A trl619 ATCCAGCGATCCGAGTTCGAGTCTCGGTGGAGCCT 255 tr904, tri 498, GGCCCCATGGTGTAATGGTTAGCACTCTGGTCTN1N2N3AA and tri 636 ACCAGCGATCCGAGTTCAAATCTCGGTGGGACCT 256 tr905 and GGTTCCATGGTGTAATGGTTAGCACTCTGGACTN1N2N3AA trl632 TCCAGCGATACGAGTTCAAATCTCGTTGGAACCT 257 tr906 and GGTTCCGTGGTGTAATGGTTAGCACTCTGGACTN1N2N3AA trl496 TCCAGCGACCCGAGTTCAAATCTCGGCGGGACCT 258 trl499, GGTCCCATGGTGTAATGGTCAGCACTCTGGACTN1N2N3A tri 629, and ATCCAGCGATCCGAGTTCAAATCTCGGTGGGACCC trl637259 tri 500, GGTCCCATGGTGTAATGGTTAGCACTCTGGTCTN1N2N3AA tri 628, and ACCAGCGATCCGAGTTCAAATCTCGGTGGGACCT tri 638260 trl501, GGCTCCATGGTGTAATGGTTAGCACTCTGGACTN1N2N3A trl633, and ATCCAGCGATCCGAGTTCAAATCTCGGTGGGACCT trl639261 tri 502, GGCCCCATGGTGTAATGGTCAGCACTCTGGTCTN1N2N3A tri 508, and AACCAGCGATCCGAGTTCAAATCTCGGTGGGACCC tri 640262 trl503, GGCCCCATGGTGTAATGGTCAGCACTCTGGACTN1N2N3A tri 630, and ATCCAGCGATCCGAGTTCAAATCTCGGTGGGACCT tri 641263 tri 504, GGCCCCATGGTGTAATGGTTAGCACTCTGGTCTN1N2N3AA tri 507, and ACCAGCGATCCGAGTTCAAATCTCGGTGGGACCC tri 642264 trl505, GGCCCCATGGTGTAATGGTTAGCACTCTGGACTN1N2N3A tri 631, and ATCCAGCGATCCGAGTTCAAATCTCGGTGGGACCC tri 643265 tri 506, GGCCCCATGGTGTAATGGTCAGCACTCTGGTCTN1N2N3A tri 634, and AACCAGCGATCCGAGTTCAAATCTCGGTGGGACCTtri 644For the sequence entry in TABLE 14, the 3-nucleotide anticodon is denoted via lowercase letters.For the sequence entries in TABLE 15, the 3-nucleotide anticodon is denoted via bolded “NIN2N3.”Attorney Docket No. TVD-013WO2 SEQ Corollary Suppressor tRNA Consensus Sequence15ID SuppressorNO: IDs266 trl511, GGTTCCATGGTGTAATGGTGAGCACTCTGGACTN1N2N3A tri 510, and ATCCAGCGATCCGAGTTCGAGTCTCGGTGGAACCC tri 646267 trl513, GGTCCCATGGTGTAATGGTTAGCACTCTGGACTN1N2N3A tri 512, and ATCCAGCAATCTGAGTTCAAATCTCGGTGGGACTT tri 647268 tri 515, GGTTCCATGGTGTAATGGTGAGCACTCTGGACTN1N2N3A tri 514, and ATCCAGCGATCCGAGTTCGAGCCTCGGTGGAACCT tri 648269 tri 517, GGTTCCATGGTGTAATGGTGAGCACTCTGGACTN1N2N3A tri 516, and ATCCAGCGACCCGAGTTCGAGTCTCGGTGGAACCT tri 649270 tri 519, GGTTCCATGGTGTAATGGTAAGCACTCTGGACTN1N2N3A tri 518, and ATCCAGCGACCCGAGTTCGAGTCTCGGTGGAACCT trl650271 tri 521, GGTCCCATGGTGTAATGGTTAGCACTCTGGACTN1N2N3A tri 520, and ATCCAGTAATCCGAGTTCGAATCTCGGTGGGACCT tri 651272 trl523, GGTTCCATGGTGTAATGGTGAGCACTCTGGACTN1N2N3A tri 522, and ATCCAGTGATCCGAGTTCGAGTCTCGGTGGAACCT trl652273 trl525, GGTCCCATGGTGTAATGGTTAGCACTCTGGACTN1N2N3A tri 524, and ATCCAGCAACCCGAGTTCGAATCTCGGTGGGACCT trl653274 trl531, GGTCCCATGGTGTAATGGTAAGCACTCTGGACTN1N2N3A tri 509, and ATCCAGCAATCCGAGTTCGAATCTCGGTGGGACCT tri 645918 tri 621, GGCCCCATGGTGTAATGGTTAGCACTCTGGACTN1N2N3A tri 732, and ATCCAGCGATCCGAGTTCGAGTCTCGGTGGGACCT trl733925 tri 622, GGCCCCATGGTGTAATGGTCAGCACTCTGGACTN1N2N3AA tri 734, and TCCAGCGaTCCGAGTTCGAGTCTCGGTGGGACCC tri 7351149 tri 626, GGCCCCATGGTGTAATGGTCAGCACTCTGGACTN1N2N3AA tri 624, and TCCAGCGaTCCGAGTTCGAGTCTCGGTGGGACCC trl8531150 tri 627, GGCGCCATGGTGTAATGGTGAGCACTCTGGACTN1N2N3A tri 625, and ATCCAGCGATCCGAGTTCGAGTCTCGGTGGCGCCTtri 854

[0117] In TABLE 15, Ni, N2, and N3 depict the anticodon of the suppressor tRNA, wherein the combination of Ni, N2, and N3 in a given sequence is one of:(i) thymine (T), thymine (T), and adenosine (A), respectively;(ii) thymine (T), cytosine (C), and adenosine (A), respectively; orAttorney Docket No. TVD-013WO2 (iii) cytosine (C), thymine (T), and adenosine (A), respectively.

[0118] Given that in SEQ IDNOs: 254-274, 918, 925, 1149, and 1150 of TABLE 15, N3is always an adenosine (A), then Ni is a thymine (T) or cytosine (C) and N2 is thymine (T) or cytosine (C), provided that when Ni is a cytosine (C) then N2 is a thymine (T).

[0119] A suppressor tRNA can be engineered to allow for more efficient suppression of premature termination. 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, 9-15, 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).

[0120] 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 Soil etal. (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 etal. (2000) ANNU. REV. BIOCHEM. 69:617-650; Johnson et al. (1995) COLD SPRING HA BOR SYMP. QUANT. BIOL. 60:71-82; Johnson et al. (1982) J.Attorney Docket No. TVD-013WO2 MOL. BIOL. 156:113-140; Crowley etal. (1994) CELL 78:61-71; Beier etal. (2001) NUCLEIC ACIDS RES. 29:4767-4782; Torres et al. (2014) TRENDS MOL. MED. 20:306-314; Bjork et al. (1987) ANNU. REV. BIOCHEM. 56:263-287; Schaffrath etal. (2017) RNABIOL. 14(9): 1209-1222; and Johansson et al. (2008) MOL. CELL. BIOL. 28(10): 3301 - 12.

[0121] tRNAs (including suppressor tRNAs) are known to undergo a wide variety of post-transcriptional modifications. In some embodiments, a suppressor tRNA comprises a naturally-occurring nucleotide modification. Naturally-occurring tRNAs modifications are described, for example, in Machnicka etal. (2014) RNABlOLOGY 11 (12): 1619-1629. For example, a base in a tRNAs that is initially transcribed as a uracil may be post-transcriptionally modified to pseudouridine, or a base that is initially transcribed as adenine may have its adenosine nucleobase be post-transcriptionally modified to inosine.Accordingly, in some embodiments, a suppressor tRNA of the disclosure comprises a naturally-occurring modification. In some embodiments, the tRNA comprises one or more of the modifications 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-m ethylguanosine 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-m ethylguanosine or G at position 18; 3-(3-amino-3-carboxypropyl)uridine, D, orU 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-Attorney Docket No. TVD-013WO2 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-m ethylguanosine, peroxy wybutosine, wybutosine, G, or a modified G at position 37; pseudouridine, U, 5-methylcytidine, C, or a modified C at position 38; 1-methylpseudouridine, 2'-O-methylpseudouridine, 2'-O-methyluridine, pseudouridine, U, 2'-O-methylguanosine, or G at position 39; pseudouridine, U, 5-methylcytidine, or C at position 40; 2'-O-methyluridine, U, or a modified U at position 44; pseudouridine or U at position ell; pseudouridine or U at position el 2; pseudouridine or U at position el 4; 3-methylcytidine or C at position e2; 7-m ethylguanosine 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. 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. For the avoidance of doubt, unless otherwise stated or implied from context, a tRNA that was initially transcribed as a pre-tRNA comprising a nucleic acid sequence (e.g., SEQ ID NO: X) and that has subsequently undergone one or more naturally-occurring post-transcriptional modifications is understood to be within the scope of “a tRNA comprising the nucleic acid sequence of SEQ ID NO: X.”

[0122] In some embodiments, multiple (e.g, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more) 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 diseaseAttorney Docket No. TVD-013WO2 mediated by multiple, different PTCs in the same subject and / or treat a disease mediated by multiple, different PTCs in multiple, different subjects.

[0123] 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 (CCA) 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. Set. 105(23): 7953-7958). Accordingly, in some embodiments of any of the tRNAs disclosed herein, a CCA sequence may be added to the 3' end of the tRNA.Methods of Making Engineered Suppressor tRNAs

[0124] 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.

[0125] For example, DNA molecules encoding a tRNA of the disclosure can be synthesized chemically or by recombinant DNA methodologies. For example, a nucleic acid, e.g., DNA, encoding engineered tRNA can be chemically synthesized on an automated synthesizer 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 ( / .<., 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.Attorney Docket No. TVD-013WO2

[0126] 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.Expression Vectors

[0127] The suppressor tRNAs of the disclosure may be expressed in a cell of interest by incorporating a gene or nucleic acid, e.g., DNA, encoding the tRNA of interest into an appropriate expression vector.

[0128] 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.

[0129] 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).

[0130] 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 16.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 (e.g., SEQ ID NOs: 1153-1159), a promoter element (e.g., SEQ ID NOs: 917, 1132-1134, and 1151-1152), 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-013WO2TABLE 16LocationSEQ ID(Relative to Nucleotide SequenceNO:tRNA)CTACCCAGAGGCAGGCGGGAGACTCCCCCGAGCGTCCAAT AAGAGCGCCGCCAATGGAGCCGCCCGCCCGCGGGGGTGC AGAGGGACTTCCGGGTGAGGTCCTCCGCTACTTCCCTCCCC26 5'ACGGAAAAGATAGACCAGTCTGACGCGAGCCTGAAGGCG GCTACACGCTTTAAGCTAAGTAAAGGCACCTTCTCGCTGG C ACTTGTATGTTGTTTTTATCTGTCAGTTTGTTAATCCCAAGA TTCCCTTTGGAAATAAAGCGAAATTGACCGTAGTGGTTAT27 3' GACCAACTTCTAGTCTAAACTTAATTCTTGGAACTCAAGGA TCTGAGCAAACAACTGTCAGGGTGACACATTGCTTAAACG GTGACAGCGGTCGAGAGCCTTGTCCCGGATGGAGAGT ACTTGTATGTTGTTTTTATCTGTCAGTTTGTTAATCCCAAGA32 3' TTCCCTTTGGAAATAAAGCGAAATTGACCGTAGTGGTTAT GACCAACTTCTAGTCTAAACTT GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACG ATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACT GTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAA33 5' AGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGT TTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTA TTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGGG CGGAGGAAGGCACCTTCTCGCTGGC ACTTGTATGTTGTTTTTATCTGTCAGTTTGTTAATCCCAAGA34 3'TTCC GATCACCGGAAGAGGTGACAGACACCTCGGGGCCCATGA ACGTTTGGAATTCGTAAGGACATGAGAATCTCGGTGGTTC CGTGTCTGCCCGCCATCGCGGCCACCGGCCACGGGCCCAA173 5'GCCAAGTGTAGCGAAGCTTAGAAAAGGTTGCCCAACGTCA TGTGGCTTGAGAAGGCTGCCGGGCGCCTTAAGCCGCCAGC A CACTGAACCTTTTTTTGGCCTTAGAATCCCTGTTTTGGGGC CTGCAGGAAGTAGCAACCAACCCGAGCCTCCGCAGGGAAT174 3' GCACTGACCTGTAGAATGGACGTTCAGCTTCCCTCCCTGTG TCTCAACACGATTACATTTCAGGAACAGCCTGGGCTGGGA GGCACTGCGCACGCGCGCCGAGTCGGGCGGAAAAATAA CCAAAACATCTTTTACTGTAGTATCTACTTACCATACTACC869 5' CAAGAATGGCACACTGCTCACATCTTCAAAAGCTTAAACC AAGAGCACTACACAGGTGC TGTGTGTCGGGGCCGGTACCCTGCTTCCGGTTCCCGCACGC870 5' ATTCCCGGATTGCAGTGCGGACCCCTTCTGTAAGCGCGCG ATAAAGCGCGGTTTTGGAA TCATGTCATATAAGTAGAACCATACAATATATATATAAAA871 5' TCCAGGTTAATAGCCAATCTTACAACATTTCTCATATTTTTTGCAGTTGCTAAGCCATGGAttorney Docket No. TVD-013WO2 LocationSEQ ID(Relative to Nucleotide SequenceNO:tRNA)ACATTACAATACATATCAACATATCACCATAATTAAATTGC872 5' AAGTCTTCGTCAAAAGCAAGCCTTAAAGGAGTATCCCAAA AACACATTTTCCCCAGAAG AGACCTTTAGAGCGTGGTTAAACCCATATGTTGGGATTTAT873 5' GCTGCTTTTATGGTAGCAATACCCTATATTAAGATTTGAAG TAGACCCGGAAAGTTAGT GTTCATGAAAGAATAAATAAATGTTTAAAAAAAAAAAAA874 5' ACTGAGGTAAATTTCTATATTCTTTCATAAAAGCAGTTTAA AGACGAACGTTTTTCGAGGT GCTGGGTCTCGGTGACACTGACGACGGGAGGCGCGGTCGG875 5' AAGAGCGCGGGGCCGTCGCCTCTGGCTTAACATAGCAGAT GCGCTGAGACTCCAACAGGT CAGTGGCGGCGAAAACTCTCTGCGTTCTGGAGGGAGGGTG876 5' CGGGCAGGAGGAGGTAGAGGATGCCTTGTAAGCGGAGCA AAAACAAGGTTCAACGTCTGC CAAATCACTTGCCTCTCGGCGCGAGACCGCGATGCGCGGG877 5' GGCGGGAGCGTGATGATGGCATCGCGTAAGGAGAGGGTG TGAGAAGCCGGATCCTGTGGT CCCTGTGTCCGAAGAGGTCTGCGTTGCGACTTACGTGGTA878 5' GTGCTTGGAAGGTGCGGAGTAGATGAGAGATAAGTGAATG TGGACAAACCTGTCACGTAG GAGCGGAGCTCAGAGGGTGCGCGCTCCGCCCTTTCGCGGG879 5' CCTGGCATGAGCGCAGTGGTTGTTACACTAAAGTGTCTCC GCCTGTCGAATATTCTCGTG GTGTCACTGGTTTCAAATCAACCTCAATTTTTTTGGAGACG880 5' TGAGTGCTGAGCATTTTTTCTTCAGTGAAGTGACTTGGCAG CCAAAATCGCCAACGCCC TCCTGGCATGTCCCGCCCAAGTCCCTTAGCCCCGCTCCCCA881 5' ACCCTGCCCCATTCCCACTCTAGTACCCGTAAGCTACAAGA CGCCGCCGTTCGTCGGGT TGCTCAGTCGTCCTGCCGGGCGGGCCCTGAGGTTGCAAGG882 5' GACGGAGGAAGTTTCGTGCGTGCGCCCTTCCTATAGCGCC CAGTAGAACTGACAGTACCT TCCTCGGATTACGCATGCTCAGTGCAATCTTCGGTTGCCTG883 5' GACTAGCGCTCCGGTTTTTCTGTGCTGAACCTCAGGGGACG CCGACACACGTACACGTC GATAATTTCCTGAAAGAAAAGATCAATTCGATGTTACCAA884 5' ATCTGGGATATCCAGAAAAATTTTCTTCTTCTCCTAGGAGA AAAACTATCAAATGTCAGG TCTCTCACGGCAAACTGTTGCAGACTGTAGAGACGCTATG885 5' CCAAGAATCTTTTACTTAAAAGCAGGAATAGATTCAATAG GCAACTTCACTGCACATGTA CAACCTCCCCTTCTCAAGGAGCAGGTGGATTGGTCCCGAG886 5' CTAGCTGGTGGGCGGAGGTGACGTTTTTATAAGTTGCTCAAGAGACGGTAACAACCGACGAttorney Docket No. TVD-013WO2 LocationSEQ ID(Relative to Nucleotide SequenceNO:tRNA)GTGGAACTTCCACTGAATTACTCTTTTCGCATGTAAGATCA887 5' CTGAACCGTGATAATCATTGATCCTATTTGTAGAACTGTAT GAAACAGTTCCCTAAGGA TCGCTCAACAGGCGGCCAGGGTGCGAGCAGTGAAGCTGCG888 5' GCACGCCGGAGCGTTTAATGGCCATCAAATTGGCCTCTCT AGGAGGTAGCTGCAGCCGGA895 5' AAAGGCACCTTCTCGCTGGC896 3' ACTTGTATGTTGTTTTT897 5' TCTCGCTGGC AGCGCTCCGGTTTTTCTGTGCTGAACCTCAGGGGACGCCG900 5'ACACACGTACACGTC GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACG ATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACT GTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAA917 5' AGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGT TTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTA TTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAA ACACC GCTCCGCGGCACGAGAACTCAAAGCCCCGGGGCCTGGGTC CCACGCGGGGTCCCTTACCCAGGGTGCCCCGGGCGCTCAT TTGCATGTCCCACCCAACAGGTAAACCTGACAGGTCATCG CGGCCAGGTACGACCTGGCGGTCAGAGCACCAAACATACG1132 5'AGCCTTGTGATGAGTTCCGTTGCATGAAATTCTCCCAAAGG CTCCAAGATGGACAGGAAAGGGCGCGGTTCGGTCACCGTA AGTAGAATAGGTGAAAGACTCCCGTGCCTTATAAGGCCTG TGGGTGACTTCTTCTCAAC GCTCCGCGGCACGAGAACTCAAAGCCCCCGGCCTGGGTCC TACGCGGGGCCCTTTACCCAGGGTGCCCCGGGCGCTCATTT GCATGTCCCACCCAACAGGTAAACCTGACAGGTCATCGCG GCCAGGTACGACCTGGCGGTCAGAGCACCAAACATACGAG1133 5'CCTTGTGATGAGTTCCGTTGCATGAAATTCTCCCAAAGGCT CCAAGATGGACAGGAAAGGGCGCGGTTCGGTCACCGTAA GTAGAATAGGTGAAAGACTCCCGTGCCTTATAAGGCCTGT GGGTGACTTCTTCTCAAC GCCTGAGGCGTGGGGCCGCCTCCCAAAGACTTCTGGGAGG GCGGTGCGGCTCAGGCTCTGCCCCGCCTCCGGGGCTATTTG CATACGACCATTTCCAGTAATTCCCAGCAGCCACCGTAGCT ATATTTGGTAGAACAACGAGCACTTTCTCAACTCCAGTCA1134 5'ATAACTACGTTAGTTGCATTACACATTGGGCTAATATAAAT AGAGGTTAAATCTCTAGGTCATTTAAGAGAAGTCGGCCTA TGTGTACAGACATTTGTTCCAGGGGCTTTAAATAGCTGGTGGTGGAACTCAATATTCAttorney Docket No. TVD-013WO2 LocationSEQ ID(Relative to Nucleotide SequenceNO:tRNA)GCTCCGCGGCACGAGAACTCAAAGCCCCGGGGCCTGGGTC CCACGCGGGGTCCCTTACCCAGGGTGCCCCGGGCGCTCAT TTGCATGTCCCACCCAACAGGTAAACCTGACAGGTCATCG CGGCCAGGTACGACCTGGCGGTCAGAGCACCAAACATACG1151 5'AGCCTTGTGATGAGTTCCGTTGCATGAAATTCTCCCAAAGG CTCCAAGATGGACAGGAAAGGGCGCGGTTCGGTCACCGTA AGTAGAATAGGTGAAAGACTCCCGTGCCTTATAAGGCCTG TGGGTGACTTCTTCTCAACAACTT GCTCCGCGGCACGAGAACTCAAAGCCCCGGGGCCTGGGTC CCACGCGGGGTCCCTTACCCAGGGTGCCCCGGGCGCTCAT TTGCATGTCCCACCCAACAGGTAAACCTGACAGGTCATCG CGGCCAGGTACGACCTGGCGGTCAGAGCACCAAACATACG1152 5'AGCCTTGTGATGAGTTCCGTTGCATGAAATTCTCCCAAAGG CTCCAAGATGGACAGGAAAGGGCGCGGTTCGGTCACCGTA AGTAGAATAGGTGAAAGACTCCCGTGCCTTATAAGGCCTG TGGGTGACTTCTTCTCAACAGTGA CGACGCTGTGGCTGCTGGGATAGGGTGAAGACTGTGCCCT CGGCTGCTGGCTACTGACTTTAGGCACACTCCCACGCACG GAAAAAGTTCTATCTTTCGGGTCACGGGCTCCGGCAGCGT1153 5' CCGGATGCGGGCATTCGTCACTATTTCCTGGGAAGCGAAG ATGCTGAAGATTTTGGAGGGTGGAGTTTGCACAAGCGGCC CTAATGACGTAGGTAACATAAGAGCAAAGGAGAGACACC TGCCGGCAGC AGCTCTGTGAGCTTCGCGGTGGAAGGAGCGAGGGGAAAA CAAAAAATCTCACTCACACAATCCCTGGTGTTGGTAGAAA AAAGTAAACAGAAAATGAAAGGAAGTAAGGGGGATGATC1154 5' TGGTGCCAACGGCTGGATTTTTACATTTCTGAGTTTTGGGA ACTTTTTCTTTATTTCCAAAAAATCTACTTAGGGATTGATCTGAGGCCACATCAAAATAATAGATAACGTGAACCGTTGTATCACACAGC GGTCTCTAGTGGTGTACCCGCCGGGCTTAGCAAAACAAGA AAAAGACAGAATGAATGAAAGGAAGGAAGGGAGAAAAA GAAATTGCAAACAAGTTCAAACTTTTAAATAGTCTCAGCC1155 5' TGTTAAGTTAACTGTGCTTTGTTAATATTTTTTCTCTCTCCT CATTGAAAAATGCATAAACCTTAAACAGGTGTGAAACGTA TATGACGACATCACATAAGAAGAACATTTCAACACACCAG TGCAACCAA TAAAAGGGTGGTCACTGAAACCCCTTGTCATGAAACAGTG AAGCAGGTGACATTTGAACTTTCCTTCCTCAGAATTGTATT TTAATTGAAATGTCAGGGTTCGGAGAAATTTGCCGAGGAT1156 5' CATAAACAAGTTAGCAAAGATCGCAGGTTCTTTCCAGTTC CGAGAAGTTCAGAAAAGTTTCTTTGGTGATTGAAATAACG TTCGCCTTTAAACTTCTCAAGAGGTTTAGGGGGGGTTTTAGTAAGCGGAttorney Docket No. TVD-013WO2 LocationSEQ ID(Relative to Nucleotide SequenceNO:tRNA)TCCGGAAGTTTCTACCCGGAGCCAGTTGCCTTCTGGTAACA GAATTATATTGCACCTACTGCTTCCTATTCCCTGAATCACT AGCGCTCCCGAAGGCTTGGAGGGAGGAGTCTCTGGGCACC1157 5' CGGGTGAAAGGAAGGTTCACGTGCAACCGCCGCGTCTTTT TTTCCCTGAAGCGCTTTCATGGAGGCAGATGTTTGTCAGTC AAGGGAAGTAAGAAAGGCATTGGATGAAAACGAAGCCCT AAGCCTC TCTGTTAAGCTGGGACAGCCAGTGACTGCCCACTGCAGGG CAGATGGAACAGGGGTGGCAGAGGTAAAGATATGCCTGG CACATGAAATATGGTCAGGTGTTCACAGGTAGACCAGGGA1158 5' GTGAGTGATGGCAGTGAGGGCCAGGCTATCATGGGCAGGC TCCCCACACCCCTCAGGAACATGGCTGAGTCCAGAAACAG CCATCAGCTGCACAGTTGTAAAGGTGAAATTAAGATGTAG AACAAGGCCT GCTTTTGCGCAGGCGCGGGGACTACGGCGCAGGCGCGGAG ACTATTGCGCAGGCAAGCGCGTACGCAGAAGCGTGCGCGC GCCCGTTCAACGTCCGGAGCATCGGTGCAGTTTCGAGGGT AAAGCCTTTGGCGCGGTGATGTGGACTTTTGTTCTCTAACT1159 5'ACAACTCCCAGCATACGTCACCCCTCACGTGGGCGCTAGG TGTGGTTTCGTGGGATAGGGTAAGGCGAAAATGAAGTGAC GATGACGTACCAAAGAGGAAAATAGTTGAATATGTTGTGTGAGCGCGTCGTTTGCAGAA

[0131] An expression vector of the disclosure can comprise a nucleotide sequence encoding a tRNA set forth in any of TABLES 1-15. In some embodiments, the expression vector comprises a nucleotide sequence encoding a tRNA set forth in any of TABLES 1-15, and further comprises an additional 5' nucleotide sequence, an additional 3' nucleotide sequence, or a combination of both 5' and 3' nucleotide sequences set forth in TABLE 16.

[0132] In another aspect, an expression vector of the disclosure can comprise a nucleic acid comprising: (a) a U6 promoter comprising a nucleic acid sequence selected from any one of SEQ ID NOs: 917, 1132-1134, and 1151-1152; and (b) a nucleic acid encoding a suppressor tRNA. In certain embodiments, the U6 promoter comprises a U6-1 promoter comprising the nucleic acid sequence of SEQ ID NO: 917. In certain embodiments, the U6 promoter comprises a U6-8 promoter comprising the nucleic acid sequence of SEQ ID NO: 1132, SEQ ID NO: 1133, SEQ ID NO: 1151, or SEQ ID NO: 1152. In certain embodiments, the U6 promoter comprises a U6-8 promoter comprising the nucleic acid sequence of SEQ ID NO: 1132. In certain embodiments, the U6 promoter comprises a U6-9 promoter comprising the nucleic acid sequence of SEQ ID NO: 1133. In certain embodiments, theAttorney Docket No. TVD-013WO2 nucleic acid encoding the suppressor tRNA comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 254-274, 918, 925, 1149, and 1150, wherein Ni is a thymine (T) or cytosine (C) and N2 is thymine (T) or cytosine (C), provided that when Ni is a cytosine (C) then N2 is a thymine (T). In other embodiments, the suppressor tRNA comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 175-237, 901-909, 915-916, 921-924, 1000-1119, 1122-1123, and 1125-1131, wherein each thymine in any one of SEQ ID NOs: 175-237, 901-909, 915-916, 921-924, 1000-1119, 1122-1123, and 1125-1131 is replaced by uracil. In other embodiments, the suppressor tRNA comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 238-252, 910-914, 1120, 1121, and 1124 or the nucleic acid encoding the suppressor tRNA comprises the nucleic acid sequence of any one of SEQ ID NOs: 238-253, 910-914, 1120, 1121, and 1124.

[0133] In another aspect, an expression vector of the disclosure can comprise a nucleic acid comprising: (a) a 5' flanking sequence comprising a nucleic acid sequence selected from any one of SEQ ID NOs: 1153-1156 and 1159; and (b) a nucleic acid encoding a suppressor tRNA. In certain embodiments, the nucleic acid encoding the suppressor tRNA comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 254-274, 918, 925, 1149, and 1150, wherein Ni is a thymine (T) or cytosine (C) and N2 is thymine (T) or cytosine (C), provided that when Ni is a cytosine (C) then N2 is a thymine (T). In other embodiments, the suppressor tRNA comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 175-237, 901-909, 915-916, 921-924, 1000-1119, 1122-1123, and 1125-1131, wherein each thymine in any one of SEQ ID NOs: 175-237, 901-909, 915-916, 921-924, 1000-1119, 1122-1123, and 1125-1131 is replaced by uracil. In other embodiments, the suppressor tRNA comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 238-252, 910-914, 1120, 1121, and 1124 or the nucleic acid encoding the suppressor tRNA comprises the nucleic acid sequence of any one of SEQ ID NOs: 238-253, 910-914, 1120, 1121, and 1124.

[0134] In another aspect, an expression vector of the disclosure can comprise a nucleic acid comprising: (a) a 5' flanking sequence comprising a nucleic acid sequence selected from any one of SEQ ID NO: 1157 and SEQ ID NO: 1158; and (b) a nucleic acid encoding a suppressor tRNA. In certain embodiments, the nucleic acid encoding the suppressor tRNA comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 254-274, 918, 925, 1149, and 1150, wherein Ni is a thymine (T) or cytosine (C) and N2 is thymine (T) or cytosine (C), provided that when Ni is a cytosine (C) then N2 is a thymine (T). In other embodiments, the suppressor tRNA comprises a nucleic acid sequence selected from any oneAttorney Docket No. TVD-013WO2 of SEQ ID NOs: 175-237, 901-909, 915-916, 921-924, 1000-1119, 1122-1123, and 1125-1131, wherein each thymine in any one of SEQ ID NOs: 175-237, 901-909, 915-916, 921-924, 1000-1119, 1122-1123, and 1125-1131 is replaced by uracil. In other embodiments, the suppressor tRNA comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 238-252, 910-914, 1120, 1121, and 1124 or the nucleic acid encoding the suppressor tRNA comprises the nucleic acid sequence of any one of SEQ ID NOs: 238-253, 910-914, 1120, 1121, and 1124.

[0135] In some embodiments, a gene encoding an engineered tRNA 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.

[0136] 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 P-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus 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.

[0137] 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 (AAV) 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., AAV), 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.Attorney Docket No. TVD-013WO2 Exemplary RNA viruses include bunyaviruses (e.g., hantavirus), coronaviruses, flaviviruses (e.g., yellow fever virus, West Nile virus, and dengue virus), hepatitis viruses (e.g., hepatitis A virus, hepatitis C virus, and hepatitis E virus), influenza viruses (e.g., influenza virus type A, influenza virus type B, and 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.

[0138] 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 e.g., AAV-293 or HEK293T), 911, A549, HER96, or PER-C6 cells. Specific production and purification conditions will vary depending upon the virus and the production system employed.

[0139] 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 gradient centrifugation, clarification, enzymatic treatment, e.g., benzonase or protease treatment, chromatographic steps, e.g., ion exchange chromatography or filtration steps.Retroviral Vectors Including Lentivirus Vectors

[0140] 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.

[0141] 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), felineAttorney Docket No. TVD-013WO2 immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV), and caprine arthritis encephalitis virus (CAEV).

[0142] 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 ( / .<., 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, and 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.

[0143] 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.

[0144] 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, repeat (R), and U5 regions. LTRs generally provide functions fundamental to the expression of retroviralAttorney Docket No. TVD-013WO2 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.

[0145] 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' LTR, or both 3' and 5' LTRs, are also contemplated to be useful in the practice of the disclosure.

[0146] 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.Attorney Docket No. TVD-013WO2

[0147] 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 (T) 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 (T) sequence) needed for encapsidation of the viral genome.

[0148] 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 c / .s-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.

[0149] In some embodiments, the retroviral vector (e.g., lentiviral vector) further comprises an export element. In one embodiment, retroviral vectors comprise one or more export elements. The term “export element” refers to a c / .s-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.

[0150] 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., woodchuckAttorney Docket No. TVD-013WO2 hepatitis virus posttranscriptional regulatory element (WPRE; see Zufferey etal., (1999) J. VIROL., 73:2886); the posttranscriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al., MO. CE. 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.

[0151] 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, ATT AAA, and AGTAAA), a bovine growth hormone polyadenylation sequence (BGHpA), a rabbit P-globin polyadenylation sequence (rPgpA), or another suitable heterologous or endogenous polyadenylation sequence known in the art.

[0152] Non-limiting examples of lentiviral vectors include pLVX-EFlalpha-AcGFPl-Cl (Clontech Catalog #631984), pLVX-EFlalpha-IRES-mCherry (Clontech Catalog #631987), pLVX-Puro (Clontech Catalog #632159), pLVX-IRES-Puro (Clontech Catalog #632186), pLenti6 / V5-DESTTM (Thermo Fisher), pLenti6.2 / V5-DESTTM (Thermo Fisher), pLKO.l (Plasmid #10878 at Addgene), pLKO.3G (Plasmid #14748 at Addgene), pSico (Plasmid #11578 at Addgene), pLJMl-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-mCherryAttorney Docket No. TVD-013WO2 (Plasmid #36084 at Addgene), pLionll (Plasmid #1730 at Addgene), and plnducerlO-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.Adenoviral Vectors

[0153] 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.

[0154] 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, 1 1, 14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-48), subgroup E (e.g., serotype 4), subgroup F (e.g., serotypes 40 and 41 ), an unclassified serogroup (e.g., serotypes 49 and 51), or any other adenoviral serogroup or serotype. Adenoviral serotypes 1 through 51 are available from the American Type Culture Collection (ATCC, Manassas, Virginia). Non-group C adenoviral vectors, methods of producing non-group C adenoviral vectors, and methods of using non-group C adenoviral vectors are disclosed in, for example, U. S. Patent Nos. 5,801,030; 5,837,511; and 5,849,561; and PCT Publication Nos. WO 1997 / 012986 and WO 1998 / 053087.

[0155] Non-human adenovirus (e.g., ape, simian, avian, canine, ovine, or bovine adenoviruses) can be used to generate the adenoviral vector (z.e., as a source of theAttorney Docket No. TVD-013WO2 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):el000503. 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.

[0156] 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 replicationessential 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.

[0157] Preferably, the adenoviral vector is replication-deficient, such that the replicationdeficient adenoviral vector requires complementation of at least one replication-essential gene function of one or more regions of the adenoviral genome for propagation (e.g., to form adenoviral vector particles). The adenoviral vector can be deficient in one or more replication-essential gene functions of only the early regions (i.e., E1-E4 regions) of the adenoviral genome, only the late regions (i.e., L1-L5 regions) of the adenoviral genome, both the early and late regions of the adenoviral genome, or all adenoviral genes (i.e., a high capacity adenovector (HC-Ad)). See, e.g., Morsy et al. (1998) PROC. NATL. ACAD. SCI. USA 95: 965-976, Chen et al. (1997) PROC. NATL. ACAD. Set. 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;Attorney Docket No. TVD-013WO2 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.

[0158] 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.

[0159] 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.

[0160] 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.AA V Vectors

[0161] In certain embodiments, an expression vector is an adeno-associated virus (AAV) vector. AAV is a small, nonenveloped icosahedral virus of the genus Dependoparvovirus and family Parvovirus. AAV has a single-stranded linear DNA genome of approximately 4.7Attorney Docket No. TVD-013WO2 kb. AAV are capable of infecting both dividing and quiescent cells of several tissue types, with different AAV serotypes exhibiting different tissue tropism.

[0162] AAV includes numerous serologically distinguishable types including serotypes AAV-1 to AAV-12, as well as more than 100 serotypes from nonhuman primates (See, e.g., Srivastava (2008) J. CELL BIOCHEM., 105(1): 17-24, and Gao et al. (2004) J. VIROL., 78(12), 6381-6388). The serotype of the AAV vector used can be selected by a skilled person in the art based on the efficiency of delivery, tissue tropism, and immunogenicity. For example, AAV-1, AAV-2, AAV-4, AAV-5, AAV-8, and AAV-9 can be used for delivery to the central nervous system; AAV-1, AAV-8, and AAV-9 can be used for delivery to the heart; AAV-2 can be used for delivery to the kidney; AAV-7, AAV-8, and AAV-9 can be used for delivery to the liver; AAV-4, AAV-5, AAV-6, AAV-9 can be used for delivery to the lung, AAV-8 can be used for delivery to the pancreas, AAV-2, AAV-5, and AAV-8 can be used for delivery to the photoreceptor cells; AAV-1, AAV-2, AAV-4, AAV-5, and AAV-8 can be used for delivery to the retinal pigment epithelium; AAV-1, AAV-6, AAV-7, AAV-8, and AAV-9 can be used for delivery to the skeletal muscle. AAV serotypes identified from rhesus monkeys, e.g., rh8, rhlO, rh39, rh43, and rh74, are also contemplated. Besides the natural AAV serotypes, modified AAV capsids have been developed for improving efficiency of delivery, tissue tropism, and immunogenicity. For example, in some embodiments, the AAV used is MyoAAV 2A (Tabebordbar et al. (2021) CELL 184(19):4914-4938). Exemplary natural and modified AAV capsids are disclosed in U. S. Patent Nos. 7,906,111; 9,493,788; and 7,198,951; and PCT Publication No. WO 2017 / 189964.

[0163] The wild-type AAV genome contains two 145 nucleotide inverted terminal repeats (ITRs), which contain signal sequences directing AAV replication, genome encapsidation and integration. In addition to the ITRs, three AAV promoters, p5, pl 9, and p40, drive expression of two open reading frames encoding Rep and Cap genes. Two Rep promoters, coupled with differential splicing of the single AAV intron, result in the production of four rep proteins (Rep 78, Rep 68, Rep 52, and Rep 40) from the Rep gene. Rep proteins are responsible for genomic replication. The Cap gene is expressed from the p40 promoter, and encodes three capsid proteins (VP1, VP2, and VP3) which are splice variants of the Cap gene. These proteins form the capsid of the AAV particle.

[0164] Because the c / .s-acting signals for replication, encapsidation, and integration areAttorney Docket No. TVD-013WO2 contained within the ITRs, some or all of the 4.3 kb internal genome may be replaced with foreign DNA, for example, an expression cassette for an exogenous gene of interest e.g., a suppressor tRNA. Accordingly, in certain embodiments, the AAV vector comprises a genome comprising an expression cassette for an exogenous gene flanked by a 5' ITR and a 3' ITR. The ITRs may be derived from the same serotype as the capsid or a derivative thereof. Alternatively, the ITRs may be of a different serotype from the capsid, thereby generating a pseudotyped AAV. At least one of the ITRs may be modified to mutate or delete the terminal resolution site, thereby allowing production of a self-complementary AAV vector.

[0165] The Rep and Cap proteins can be provided in trans, for example, on a plasmid, to produce an AAV vector. A host cell line permissive of AAV replication must express the Rep and Cap genes, the ITR-flanked expression cassette, and helper functions provided by a helper virus, for example adenoviral genes Ela, Elb55K, E2a, E4orf6, and VA (Weitzman et al., (2011) ABEND- ASSOCIATED VIRUS: METHODS AND PROTOCOLS, pp. 1-23). Methods for generating and purifying AAV vectors have been described in detail (See e.g., Mueller et al., (2012) CURRENT PROTOCOLS IN MICROBIOLOGY, 14D.1.1-14D.1.21, Production and Discovery of Novel Recombinant Adeno-Associated Viral Vectors). Numerous cell types are suitable for producing AAV vectors, including HEK293 cells, COS cells, HeLa cells, BHK cells, Vero cells, as well as insect cells (See e.g., U. S. Patent Nos. 6,156,303;5,387,484; 5,741,683; 5,691,176; 5,688,676; and 8,163,543; U. S. Patent Publication No. 20020081721; and PCT Publication Nos. WOOO / 47757, WOOO / 24916; and WO96 / 17947). AAV vectors are typically produced in these cell types by one plasmid containing the ITR-flanked expression cassette, and one or more additional plasmids providing the additional AAV and helper virus genes.

[0166] AAV of any serotype may be used. Similarly, it is contemplated that any adenoviral type may be used, and a person of skill in the art will be able to identify AAV and adenoviral types suitable for the production of their desired recombinant AAV vector (rAAV). AAV particles may be purified, for example by affinity chromatography, iodixonal gradient, or CsCl gradient. Both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors may be used.

[0167] Non-limiting examples of AAV vectors include pAAV-MCS (Agilent Technologies), pAAVK-EFla-MCS (System Bio Catalog # AAV502A-1), pAAVK-EFla-Attorney Docket No. TVD-013WO2 MCS1-CMV-MCS2 (System Bio Catalog # AAV503A-1), pAAV-ZsGreenl (Clontech Catalog #6231), pAAV-MCS2 (Addgene Plasmid #46954), AAV-Stuffer (Addgene Plasmid #106248), pAAVscCBPIGpluc (Addgene Plasmid #35645), AAVSl_Puro_PGKl_3xFLAG_Twin_Strep (Addgene Plasmid #68375), pAAV-RAM-d2TTA:: TRE-MCS-WPRE-pA (Addgene Plasmid #63931), pAAV-UbC (Addgene Plasmid #62806), pAAVSl-P-MCS (Addgene Plasmid #80488), pAAV-Gateway (Addgene Plasmid #32671), pAAV-Puro_siKD (Addgene Plasmid #86695), pAAVSl-Nst-MCS (Addgene Plasmid #80487), pAAVSl-Nst-CAG-DEST (Addgene Plasmid #80489), pAAVSl-P-CAG-DEST (Addgene Plasmid #80490), pAAVf-EnhCB-lacZnls (Addgene Plasmid #35642), and pAAVSl-shRNA (Addgene Plasmid #82697). These vectors can be modified to be suitable for therapeutic use. For example, an exogenous gene of interest can be inserted in a multiple cloning site, and a selection marker (e.g., puro or a gene encoding a fluorescent protein) can be deleted or replaced with another (same or different) exogenous gene of interest. Further examples of AAV vectors are disclosed in U. S. Patent Nos. 5,871,982; 6,270,996; 7,238,526; 6,943,019; 6,953,690; 9,150,882; and 8,298,818; U. S. Patent Publication No. 2009 / 0087413; and PCT Publication Nos. WO 2017 / 075335; WO 2017 / 075338; and WO 2017 / 201258.II. PHARMACEUTICAL COMPOSITIONS, MEDICAMENTS, AND ROUTES OF ADMINISTRATION

[0168] For therapeutic use, a nucleic acid, a tRNA, and / or an expression vector (including viral particles that contain a nucleic acid encoding one or more genes of interest) 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 or 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 withAttorney Docket No. TVD-013WO2 pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.

[0169] 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, and tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol, and sorbitol); delivery vehicles; diluents; excipients; and / or pharmaceutical adjuvants (see, REMINGTON’S PHARMACEUTICAL SCIENCES, 18thed. (Mack Publishing Company, 1990).

[0170] In some embodiments, a pharmaceutical composition may contain nanoparticles, e.g., polymeric nanoparticles, liposomes, or micelles (see, Anselmo et al. (2016) BlOENG. TRANSL. MED. 1: 10-29).

[0171] 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 andAttorney Docket No. TVD-013WO2 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.

[0172] 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 intramuscular (IM), 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 intramuscular 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.

[0173] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0174] 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 PCTAttorney Docket No. TVD-013WO2 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 exonucleases 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) J. CONTROL. 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 etal. (2003) supra, Verma etal. (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 etal. (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 poly amidoamines (Tomalia et al. (2007) BIOCHEM. Soc. TRANS. 35:61-67; Noo etal. (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.

[0175] 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.

[0176] The compositions described herein may be administered locally or systemically. In some embodiments, the pharmaceutical composition is administered intramuscularly. In some embodiments, the pharmaceutical composition is administered parenterally. In someAttorney Docket No. TVD-013WO2 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.

[0177] 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 and 1 mg / kg to 10 mg / kg. In certain embodiments, a therapeutically effective amount of a viral expression vector is in the range of 102to 1015plaque forming units (pfus), e.g., 102to 1010102to 105, 105to 1015, 105to 1010, or 1010to 1015plaque forming units. The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the 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-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.

[0178] 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

[0179] The disclosure provides a method of treating a disease or disorder associated with a protein encoded by a gene including a PTC (e.g., a muscular dystrophy or a cardiology disorder) 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.

[0180] In some embodiments, the tRNA permits an amino acid to be incorporated into theAttorney Docket No. TVD-013WO2 gene product at a position corresponding to a PTC i.e., the tRNA permits read-through of the PTC), but the tRNA does not permit a substantial amount of amino acid to be incorporated into a gene product at a position corresponding to a native stop codon (i.e., the tRNA does not permit read-through of a native stop codon). For example, in 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.

[0181] 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 17 or TABLE 18 below, and / or the gene with a premature termination codon is a gene listed in the corresponding row of TABLE 17 or TABLE 18 below.TABLE 17Gene DisorderDMD Duchenne muscular dystrophy (DMD)CAPN3 Limb GirdleFKRP Limb GirdleDNAJB6 Limb GirdleGMPPB Limb Girdle & CongenitalDYSF Limb GirdleDAG1 Limb GirdlePOMT2 Limb GirdlePOMT1 Limb Girdle & CongenitalFKTN Limb Girdle etc.LMNA L-CMD, Emery-DreifussFHL1 Emery-DreifussSYNE1 Emery-DreifussEMD Emery-DreifussSYNE2 Emery-DreifussPYGM McArdle / GSDVAttorney Docket No. TVD-013WO2Gene DisorderDES DesminopathyCACNA1S Hypokalemic Periodic ParalysisRYR1 Malignant HyperthermiaACTA1 Actin-accumulation myopathyMYBPC1 Congenital MyopathyTPM3 Congenital MyopathySTAC3 Congenital MyopathyMYH2 Congenital MyopathyCOLQ Congenital myasthenic syndromeDOK7 Congenital myasthenic syndromeCHRNE Congenital myasthenic syndromeRAPSN Congenital myasthenic syndromeMUSK Congenital myasthenic syndromeCHRNB1 Congenital myasthenic syndromeDPAGT1 Congenital myasthenic syndromeCRPPA Muscular Dy strophy-DystroglycanopathySGCA Limb GirdleSGCB Limb GirdleSGCD Limb GirdleSGCG Limb GirdleMYOT Limb GirdleTNPO3 Limb GirdleHNRNPDL Limb GirdlePLEC Limb GirdleTRAPPC11 Limb GirdleTCAP Limb GirdleANO5 Limb GirdlePOMK DystroglycanopathyLAMA2 Limb GirdleCAPN3 Muscular dystrophy, limb-girdle, autosomal dominant 4 FKRP Autosomal recessive limb-girdle muscular dystrophyAttorney Docket No. TVD-013WO2 Gene DisorderDNAJB6 Autosomal dominant limb-girdle muscular dystrophy type ID Muscular dystrophy-dystroglycanopathy (congenital with brain and eye GMPPB anomalies), type A14DAG1 Autosomal recessive limb-girdle muscular dystrophy type 2P P0MT2 Autosomal recessive limb-girdle muscular dystrophy type 2N DYSF Autosomal recessive limb-girdle muscular dystrophy type 2B Muscular dystrophy-dystroglycanopathy (congenital with brain and eye P0MT1 anomalies), type AlMuscular dystrophy-dystroglycanopathy (congenital with brain and eye FKTN anomalies), type A, 4LMNA Emery-Dreifuss muscular dystrophy 3, autosomal recessive FHL1 X-linked myopathy with postural muscle atrophySYNE1 Emery-Dreifuss muscular dystrophy 4, autosomal dominantEMD X-linked Emery-Dreifuss muscular dystrophySYNE2 Emery-Dreifuss muscular dystrophy 5, autosomal dominant PYGM Glycogen storage disease, type V, McArdle diseaseDES Desmin-related myofibrillar myopathyCACNA1S Congenital myopathy 18RYR1 Congenital myopathy with fiber type disproportionACTA1 Actin accumulation myopathyHDAC1 Congenital myopathy 11MYBPC1 Myopathy, congenital, with tremorTPM3 Congenital myopathy with fiber type disproportionSTAC3 Bailey-Bloch congenital myopathyMYH2 Myopathy, proximal, and ophthalmoplegiaCOLQ Congenital myasthenic syndrome 5D0K7 Congenital myasthenic syndrome 10CHRNE Congenital myasthenic syndrome 4ARAPSN Congenital myasthenic syndrome 11MUSK Congenital myasthenic syndrome 9CHRNB1 Congenital myasthenic syndrome 2AAttorney Docket No. TVD-013WO2 Gene DisorderDPAGT1 Congenital myasthenic syndrome 13Muscular dystrophy-dystroglycanopathy (congenital with brain and eye CRPPA anomalies), type A, 7SGCA Autosomal recessive limb-girdle muscular dystrophy type 2D SGCB Autosomal recessive limb-girdle muscular dystrophy type 2E SGCD Autosomal recessive limb-girdle muscular dystrophy type 2F MYOT Myofibrillar myopathy 3TNPO3 Autosomal dominant limb-girdle muscular dystrophy type IFTTN Autosomal recessive limb-girdle muscular dystrophy type 2J HNRNPDL Autosomal dominant limb-girdle muscular dystrophy type 1G PLEC Autosomal recessive limb-girdle muscular dystrophy type 2Q TRAPPC11 Autosomal recessive limb-girdle muscular dystrophy type R18 TCAP Autosomal recessive limb-girdle muscular dystrophy type 2G SGCG Autosomal recessive limb-girdle muscular dystrophy type 2C AN05 Autosomal recessive limb-girdle muscular dystrophy type 2L POMK Limb-girdle muscular dystrophy due to POMK deficiency LAMA2 Merosin deficient congenital muscular dystrophyTABLE 18Gene DisorderTTN TitinopathyTTN Dilated cardiomyopathy (DCM)LMNA Dilated cardiomyopathy (DCM)FLNC Dilated cardiomyopathy (DCM)MYBPC3 Hypertrophic cardiomyopathy (HCM)BAG3 Dilated cardiomyopathy (DCM)RBM20 Dilated cardiomyopathy (DCM)LAMP2 Dilated cardiomyopathy (DCM)SCN5A Brugada syndromeKCNH2 Long QT syndrome (LQTS)KCNQ1 Long QT syndrome (LQTS)Attorney Docket No. TVD-013WO2 Gene DisorderFXN Friedreich's ataxiaLDLR Cholesterolemia

[0182] For example, in some embodiments, the disorder is a cardiology disorder e.g., a titinopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, Brugada syndrome, long QT syndrome, Friderich’s ataxia, or cholesterol emia).

[0183] For example, in certain embodiments, the subject has a TTN gene with a PTC, and the method comprises administering to the subject an effective amount of a suppressor tRNA comprising a nucleotide sequence selected from any one of SEQ ID NOs: 175-274, 901-916, 918, 921-925, 1000-1119, 1122-1123, 1125-1131, 1149, and 1150 or a nucleic acid or expression vector encoding the same. In certain embodiments, the suppressor tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 175-253, 901-916, 921-924, 1000-1119, 1122-1123, and 1125-1131. In certain embodiments, the suppressor tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 254-274, 918, 925, 1149, and 1150. In certain embodiments, the suppressor tRNA is tri 511 (SEQ ID NO: 187). In certain embodiments, the suppressor tRNA is trl622 (SEQ ID NO: 916). In certain embodiments, the suppressor tRNA is trl723 (SEQ ID NO: 1088). In certain embodiments, the suppressor tRNA is trl773 (SEQ ID NO: 1091). In certain embodiments, the suppressor tRNA is trl794 (SEQ ID NO: 1104). In certain embodiments, the suppressor tRNA is trl806 (SEQ ID NO: 1106). In certain embodiments, the suppressor tRNA is tri 821 (SEQ ID NO: 1112). In certain embodiments, the suppressor tRNA is trl624 (SEQ ID NO: 1122). In certain embodiments, the suppressor tRNA is trl625 (SEQ ID NO: 1123). In certain embodiments, the suppressor tRNA is trl626 (SEQ ID NO: 1125). In certain embodiments, the suppressor tRNA is trl627 (SEQ ID NO: 1126). In certain embodiments, the suppressor tRNA is tri 853 (SEQ ID NO: 1127). In certain embodiments, the suppressor tRNA is tri 854 (SEQ ID NO: 1128). In certain embodiments, the suppressor tRNA is tri 857 (SEQ ID NO: 1129). In certain embodiments, the suppressor tRNA is tri 858 (SEQ ID NO: 1130).

[0184] In certain cases, production of the tRNA in the subject increases expression of the TTN gene, and the increased expression ameliorates at least one symptom of the cardiology disorder (e.g., ameliorates at least one symptom of a titinopathy or dilated cardiomyopathy). In certain embodiments, production of the tRNA in the subject increases expression of the TTN gene by at least about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, aboutAttorney Docket No. TVD-013WO2 15%, about 20%, about 25%, about 30% or more, as compared to a suitable control. For example, in some embodiments, production of the tRNA in the subject increases expression of the TTN gene by at least about 5%, as compared to expression of the TTN in the subject prior to administration. In certain embodiments, production of the tRNA in the subject increases expression of full-length titin encoded by the TTN gene, and the increased expression ameliorates at least one symptom of the cardiology disorder. Other suitable controls may include, for example, the average expression of TTN in subjects having a TTN gene with a PTC and not administered a suppressor tRNA described herein. In certain embodiments, production of the tRNA in the subject increases expression of the TTN gene by about 5-30%, about 10-25%, or about 15-20%, as compared to expression of the TTN in the subject prior to administration.

[0185] Alternatively, in certain embodiments, the disorder is a muscular dystrophy (e.g., Duchenne muscular dystrophy, limb girdle syndrome, congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy, McArdle disease, Glycogen storage disease type V, a desminopathy, hypokalemic periodic paralysis, malignant hyperthermia, actin-accumulation myopathy, congenital myopathy, congenital myasthenic syndrome, dystroglycanopathy, Muscular dystrophy, limb-girdle, autosomal dominant 4, autosomal recessive limb-girdle muscular dystrophy, autosomal dominant limb-girdle muscular dystrophy type ID, muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A14, autosomal recessive limb-girdle muscular dystrophy type 2P, autosomal recessive limb-girdle muscular dystrophy type 2N, autosomal recessive limb-girdle muscular dystrophy type 2B, muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type Al, muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 4, emery -Dreifuss muscular dystrophy 3, autosomal recessive, X-linked myopathy with postural muscle atrophy, emery-Dreifuss muscular dystrophy 4, autosomal dominant, X-linked Emery-Dreifuss muscular dystrophy, Emery-Dreifuss muscular dystrophy 5, autosomal dominant, glycogen storage disease, type V, McArdle disease, desmin-related myofibrillar myopathy, congenital myopathy 18, congenital myopathy with fiber type disproportion, actin accumulation myopathy, congenital myopathy 11, myopathy, congenital, with tremor, congenital myopathy with fiber type disproportion, Bailey-Bloch congenital myopathy, myopathy, proximal, and ophthalmoplegia, congenital myasthenic syndrome 5, congenital myasthenic syndrome 10, congenital myasthenic syndrome 4 A, congenital myasthenic syndrome 11, congenital myasthenic syndrome 9, congenital myasthenicAttorney Docket No. TVD-013WO2 syndrome 2A, congenital myasthenic syndrome 13, muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 7, autosomal recessive limb-girdle muscular dystrophy type 2D, autosomal recessive limb-girdle muscular dystrophy type 2E, autosomal recessive limb-girdle muscular dystrophy type 2F, myofibrillar myopathy 3, autosomal dominant limb-girdle muscular dystrophy type IF, autosomal recessive limbgirdle muscular dystrophy type 2J, autosomal dominant limb-girdle muscular dystrophy type 1G, autosomal recessive limb-girdle muscular dystrophy type 2Q, autosomal recessive limbgirdle muscular dystrophy type R18, autosomal recessive limb-girdle muscular dystrophy type 2G, autosomal recessive limb-girdle muscular dystrophy type 2C, autosomal recessive limb-girdle muscular dystrophy type 2L, limb-girdle muscular dystrophy due to POMK deficiency, limb-girdle muscular dystrophy type 2A, or merosin deficient congenital muscular dystrophy).

[0186] For example, in some embodiments, the subject has a DMD gene with a PTC, and the method comprises administering to the subject an effective amount of a suppressor tRNA comprising a nucleotide sequence selected from any one of SEQ ID NOs: 175-274, 901-916, 918, 921-925, 1000-1119, 1122-1123, 1125-1131, 1149, and 1150 or a nucleic acid or expression vector encoding the same. In some embodiments, the suppressor tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 175-253, 901-916, 921-924, 1000-1119, 1122-1123, and 1125-1131. In some embodiments, the suppressor tRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 254-274, 918, 925, 1149, and 1150. In certain embodiments, the suppressor tRNA is tri 511 (SEQ ID NO: 187). In certain embodiments, the suppressor tRNA is trl622 (SEQ ID NO: 916). In certain embodiments, the suppressor tRNA is trl723 (SEQ ID NO: 1088). In certain embodiments, the suppressor tRNA is trl773 (SEQ ID NO: 1091). In certain embodiments, the suppressor tRNA is trl794 (SEQ ID NO: 1104). In certain embodiments, the suppressor tRNA is trl806 (SEQ ID NO: 1106). In certain embodiments, the suppressor tRNA is tri 821 (SEQ ID NO: 1112). In certain embodiments, the suppressor tRNA is trl624 (SEQ ID NO: 1122). In certain embodiments, the suppressor tRNA is trl625 (SEQ ID NO: 1123). In certain embodiments, the suppressor tRNA is trl626 (SEQ ID NO: 1125). In certain embodiments, the suppressor tRNA is trl627 (SEQ ID NO: 1126). In certain embodiments, the suppressor tRNA is tri 853 (SEQ ID NO: 1127). In certain embodiments, the suppressor tRNA is tri 854 (SEQ ID NO: 1128). In certain embodiments, the suppressor tRNA is tri 857 (SEQ ID NO: 1129). In certain embodiments, the suppressor tRNA is tri 858 (SEQ ID NO: 1130).Attorney Docket No. TVD-013WO2

[0187] In certain cases, production of the tRNA in the subject increases expression of the DMD gene, and the increased expression ameliorates at least one symptom of DMD. In some embodiments, production of the tRNA in the subject increases expression of the DMD gene by at least about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30% or more, as compared to a suitable control. For example, in some embodiments, production of the tRNA in the subject increases expression of the DMD gene by at least about 5% of a DMD gene expression level in a subject not having DMD or the subject that is being treated prior to the start of treatment.

[0188] In some embodiments, production of the tRNA in the subject increases expression of the DMD gene by about 5-30%, about 10-25%, or about 15-20% of a DMD gene expression level in a subject not having DMD or the subject that is being treated prior to the start of treatment.

[0189] In certain cases, production of the suppressor tRNA in the subject increases expression of full-length dystrophin encoded by the DMD gene, whereupon the increased expression ameliorates at least one symptom of DMD. In some embodiments, production of the tRNA in the subject increases expression of the full-length dystrophin in at least one muscle by at least about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35% or more, as compared to a suitable control. For example, in some embodiments, production of the suppressor tRNA in the subject increases expression of the full-length dystrophin in at least one muscle by at least about 3% of a DMD gene expression level in a subject without DMD or the subject that is being treated prior to the start of treatment. In certain embodiments, the muscle is gastrocnemius, quadriceps, diaphragm, triceps, heart, or tibialis anterior.

[0190] In certain embodiments, production of the suppressor tRNA in the subject increases expression of full-length dystrophin in at least one muscle by about 3-30%, about 5-25%, or about 10-20% of a full-length dystrophin expression level in a subject without DMD or the subject that is being treated prior to the start of treatment. In certain embodiments, the muscle is gastrocnemius, quadriceps, diaphragm, triceps, heart, or tibialis anterior. For example, in certain embodiments, the subject has a DMD gene with a PTC, and the method comprises administering to the subject an effective amount of a suppressor tRNA comprising a nucleotide sequence selected from any one of SEQ ID NOs: 175, 176, 177, 178, 236, 238, 240, 241, 242, 249, 250, 251, and 252, or a nucleic acid or expression vectorAttorney Docket No. TVD-013WO2 encoding the same. In certain embodiments, the subject has a DMD gene with a PTC, and the method comprises administering to the subject an effective amount of a suppressor tRNA comprising a nucleotide sequence selected from any one of SEQ ID NOs: 176, 179, 180, 181, 182, 183, 184, 185, 186, 239, and 241 or a nucleic acid or expression vector encoding the same. In some embodiments, the subject has a DMD gene with a PTC, and the method comprises administering to the subject an effective amount of a suppressor tRNA comprising a nucleotide sequence selected from any one of SEQ ID NOs: 187, 188, 189, 190, 191, 192, 193, 194, 195, 915, and 916 or a nucleic acid or expression vector encoding the same.

[0191] In some embodiments, wherein the gene is a TTN gene, the TTN gene product produced with the suppressor tRNA is a functional TTN gene product. In some embodiments, the functional TTN gene product has greater activity than the truncated TTN gene product.

[0192] In some embodiments, wherein the gene is a DMD gene, the DMD gene product produced with the suppressor tRNA is a functional DMD gene product. In some embodiments, the functional DMD gene product has greater activity than the truncated DMD gene product.

[0193] 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 PTC. In certain embodiments, the method comprises administering to the subject an effective amount of a suppressor tRNA disclosed herein (e.g., a Gin-suppressor tRNA), or a nucleic acid or expression vector disclosed herein encoding the same. Exemplary Gin-suppressor tRNAs include tRNAs described in TABLE 1, TABLE 2, TABLE 3, TABLE 7, TABLE 8, TABLE 9, and TABLE 15. In certain embodiments, the suppressor tRNA is selected from any one of SEQ ID NOs: 254-274, 918, 925, 1149, and 1150. In certain embodiments, the suppressor tRNA is tr0904 (SEQ ID NO: 176). In certain embodiments, the suppressor tRNA is tr0524 (SEQ ID NO: 241). In certain embodiments, the suppressor tRNA is trl344 (SEQ ID NO: 236). In certain embodiments, the suppressor tRNA is tri 519 (SEQ ID NO: 191). In certain embodiments, the suppressor tRNA is tri 517 (SEQ ID NO: 190). In certain embodiments, the suppressor tRNA is tri 511 (SEQ ID NO: 187). In certain embodiments, the suppressor tRNA is tr0453 (SEQ ID NO: 217). In certain embodiments, the suppressor tRNA is trl622 (SEQ ID NO: 916). In certain embodiments, the suppressor tRNA is trl723 (SEQ ID NO: 1088). In certain embodiments, the suppressor tRNA is trl773 (SEQ ID NO:Attorney Docket No. TVD-013WO2 1091). In certain embodiments, the suppressor tRNA is trl794 (SEQ ID NO: 1104). In certain embodiments, the suppressor tRNA is trl806 (SEQ ID NO: 1106). In certain embodiments, the suppressor tRNA is tri 821 (SEQ ID NO: 1112). In certain embodiments, the suppressor tRNA is trl624 (SEQ ID NO: 1122). In certain embodiments, the suppressor tRNA is trl625 (SEQ ID NO: 1123). In certain embodiments, the suppressor tRNA is trl626 (SEQ ID NO: 1125). In certain embodiments, the suppressor tRNA is trl627 (SEQ ID NO: 1126). In certain embodiments, the suppressor tRNA is tri 853 (SEQ ID NO: 1127). In certain embodiments, the suppressor tRNA is tri 854 (SEQ ID NO: 1128). In certain embodiments, the suppressor tRNA is tri 857 (SEQ ID NO: 1129). In certain embodiments, the suppressor tRNA is tri 858 (SEQ ID NO: 1130).

[0194] For example, in some embodiments, the subject has a TTN gene with a PTC R30,227X, and the method comprises administering to the subject an effective amount of a suppressor tRNA comprising a nucleotide sequence selected from any one of SEQ ID NOs: 175-274, 901-916, 918, 921-925, 1000-1119, 1122-1123, 1125-1131, 1149, and 1150 or a nucleic acid or expression vector encoding the same.

[0195] In certain 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 entries #Q8WZ42 (human) or #A2ASS6 (mouse), which sequences are hereby incorporated by reference in their entirety. In certain embodiments, the functional TTN gene product is the TTN Q8WZ42-1 or A2ASS6-1 isoform (as set forth in UniProt No. Q8WZ42 (human) or A2ASS6 (mouse), respectively), 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 Q8WZ42-1 or A2ASS6-1 isoform thereof, provided below as SEQ ID NOs: 306 and 307, respectively:MTTQAPTFTQPLQSVWLEGSTATFEAHISGFPVPEVSWFRDGQVISTSTLPGVQISFSDGR AKLTIPAVTKANSGRYSLKATNGSGQATSTAELLVKAETAPPNFVQRLQSMTVRQGSQVRLQ VRVTGIPTPWKFYRDGAEIQSSLDFQISQEGDLYSLLIAEAYPEDSGTYSVNATNSVGRAT STAELLVQGEEEVPAKKTKTIVSTAQISESRQTRIEKKIEAHFDARSIATVEMVIDGAAGQQ LPHKTPPRIPPKPKSRSPTPPSIAAKAQLARQQSPSPIRHSPSPVRHVRAPTPSPVRSVSPA ARISTSPIRSVRSPLLMRKTQASTVATGPEVPPPWKQEGYVASSSEAEMRETTLTTSTQIRT EERWEGRYGVQEQVT I SGAAGAAASVSASAS YAAEAVATGAKEVKQDADKSAAVATWAAVD MARVREPVISAVEQTAQRTTTTAVHIQPAQEQVRKEAEKTAVTKVWAADKAKEQELKSRTK EVITTKQEQMHVTHEQIRKETEKTFVPKWISAAKAKEQETRISEEITKKQKQVTQEAIRQEAttorney Docket No. TVD-013WO2 TEITAASMVWATAKSTKLETVPGAQEETTTQQDQMHLSYEKIMKETRKTWPKVIVATPKV KEQDLVSRGREGITTKREQVQITQEKMRKEAEKTALSTIAVATAKAKEQETILRTRETMATR QEQIQVTHGKVDVGKKAEAVATWAAVDQARVREPREPGHLEESYAQQTTLEYGYKERISAA KVAEPPQRPASEPHWPKAVKPRVIQAPSETHIKTTDQKGMHISSQIKKTTDLTTERLVHVD KRPRTASPHFTVSKISVPKTEHGYEASIAGSAIATLQKELSATSSAQKITKSVKAPTVKPSE T RVRAE PTPLPQFP FAD T P D T YKS E AGVE VKKE VGVS I T G T T VRE E R EE VLHGRE AKVT E TA RVPAPVEIPVTPPTLVSGLKNVTVIEGESVTLECHISGYPSPTVTWYREDYQIESSIDFQIT FQSGIARLMIREAFAEDSGRFTCSAVNEAGTVSTSCYLAVQVSEEFEKETTAVTEKFTTEEK RFVESRDWMTDTSLTEEQAGPGEPAAPYFITKPWQKLVEGGSWFGCQVGGNPKPHVYWK KSGVPLTTGYRYKVSYNKQTGECKLVISMTFADDAGEYTIWRNKHGETSASASLLEEADYE LLMKSQQEMLYQTQVTAFVQEPKVGETAPGFVYSEYEKEYEKEQALIRKKMAKDTVWRTYV EDQEFHISSFEERLIKEIEYRI IKTTLEELLEEDGEEKMAVDISESEAVESGFDSRIKNYRI LEGMGVTFHCKMSGYPLPKIAWYKDGKRIKHGERYQMDFLQDGRASLRIPWLPEDEGIYTA FASNIKGNAICSGKLYVEPAAPLGAPTYIPTLEPVSRIRSLSPRSVSRSPIRMSPARMSPAR MSPARMSPARMSPGRRLEETDESQLERLYKPVFVLKPVSFKCLEGQTARFDLKWGRPMPET FWFHDGQQIVNDYTHKWIKEDGTQSLIIVPATPSDSGEWTWAQNRAGRSSISVILTVEAV EHQVKPMFVEKLKNVNIKEGSRLEMKVRATGNPNPDIVWLKNSDI IVPHKYPKIRIEGTKGE AALKIDSTVSQDSAWYTATAINKAGRDTTRCKVNVEVEFAEPEPERKLI IPRGTYRAKEIAA PELEPLHLRYGQEQWEEGDLYDKEKQQKPFFKKKLTSLRLKRFGPAHFECRLTPIGDPTMW EWLHDGKPLEAANRLRMINEFGYCSLDYGVAYSRDSGI ITCRATNKYGTDHTSATLIVKDEK SLVEESQLPEGRKGLQRIEELERMAHEGALTGVTTDQKEKQKPDIVLYPEPVRVLEGETARF RCRVTGYPQPKVNWYLNGQLIRKSKRFRVRYDGIHYLDIVDCKSYDTGEVKVTAENPEGVIE HKVKLEIQQREDFRSVLRRAPEPRPEFHVHEPGKLQFEVQKVDRPVDTTETKEWKLKRAER ITHEKVPEESEELRSKFKRRTEEGYYEAITAVELKSRKKDESYEELLRKTKDELLHWTKELT EEEKKALAEEGKITIPTFKPDKIELSPSMEAPKIFERIQSQTVGQGSDAHFRVRWGKPDPE CEWYKNGVKIERSDRI YWYWPEDNVCELVIRDVTAEDSAS IMVKAINIAGETSSHAFLLVQA KQLITFTQELQDWAKEKDTMATFECETSEPFVKVKWYKDGMEVHEGDKYRMHSDRKVHFLS ILTIDTSDAEDYSCVLVEDENVKTTAKLIVEGAWEFVKELQDIEVPESYSGELECIVSPEN IEGKWYHNDVELKSNGKYTITSRRGRQNLTVKDVTKEDQGEYSFVIDGKKTTCKLKMKPRPI AILQGLSDQKVCEGDIVQLEVKVSLESVEGVWMKDGQEVQPSDRVHIVIDKQSHMLLIEDMT KEDAGNYSFTIPALGLSTSGRVSVYSVDVITPLKDVNVIEGTKAVLECKVSVPDVTSVKWYL NDEQIKPDDRVQAIVKGTKQRLVINRTHASDEGPYKLIVGRVETNCNLSVEKIKI IRGLRDL TCTETQNWFEVELSHSGIDVLWNFKDKEIKPSSKYKIEAHGKIYKLTVLNMMKDDEGKYTF YAGENMTSGKLTVAGGAISKPLTDQTVAESQEAVFECEVANPDSKGEWLRDGKHLPLTNNIRAttorney Docket No. TVD-013WO2 SESDGHKRRLIIAATKLDDIGEYTYKVATSKTSAKLKVEAVKIKKTLKNLTVTETQDAVFTV ELTHPNVKGVQWIKNGWLESNEKYAISVKGTIYSLRIKNCAIVDESVYGFRLGRLGASARL HVETVKI IKKPKDVTALENATVAFEVSVSHDTVPVKWFHKSVEIKPSDKHRLVSERKVHKLM LQNISPSDAGEYTAWGQLECKAKLFVETLHITKTMKNIEVPETKTASFECEVSHFNVPSMW LKNGVEIEMSEKFKIWQGKLHQLI IMNTSTEDSAEYTFVCGNDQVSATLTVTPIMITSMLK DINAEEKDTITFEVTVNYEGISYKWLKNGVEIKSTDKCQMRTKKLTHSLNIRNVHFGDAADY T FVAGKAT S TAT L YVEARH I E FRKH I KD I KVLEKKRAMFE GE VS E PD I T VQWMKDDQE LQ I T DRIKIQKEKYVHRLLIPSTRMSDAGKYTWAGGNVSTAKLFVEGRDVRIRSIKKEVQVIEKQ RAWEFEVNEDDVDAHWYKDGIEINFQVQERHKYWERRIHRMFISETRQSDAGEYTFVAGR NRSSVTLYVNAPEPPQVLQELQPVTVQSGKPARFCAVISGRPQPKISWYKEEQLLSTGFKCK FLHDGQEYTLLLIEAFPEDAAVYTCEAKNDYGVATTSASLSVEVPEWSPDQEMPVYPPAI I TPLQDTVTSEGQPARFQCRVSGTDLKVSWYSKDKKIKPSRFFRMTQFEDTYQLEIAEAYPED EGTYTFVASNAVGQVSSTANLSLEAPESILHERIEQEIEMEMKEFSSSFLSAEEEGLHSAEL QLSKINETLELLSESPVYPTKFDSEKEGTGPIFIKEVSNADISMGDVATLSVTVIGIPKPKI QWFFNGVLLTPSADYKFVFDGDDHSLI ILFTKLEDEGEYTCMASNDYGKTICSAYLKINSKG EGHKDTETESAVAKSLEKLGGPCPPHFLKELKPIRCAQGLPAI FEYTWGEPAPTVTWFKEN KQLCTSVYYTI IHNPNGSGTFIVNDPQREDSGLYICKAENMLGESTCAAELLVLLEDTDMTD TPCKAKSTPEAPEDFPQTPLKGPAVEALDSEQEIATFVKDTILKAALITEENQQLSYEHIAK ANELSSQLPLGAQELQSILEQDKLTPESTREFLCINGSIHFQPLKEPSPNLQLQIVQSQKTF SKEGILMPEEPETQAVLSDTEKI FPSAMS IEQINSLTVEPLKTLLAEPEGNYPQSS IEPPMH SYLTSVAEEVLSPKEKTVSDTNREQRVTLQKQEAQSALILSQSLAEGHVESLQSPDVMISQV NYEPLVPSEHSCTEGGKILIESANPLENAGQDSAVRIEEGKSLRFPLALEEKQVLLKEEHSD NWMPPDQI IESKREPVAIKKVQEVQGRDLLSKESLLSGIPEEQRLNLKIQICRALQAAVAS EQPGLFSEWLRNIEKVEVEAVNITQEPRHIMCMYLVTSAKSVTEEVTI I IEDVDPQMANLKM ELRDALCAI IYEEIDILTAEGPRIQQGAKTSLQEEMDSFSGSQKVEPITEPEVESKYLISTE EVSYFNVQSRVKYLDATPVTKGVASAWSDEKQDESLKPSEEKEESSSESGTEEVATVKIQE AEGGLIKEDGPMIHTPLVDTVSEEGDIVHLTTSITNAKEVNWYFENKLVPSDEKFKCLQDQN TYTLVIDKVNTEDHQGEYVCEALNDSGKTATSAKLTWKRAAPVIKRKIEPLEVALGHLAKF TCEIQSAPNVRFQWFKAGREIYESDKCSIRSSKYISSLEILRTQWDCGEYTCKASNEYGSV SCTATLTVTEAYPPTFLSRPKSLTTFVGKAAKFICTVTGTPVIETIWQKDGAALSPSPNWRI SDAENKHILELSNLTIQDRGVYSCKASNKFGADICQAELI I IDKPHFIKELEPVQSAINKKV HLECQVDEDRKVTVTWSKDGQKLPPGKDYKICFEDKIATLEIPLAKLKDSGTYVCTASNEAG SSSCSATVTVREPPSFVKKVDPSYLMLPGESARLHCKLKGSPVIQVTWFKNNKELSESNTVR MYFVNSEAILDITDVKVEDSGSYSCEAVNDVGSDSCSTEIVIKEPPSFIKTLEPADIVRGTNAttorney Docket No. TVD-013WO2 ALLQCEVSGTGPFEISWFKDKKQIRSSKKYRLFSQKSLVCLEIFSFNSADVGEYECWANEV GKCGCMATHLLKEPPTFVKKVDDLIALGGQTVTLQAAVRGSEPISVTWMKGQEVIREDGKIK MSFSNGVAVLI IPDVQISFGGKYTCLAENEAGSQTSVGELIVKEPAKI IERAELIQVTAGDP ATLE YTVAGTPELKPKWYKDGRPLVASKKYRI S FKNNVAQLKFYSAELHDSGQYT EE I SNEV GSSSCETTFTVLDRDIAPFFTKPLRNVDSWNGTCRLDCKIAGSLPMRVSWFKDGKEIAASD RYRIAFVEGTASLEI IRVDMNDAGNFTCRATNSVGSKDSSGALIVQEPPSFVTKPGSKDVLP GSAVCLKSTFQGSTPLTIRWFKGNKELVSGGSCYITKEALESSLELYLVKTSDSGTYTCKVS NVAGGVECSANLFVKEPATFVEKLEPSQLLKKGDATQLACKVTGTPPIKITWFANDREIKES SKHRMSFVESTAVLRLTDVGIEDSGEYMCEAQNEAGSDHCSSIVIVKESPYFTKEFKPIEVL KEYDVMLLAEVAGTPPFEITWFKDNTILRSGRKYKTFIQDHLVSLQILKFVAADAGEYQCRV TNEVGSSICSARVTLREPPSFIKKIESTSSLRGGTAAFQATLKGSLPITVTWLKDSDEITED DN I RMT FENNVAS L YL S G I E VKHDGKYVCQAKNDAG I QRC S ALL S VKE PAT I TEEAVS I DVT QGDPATLQVKFSGTKEITAKWFKDGQELTLGSKYKISVTDTVSILKIISTEKKDSGEYTFEV QNDVGRSSCKARINVLDLIIPPSFTKKLKKMDSIKGSFIDLECIVAGSHPISIQWFKDDQEI SASEKYKFSFHDNTAFLEISQLEGTDSGTYTCSATNKAGHNQCSGHLTVKEPPYFVEKPQSQ DVNPNTRVQLKALVGGTAPMTIKWFKDNKELHSGAARSVWKDDTSTSLELFAAKATDSGTYI CQLSNDVGTATSKATLFVKEPPQFIKKPSPVLVLRNGQSTTFECQITGTPKIRVSWYLDGNE ITAIQKHGISFIDGLATFQISGARVENSGTYVCEARNDAGTASCSIELKVKEPPTFIRELKP VEWKYSDVELECEVTGTPPFEVTWLKNNREIRSSKKYTLTDRVSVFNLHITKCDPSDTGEY QCIVSNEGGSCSCSTRVALKEPPSFIKKIENTTTVLKSSATFQSTVAGSPPISITWLKDDQI LDEDDNVYISFVDSVATLQIRSVDNGHSGRYTCQAKNESGVERCYAFLLVQEPAQIVEKAKS VDVTEKDPMTLECWAGTPELKVKWLKDGKQIVPSRYFSMS FENNVAS FRIQSVMKQDSGQY TFKVENDFGSSSCDAYLRVLDQNIPPSFTKKLTKMDKVLGSSIHMECKVSGSLPISAQWFKD GKEISTSAKYRLVCHERSVSLEVNNLELEDTANYTCKVSNVAGDDACSGILTVKEPPSFLVK PGRQQAIPDSTVEFKAILKGTPPFKIKWFKDDVELVSGPKCFIGLEGSTSFLNLYSVDASKT GQYTCHVTNDVGSDSCTTMLLVTEPPKFVKKLEASKIVKAGDSSRLECKIAGSPEIRWWFR NEHELPASDKYRMTFIDSVAVIQMNNLSTEDSGDFICEAQNPAGSTSCSTKVIVKEPPVFSS FPPIVETLKNAEVSLECELSGTPPFEWWYKDKRQLRSSKKYKIASKNFHTS IHILNVDTSD IGEYHCKAQNEVGSDTCVCTVKLKEPPRFVSKLNSLTWAGEPAELQAS IEGAQPI FVQWLK EKEEVIRESENIRITFVENVATLQFAKAEPANAGKYICQIKNDGGMRENMATLMVLEPAVIV EKAGPMTVTVGETCTLECKVAGTPELSVEWYKDGKLLTSSQKHKFSFYNKISSLRILSVERQ DAGTYTFQVQNNVGKSSCTAWDVSDRAVPPSFTRRLKNTGGVLGASCILECKVAGSSPISV AWFHEKTKIVSGAKYQTTFSDNVCTLQLNSLDSSDMGNYTCVAANVAGSDECRAVLTVQEPP SFVKEPEPLEVLPGKNVTFTSVIRGTPPFKVNWFRGARELVKGDRCNIYFEDTVAELELFNIAttorney Docket No. TVD-013WO2 DISQSGEYTCWSNNAGQASCTTRLFVKEPAAFLKRLSDHSVEPGKSIILESTYTGTLPISV TWKKDGFNITTSEKCNIVTTEKTCILEILNSTKRDAGQYSCEIENEAGRDVCGALVSTLEPP YFVTELEPLEAAVGDSVSLQCQVAGTPEITVSWYKGDTKLRPTPEYRTYFTNNVATLVFNKV NINDSGEYTCKAENSIGTASSKTVFRIQERQLPPSFARQLKDIEQTVGLPVTLTCRLNGSAP IQVCWYRDGVLLRDDENLQTSFVDNVATLKILQTDLSHSGQYSCSASNPLGTASSSARLTAR EPKKSPFFDIKPVSIDVIAGESADFECHVTGAQPMRITWSKDNKEIRPGGNYTITCVGNTPH LRILKVGKGDSGQYTCQATNDVGKDMCSAQLSVKEPPKFVKKLEASKVAKQGESIQLECKIS GSPEIKVSWFRNDSELHESWKYNMSFINSVALLTINEASAEDSGDYICEAHNGVGDASCSTA LTVKAPPVFTQKPSPVGALKGSDVILQCEISGTPPFEWWVKDRKQVRNSKKFKITSKHFDT SLHILNLEASDVGEYHCKATNEVGSDTCSCSVKFKEPPRFVKKLSDTSTLIGDAVELRAIVE GFQPISWWLKDRGEVIRESENTRISFIDNIATLQLGSPEASNSGKYICQIKNDAGMRECSA VLTVLEPARIIEKPEPMTVTTGNPFALECWTGTPELSAKWFKDGRELSADSKHHITFINKV ASLKIPCAEMSDKGLYSFEVKNSVGKSNCTVSVHVSDRIVPPSFIRKLKDVNAILGASWLE CRVSGSAPISVGWFQDGNEIVSGPKCQSSFSENVCTLNLSLLEPSDTGIYTCVAANVAGSDE CSAVLTVQEPPSFEQTPDSVEVLPGMSLTFTSVIRGTPPFKVKWFKGSRELVPGESCNISLE DFVTELELFEVQPLESGDYSCLVTNDAGSASCTTHLFVKEPATFVKRLADFSVETGSPIVLE ATYTGTPPISVSWIKDEYLISQSERCSITMTEKSTILEILESTIEDYAQYSCLIENEAGQDI CEALVSVLEPPYFIEPLEHVEAVIGEPATLQCKVDGTPEIRISWYKEHTKLRSAPAYKMQFK NNVASLVINKVDHSDVGEYSCKADNSVGAVASSAVLVIKARKLPPFFARKLKDVHETLGFPV AFECRINGSEPLQVSWYKDGVLLKDDANLQTSFVHNVATLQILQTDQSHIGQYNCSASNPLG TASSSAKLILSEHEVPPFFDLKPVSVDLALGESGTFKCHVTGTAPIKITWAKDNREIRPGGN YKMTLVENTATLTVLKVGKGDAGQYTCYASNIAGKDSCSAQLGVQEPPRFIKKLEPSRIVKQ DEFTRYECKIGGSPEIKVLWYKDETEIQESSKFRMSFVDSVAVLEMHNLSVEDSGDYTCEAH NAAGSASSSTSLKVKEPPI FRKKPHPIETLKGADVHLECELQGTPPFHVSWYKDKRELRSGK KYKIMSENFLTSIHILNVDAADIGEYQCKATNDVGSDTCVGSIALKAPPRFVKKLSDISTW GKEVQLQTTIEGAEPISWWFKDKGEIVRESDNIWISYSENIATLQFSRVEPANAGKYTCQI KNDAGMQECFATLSVLEPATIVEKPESIKVTTGDTCTLECTVAGTPELSTKWFKDGKELTSD NKYKISFFNKVSGLKI INVAPSDSGVYSFEVQNPVGKDSCTASLQVSDRTVPPSFTRKLKET NGLSGSSWMECKVYGSPPISVSWFHEGNEISSGRKYQTTLTDNTCALTVNMLEESDSGDYT CIATNMAGSDECSAPLTVREPPSFVQKPDPMDVLTGTNVTFTSIVKGTPPFSVSWFKGSSEL VPGDRCNVSLEDSVAELELFDVDTSQSGEYTCIVSNEAGKASCTTHLYIKAPAKFVKRLNDY SIEKGKPLILEGTFTGTPPISVTWKKNGINVTPSQRCNITTTEKSAILEIPSSTVEDAGQYN CYIENASGKDSCSAQILILEPPYFVKQLEPVKVSVGDSASLQCQLAGTPEIGVSWYKGDTKL RPTTTYKMHFRNNVATLVFNQVDINDSGEYICKAENSVGEVSASTFLTVQEQKLPPSFSRQLAttorney Docket No. TVD-013WO2 RDVQETVGLPWFDCAISGSEPISVSWYKDGKPLKDSPNVQTSFLDNTATLNIFKTDRSLAG QYSCTATNPIGSASSSARLILTEGKNPPFFDIRLAPVDAWGESADFECHVTGTQPIKVSWA KDSREIRSGGKYQISYLENSAHLTVLKVDKGDSGQYTCYAVNEVGKDSCTAQLNIKERLIPP SFTKRLSETVEETEGNSFKLEGRVAGSQPITVAWYKNNIEIQPTSNCEITFKNNTLVLQVRK AGMNDAGLYTCKVSNDAGSALCTSS IVIKEPKKPPVFDQHLTPVTVSEGEYVQLSCHVQGSE PIRIQWLKAGREIKPSDRCSFSFASGTAVLELRDVAKADSGDYVCKASNVAGSDTTKSKVTI KDKPAVAPATKKAAVDGRLFFVSEPQSIRWEKTTATFIAKVGGDPIPNVKWTKGKWRQLNQ GGRVFIHQKGDEAKLEIRDTTKTDSGLYRCVAFNEHGEIESNVNLQVDERKKQEKIEGDLRA MLKKTPILKKGAGEEEEIDIMELLKNVDPKEYEKYARMYGITDFRGLLQAFELLKQSQEEET HRLEIEEIERSERDEKEFEELVSFIQQRLSQTEPVTLIKDIENQTVLKDNDAVFEIDIKINY PEIKLSWYKGTEKLEPSDKFEISIDGDRHTLRVKNCQLKDQGNYRLVCGPHIASAKLTVIEP AWERHLQDVTLKEGQTCTMTCQFSVPNVKSEWFRNGRILKPQGRHKTEVEHKVHKLTIADVR AEDQGQYTCKYEDLETSAELRIEAEPIQFTKRIQNIWSEHQSATFECEVSFDDAIVTWYKG PTELTESQKYNFRNDGRCHYMTIHNVTPDDEGVYSVIARLEPRGEARSTAELYLTTKEIKLE LKPPDIPDSRVPIPTMPIRAVPPEEIPPWAPPIPLLLPTPEEKKPPPKRIEVTKKAVKKDA KKWAKPKEMTPREE I VKKPPPPTTL I PAKAPE 11 DVS SKAEEVKIMT I TRKKEVQKEKEAV YEKKQAVHKEKRVFIESFEEPYDELEVEPYTEPFEQPYYEEPDEDYEEIKVEAKKEVHEEWE EDFEEGQEYYEREEGYDEGEEEWEEAYQEREVIQVQKEVYEESHERKVPAKVPEKKAPPPPK VIKKPVIEKIEKTSRRMEEEKVQVTKVPEVSKKIVPQKPSRTPVQEEVIEVKVPAVHTKKMV ISEEKMFFASHTEEEVSVTVPEVQKEIVTEEKIHVAISKRVEPPPKVPELPEKPAPEEVAPV PIPKKVEPPAPKVPEVPKKPVPEEKKPVPVPKKEPAAPPKVPEVPKKPVPEEKIPVPVAKKK EAPPAKVPEVQKGWTEEKI T I VTQREES PPPAVPE I PKKKVPEERKPVPRKEEEVPPPPKV PALPKKPVPEEKVAVPVPVAKKAPPPRAEVSKKTWEEKRFVAEEKLSFAVPQRVEVTRHEV SAEEEWSYSEEEEGVSISVYREEEREEEEEAEVTEYEVMEEPEEYWEEKLHIISKRVEAEP AEVTERQEKKIVLKPKIPAKIEEPPPAKVPEAPKKIVPEKKVPAPVPKKEKVPPPKVPEEPK KPVPEKKVPPKVIKMEEPLPAKVTERHMQITQEEKVLVAVTKKEAPPKARVPEEPKRAVPEE KVLKLKPKREEEPPAKVTEFRKRWKEEKVSIEAPKREPQPIKEVTIMEEKERAYTLEEEAV SVQREEEYEEYEEYDYKEFEEYEPTEEYDQYEEYEEREYERYEEHEEYITEPEKPIPVKPVP EEPVPTKPKAPPAKVLKKAVPEEKVPVPIPKKLKPPPPKVPEEPKKVFEEKIRISITKREKE QVTEPAAKVPMKPKRWAEEKVPVPRKEVAPPVRVPEVPKELEPEEVAFEEEWTHVEEYLV EEEEEYIHEEEEFITEEEWPVIPVKVPEVPRKPVPEEKKPVPVPKKKEAPPAKVPEVPKKP EEKVPVLIPKKEKPPPAKVPEVPKKPVPEEKVPVPVPKKVEAPPAKVPEVPKKPVPEKKVPV PAPKKVEAPPAKVPEVPKKLIPEEKKPTPVPKKVEAPPPKVPKKREPVPVPVALPQEEEVLF EEEIVPEEEVLPEEEEVLPEEEEVLPEEEEVLPEEEEIPPEEEEVPPEEEYVPEEEEFVPEEAttorney Docket No. TVD-013WO2 EVLPEVKPKVPVPAPVPEIKKKVTEKKWIPKKEEAPPAKVPEVPKKVEEKRI ILPKEEEVL PVEVTEEPEEEPISEEEIPEEPPSIEEVEEVAPPRVPEVIKKAVPEAPTPVPKKVEAPPAKV SKKIPEEKVPVPVQKKEAPPAKVPEVPKKVPEKKVLVPKKEAVPPAKGRTVLEEKVSVAFRQ EVWKERLELEWEAEVEEIPEEEEFHEVEEYFEEGEFHEVEEFIKLEQHRVEEEHRVEKVH RVIEVFEAEEVEVFEKPKAPPKGPEISEKI IPPKKPPTKWPRKEPPAKVPEVPKKIWEEK VRVPEEPRVPPTKVPEVLPPKEWPEKKVPVPPAKKPEAPPPKVPEAPKEWPEKKVPVPPP KKPEVPPTKVPEVPKAAVPEKKVPEAIPPKPESPPPEVPEAPKEWPEKKVPAAPPKKPEVT PVKVPEAPKEWPEKKVPVPPPKKPEVPPTKVPEVPKVAVPEKKVPEAIPPKPESPPPEVFE EPEEVALEEPPAEWEEPEPAAPPQVTVPPKKPVPEKKAPAWAKKPELPPVKVPEVPKEW PEKKVPLWPKKPEAPPAKVPEVPKEWPEKKVAVPKKPEVPPAKVPEVPKKPVLEEKPAVP VPERAESPPPEVYEEPEEIAPEEEIAPEEEKPVPVAEEEEPEVPPPAVPEEPKKI IPEKKVP VIKKPEAPPPKEPEPEKVIEKPKLKPRPPPPPPAPPKEDVKEKIFQLKAIPKKKVPEKPQVP EKVELTPLKVPGGEKKVRKLLPERKPEPKEEWLKSVLRKRPEEEEPKVEPKKLEKVKKPAV PEPPPPKPVEEVEVPTVTKRERKIPEPTKVPEIKPAIPLPAPEPKPKPEAEVKTIKPPPVEP EPTPIAAPVTVPWGKKAEAKAPKEEAAKPKGPIKGVPKKTPSPIEAERRKLRPGSGGEKPP DEAPFTYQLKAVPLKFVKEIKDIILTESEFVGSSAIFECLVSPSTAITTWMKDGSNIRESPK HRFIADGKDRKLHI IDVQLSDAGEYTCVLRLGNKEKTSTAKLWEELPVRFVKTLEEEVTW KGQPLYLSCELNKERDWWRKDGKIWEKPGRIVPGVIGLMRALTINDADDTDAGTYTVTVE NANNLE C S S CVKWE VI RDWLVKP I RDQHVKPKGTAI FACD I AKDT PN I KW FKG YDE I PAE P NDKTEILRDGNHLYLKIKNAMPEDIAEYAVEIEGKRYPAKLTLGEREVELLKPIEDVTIYEK ESAS FDAE I SEADI PGQWKLKGELLRPS PTCE IKAEGGKRFLTLHKVKLDQAGEVLYQALNA ITTAILTVKEIELDFAVPLKDVTVPERRQARFECVLTREANVIWSKGPDI IKSSDKFDI IAD GKKHILVINDSQFDDEGVYTAEVEGKKTSARLFVTGIRLKFMSPLEDQTVKEGETATFVCEL SHEKMHWWFKNDAKLHTSRTVLISSEGKTHKLEMKEVTLDDISQIKAQVKELSSTAQLKVL EADPYFTVKLHDKTAVEKDEITLKCEVSKDVPVKWFKDGEEIVPSPKYSIKADGLRRILKIK KADLKDKGEYVCDCGTDKTKANVTVEARLIKVEKPLYGVEVFVGETAHFEIELSEPDVHGQW KLKGQPLTASPDCEIIEDGKKHILILHNCQLGMTGEVSFQAANAKSAANLKVKELPLIFITP LSDVKVFEKDEAKFECEVSREPKTFRWLKGTQEITGDDRFELIKDGTKHSMVIKSAAFEDEA KYMFEAEDKHTSGKLI lEGIRLKFLTPLKDVTAKEKESAVFTVELSHDNIRVKWFKNDQRLH TTRSVSMQDEGKTHSITFKDLSIDDTSQIRVEAMGMSSEAKLTVLEGDPYFTGKLQDYTGVE KDEVILQCEISKADAPVKWFKDGKEIKPSKNAVIKADGKKRMLILKKALKSDIGQYTCDCGT DKTSGKLDIEDREIKLVRPLHSVEVMETETARFETEISEDDIHANWKLKGEALLQTPDCEIK EEGKIHSLVLHNCRLDQTGGVDFQAANVKSSAHLRVKPRVIGLLRPLKDVTVTAGETATFDC ELSYEDIPVEWYLKGKKLEPSDKWPRSEGKVHTLTLRDVKLEDAGEVQLTAKDFKTHANLFAttorney Docket No. TVD-013WO2 VKEPPVEFTKPLEDQTVEEGATAVLECEVSRENAKVKWFKNGTEILKSKKYEIVADGRVRKL VIHDCTPEDIKTYTCDAKDFKTSCNLNWPPHVEFLRPLTDLQVREKEMARFECELSRENAK VKWFKDGAEIKKGKKYDI ISKGAVRILVINKCLLDDEAEYSCEVRTARTSGMLTVLEEEAVF TKNLANIEVSETDTIKLVCEVSKPGAEVIWYKGDEEI IETGRYEILTEGRKRILVIQNAHLE DAGNYNCRLPSSRTDGKVKVHELAAEFISKPQNLEILEGEKAEFVCSISKESFPVQWKRDDK TLESGDKYDVIADGKKRVLWKDATLQDMGTYWMVGAARAAAHLTVIEKLRIWPLKDTRV KEQQEWFNCEVNTEGAKAKWFRNEEAI FDSSKYI ILQKDLVYTLRIRDAHLDDQANYNVSL TNHRGENVKSAANLIVEEEDLRIVEPLKDIETMEKKSVTFWCKVNRLNVTLKWTKNGEEVPF DNRVSYRVDKYKHMLTIKDCGFPDEGEYIVTAGQDKSVAELLI IEAPTEFVEHLEDQTVTEF DDAVFSCQLSREKANVKWYRNGREIKEGKKYKFEKDGSIHRLIIKDCRLDDECEYACGVEDR KSRARLFVEEIPVEI IRPPQDILEAPGADWFLAELNKDKVEVQWLRNNMVWQGDKHQMMS EGKIHRLQICDIKPRDQGEYRFIAKDKEARAKLELAAAPKIKTADQDLWDVGKPLTMWPY DAYPKAEAEWFKENEPLSTKTIDTTAEQTSFRILEAKKGDKGRYKIVLQNKHGKAEGFINLK VIDVPGPVRNLEVTETFDGEVSLAWEEPLTDGGSKI IGYWERRDIKRKTWVLATDRAESCE FTVTGLQKGGVEYLFRVSARNRVGTGEPVETDNPVEARSKYDVPGPPLNVTITDVNRFGVSL TWEPPEYDGGAEITNYVIELRDKTSIRWDTAMTVRAEDLSATVTDWEGQEYSFRVRAQNRI GVGKPSAATPFVKVADPIERPSPPVNLTSSDQTQSSVQLKWEPPLKDGGSPILGYI IERCEE GKDNWIRCNMKLVPELTYKVTGLEKGNKYLYRVSAENKAGVSDPSEILGPLTADDAFVEPTM DLSAFKDGLEVIVPNPITILVPSTGYPRPTATWCFGDKVLETGDRVKMKTLSAYAELVISPS ERSDKGIYTLKLENRVKTISGEIDVNVIARPSAPKELKFGDITKDSVHLTWEPPDDDGGSPL TGYWEKREVSRKTWTKVMDFVTDLEFTVPDLVQGKEYLFKVCARNKCGPGEPAYVDEPVNM STPATVPDPPENVKWRDRTANS I FLTWDPPKNDGGSRIKGYIVERCPRGSDKWVACGEPVAE TKMEVTGLEEGKWYAYRVKALNRQGASKPSRPTEEIQAVDTQEAPEIFLDVKLLAGLTVKAG TKIELPATVTGKPEPKITWTKADMILKQDKRITIENVPKKSTVTIVDSKRSDTGTYI IEAVN VCGRATAWEVNVLDKPGPPAAFDITDVTNESCLLTWNPPRDDGGSKITNYWERRATDSEV WHKLSSTVKDTNFKATKLIPNKEYIFRVAAENMYGVGEPVQASPITAKYQFDPPGPPTRLEP SDITKDAVTLTWCEPDDDGGSPITGYWVERLDPDTDKWVRCNKMPVKDTTYRVKGLTNKKKY RFRVLAENLAGPGKPSKSTEPILIKDPIDPPWPPGKPTVKDVGKTSVRLNWTKPEHDGGAKI ESYVIEMLKTGTDEWVRVAEGVPTTQHLLPGLMEGQEYSFRVRAVNKAGESEPSEPSDPVLC REKLYPPSPPRWLEVINITKNTADLKWTVPEKDGGSPITNYIVEKRDVRRKGWQTVDTTVKD TKCTVTPLTEGSLYVFRVAAENAIGQSDYTEIEDSVLAKDTFTTPGPPYALAWDVTKRHVD LKWEPPKNDGGRPIQRYVIEKKERLGTRWVKAGKTAGPDCNFRVTDVIEGTEVQFQVRAENE AGVGHPSEPTEILSIEDPTSPPSPPLDLHVTDAGRKHIAIAWKPPEKNGGSPIIGYHVEMCP VGTEKWMRVNSRPIKDLKFKVEEGWPDKEYVLRVRAVNAIGVSEPSEISENWAKDPDCKPAttorney Docket No. TVD-013WO2 TIDLETHDIIVIEGEKLSIPVPFRAVPVPTVSWHKDGKEVKASDRLTMKNDHISAHLEVPKS VRADAGIYTITLENKLGSATASINVKVIGLPGPCKDIKASDITKSSCKLTWEPPEFDGGTPI LHYVLERREAGRRTYIPVMSGENKLSWTVKDLIPNGEYFFRVKAVNKVGGGEYIELKNPVIA QDPKQPPDPPVDVEVHNPTAEAMTITWKPPLYDGGSKIMGYI IEKIAKGEERWKRCNEHLVP ILTYTAKGLEEGKEYQFRVRAENAAGISEPSRATPPTKAVDPIDAPKVILRTSLEVKRGDEI ALDASISGSPYPTITWIKDENVIVPEEIKKRAAPLVRRRKGEVQEEEPFVLPLTQRLSIDNS KKGESQLRVRDSLRPDHGLYMIKVENDHGIAKAPCTVSVLDTPGPPINFVFEDIRKTSVLCK WEPPLDDGGSEI INYTLEKKDKTKPDSEWIWTSTLRHCKYSVTKLIEGKEYLFRVRAENRF GPGPPCVSKPLVAKDPFGPPDAPDKPIVEDVTSNSMLVKWNEPKDNGSPILGYWLEKREVNS THWSRVNKSLLNALKANVDGLLEGLTYVFRVCAENAAGPGKFSPPSDPKTAHDPISPPGPPI PRVTDTSSTTIELEWEPPAFNGGGEIVGYFVDKQLVGTNEWSRCTEKMIKVRQYTVKEIREG ADYKLRVSAVNAAGEGPPGETQPVTVAEPQEPPAVELDVSVKGGIQIMAGKTLRIPAWTGR PVPTKVWTKEEGELDKDRWIDNVGTKSELIIKDALRKDHGRYVITATNSCGSKFAAARVEV FDVPGPVLDLKPWTNRKMCLLNWSDPEDDGGSEITGFI IERKDAKMHTWRQPIETERSKCD ITGLLEGQEYKFRVIAKNKFGCGPPVEIGPILAVDPLGPPTSPERLTYTERTKSTITLDWKE PRSNGGSPIQGYI lEKRRHDKPDFERVNKRLCPTTSFLVENLDEHQMYEFRVKAVNEIGESE PSLPLNWIQDDEVPPTIKLRLSVRGDTIKVKAGEPVHIPADVTGLPMPKIEWSKNETVIEK PTDALQITKEEVSRSEAKTELSIPKAVREDKGTYTVTASNRLGSVFRNVHVEVYDRPSPPRN LAVTDIKAESCYLTWDAPLDNGGSEITHYVIDKRDASRKKAEWEEVTNTAVEKRYGIWKLIP NGQYEFRVRAVNKYGISDECKSDKWIQDPYRLPGPPGKPKVLARTKGSMLVSWTPPLDNGG SPITGYWLEKREEGSPYWSRVSRAPITKVGLKGVEFNVPRLLEGVKYQFRAMAINAAGIGPP SEPSDPEVAGDPIFPPGPPSCPEVKDKTKSSISLGWKPPAKDGGSPIKGYIVEMQEEGTTDW KRVNEPDKLITTCECWPNLKELRKYRFRVKAVNEAGESEPSDTTGEIPATDIQEEPEVFID IGAQDCLVCKAGSQIRIPAVIKGRPTPKSSWEFDGKAKKAMKDGVHDIPEDAQLETAENSSV IIIPECKRSHTGKYSITAKNKAGQKTANCRVKVMDVPGPPKDLKVSDITRGSCRLSWKMPDD DGGDRIKGYVIEKRTIDGKAWTKVNPDCGSTTFWPDLLSEQQYFFRVRAENRFGIGPPVET IQRTTARDPIYPPDPPIKLKIGLITKNTVHLSWKPPKNDGGSPVTHYIVECLAWDPTGTKKE AWRQCNKRDVEELQFTVEDLVEGGEYEFRVKAVNAAGVSKPSATVGPVTVKDQTCPPS IDLK EFMEVEEGTNVNIVAKIKGVPFPTLTWFKAPPKKPDNKEPVLYDTHVNKLWDDTCTLVIPQ SRRSDTGLYTITAVNNLGTASKEMRLNVLGRPGPPVGPIKFESVSADQMTLSWFPPKDDGGS KITNYVIEKREANRKTWVHVSSEPKECTYTIPKLLEGHEYVFRIMAQNKYGIGEPLDSEPET ARNLFSVPGAPDKPTVSSVTRNSMTVNWEEPEYDGGSPVTGYWLEMKDTTSKRWKRVNRDPI KAMTLGVSYKVTGLIEGSDYQFRVYAINAAGVGPASLPSDPATARDPIAPPGPPFPKVTDWT KSSADLEWSPPLKDGGSKVTGYIVEYKEEGKEEWEKGKDKEVRGTKLWTGLKEGAFYKFRVAttorney Docket No. TVD-013WO2 RAVNIAGIGEPGEVTDVIEMKDRLVSPDLQLDASVRDRIWHAGGVIRI IAYVSGKPPPTVT WNMNERTLPQEATIETTAISSSMVIKNCQRSHQGVYSLLAKNEAGERKKTIIVDVLDVPGPV GTPFLAHNLTNESCKLTWFSPEDDGGSPITNYVIEKRESDRRAWTPVTYTVTRQNATVQGLI QGKAYFFRIAAENSIGMGPFVETSEALVIREPITVPERPEDLEVKEVTKNTVTLTWNPPKYD GGSEI INYVLESRLIGTEKFHKVTNDNLLSRKYTVKGLKEGDTYEYRVSAVNIVGQGKPSFC TKPITCKDELAPPTLHLDFRDKLTIRVGEAFALTGRYSGKPKPKVSWFKDEADVLEDDRTHI KTTPATLALEKIKAKRSDSGKYCVWENSTGSRKGFCQVNWDRPGPPVGPVSFDEVTKDYM VISWKPPLDDGGSKITNYIIEKKEVGKDVWMPVTSASAKTTCKVSKLLEGKDYIFRIHAENL YGISDPLVSDSMKAKDRFRVPDAPDQPIVTEVTKDSALVTWNKPHDGGKPITNYILEKRETM SKRWARVTKDPIHPYTKFRVPDLLEGCQYEFRVSAENEIGIGDPSPPSKPVFAKDPIAKPSP PVNPEAIDTTCNSVDLTWQPPRHDGGSKILGYIVEYQKVGDEEWRRANHTPESCPETKYKVT GLRDGQTYKFRVLAVNAAGESDPAHVPEPVLVKDRLEPPELILDANMAREQHIKVGDTLRLS AIIKGVPFPKVTWKKEDRDAPTKARIDVTPVGSKLEIRNAAHEDGGIYSLTVENPAGSKTVS VKVLVLDKPGPPRDLEVSEIRKDSCYLTWKEPLDDGGSVITNYWERRDVASAQWSPLSATS KKKSHFAKHLNEGNQYLFRVAAENQYGRGPFVETPKPIKALDPLHPPGPPKDLHHVDVDKTE VSLVWNKPDRDGGSPITGYLVEYQEEGTQDWIKFKTVTNLECWTGLQQGKTYRFRVKAENI VGLGLPDTTIPIECQEKLVPPSVELDVKLIEGLWKAGTTVRFPAI IRGVPVPTAKWTTDGS EIKTDEHYTVETDNFSSVLTIKNCLRRDTGEYQITVSNAAGSKTVAVHLTVLDVPGPPTGPI NILDVTPEHMTISWQPPKDDGGSPVINYIVEKQDTRKDTWGWSSGSSKTKLKIPHLQKGCE YVFRVRAENKIGVGPPLDSTPTVAKHKFSPPSPPGKPWTDITENAATVSWTLPKSDGGSPI TGYYMERREVTGKWVRVNKTPIADLKFRVTGLYEGNTYEFRVFAENLAGLSKPSPSSDPIKA CRPIKPPGPPINPKLKDKSRETADLVWTKPLSDGGSPILGYWECQKPGTAQWNRINKDELI RQCAFRVPGLIEGNEYRFRIKAANIVGEGEPRELAESVIAKDILHPPEVELDVTCRDVITVR VGQTIRILARVKGRPEPDITWTKEGKVLVREKRVDLIQDLPRVELQIKEAVRADHGKYI ISA KNS SGHAQGSAI VNVLDRPGPCQNLKVTNVTKENCT I SWENPLDNGGSE I TNFI VE YRKPNQ KGWSIVASDVTKRLIKANLLANNEYYFRVCAENKVGVGPTIETKTPILAINPIDRPGEPENL HIADKGKTFVYLKWRRPDYDGGSPNLSYHVERRLKGSDDWERVHKGS IKETHYMVDRCVENQ IYEFRVQTKNEGGESDWVKTEEVWKEDLQKPVLDLKLSGVLTVKAGDTIRLEAGVRGKPFP EVAWTKDKDATDLTRSPRVKIDTRADSSKFSLTKAKRSDGGKYWTATNTAGSFVAYATVNV LDKPGPVRNLKIVDVSSDRCTVCWDPPEDDGGCEIQNYILEKCETKRMVWSTYSATVLTPGT TVTRLIEGNEYI FRVRAENKIGTGPPTESKPVIAKTKYDKPGRPDPPEVTKVSKEEMTWWN PPEYDGGKSITGYFLEKKEKHSTRWVPVNKSAIPERRMKVQNLLPDHEYQFRVKAENEIGIG EPSLPSRPWAKDPIEPPGPPTNFRWDTTKHSITLGWGKPVYDGGAPIIGYWEMRPKIAD AS PDE GWKRCNAAAQLVRKE FT VT S LDENQE YE FRVCAQNQVG I GRPAE LKEAI KPKE I LE PAttorney Docket No. TVD-013WO2 PEIDLDASMRKLVIVRAGCPIRLFAIVRGRPAPKVTWRKVGIDNWRKGQVDLVDTMAFLVI PNSTRDDSGKYSLTLVNPAGEKAVFVNVRVLDTPGPVSDLKVSDVTKTSCHVSWAPPENDGG SQVTHYIVEKREADRKTWSTVTPEVKKTSFHVTNLVPGNEYYFRVTAVNEYGPGVPTDVPKP VLASDPLSEPDPPRKLEVTEMTKNSATLAWLPPLRDGGAKIDGYITSYREEEQPADRWTEYS WKDLSLWTGLKEGKKYKFRVAARNAVGVSLPREAEGVYEAKEQLLPPKILMPEQITIKAG KKLRIEAHVYGKPHPTCKWKKGEDEWTSSHLAVHKADSSS ILI IKDVTRKDSGYYSLTAEN SSGTDTQKIKWVMDAPGPPQPPFDISDIDADACSLSWHIPLEDGGSNITNYIVEKCDVSRG DWVTALASVTKTSCRVGKLIPGQEYIFRVRAENRFGISEPLTSPKMVAQFPFGVPSEPKNAR VTKVNKDC I FVAWDRPDS DGGS P I I GYL I ERKERNS LLWVKANDTLVRS TE YPCAGLVEGLE YSFRIYALNKAGSSPPSKPTEYVTARMPVDPPGKPEVIDVTKSTVSLIWARPKHDGGSKIIG YFVEACKLPGDKWVRCNTAPHQIPQEEYTATGLEEKAQYQFRAIARTAVNISPPSEPSDPVT ILAENVPPRIDLSVAMKSLLTVKAGTNVCLDATVFGKPMPTVSWKKDGTLLKPAEGIKMAMQ RNLCTLELFSVNRKDSGDYTITAENSSGSKSATIKLKVLDKPGPPASVKINKMYSDRAMLSW EPPLEDGGSEITNYIVDKRETSRPNWAQVSATVPITSCSVEKLIEGHEYQFRICAENKYGVG DPVFTEPAIAKNPYDPPGRCDPPVISNITKDHMTVSWKPPADDGGSPITGYLLEKRETQAVN WTKVNRKPI IERTLKATGLQEGTEYEFRVTAINKAGPGKPSDASKAAYARDPQYPPGPPAFP KVYDTTRSSVSLSWGKPAYDGGSPI IGYLVEVKRADSDNWVRCNLPQNLQKTRFEVTGLMED TQYQFRVYAVNKIGYSDPSDVPDKHYPKDILIPPEGELDADLRKTLILRAGVTMRLYVPVKG RPPPKITWSKPNVNLRDRIGLDIKSTDFDTFLRCENVNKYDAGKYILTLENSCGKKEYTIW KVLDTPGPPVNVTVKEISKDSAYVTWEPPI IDGGSPI INYWQKRDAERKSWSTVTTECSKT SFRVANLEEGKSYFFRVFAENEYGIGDPGETRDAVKASQTPGPWDLKVRSVSKSSCSIGWK KPHSDGGSRI IGYWDFLTEENKWQRVMKSLSLQYSAKDLTEGKEYTFRVSAENENGEGTPS E I TWARDDWAPDLDLKGLPDLCYLAKENSNFRLKI P IKGKPAPSVSWKKGEDPLATDTRV SVESSAVNTTLIVYDCQKSDAGKYTITLKNVAGTKEGTISIKWGKPGIPTGPIKFDEVTAE AMTLKWAPPKDDGGSEITNYILEKRDSVNNKWVTCASAVQKTTFRVTRLHEGMEYTFRVSAE NKYGVGEGLKSEPIVARHPFDVPDAPPPPNIVDVRHDSVSLTWTDPKKTGGSPITGYHLEFK ERNSLLWKRANKTPIRMRDFKVTGLTEGLEYEFRVMAINLAGVGKPSLPSEPWALDPIDPP GKPEVINITRNSVTLIWTEPKYDGGHKLTGYIVEKRDLPSKSWMKANHVNVPECAFTVTDLV EGGKYEFRIRAKNTAGAI SAPSESTET I ICKDEYEAPTIVLDPTIKDGLTIKAGDTIVLNAI SILGKPLPKSSWSKAGKDIRPSDITQITSTPTSSMLTIKYATRKDAGEYTITATNPFGTKVE HVKVTVLDVPGPPGPVEISNVSAEKATLTWTPPLEDGGSPIKSYILEKRETSRLLWTWSED IQSCRHVATKLIQGNEYI FRVSAVNHYGKGEPVQSEPVKMVDRFGPPGPPEKPEVSNVTKNT ATVSWKRPVDDGGSEITGYHVERREKKSLRWVRAIKTPVSDLRCKVTGLQEGSTYEFRVSAE NRAGIGPPSEASDSVLMKDAAYPPGPPSNPHVTDTTKKSASLAWGKPHYDGGLEITGYWEHAttorney Docket No. TVD-013WO2 QKVGDE AW I KD T T G T ALR I T Q FWP DL Q T KE K YN ER I S Al NDAGVGE PAV I P DVE I VE REMA PDFELDAELRRTLWRAGLSIRIFVPIKGRPAPEVTWTKDNINLKNRANIENTESFTLLI IP ECNRYDTGKFVMTIENPAGKKSGFVNVRVLDTPGPVLNLRPTDITKDSVTLHWDLPLIDGGS RITNYIVEKREATRKSYSTATTKCHKCTYKVTGLSEGCEYFFRVMAENEYGIGEPTETTEPV KASEAPSPPDSLNIMDITKSTVSLAWPKPKHDGGSKITGYVIEAQRKGSDQWTHITTVKGLE CWRNLTEGEEYTFQVMAVNSAGRSAPRESRPVIVKEQTMLPELDLRGI YQKLVIAKAGDNI KVEIPVLGRPKPTVTWKKGDQILKQTQRVNFETTATSTILNINECVRSDSGPYPLTARNIVG EVGDVITIQVHDIPGPPTGPIKFDEVSSDFVTFSWDPPENDGGVPISNYWEMRQTDSTTWV ELATTVIRTTYKATRLTTGLEYQFRVKAQNRYGVGPGITSACIVANYPFKVPGPPGTPQVTA VTKDSMTISWHEPLSDGGSPILGYHVERKERNGILWQTVSKALVPGNIFKSSGLTDGIAYEF RVIAENMAGKSKPSKPSEPMLALDPIDPPGKPVPLNITRHTVTLKWAKPEYTGGFKITSYIV EKRDLPNGRWLKANFSNILENEFTVSGLTEDAAYEFRVIAKNAAGAISPPSEPSDAITCRDD VEAPKIKVDVKFKDTVILKAGEAFRLEADVSGRPPPTMEWSKDGKELEGTAKLEIKIADFST NLVNKDSTRRDSGAYTLTATNPGGFAKHIFNVKVLDRPGPPEGPLAVTEVTSEKCVLSWFPP LDDGGAKIDHYIVQKRETSRLAWTNVASEVQVTKLKVTKLLKGNEYI FRVMAVNKYGVGEPL ESEPVLAVNPYGPPDPPKNPEVTTITKDSMWCWGHPDSDGGSEI INYIVERRDKAGQRWIK CNKKTLTDLRYKVSGLTEGHEYEFRIMAENAAGISAPSPTSPFYKACDTVFKPGPPGNPRVL DTSRSSISIAWNKPIYDGGSEITGYMVEIALPEEDEWQIVTPPAGLKATSYTITGLTENQEY KIRIYAMNSEGLGEPALVPGTPKAEDRMLPPEIELDADLRKWTIRACCTLRLFVPIKGRPA PEVKWARDHGESLDKAS IESTSSYTLLIVGNVNRFDSGKYILTVENSSGSKSAFVNVRVLDT PGPPQDLKVKEVTKTSVTLTWDPPLLDGGSKIKNYIVEKRESTRKAYSTVATNCHKTSWKVD QLQEGCSYYFRVLAENEYGIGLPAETAESVKASERPLPPGKITLMDVTRNSVSLSWEKPEHD GGSRILGYIVEMQTKGSDKWATCATVKVTEATITGLIQGEEYSFRVSAQNEKGISDPRQLSV PVIAKDLVIPPAFKLLFNTFTVLAGEDLKVDVPFIGRPTPAVTWHKDNVPLKQTTRVNAEST ENNSLLTIKDACREDVGHYWKLTNSAGEAIETLNVIVLDKPGPPTGPVKMDEVTADSITLS WGPPKYDGGSSINNYIVEKRDTSTTTWQIVSATVARTTIKACRLKTGCEYQFRIAAENRYGK STYLNSEPTVAQYPFKVPGPPGTPWTLSSRDSMEVQWNEPISDGGSRVIGYHLERKERNSI LWVKLNKTPIPQTKFKTTGLEEGVEYEFRVSAENIVGIGKPSKVSECYVARDPCDPPGRPEA11 VTRNSVTLQWKKPT YDGGSKI TGYI VEKKELPEGRWMKAS FTNI I DTHFEVTGLVEDHRY EFRVIARNAAGVFSEPSESTGAITARDEVDPPRISMDPKYKDTIWHAGESFKVDADIYGKP IPTIQWIKGDQELSNTARLEIKSTDFATSLSVKDAVRVDSGNYILKAKNVAGERSVTVNVKV LDRPGPPEGPWISGVTAEKCTLAWKPPLQDGGSDI INYIVERRETSRLVWTWDANVQTLS CKVTKLLEGNEYTFRIMAVNKYGVGEPLESEPWAKNPFWPDAPKAPEVTTVTKDSMIWW ERPAS DGGSE I LGYVLEKRDKEG I RWTRCHKRL I GELRLRVTGL I ENHDYE FRVSAENAAGLAttorney Docket No. TVD-013WO2 SEPSPPSAYQKACDPIYKPGPPNNPKVIDITRSSVFLSWSKPIYDGGCEIQGYIVEKCDVSV GEWTMCTPPTGINKTNIEVEKLLEKHEYNFRICAINKAGVGEHADVPGPI IVEEKLEAPDID LDLELRKI INIRAGGSLRLFVPIKGRPTPEVKWGKVDGEIRDAAI IDVTSSFTSLVLDNVNR YDSGKYTLTLENSSGTKSAFVTVRVLDTPSPPVNLKVTEITKDSVSITWEPPLLDGGSKIKN YIVEKREATRKSYAAWTNCHKNSWKIDQLQEGCSYYFRVTAENEYGIGLPAQTADPIKVAE VPQPPGKITVDDVTRNSVSLSWTKPEHDGGSKI IQYIVEMQAKHSEKWSECARVKSLQAVIT NLTQGEEYLFRWAVNEKGRSDPRSLAVPIVAKDLVIEPDVKPAFSSYSVQVGQDLKIEVPI SGRPKPTITWTKDGLPLKQTTRINVTDSLDLTTLSIKETHKDDGGQYGITVANWGQKTASI EIVTLDKPDPPKGPVKFDDVSAESITLSWNPPLYTGGCQITNYIVQKRDTTTTVWDWSATV ARTTLKVTKLKTGTEYQFRIFAENRYGQSFALESDPIVAQYPYKEPGPPGTPFATAISKDSM VI QWHE PVNNGGS PVI GYHLERKERNS I LWTKVNKT 11 HDTQFKAQNLEEG I E YE FRVYAEN IVGVGKASKNSECYVARDPCDPPGTPEPIMVKRNEITLQWTKPVYDGGSMITGYIVEKRDLP DGRWMKASFTNVIETQFTVSGLTEDQRYEFRVIAKNAAGAISKPSDSTGPITAKDEVELPRI SMDPKFRDTIWNAGETFRLEADVHGKPLPTIEWLRGDKEIEESARCEIKNTDFKALLIVKD AIRIDGGQYILRASNVAGSKSFPVNVKVLDRPGPPEGPVQVTGVTSEKCSLTWSPPLQDGGS DISHYWEKRETSRLAWTWASEWTNSLKVTKLLEGNEYVFRIMAVNKYGVGEPLESAPVL MKNPFVLPGPPKSLEVTNIAKDSMTVCWNRPDSDGGSEI IGYIVEKRDRSGIRWIKCNKRRI TDLRLRVTGLTEDHEYEFRVSAENAAGVGEPSPATVYYKACDPVFKPGPPTNAHIVDTTKNS ITLAWGKPIYDGGSEILGYWEICKADEEEWQIVTPQTGLRVTRFEISKLTEHQEYKIRVCA LNKVGLGEATSVPGTVKPEDKLEAPELDLDSELRKGIWRAGGSARIHIPFKGRPTPEITWS REEGEFTDKVQIEKGVNYTQLS IDNCDRNDAGKYILKLENSSGSKSAFVTVKVLDTPGPPQN LAVKE VRKD S AFL VWE P P 11 DGGAKVKN YV I DKRE S T RKAYANVS S KG S KT S FKVENL T E GA I YYFRVMAENEFGVGVPVETVDAVKAAEPPSPPGKVTLTDVSQTSASLMWEKPEHDGGSRVL GYWEMQPKGTEKWS IVAESKVCNAWTGLSSGQEYQFRVKAYNEKGKSDPRVLGVPVIAKD LTIQPSLKLPFNTYSIQAGEDLKIEIPVIGRPRPNISWVKDGEPLKQTTRVNVEETATSTVL HIKEGNKDDFGKYTVTATNSAGTATENLSVIVLEKPGPPVGPVRFDEVSADFWI SWEPPAY TGGCQISNYIVEKRDTTTTTWHMVSATVARTTIKITKLKTGTEYQFRIFAENRYGKSAPLDS KAVIVQYPFKEPGPPGTPFVTSISKDQMLVQWHEPVNDGGTKIIGYHLEQKEKNSILWVKLN KTPIQDTKFKTTGLDEGLEYEFKVSAENIVGIGKPSKVSECFVARDPCDPPGRPEAIVITRN NVTLKWKKPAYDGGSKITGYIVEKKDLPDGRWMKASFTNVLETEFTVSGLVEDQRYEFRVIA RNAAGNFSEPSDSSGAITARDEIDAPNASLDPKYKDVIWHAGETFVLEADIRGKPIPDWW SKDGKELEETAARMEIKSTIQKTTLWKDCIRTDGGQYILKLSNVGGTKSIPITVKVLDRPG PPEGPLKVTGVTAEKCYLAWNPPLQDGGANISHYI IEKRETSRLSWTQVSTEVQALNYKVTK LLPGNEYIFRVMAVNKYGIGEPLESGPVTACNPYKPPGPPSTPEVSAITKDSMWTWARPVDAttorney Docket No. TVD-013WO2 DGGTEIEGYILEKRDKEGVRWTKCNKKTLTDLRLRVTGLTEGHSYEFRVAAENAAGVGEPSE PSVFYRACDALYPPGPPSNPKVTDTSRSSVSLAWSKPI YDGGAPVKGYWEVKEAAADEWTT CTPPTGLQGKQFTVTKLKENTEYNFRICAINSEGVGEPATLPGSWAQERIEPPEIELDADL RKVWLRASATLRLFVTIKGRPEPEVKWEKAEGILTDRAQIEVTSSFTMLVIDNVTRFDSGR YNLTLENNSGSKTAFVNVRVLDSPSAPVNLTIREVKKDSVTLSWEPPLIDGGAKITNYIVEK RETTRKAYATITNNCTKTTFRIENLQEGCSYYFRVLASNEYGIGLPAETTEPVKVSEPPLPP GRVTLVDVTRNTATIKWEKPESDGGSKITGYWEMQTKGSEKWSTCTQVKTLEATISGLTAG EEYVFRVAAVNEKGRSDPRQLGVPVIARDIEIKPSVELPFHTFNVKAREQLKIDVPFKGRPQ ATVNWRKDGQTLKETTRVNVSSSKTVTSLSIKEASKEDVGTYELCVSNSAGSITVPITIIVL DRPGPPGPIRIDEVSCDSITISWNPPEYDGGCQISNYIVEKKETTSTTWHIVSQAVARTSIK IVRLTTGSEYQFRVCAENRYGKSSYSESSAWAEYPFSPPGPPGTPKWHATKSTMLVTWQV PVNDGGSRVIGYHLEYKERSS ILWSKANKILIADTQMKVSGLDEGLMYEYRVYAENIAGIGK CSKSCEPVPARDPCDPPGQPEVTNITRKSVSLKWSKPHYDGGAKITGYIVERRELPDGRWLK CNYTNIQETYFEVTELTEDQRYEFRVFARNAADSVSEPSESTGPI IVKDDVEPPRVMMDVKF RDVIWKAGEVLKINADIAGRPLPVISWAKDGIEIEERARTEI ISTDNHTLLTVKDCIRRDT GQYVLTLKNVAGTRSVAVNCKVLDKPGPPAGPLEINGLTAEKCSLSWGRPQEDGGADIDYYI VEKRETSHLAWTICEGELQMTSCKVTKLLKGNEYIFRVTGVNKYGVGEPLESVAIKALDPFT VPSPPTSLEITSVTKESMTLCWSRPESDGGSEISGYI IERREKNSLRWVRVNKKPVYDLRVK STGLREGCEYEYRVYAENAAGLSLPSETSPLIRAEDPVFLPSPPSKPKIVDSGKTTITIAWV KPLFDGGAPITGYTVEYKKSDDTDWKTSIQSLRGTEYTISGLTTGAEYVFRVKSVNKVGASD PSDSSDPQIAKEREEEPLFDIDSEMRKTLIVKAGASFTMTVPFRGRPVPNVLWSKPDTDLRT RAYVDTTDSRTSLTIENANRNDSGKYTLTIQNVLSAASLTLWKVLDTPGPPTNITVQDVTK ESAVLSWDVPENDGGAPVKNYHIEKREASKKAWVSVTNNCNRLSYKVTNLQEGAI YYFRVSG ENEFGVGIPAETKEGVKITEKPSPPEKLGVTSISKDSVSLTWLKPEHDGGSRIVHYWEALE KGQKNWVKCAVAKSTHHWSGLRENSEYFFRVFAENQAGLSDPRELLLPVLIKEQLEPPEID MKNFPSHTVYVRAGSNLKVDI P I SGKPLPKVTLSRDGVPLKATMRFNTE I TAENLT INLKES VTADAGRYEITAANSSGTTKAFINIWLDRPGPPTGPWISDITEESVTLKWEPPKYDGGSQ VTNYILLKRETSTAVWTEVSATVARTMMKVMKLTTGEEYQFRIKAENRFGISDHIDSACVTV KLPYTTPGPPSTPWVTNVTRESITVGWHEPVSNGGSAWGYHLEMKDRNSILWQKANKLVIR TTHFKVTTISAGLIYEFRVYAENAAGVGKPSHPSEPVLAIDACEPPRNVRITDISKNSVSLS WQQPAFDGGSKITGYIVERRDLPDGRWTKASFTNVTETQFI ISGLTQNSQYEFRVFARNAVG SISNPSEWGPITCIDSYGGPVIDLPLEYTEWKYRAGTSVKLRAGISGKPAPTIEWYKDDK ELQTNALVCVENTTDLASILIKDADRLNSGCYELKLRNAMGSASATIRVQILDKPGPPGGPI EFKTVTAEKITLLWRPPADDGGAKITHYIVEKRETSRWWSMVSEHLEECI ITTTKI IKGNEAttorney Docket No. TVD-013WO2 YIFRVRAVNKYGIGEPLESDSWAKNAFVTPGPPGIPEVTKITKNSMTWWSRPIADGGSDI SGYFLEKRDKKSLGWFKVLKETIRDTRQKVTGLTENSDYQYRVCAVNAAGQGPFSEPSEFYK AADPIDPPGPPAKIRIADSTKSSITLGWSKPVYDGGSAVTGYWEIRQGEEEEWTTVSTKGE VRTTEYWSNLKPGVNYYFRVSAVNCAGQGEPIEMNEPVQAKDILEAPEIDLDVALRTSVIA KAGEDVQVLIPFKGRPPPTVTWRKDEKNLGSDARYSIENTDSSSLLTIPQVTRNDTGKYILT I ENGVGE PKS S TVS VKVLDT PAACQKLQVKHVSRGTVTLLWDPPL I DGGS P I INYVI EKRDA TKRTWSWSHKCSSTSFKLIDLSEKTPFFFRVLAENEIGIGEPCETTEPVKAAEVPAPIRDL SMKDSTKTSVILSWTKPDFDGGSVITEYWERKGKGEQTWSHAGISKTCEIEVSQLKEQSVL EFRVFAKNEKGLSDPVTIGPITVKELI ITPEVDLSDIPGAQVTVRIGHNVHLELPYKGKPKP SISWLKDGLPLKESEFVRFSKTENKITLSIKNAKKEHGGKYTVILDNAVCRIAVPITVITLG PPSKPKGPIRFDEIKADSVILSWDVPEDNGGGEITCYSIEKRETSQTNWKMVCSSVARTTFK VPNLVKDAEYQFRVRAENRYGVSQPLVSSIIVAKHQFRIPGPPGKPVIYNVTSDGMSLTWDA PVYDGGSEVTGFHVEKKERNSILWQKVNTSPISGREYRATGLVEGLDYQFRVYAENSAGLSS PSDPSKFTLAVSPVDPPGTPDYIDVTRETITLKWNPPLRDGGSKIVGYSIEKRQGNERWVRC NFTDVSECQYTVTGLSPGDRYEFRI IARNAVGTISPPSQSSGI IMTRDENVPPIVEFGPEYF DGLIIKSGESLRI KALVQGRPVPRVT W FKDGVE I EKRMNME I T DVLGS T S L FVRDATRDHRG VYTVEAKNASGSAKAEIKVKVQDTPGKWGPIRFTNITGEKMTLWWDAPLNDGCAPITHYI I EKRETSRLAWALIEDKCEAQSYTAIKLINGNEYQFRVSAVNKFGVGRPLDSDPWAQIQYTV PDAPGIPEPSNITGNSITLTWARPESDGGSEIQQYILERREKKSTRWVKVISKRPISETRFK VTGLTEGNEYEFHVMAENAAGVGPASGISRLIKCREPVNPPGPPTWKVTDTSKTTVSLEWS KPVFDGGMEI IGYI IEMCKADLGDWHKVNAEACVKTRYTVTDLQAGEEYKFRVSAINGAGKG DSCEVTGTIKAVDRLTAPELDIDANFKQTHWRAGASIRLFIAYQGRPTPTAVWSKPDSNLS LRADIHTTDSFSTLTVENCNRNDAGKYTLTVENNSGSKSITFTVKVLDTPGPPGPITFKDVT RGSATLMWDAPLLDGGARIHHYWEKREASRRSWQVISEKCTRQIFKVNDLAEGVPYYFRVS AVNEYGVGEPYEMPEPIVATEQPAPPRRLDWDTSKSSAVLAWLKPDHDGGSRITGYLLEMR QKGSDFWVEAGHTKQLTFTVERLVEKTEYEFRVKAKNDAGYSEPREAFSSVI IKEPQIEPTA DLTGITNQLITCKAGSPFTIDVPISGRPAPKVTWKLEEMRLKETDRVSITTTKDRTTLTVKD SMRGDSGRYFLTLENTAGVKTFSVTVWIGRPGPVTGPIEVSSVSAESCVLSWGEPKDGGGT EITNYIVEKRESGTTAWQLVNSSVKRTQIKVTHLTKYMEYSFRVSSENRFGVSKPLESAPI I AEHPFVPPSAPTRPEVYHVSANAMSIRWEEPYHDGGSKIIGYWVEKKERNTILWVKENKVPC LECNYKVTGLVEGLEYQFRTYALNAAGVSKASEASRPIMAQNPVDAPGRPEVTDVTRSTVSL IWSAPAYDGGSKWGYI lERKPVSEVGDGRWLKCNYTIVSDNFFTVTALSEGDTYEFRVLAK NAAGVISKGSESTGPVTCRDEYAPPKAELDARLHGDLVTIRAGSDLVLDAAVGGKPEPKIIW TKGDKELDLCEKVSLQYTGKRATAVIKFCDRSDSGKYTLTVKNASGTKAVSVMVKVLDSPGPAttorney Docket No. TVD-013WO2 CGKLTVSRVTQEKCTLAWSLPQEDGGAEITHYIVERRETSRLNWVIVEGECPTLSYWTRLI KNNEYIFRVRAVNKYGPGVPVESEPIVARNSFTIPSPPGIPEEVGTGKEHIIIQWTKPESDG GNEISNYLVDKREKKSLRWTRVNKDYWYDTRLKVTSLMEGCDYQFRVTAVNAAGNSEPSEA SNFISCREPSYTPGPPSAPRWDTTKHSISLAWTKPMYDGGTDIVGYVLEMQEKDTDQWYRV HTNATIRNTEFTVPDLKMGQKYSFRVAAVNVKGMSEYSESIAEIEPVERIEIPDLELADDLK KTVTIRAGASLRLMVSVSGRPPPVITWSKQGIDLASRAI IDTTESYSLLIVDKVNRYDAGKY T IEAENQSGKKSATVLVKVYDTPGPCPSVKVKEVSRDSVT I TWE I PT I DGGAPVNNYI VEKR EAAMRAFKTVTTKCSKTLYRISGLVEGTMYYFRVLPENIYGIGEPCETSDAVLVSEVPLVPA KLEWDVTKSTVTLAWEKPLYDGGSRLTGYVLEACKAGTERWMKWTLKPTVLEHTVTSLNE GEQYLFRIRAQNEKGVSEPRETVTAVTVQDLRVLPTIDLSTMPQKTIHVPAGRPVELVIPIA GRPPPAASWFFAGSKLRESERVTVETHTKVAKLT TRETT IRDTGEYTLELKNVTGTTSET IK VIILDKPGPPTGPIKIDEIDATSITISWEPPELDGGAPLSGYWEQRDAHRPGWLPVSESVT RSTFKFTRLTEGNEYVFRVAATNRFGIGSYLQSEVIECRSSIRIPGPPETLQIFDVSRDGMT LTWYPPEDDGGSQVTGYIVERKEVRADRWVRVNKVPVTMTRYRSTGLTEGLEYEHRVTAINA RGSGKPSRPSKPIVAMDPIAPPGKPQNPRVTDTTRTSVSLAWSVPEDEGGSKVTGYLIEMQK VDQHEWTKCNTTPTKIREYTLTHLPQGAEYRFRVLACNAGGPGEPAEVPGTVKVTEMLEYPD YELDERYQEGIFVRQGGVIRLTIPIKGKPFPICKWTKEGQDISKRAMIATSETHTELVIKEA DRGDSGTYDLVLENKCGKKAVYIKVRVIGSPNSPEGPLEYDDIQVRSVRVSWRPPADDGGAD ILGYILERREVPKAAWYTIDSRVRGTSLWKGLKENVEYHFRVSAENQFGISKPLKSEEPVT PKTPLNPPEPPSNPPEVLDVTKSSVSLSWSRPKDDGGSRVTGYYIERKETSTDKWVRHNKTQ ITTTMYTVTGLVPDAEYQFRIIAQNDVGLSETSPASEPWCKDPFDKPSQPGELEILSISKD SVTLQWEKPECDGGKEILGYWVEYRQSGDSAWKKSNKERIKDKQFTIGGLLEATEYEFRVFA ENETGLSRPRRTAMS IKTKLTSGEAPGIRKEMKDVTTKLGEAAQLSCQIVGRPLPDIKWYRF GKELIQSRKYKMSSDGRTHTLTVMTEEQEDEGVYTCIATNEVGEVETSSKLLLQATPQFHPG YPLKEKYYGAVGSTLRLHVMYIGRPVPAMTWFHGQKLLQNSENITIENTEHYTHLVMKNVQR KTHAGKYKVQLSNVFGTVDAILDVEIQDKPDKPTGPIVIEALLKNSAVISWKPPADDGGSWI TNYWEKCEAKEGAEWQLVSSAISVTTCRIVNLTENAGYYFRVSAQNTFGISDPLEVSSWI IKSPFEKPGAPGKPTITAVTKDSCWAWKPPASDGGAKIRNYYLEKREKKQNKWISVTTEEI RETVFSVKNLIEGLEYEFRVKCENLGGESEWSEISEPITPKSDVPIQAPHFKEELRNLNVRY QSNATLVCKVTGHPKPIVKWYRQGKEI IADGLKYRIQEFKGGYHQLI IASVTDDDATVYQVR ATNQGGSVSGTASLEVEVPAKIHLPKTLEGMGAVHALRGEWSIKIPFSGKPDPVITWQKGQ DLIDNNGHYQVIVTRSFTSLVFPNGVERKDAGFYWCAKNRFGIDQKTVELDVADVPDPPRG VKVSDVSRDSVNLTWTEPASDGGSKITNYIVEKCATTAERWLRVGQARETRYTVINLFGKTS YQFRVIAENKFGLSKPSEPSEPTITKEDKTRAMNYDEEVDETREVSMTKASHSSTKELYEKYAttorney Docket No. TVD-013WO2 MIAEDLGRGEFGIVHRCVETSSKKTYMAKFVKVKGTDQVLVKKEISILNIARHRNILHLHES FESMEELVMIFEFISGLDIFERINTSAFELNEREIVSYVHQVCEALQFLHSHNIGHFDIRPE NIIYQTRRSSTIKIIEFGQARQLKPGDNFRLLFTAPEYYAPEVHQHDWSTATDMWSLGTLV YVLLSGINPFLAETNQQIIENIMNAEYTFDEEAFKEISIEAMDFVDRLLVKERKSRMTASEA LQHPWLKQKIERVSTKVIRTLKHRRYYHTLIKKDLNMWSAARISCGGAIRSQKGVSVAKVK VASIEIGPVSGQIMHAVGEEGGHVKYVCKIENYDQSTQVTWYFGVRQLENSEKYEITYEDGV AILYVKDITKLDDGTYRCKWNDYGEDSSYAELFVKGVREVYDYYCRRTMKKIKRRTDTMRL LERPPEFTLPLYNKTAYVGENVRFGVTITVHPEPHVTWYKSGQKIKPGDNDKKYTFESDKGL YQLTINSVTTDDDAEYTWARNKYGEDSCKAKLTVTLHPPPTDSTLRPMFKRLLANAECQEG QSVCFEIRVSGIPPPTLKWEKDGQPLSLGPNIEI IHEGLDYYALHIRDTLPEDTGYYRVTAT NTAGSTSCQAHLQVERLRYKKQEFKSKEEHERHVQKQIDKTLRMAEILSGTESVPLTQVAKE ALREAAVLYKPAVSTKTVKGEFRLEIEEKKEERKLRMPYDVPEPRKYKQTTIEEDQRIKQFV PMSDMKWYKKIRDQYEMPGKLDRWQKRPKRIRLSRWEQFYVMPLPRITDQYRPKWRIPKLS QDDLEIVRPARRRTPSPDYDFYYRPRRRSLGDISDEELLLPIDDYLAMKRTEEERLRLEEEL ELGFSASPPSRSPPHFELSSLRYSSPQAHVKVEETRKDFRYSTYHIPTKAEASTSYAELRER HAQAAYRQPKQRQRIMAEREDEELLRPVTTTQHLSEYKSELDFMSKEEKSRKKSRRQREVTE ITEIEEEYEISKHAQRESSSSASRLLRRRRSLSPTYIELMRPVSELIRSRPQPAEEYEDDTE RRSPTPERTRPRSPSPVSSERSLSRFERSARFDI FSRYESMKAALKTQKTSERKYEVLSQQP FTLDHAPRITLRMRSHRVPCGQNTRFILNVQSKPTAEVKWYHNGVELQESSKIHYTNTSGVL TLEILDCHTDDSGTYRAVCTNYKGEASDYATLDVTGGDYTTYASQRRDEEVPRSVFPELTRT EAYAVSSFKKTSEMEASSSVREVKSQMTETRESLSSYEHSASAEMKSAALEEKSLEEKSTTR KIKTTLAARILTKPRSMTVYEGESARFSCDTDGEPVPTVTWLRKGQVLSTSARHQVTTTKYK STFEISSVQASDEGNYSVWENSEGKQEAEFTLTIQKARVTEKAVTSPPRVKSPEPRVKSPE AVKSPKRVKSPEPSHPKAVSPTETKPTPTEKVQHLPVSAPPKITQFLKAEASKEIAKLTCW ESSVLRAKEVTWYKDGKKLKENGHFQFHYSADGTYELKINNLTESDQGEYVCEISGEGGTSK TNLQFMGQAFKSIHEKVSKISETKKSDQKTTESTVTRKTEPKAPEPISSKPVIVTGLQDTTV SSDSVAKFAVKATGEPRPTAIWTKDGKAITQGGKYKLSEDKGGFFLEIHKTDTSDSGLYTCT VKNSAGSVSSSCKLTIKAIKDTEAQKVSTQKTSEITPQKKAWQEEISQKALRSEEIKMSEA KSQEKLALKEEASKVLISEEVKKSAATSLEKSIVHEEITKTSQASEEVRTHAEIKAFSTQMS INEGQRLVLKANIAGATDVKWVLNGVELTNSEEYRYGVSGSDQTLTIKQASHRDEGILTCIS KTKEGIVKCQYDLTLSKELSDAPAFISQPRSQNINEGQNVLFTCEISGEPSPEIEWFKNNLP ISISSNVSISRSRNVYSLEIRNASVSDSGKYTIKAKNFRGQCSATASLMVLPLVEEPSREW LRTSGDTSLQGSFSSQSVQMSASKQEASFSSFSSSSASSMTEMKFASMSAQSMSSMQESFVE MSSSSFMGISNMTQLESSTSKMLKAGIRGIPPKIEALPSDISIDEGKVLTVACAFTGEPTPEAttorney Docket No. TVD-013WO2 VTWSCGGRKIHSQEQGRFHIENTDDLTTLI IMDVQKQDGGLYTLSLGNEFGSDSATVNIHIR SI (SEQ IDNO: 306);MTTQAPMFTQPLQSVWLEGSTATFEAHVSGSPVPEVSWFRDGQVISTSTLPGVQISFSDGR ARLMIPAVTKANSGRYSLRATNGSGQATSTAELLVTAETAPPNFSQRLQSMTVRQGSQVRLQ VRVTGIPTPWKFYRDGAEIQSSLDFQISQEGDLYSLLIAEAYPEDSGTYSVNATNSVGRAT STAELWQGEEWPAKKTKTIVSTAQISETRQTRIEKKIEAHFDARSIATVEMVIDGATGQL PHKTPPRIPPKPKSRSPTPPSIAAKAQLARQQSPSPIRHSPSPVRHVRAPTPSPVRSVSPAG RISTSPIRSVKSPLLIRKTQTTTMATGPEVPPPWKQEGYVASSTEAEMRETTMTSSTQIRRE ERWEGRYGVQEQVT I SGAAAAAASASVS S S FTAGAI TTGTKEVKQETDKSAAVATWAAVDM ARVREPAISAVEQTAQRTTTTAVHIQPAQEQARKEAEKTAVTKVWAADKAKEQELKSRTRE VMVTTQEQTHISHEQIRKETEKAFVPKWISATKAKEQETRITGEITTKQEQKRITQETIRQ ETEEIAASMVWATAKSTKLEAAVGVQEETAIQQDQMHLTHEQMMKETRKTWPKVIVATPK I KE QDLVS RS REAI T TKRDQVQ I T QEKKRKE VE T TAL S T I AVATAKAKE QE T VLRS REAMAT RQEHIQVTHGQVGVGKKAEAVATWAAVDQARVREPREPTHVEESHSQQTTLEYGYKEHIST TKVPEQPRRPASEPHWPQAVKPAVIQAPSETHIKTTDQMGMHISSQVKKTTDISTERLVHV DKRPRTASPHFTVSKISVPKTEHGYEASIAGSAIATLQKELSATSSTQKITKSVKAPTVKPG ETRVRAEPTPSPQFPFADMPPPDTYKSQAGIEVKKEVGVSISGSTVREEHFEVLRGREAKVT ETARVPAPAEVPVTPPTLVSGLKNVTVIEGESVTLECHISGYPSPKVTWYREDYQIESSIDF QITFQGGIARLMIREAFAEDSGRFTCSAVNEAGTVSTSCYLAVQVSEEFDKETTLTEKFATE EKRFVESRDWMTDTSITEEQAGPGEPAAPFFISKPWQKLVEGGSWFECQIGGNPKPHVY WKKSGVPLTTGYRYKVSYNKQTGECRLVISMTFADDAGEYTIVIRNKHGETSASASLLEEAD YEALVKTQQEMLYQTQMSTFIQEPKVGEIAPGFAYSEYEKEYEKEQALIRKKMAKDTVMVRT FVEDQEFHISSFEERLIKEIEYRI IKTTLEELLEEDGEEKMAVDISESEAIESGFDIRIKNY RILEGMGVTFHCKMSGYPLPKIAWYKDGKRIRHGERYQMDFLQDGRASLRIPWLPEDEGIY TAFASNIKGNAICSGKLYVEPAAPFSAPTYMPTPEAVSRIRSVSPRSLSRSPIRMSPAMSPA RMS FARMS FARMS FARMS PGRRLEE TDE S QLERL YKPVFVLKPAS FKCLEGQTARFDLKWG RPMPETFWFHNGQQIVNDYTHKWIKEDGTQSLI IVPASPSDSGEWTWAQNRAGKSTISVT LTVEAVEHQIKPAFVEKLKNVNIKEGSRLEMKVRATGNPNPDIVWLKNSDI IVPHKYPRIRI EGTRGEAALKIDSIISQDSAWYTATAINKAGRDTTRCKVNVEVEFAEPEPERKLIIPRGTYR AKEIAAPELEPLHLRYGQEQWEEGDLYDKEKQQKPFFKKKLTSLRLKRFGPAHFECRLTPIG DPTMWEWLHDGKPLEAANRLRMINEFGYCSLDYGAAYSRDSGVITCRATNKYGTDHTSATL IVKDEKSLVEESQLPDGKKGLQRIEELERMAHEGALTGVTTDQKEKQKPDIVLFPEPVRVLE GETARFRCRVTGYPQPKVNWYLNGQLIRKSKRFRVRYDGIHYLDIVDCKSYDTGEVKVTAENAttorney Docket No. TVD-013WO2 PEGVTEHKVKLEIQQREDFRSVLRRAPEPKPEFHVHEPGKLQFEVQKVDRPVDTSETKEWK LKRAERITHEKVSEESEELRSKFKRRTEEGYYEAITAVELKSRKKDESYEELLKKTKDELLH WTKELTEEEKKALAEEGKITIPTFKPERIELSPSMEAPKIFERIQSQTVGQGSDAHFRVRW GKPDPECEWYKNGVKIERSDRI YWYWPEDNVCELVIRDVTAEDSAS IMVKAINIAGETSSHA FLLVQAKQLITFTQELQDWAKEKDTMATFECETSEPFIKVKWYKDGIEVHAGDKYRMHSDR KVHFLSVLTIDTSDAEDYSCVLVEDENIKTTAKLIVEGAWEFVKELQDIEVPESYSGELEC IISPENIEGKWYHNDVELKSNGKYSITSRRGRQNLTVKDVTKEDQGEYSFWDGKKTTCKLK MKPRPIAILQGLSDQKVCEGDIVQLEVKVSLENVEGVWMKDGQEVQHSDRVHIVIDKQSHML LIEDMTKEDAGNYSFTIPALGLSTSGNVSVYSVDVITPLKDVNVIEGTKAVLECKVSVPDVT SVKWYLNDEQIKPDDRVQS IVKGTKQRLVINRTHASDEGPYKLMVGRVETSCNLSVEKIKI I RGLRDLTCTETQNWFEVELSHSGIDWWNFKGKEIKPSSKYKIEAHGKIYKLTVLNMMKDD EGEYAFYAGENTTSGKLTVAGGAISTPLTDQTVAESQEAVFECEVANPESEGEWLKDGKHLA LSNNFRGESDGHKRRLVIAAAKLDDAGEYTYKVATSKTSAKLKVEAVKIKKTLRNLTVTETQ DAVFSVELTHPDVKGVQWIKNGWLDSNDKYEISVKGTLYSLKIKNCAMADESVYGFKLGRL GASARLHVETVKI IKKPKDVTALENATVTFEVSVSHDTVPVKWFHKNVEIKPSDKHRLVSER KVHKLMLQSISPSDAGEYTAMVGQLECKAKLFVETLHITKTMKSIEVPETKAASFECEVSHF NVPSMWLKNGVEIEMSEKFKIWQGKLHQLI IMNTSTEDSAEYTFVCGNDQVSATLTVTPIM ITSMLKDINAEEKDTITFEVTVNYEGISYKWLKNGVEIKSTDRCQMRTKKLTHSLNIRNVHF GDAAD YT FVAGKAT S TAT L YVEARH I E FRKH I KD I KVLEKKRAMFE GE VS E PD I T VQWMKDG QELQIADRIKIQKEKYVHRLLIPSTRMSDAGKYTWAGGNMSTANLFVEGRDVRIRSIKKEV QVIEKQRAWEFEVNEDDVDAHWYKDGIEINFQVQERHQYWERRIHRMFISETRHSDAGEY TFVAGRNRSSVTLYVNAPEPPQVLQELQPVTVQSGKPARFCAVISGRPQPKISWYKEEQLLS TGFKCKFLHDGQEYTLLLIEAFPEDAAVYTCEAKNDYGVATTSASLSVEVPEWSPDQEMPV YPPAIVTPLQDTVTSEGRPARFQCQVSGTDLKVSWYCKDKKIKPSRFFRMTQFEDTYQLEIA EAYPEDEGTYAFVANNAVGQVSSTATLRLEAPESILHERIGQQIEMEMKEIASLLSAEEDFQ TYSSDLRLPNANETLELLSEPPARSTQFDSRQEGAAPVFIREISDVEISVEDVAKLSVTVTG CPKPKIQWFFNGMLLTPSADYKFVFDGDTHSLI ILFTRFQDEGEYTCLASNEYGKAVCSAHL RISPRGERSTEMESGEKKALEKPKGPCPPYFFKELKPVHCGPGIPAVFEYSVHGEPAPTVLW FKEDMPLYTSVCYTI IHSPDGSGTFIVNDPQRGDSGLYLCKAQNLWGESTCAAELLVLPEDT DVPDASCKEESTLGVPGDFLETSARGPLVQGVDSRQEITAFAEGTISKAALIAEETLQLSYE RSVDDSEVGTGVTIGAQKLPPWLSTPQGTGELPSIDGAVHTQPGRGPPPTLNLQAVQAQTT LPKEATLQFEEPEGVFPGASSAAQVSPVTIKPLITLTAEPKGNYPQSSTAAPDHALLSSVAA ETLQLGEKKIPEVDKAQRALLLSQSLAEGCVESLEVPDVAVSNMRSEPQVPFQHTCTEGKIL MASADTLKSTGQDVALRTEEGKSLSFPLALEEKQVLLKEEQSEWAVPTSQTSKSEKEPEAIAttorney Docket No. TVD-013WO2 KGVKEVREQELLSKETLFPSMPEEQRLHLKTQVRRALQAAVAREQANLFSEWLRNIDKVEVT AVNFTQEPKRILCTYLITSVSSLTEELTVTIEDIDPQMANLETGLKDALCSIVCEERNILMA EDPRIHEEDKIDVQGGRDHLSDAQKVETVIEAEADSKYLVSKEEVSWSKVESQLKDGDTNEV PQAETLKLAEESGTQKTSTEMSQEEAEGTLADLCPAVLKHLVDTISEEGDTVHLTSSISNAK EVHWYFKGNLVPSDGKFKCLKEQNAYTLVIEAVKTEDEGEYVCEASNDSGKAKTSAKLTVGE RAAPVIKRRIEPLEVALGHLAKFTCEIQGAPNVRFQWFKAGREIYESDKCSIRSSNYVSSLE ILRTQWDCGEYTCKASNEYGSVSCTATLTVTEAYPPTFLSRPKALTTFVGKAAKFLCTVSG TPVIEI IWQKDGAALSPSPDCRVTDADNKHSLELSNLTVQDRGI YSCKASNKFGADICQAEL TIIDKPHFIKELEAVQSAINKKIHLECQVDEDRKVTITWSKDGQKLPAGKDYKIYFEDKIAS LEIPLAKLKDSGTYTCTASNEAGSSSSSAAVAVREPPSFVKKVDPSYLMLPGESARLHCKLK GSPVIQVTWFKNNKELSESNTVRMSFVNSEAILDITDVKVDDSGTYSCEATNDVGSDSCSTE WIKEPPSFIKTLEPADIVRGANALLQCEIAGTGPFEVNWFKDKKQIRSSKKYRLFTQKTFV YLEISSFNSADVGDYECWANEVGKCGCVATHLLKEPPTFVKKVDDFTALAGQTVTLQAAVR GSEPISVMWMKGQEVIKEDGKIKMSFSNGVAVLTIPDVQISLGGKYTCLAENEAGSQTSVGE LIVKEPAKI lERAELIQVTAGDPATLEYTVSGTPELKPKWYKDGRPLVASKKYRISFKNNIA QLKFYSAELHDSGQYTFEISNEVGSSSCETTFTVLDRDIAPLFTKPLRNVDSWGGACRLDC KIAGSLPMRVSWFKDGKELTASDRYQIAFVEGTASLEISRVDMNDAGNFTCRATNSVGSKDS SGALIVQEPPSFVTKPGSRDVLPGSAVCLKSAFQGSAPLTIKWFKGDKELVSGGSCYITKET SESSLELYAVKTSDSGTYTCKVSNVAGSVECSADLFVKEPATFIEKLEPSQLLKKGDGTQLA CKVTGTPPIKITWFANDRELRESSKHKMSFAESTAVLRLTDVAIEDSGEYMCEAQNEAGSDH CTGIVIVKESPYFTKEFKSIEVLKEYDVMLLAEVAGTPPFEITWFKDNTTLRSGRKYKTFLQ DQLVSLQVLKFVAADAGEYQCRVTNEVGSSTCSARVTLREPPSFIKKIEATSSLRGGTAAFQ ATLKGSLPITVTWLKDNDEITEDDNIRMTFENNVASLYLSGIEVKHDGKYVCQAKNDAGIQR CSALLSVKEPAT IMEEAVS I DVTQGDPATLQVKFSGTKE I SAKWFKDGQELTLGPKYKI SVT DTVSILKIISTEKKDSGEYTFEVQNDVGRSSCKASINVLDLIIPPSFTKKLRKMDSIKGSFI DLEC I VAGSHP I S I QWFKDDQE I SASDKHKFS FHDNTAFLE I SQLEGTDSGT YTCSATNKAG HSQCSGHLTVKEPPYFVEKPQSQDVNPGTRVQLKALVGGTAPMTIKWFKDNKELHPGAARSV WKDDTSTILELFSAKAADSGTYICQLSNDVGTTSSKATIFVKEPPQFIKKPSPVLVLRNGQS TTFECQVTGTPEIRVSWYLDGNEITDLRKYGISFVDGLATFQISNARVENSGTYVCEARNDA GTASCSIELKVKEPPIFIRELEPVEWKDSDVELECEVMGTTPFEVTWLKNNKEIRSGKKYT MSEKMSVFYLHITKCDPSDVGEYQCI IANEGGSCACSARVALKEPPSFIKKIENVTTVLKSS AT EQS TVAGS PP I S I TWLKDDQ I LEENDNVH I S FEDS VATLQVRS VDNGHS GRYTCQAKNE S GIERCYAFLLVQEPAQI IEKAKSVDVTEKDPVTLECWAGTPELKVKWLKDGKQIVPSRYFS MSFENNVASFRIQSVMKQDSGQYTFKVENDFGSSSCDAYLRVLDQDIPPSFTKKLTKMDKVLAttorney Docket No. TVD-013WO2 GSSIHMECKVSGSLPISAQWFKDGKEISTSAKYRLVCHENTVSLEVSNLELEDTANYTCKVS NVAGDNACSGILTVKEPPSFLVKPERQQAIPDSTVEFKAVLKGTPPFKIKWLKDDVELVSGP KCFIGLEGSTSFLNLYSVDSSKTGQYTCQVTNDVGSDSCTTMLLVTEPPKFVKKLEASKI IK AGDSARLECKITGSPEIQWWYRNEHELTASDKYQMTFIDSVAVIQMNSLGTEDSGDFICEA QNPAGSTSCSTKVIVKEPPVFSSFPPIVETLKNTEVSLECELSGTPPFEWWYKDKRQLRSS KKYKVAS KNFHAS I H I LNVE S T D I GE YHCKAQNE VGS DACVCAVKLKE P PKF I S KLNS L T W AGE PAE L QAS I E GAQ P I S VQ W L KE KE E V I RE S E N I R I S FVNNVAT L Q FAKVE P ANAGK Y ICQ VKNDGGVRENMATLTVLEPAVI IEKAGSMTVTVGETCALECKVAGTPELSVEWYKDGKLLTS SQKHKFSFYNKISSLKILSVEKEDAGTYTFQVQNNVGKSSCTAWDVSDRMVPPSFTRRLKD TGGVLGTSCILECKVAGSSPISIAWFHEKTKIVSGAKYQTTFSDNVCTLQLNSLDSSDMGSY TCVAANVAGSDECRALLTVQEPPSFVKEPEPLEVLPGKNITFTSVIRGTPPFKVGWFRGARE L VKGNRCN I Y EE D T VAE LE L FNIDISQSGEYT C WS NNAGQAS C T T RL FVKE PAT FVKKL S D HSVEPGKSI ILEGTYTGTLPISVTWKKDGVSITPSERCNIVTTEKTCILEILSSTKGDAGHY SCEIENEAGRDACDALVSTLEPPYFVTELEPLEASVGDSVSLQCQVAGTPEITVSWFKGDTK LRSTPEYRTYFTNNVATLVFNKVSINDSGEYTCMAENSIGTAASKTIFRIQERQLPPSFARQ LKDIEQTVGLPVTLTCRLNGSAPIQVCWYRDGVLLRDDENLQMSFVDNVATLKILQTDLSHS GQYSCSASNPLGTASSTARLTAREPKKSPFFDIKPVS IDVIAGESADFECHVTGAQPMRVTW SKDNKEIRPGGNYTITCVGNTPHLRILKVGKGDSGQYTCQATNDVGKDMCSAQLSVKEPPKF IKKLDASKVAKQGESIQLECKISGSPEIKWWFRNDSELHESWKYNMSFVNSVALLTINEAS AEDTGDYICEAHNGVGDASCSTALKVKAPPVFTQKPPPVGALKGSDVILQCEISGTPPFEW WVKDRKQVRSSKKFKITSKNFDTSLHIFNLEAPDIGEYHCKATNEVGSDTCACTVKFKEPPR FVKKLSDASTLIGDPVELQAWEGFQPISWWLKDKGEVIRESENVRISFVDNIATLQLGSP EASQSGKYVCQIKNDAGMRECSAVLTVLEPATIVEKPEPMTVTTGNPFTLECWAGTPELSA KWFKDGRELSSGSRHHITFVRNLASLKIPSAEMNDKGLYTFEVENRVGKSSCTVSVHVSDRV VPPS FVRRLKDTSATLGASWLECRVSGSAP I SVGWFLDGNE 11 S S PKCQS S FADNVCTLTL SSLEPSDTGAYTCVAANVAGQDESSAVLTVQEPPSFEQTPDSVEVLPGMSLTFTSVIRGTPP FKVKWFKGSRELVSGEACTISLEDFVTELELLEVEPGQSGDYSCLVTNDAGSASCTTHLFVK EPATFVKRLADTSVETGSPIVLEATYSGTPPISVSWMKNEYPLSQSPNCGITTTEKSSILEI LESTIEDYAQYACLIENEAGQDICEALVSVLEPPYFIEPLEHVEAAIGEPITLQCKVDGTPE IRISWYKEHTKLRSAPAYKMQFKNNVASLVINKVDHSDVGEYTCKAENSVGAVASSAVLVIK ERKLPPSFARKLKDVHETLGFPVAFECRINGSEPLQVSWYKDGELLKDDANLQMSFVHHVAT LQILQTDQSHVGQYNCSASNPLGTASSSAKLILSEHEVPPFFDLKPVSVDLALGESGSFKCH VTGTAPIKITWAKDNREIRPGGNYKMTLVENTATLTVLKVAKGDAGQYTCYASNVAGKDSCS AQLGVQEPPRFIKKLDQSRIVKQDEYTRYECKIGGSPEIKVLWYKDEVEIQESSKFRMSFEDAttorney Docket No. TVD-013WO2 SVAILEMHSLSVEDSGDYTCEARNAAGSASSSTSLKVKEPPVFRKKPFPVETLKGADVHLEC ELQGTPPFQVSWHKDKRELRSGKKYKIMSENLLTS IHILNVDTADIGEYQCKATNDVGSDTC VGSVTMKAPPQFVKKLTDISTI IGKEVQLQTTIEGAEPISVAWFKDKGEIVRESDNIWISYS ENIATLQFSRAEPANAGKYTCQIKNDAGMQECYATLSVLEPAAIVEKPES IKVTTGDTCTLE CTVSGTPELSTKWFKDGKELTSDNKYKISFFNKVSGLKI INWPGDSGVYSFEVQNPVGKDS CKVSIQVSDRIIPPSFTRKLKETNGLSGSSWMECKVYGSPPISVLWFHDGNEISSGRKYQT TLTDNTCALTVNMLEEADAGDYTCIATNVAGSDECSAPLTVREPPSFVQKPDPMDVLTGSNV TFTS IVKGSPPFTVSWFKGSTELVPGARCNVSLQDSVGELELFDVDTSQSGEYTCIVSNEAG RASCTTRLFVKAPAIFVKRLNDYSIEKGKPLILEGTFSGTPPISVTWKKNGINVIASQRCNI TTTEKSAILEILSSTVEDSGQYNCYIENASGKDSCSAQILILEPPYFVKQLEPVKVTVGDSA SLQCQLAGTPEIGVSWYKGDTKLRPTATCKMHFKNNVATLVFTQVDSSDSGEYICRAENSVG EVSSSTFLTVQEQKLPPSFSRQLRDVQETVGLPWFECAVSGSEPISVSWYKDGKPLKDSPN IQTSFLDNIATLNIFKTDRSLSGQYSCTATNPIGSASSGAKLILTEGKNPPFFDIPLAPMDA WGESADLECHVTGTQPIKVTWAKDNREIRSGGNYQISYLENSAHLTIVKVDKGDSGQYTCY AVNEVGKDSCTAQLNIKERLIPPSFTKKLSETVEETEGNSFKLEGRVAGSQPITVAWYKNNV EIHPTSNCEIMFKNNALLLQVKRASMADAGLYTCKATNDAGSALCTSSIVIKEPKKPPVFDQ HLAPVTASEGDSVQLSCHVQGSEPIRIQWLKAGREVKPSDRCSFSFASGTAMLELKETAKAD SGDYVCKASNVAGSDTSKCKVTIKEKPAAAPAAKKAAVDGKLFFVSEPQSIRWEKTTATFI AKVGGDPIPNVKWTKGKWRQLNQGGRILIHQKGDEAKLEIRDTTKTDSGLYRCVAFNKHGEI ESNVNLQVDERKKQEKIEGDLRAMLKKTPALKKGSGEEEEIDIMELLKNVDPKEYEKYARMY GITDFRGLLQAFELLKQSQEEETHRLEIEELEKSERDEKEFEELVAFIQQRLTQTEPVTLIK DIENQTVLKDNDAIFEIDIKINYPEIKLSWYKGTEKLEPSNKYEISIDGDRHTLRVKNCQPK DQGNYRLVCGPHIASAKLTVIEPAWERHLQDVTLKEGQTCTMTCQFSVPNVKSEWFRNGRVL KPQGRVKTEVEHKVHKLTIADVRAEDQGQYTCKHEDLETSAELRIEAEPIQFTKRIQNIWS EHQSATFECEVSFDDAIVTWYKGPTELTESQKYNFRNDGRCHYMTIHNVTPDDEGVYSVIAR LEPRGEARSTAELYLTTKEIKLEMKPPDIPDSRVPIPTMPIRAVPPEEIPPAVAPSIPLLLP LPEEKKPPAKRIEVTKKGVKKDTKKWTKPKEEAPPPPVAKKPPPPTPMIPAKASEI IDVSS KAEEVKITTITRKKEVHKEKEAVYEREEAVYEKKVHIEPWEEPYEELETEPYTEPYEEPYYE EPDEDYEEIKVEAKKQVHEEWEEDFEEGQEYYEREEGYDEGEEEWEEIYHEREI IQVQKEVH EELHEKKIPAKVPEKKVPPPKWKKPWEKVEKTTRRMEEEKVQVIKVPEVSKKIVPQKPSR TPVQEEI lEVKVPAVHTKKMVISEEKMFFASHTEEEVSVSVPEVQKKTVTEEKIHVAVSKKI EPPPKVPEPPKKPVPEEWPVPIPKKVEPPAAKVPEAPKKPVPEEKKPVPIPKKKEPAAPPQ VPEAPKKPAPEEKI PVPVTKKKEAPPAKVPEVQKKWTEEKIAI I TQREES PPPAVPE I PKK KVPEEKRPVPRKEEVPPPKVPVPPKKAVPEAWPAPIPKKAPPRAEVSKKTWEEKRFAAEEAttorney Docket No. TVD-013WO2 KLSVAVPQRVELMRHEEEEWTYSEEEERVSVSVYREEERDEEEAEITEYEVLEEPEEYWEE KMHFISKKVEVEPAKVPEKKI IPKPKVPAKIEEPPPTKVPEPPKKIVPEKKVPAPAPKKVPP AKAPEESKRPVPEKRAPAEEVGIEEPPPTKVAERHMKITQEEKVLVAVTKKEAPPRARVPEE PKKVAPEERFPKLKPRREEEPPAKVTEVRKRAVKEEKVSIEVPKREPRPTKEVTVTEEKKWS YTREEETVSEHREEEYEDYEDYEEYKEFEEYEPTEEYDQYDEYAEREVEHYEEHEEYVTEPK KPVPVKPAQEPVPAKPKAPPPKVLKKAVPEEKAPLPIQKKLKPLPPKAPEEPKKWEEKIQI SITKREKQQVTEPVAKVPMKPKRWPEAKIPAPTKEVAVPVRVPGVPKKRELEEVWFKEEV EAHEEYIVEEEEEYVHEEEYVHKEEYVHEEEYVHKEEYIHEEEEHLHEEEETIAEEEWPVA PVKVPWPKKPVPEEKKPVPVPKKKEAPPAKVPE I PKKPEEKVPVP I PKKEKAPPAKVPEVP KKPVPEEKPPVPVPKKVEPPPAKVPEVPKKPVPEKKVPAPTPKKVEAPPAKVPEVPKKPIPE EKKPTALLKKMEAPPPKAPKKREWPVPVALPREEEEEEVPFEEVPEEEILPEEEVPSEEEA PPEEVPPEEEEVLPEEEEVLPEEEEVLPEEEEVQPEEEALPEIKPKVPKPAPVPKKTVPEKK VPVPVPKKVEPPPPPKVPEIKKKVPEKKVWPKKEEAPPTKVPEVSKKVEERRI IPPKEEEV PPAEVYEEAEEPTPEEIPEEPPSIEEEEIVEEEEEEEEVLPPRAPEWKKAVPEAPTPVPKK AEAPPAKVPKKI PEEKVPVPVQKKEAPPAKVPEKKKI PEKKVPVPKKEAVPPAKGKAVFEEK ISVAYQQEELVQERIELELVEAKVEEAFEEEEFHEVQEYFEEEEFHEVEEFIRVEERRFQEE HKVEEVHRVIEFLEAEEVEVYEKPKIPPKKGPEVSEKVIPPKKPPTKVIPRKEPPAKVPEVT KKTWEEKIRAPEEPKVPAPKAPEVPKKITPEEKVREAVPKKPEVPPPKVPEVPKKI IQEEK LPWLPEDTEIYMYEASEETVIEEEHVTLPQKARLKVAKVPAPPQTWTEEKTYVTIRKTRE TLALKESETTREAEPELKSYKAVPEIPEPPSPEDLEI IEDVLPEKRPPASKRRKTQLPTAPE APREMPPEMNTFEEISVEPEMLPTQVLDTYQEATVEKKTLRISRKKPELPSDEEVPEAPREV VAKKKVLPPQVPEWPVKVPGAPKEWSERKSLEEPPKKPAVRPVTVPEEPKEVIPEKKVSL VPPKKPAAPPVTVPEAPEEVFSEDEETLAPPQEPEAPPAKVPEAPKEWPEKKVSWPPKKP EAPPAKVPEAPKEAAPEKKVPVAPKKKPEAPPVKVPEAPKKWPEKKLPVAAPKKPEAPAAE VPEVPKTAVPQKKI PEAI PPKPES PPLEVPEVPPKEVTPEKKVPAAPPTKPE I PPPKVPEAP QAAWEEKTPEALPKKAEAAPVPVPQVQETVPEKTRPVGPPKKPEATTVPVPKVQKTIPEKT RPEAPPKRPEARTVPETVPEKTRPMAPPKKPEATTLPVPEVQETVPEKTRPVGPPKKPEATT VPVPEVQETIPEKTRPPKKPEATPVPVPEVQETVPEKTRPVGPPKKPEATTVSVPEVQETIP EKTRPAAPPKKPEATAVPETIPEKTRPEAPPKRPEATTVPVPEADQAWPEKKVPRVPPKKV EAPPITVPEEPKEVIPEKKVSLVPPKKPAAPPVTVPEAPEEVFSEDEETLAPPQEPEAPPAK VPEAPKEWPEKKVSWPPKKPEAPPAKVPEAPKEAAPEKKVPVAPKKKPEAPPVKVPEAPK KWPEKKLPVAAPKKPEAPAAEVPEVPKAAVPQKKIPEAIPPKPESPPPEVYEEPEEEIVPE EPPEEAVEEPVPAPPPKVTEPPKKPVPEKKAPPAWKKPEPPPAKVPEVPKEAPPEKKVPPK KPEAPPAKVPEVPKEWTEKKVAVPKKPEVPPAKVPEVPKKPVIEEKPAIPWEKVASPPAEAttorney Docket No. TVD-013WO2 VYEEPEEVTAEEEEPAPAVEEEEYEAPPPPAPVPEEPKKWPEKKFPVIKKPEAPPPKVPEV PKKAVPVKKVPWKKPEPPEAEVPEVPKKLVPVKKEPVPVTKKTEVLPEKVPEAPKKITPEK KESVPVPEEPEAPPASVEETPEETIYEEKATITIGRKETPPVEEREIEKFIQPEEPELEPEP EEIPVQEPEPEKKVIEKPKLKPRPPARPPSPPKEDVKEKMFQLKAVSKKKVPEKPEWEKVE PAPLKVPTAEKKVRKLLPEPKPQPKEEWLKSVLRKKPEEEEPKVEPKKVEKAKKPEEPQPP PKAVEVEAPPEPTPKERKVPEPAKVPEIKPAIPLPGPEPKPKPEPEVKTMKAPPIEPAPTPI AAPVTAPWGKKAEAKPKDEAAKPKGPIKGVAKKTPSPIEAERKKLRPGSGGEKPPDEAPFT YQLKAVPLKFVKEIKDIVLTEAESVGSSAIFECLVSPSTAITTWMKDGSNIRESPKHRFIAD GKDRKLHI IDVQLSDAGEYTCVLRLGNKEKTSTAKLIVEELPVRFVKTLEEEVTWKGQPLY LSCELNKERDWWRKDGKIWEKPGRIVPGVIGLMRALTINDADDTDAGTYTVTVENANNLE CSSCVKWEI IREWLVKPIRDQHVKPKGTAVFTCDIAKDTPNIKWFKGYDEIPLEPNDKTEI LKEGNHLFLKVKNAMPEDIDEYAVEIEGKRYPAKLTLGEREVELLKPIEDVTIYEKESASFD AE I SEEDI PGEWKLKGELLRPS PTCE IKAEGGKRFLTLHKVKLDQAGEVLYQACNAI TTAI L TVKEIELDFAVPLKDVTVPEKRQARFECVLTREANVIWSKGPDI IKASDKFDI IADGKKHIL VINDSQFDDEGVYTAEVEGKKTSAQLFVTGIRLKFISPLEDQTVKEGQTATFVCELSHEKMH WWFKNDVKLHTTRTVLMSSEGKTYKLEIRETTLDDISQIKAQVKNLSSTANLKVLEADPYF TVKLHDKTGVEKDEI ILKCEVSKDVPVKWFKDGEEIVPSPKHSVKTDGLRRILKIKKAELKD KGEYVCDCGTDTTKANVTVEARLIKVEKPLYGVEVFVGETARFEIELSEPDVHGQWKLKGEP LTAS PDCE I IEDGKKHVLVLYNCQLDMTGE I S FQAANAKSAANLKVKELPL I FI TPLSDVKV FEKDEAKFECEVSREPKTFRWLKGTQEITGDDRFELIKDGTRHSLVIKSAAFEDEAKYMFEA EDKRTSGKLI lEGIRLKFLTPLKDVTAKERENAVFTVELSHDNIPVSWFKNDQRLHTSKRVS MHDEGKTHSITFKDLSIDDTSQIRVEAMGISSEAKLTVLEGDPYFTGKLQDYTGVEKDEVIL QCEISKADAPVKWFKDGKEIKPSKNWIKADGKKRMLILKKALKSDIGQYTCDCGTDQTSGK LDIEDREIKLVRPLYSVEVMETETARFETEISEDDIHANWKLKGEALLQTPECEIKEEGKIH VLILHNCRLDQTGGVDFQAANVKSSAHLRVKPRVIGLLRPLKDVTVTAGETATFDCELSYED IPVEWYLKGKKLEPNDKWTRSEGRVHTLTLRDVKLEDAGEVQLTAKDFKTQANLFVKEPPV EFTKPLEDQTVEEEATAVLECEVSRENAKVKWFKNGTEILKSKKYEIVADGRVRKLI IHGCT PEDIKTYTCDAKDFKTSCNLNWPPHVEFLRPLTDLQVKEKETARFECEISKENEKVQWFKD GAEIKKGKKYDI ISKGAVRILVINKCLLNDEAEYSCEVRTARTSGMLTVLEEEAVFTKNLAN LEVSEGDTIKLVCEVSKPGAEVIWYKGDEEI IETGRFEILTDGRKRILI IQNAQLEDAGSYN CRLPSSRTDSKVKVHELAAEFISKPQNLEILEGEKAEFVCTISKESFEVQWKRDDQTLESGD KYDI IADGKKRVLWKDATLQDMGTYWMVGAARAAAHLTVIEKLRI IVPLKDTKVKEQQEV VFNCEVNTEGAKAKWFRNEEAI EDSSKYI ILQKDLVYTLRIRDARLDDQANFNVSLTNHRGE NVKSAANLIVEEEDLRIVEPLKDIETMEKKSVTFWCKVNRLNVTLKWTKNGEEVAFDNRISYAttorney Docket No. TVD-013WO2 RIDKYKHSLI IKDCGFPDEGEYWTAGQDKSVAELLI IEAPTEFVEHLEDQTVTEFDDAVFS CQLSREKANVKWYRNGREIKEGKKYKFEKDGSIHRLIIKDCRLEDECEYACGVEDRKSRARL FVEEIPVEIIRPPQDILEAPGADVIFLAELNKDKVEVQWLRNNMIWQGDKHQMMSEGKIHR LQICDIKPRDQGEYRFIAKDKEARAKLELAAAPKIKTADQDLWDAGQPLTMWPYDAYPKA EAEWFKENEPLSTKTVDTTAEQTSFRISEAKKDDKGRYKIVLQNKHGKAEGFINLQVIDVPG PVRNLEVTETFDGEVSLAWEEPLTDGGSKI IGYWERRDIKRKTWVLVTDRADSCEFTVTGL QKGGVEYLFRVSARNRVGTGEPVETDSPVEARSKYDVPGPPLNVTITDVNRFGVSLTWEPPE YDGGAEITNYVIELRDKTSIRWDTAMTVRAEDLSATVTDWEGQEYSFRVRAQNRIGVGKPS AATPFVKVADPIERPSPPVNLNASEQTQSSVQLTWEPPLKDGGSPILGYI IERREEGKDNWI RCNMKPVPELTYKVTGLQKGNKYLYRVSAENAAGVSDPSEILGPLTADDAFVEPTMDLSAFK DGLEVIVPNPIKILVPSTGYPRPKATWTFGDQVLEEGDRVKMKTISAYAELVISPSERTDKG IYTLTLENPVKSISGEINVNVIAPPSAPKELKFSDITKDSVHLTWEPPDDDGGSPLTGYWE KRDMSRKTWTKVMDFVTDLEFTVPDLVQGKEYLFKVCARNKCGPGEPAYTDEPVNMSAPATV PDPPENVKWRDRTANS I FLTWDPPKNDGGSRIKGYIVEKCPRGSDKWVACGEPVPDTKMEVT GLEEGKWYAYRVKALNRQGASKPSKPTEEIQAVDTQEAPEIFLDVKLLAGITVKAGTKIELP ATVTGKPEPKITWTKADTLLKPDQRITIENVPKKSTVTITDSKRSDTGTYI IEAVNVCGRAT AWEVNVLDKPGPPAAFDITDVTNESCLLTWNPPRDDGGSKITNYWERKATDSDVWHKLSS TVKDTNFKATKLTPNKEYIFRVAAENMYGVGEPVQAAPIIAKYQFDPPGPPTRLEPSDITKD AVTLTWCEPDDDGGSPITGYWVERLDPDTDKWVRCNKMPVKDTTYRVKGLTNKKKYRFRVLA ENLAGPGKPSRSTEPILIKDPIDPPWPPGKPTVKDIGKTSLVLNWTKPEHDGGAKIESYVIE MLKTGTDEWVRVAEGVPTTEHLLTGLMEGQEYSFRVRAVNKAGESEPSEPSDPVLCREKLYP PSPPRWLEVINITKNTADLKWTVPEKDGGSPITNYIVEKRDVRRKGWQTVDTTVKDTKCTVT PLTEGSLYVFRVAAENAIGQSDYTEIGDSVLAKDTFTTPGPPYALTWDVTKRHVDLKWEPP KNDGGRPIQRYI IEKKEKLGTRWVKAGKTSGPDCNFRVTDVIEGTEVQFQVRAENEAGVGHP SEPTEILSIEDPTSPPSPPLDLHVTDAGRKHIAIAWKPPEKNGGSPIIGYHVEMCPVGTEKW MRVNSRPIKDLKFKVEEGIVPDKEYVLRVRAVNAVGVSEPSEISENWAKDPDCKPTIDLET HD I WI E GEKLN I PVP FRAVPVP T VS WHKDGKE VKAS DRL TMKNDH I S AHLE VPKS VHADAG VYTITLENKLGSATASINVKVIGLPGPCKDIKASDITKSSCKLTWEPPEFDGGSPILHYVLE RREAGRRTYIPVMSGENKLSWTVKDLIPNGEYFFRVKAVNKIGGGEYIELKNPVIAQDPKQP PDPPVDVEVHNPTAKAMTITWKPPLYDGGSKIMGYI IEKITKGEDRWKRCNEHLVPVLTYTA KGLEEGKEYQFRVRAENAAGIGEPSRATPPTKAVDPIDAPKVILRTSLEVKRGDEIALDATI SGSPYPTITWIKDENVIVPEEIKKRAAPPVRRKKGEAEEEEPFSLPLTERLSINNSKQGESQ LRIRDSLRPDHGQYMIKVENDHGVAKAPCSVSVLDTPGPPINFVFEDIRKDSVLCKWEPPLD DGGSEI INYTLEKKDKTKPDSDWIVITSTLRNCKYSVTKLIEGKEYLFRVRAENRFGPGPPCAttorney Docket No. TVD-013WO2 VSKPLLAKDP EE PPDAPDKP I VEDVT SNSMLVKWNE PKDNGS P I LGYWLEKREVNS THWSRV NKTLLSSLKTKVDGLLEGLTYVFRVCAENAAGPGKFSPPSDPKTARDPISPPGPPVPRVADT SSTTIELEWEPPAFNGGGEIMGYFVDKQLVGTNEWSRCTEKMIKVRQFTVKEIREGADYKLR VSAVNAAGEGPPGETEPVTVAEPQEPPTVELDVSVKGGIQIMAGKTLRIPAEVTGRPVPTKV WTIEEGELDKERVI IENVGTKSELI IKNALRKDHGRYVITATNSCGSKFAAVRVEVFDVPGP VLDLKPWTNRKMCLLNWSDPADDGGSDITGFI IERKDAKMHTWRQPIETERSKCDITGLIE GQEYKFRVIAKNKFGCGPPVEIGPILAVDPLGPPTSPERLTYTERTKSTITLDWKEPRSDGG SPIQGYI lEKRRHDKPDFERVNKRLCPTTSFLVENLDEHQMYEFRVKAVNDVGESEPSLPLN WIQDDEVPPTIKLRLAVRGDTIKVKAGEPVNIPADVTGLPMPKIEWSKNEKVIDKPTDTLN I TKEEVSRSEAKTELS I PKAAREDKGT YT I TASNRLGSVFRNVHVEVYDRPS PPRNLAVTDI KAESCYLTWDAPLDNGGSEITHYI IDKRDASRKKSEWEEVTNTAVERRYGIWKLIPNGQYEF RVRAVNKYGISDECKSDKWIQDPYRLPGPPGKPKVLERTKGSMLVSWTPPLDNGGSPITGY WLEKREEGGTYWSRVSRAPITKVGLKGVEFNVPRLIEGVKYQFRAMAINAAGIGPPSEPSDP AVAGDPIYPPGPPSCPEVKDKTKSSISLAWKPPAKDGGSPIKGYIVEMQEEGTTDWKPVNEP DKLLTACECWPNLKELRKYRFRVKAVNEAGESEPSDTTGEIPATDIQEVPEVFIDIGAQDC LVCKAGS QVK I PAVI KGRP T PKS S WE FDGKAKKAMKDGVHD I PE DAQLE TAENS S VI I I PE C TRAHSGKYSITAKNKAGQKTANCRVKVMDAPGPPKDLKVSDITRGSCRLSWKMPDDDGGDRI KGYVIEKKTIDGKAWTKVNPNCGSTTFWPDLISEQQYFFRVRAENRFGIGPPAETIQRTTA RDPIYPPDPPIKLKIGLITKNTVHLSWKPPKNDGGSPVTHYIVECLAWDPTGKKKEAWRQCN RRDVEELEFTVEDLIEGGEYEFRVKAVNEAGVSKPSATVGPVIVKDQTCPPAIELKEFMEVE EGTDVNIVAKIKGVPFPTLTWFKAPPKKPDSKEPWYDTHVNKQWDDTCTLVIPQSRRSDT GLYSITAVNNLGTASKEMRLNVLGRPGPPVGPIKFESISADQMTLSWLPPKDDGGSKITNYV IEKREANRKTWVRVSSEPKECMYTIPKLLEGHEYVFRIMAQNKYGIGEPLDSEPETARNLFS VPGAPDKPTVSSVTRNSMTVNWEEPEYDGGSPVTGYWLEMKDTTSKRWKRVNRDPIKAMTLG VSYKVTGLIEGSDYQFRVYAINAAGVGPASLPSDPVTARDPVAPPGPPFPKVTDWTKSSVDL EWSPPLKDGGSKITGYIVEYKEEGKEEWEKGKDKEVRGTKLWTGLKEGAFYKFRVRAVNVA GVGEPGEVTDVIEMKDRIVSPDLQLDASVRDRIWHAGGVIRI IAYVSGKPPPTVTWSMNER ALPQEAAIETTAISSSMVIKNCQRSHQGVYSLLAKNEGGERKKTI IVDVLDVPGPVGIPFLS DNLTNDSCKLTWFSPEDDGGSPITNYVIQKREADRRAWTPVTYTVTRQNATVQGLIQGKSYF FRIAAENSIGMGPFVETPNALVIRDPITVPERPEDLEVKEVTKNTVSLTWNPPKYDGGSEII NYVLESRLIGTEKFHKVTNDNLLSRKYTVKGLKEGDTYEYRVSAVNIVGQGKPSFCTKPITC KDELAPPTLDLDFRDKLTVRVGESFALTGRYSGKPKPKIDWFKDEADVLEDDRTHIKTTPTT LALEKTKAKRSDSGKYCVWENSTGSRKGFCQVNWDRPGPPVGPVIFDEVTKEYMVISWKP PLDDGGSEITNYIIEKKELGKDIWMPVTSASAKTTCKVPKLLEGKDYIFRIHAENLYGISDPAttorney Docket No. TVD-013WO2 LVSDSMKAKDRFRVPDAPEQPWTEVTKDSALVTWNKPNDGGKPITNYILEKRETMSKRWVR VTKEPIHPYTKYRVPDLLEGCQYEFRVSAENEIGIGDPSPPSKPVFARDPIAKPSPPINPEA IDTTCNSVELTWQPPRHDGGSKILGYIVEYQKVGDEEWRRANHTPESCPETKYKVTGLRDGQ SYKFRVLAVNEAGESDPAHVPEPVLVKDRLEPPELILDANMAREQHIRVGDTLRLSAI IKGV PFPKVTWKKEDREAPTKAQIDVTPVGSKLEIRNAAHEDGGIYSLTVENPAGTKTVSVKVLVL DKPGPPRDLEVSEIRKDSCYLTWKEPLDDGGSWTNYWERKDVATAQWSPLSTTSKKKSHM AKHLTEGNQYLFRVAAENQYGRGPFVETPKPIKALDPLHPPGPPKDLHHVDVDKTEVSLVWN KPDRDGGSPITGYLVEYQEEGDKDWIKFKTVKNLDCWTGLQQGKTYRFRVKAENI IGLGLP DTTIPIECQEKLVPPSVELDVKLIEGLWKAGTTVRFPAI IRGVPVPTAKWTTDGTEIKTDD HYTVETDSFSSVLTIKNCLRKDTGEYQLTVSNAAGTKTVAVHLTVLDVPGPPTGPINILDVT PEYMTISWQPPKDDGGSPVINYIVEKQDTRKGTWGWSAGSSKLKLKVPHLQKGCEYVFRVK AENKMGVGPPLDSIPTVAKHKFSPPSPPGKPWTDITENAATVSWTLPKSDGGSPITGYYVE RREITGKWVRVNKTPIADLKFRVTGLYEGNTYEFRVFAENLAGLSNPSPSSDPIKACRPIKP PGPPINPKLKDKSKESADLVWTKPLSDGGSPILGYWEYQKPGTAQWDRINKDELIRQCAFR VPGLIEGNEYRFRIRAANIVGEGEPRELAESVIAKDILHPPEVELDVTCRDVITVRVGQTIR I LARVKGRPE PD I T WS KE GKVLVKDKRVDL I HDL PRVE LQ I KEAVRADHGKY 11 S AKNS S GH AQGSAI VNVLDRPGPCQNLKVSNVTKENCT I SWENPLDNGGSE I TNFI VE YRKPNQKGWS IV ASDVTKRLVKANLLANNEYYFRVCAENKVGVGPTIETKTPILAINPIDRPGEPENLHIADKG KTFVYLKWRRPDYDGGSPNLSYHVERRLKGSADWERVHKGS IKETHYMVDKCVENQI YEFRV QTKNEGGESDWVRTEEVWKEDLQKPVLDLKLSGVLTVKAGDTIRLEAGVRGKPFPEVAWTK DKDATDLTRSPRVKIDTSAESSKFSLTKAKRSDGGKYVITATNPAGSFVAYATVNVLDKPGP VRNLKITDVSSDRCTIRWDPPEDDGGCEIQNYILEKCESKRMVWSTYSANVLTPSATVTRLI EGNEYI FRVRAENKIGTGPPTESKPVIAKTKYDRPGRPDPPEVTKVSKEEMTWWNAPEYDG GKSITGYYLEKKEKHAVRWVPVNKSAIPERRLKVQNLLPGHEYQFRVKAENEVGIGEPSLPS RPWAKDPIEPPGPPTNFKWDTTKSSITLAWGKPVYDGGAPIIGYWEMRPKIADASPDEG WKRCNAAAQL I RME FT VT S LDENQE YE FRVCAQNQVG I GRPAE LKEAI KPKEILEPPEIDLD ASMRKLVWRAGCPIRLFAIVRGRPAPKVTWRKVGIDNWRKGQVDLVDTMAFLVIPNSTRD DSGKYSLTLVNPAGEKAVFVNVKVLDTPGPVADLKVSDVTKTSCHVSWAPPENDGGSQVTHY IVEKREAERKTWSTVTPEVKKTSFNVTNLVPGNEYFFRVTAVNEYGPGVPTDIPKPVLASDP LSEPDPPRKLEVTEMTKNSATLAWLPPLRDGGAKIDGYI ISYREEDQPADRWTEYSWKDLS LIVTGLKEGKKYKFRVAARNAVGVSMPREAEGVYEAKEQLLPPKILMPEQITIKAGKKLRVE AHVYGKPNPICKWKKGDDEWTSSHLAIHKADGSSVLI IKDVTRKDSGYYSLTAENSSGSDT QKIKVTVMDAPGPPQPPFDISEIDADACSLSWHIPLEDGGSNITNYIVEKCDVSRGDWVTAL ASVTKTSCRVGKLIPGQEYIFRVRAENRFGISEPLTSPKMLAKFPFDVPSEPKNARVTKVNKAttorney Docket No. TVD-013WO2 DCIFVAWDRPDSDGGSPITGYLIERKERNSLLWVKANDTIVRSTEYPCAGLVEGLEYSFRIY ALNKAGSSPPSKPTEYVTARMPVDPPGKPEWDVTKNSASLIWARPKHDGGSRI IGYFVEAC KLPGDKWVRCNTTPHQIPQEEYTATGLEENAQYQFRAIAKTAVNISQPSEPSDPVTILAENV PPRIELSVEMKSLLTVKAGTNVCLDATVFGKPMPTVSWKKDTTPIKQAEGIKMAMKRNLCTL ELFSVNRKDSGDYTITAENSSGSKSATIKLKVLDKPGPPASVKINKMYADRAMLSWEPPLED GGSEITNYI IDKRETSRPNWAQVSATVPITSCTVEKLIEGHEYQFRICAENKYGVGDPILTE PAIAKNPYDPPGRCDPPVISNITKDHMTVSWKAPADDGGSPITGYLVEKRETQAVNWTKVNR KPVIERTLKATGLQEGTEYEFRVTAINKAGPGKPSDASKAVYAQDPLYPPGPPAFPKVYDTT RSSVSLSWGKPAYDGGSPI IGYLVEVKRADSDHWVRCNLPEKLQKTRFEVTGLMENTEYQFR VYAVNKIGYSDPSDVPDKHCPKDILIPPEGELDAELRKTLILRAGVTMRLYVPVKGRPPPKI TWSKPNVNLRERIGLDIKSTDFDTFLRCENVNKYDAGKYILTLENSCGKKEYTIWKVLDTP GPPVNVTVKEVSKDSAYVTWDPPI IDGGSPI INYWEKRDAERKSWSTVTTECSKTSFRVSN LEEGKSYFFRVFAENEYGIGDPGETRDAVKASETPGPWDLKALAITKSSCTIGWKKPRSDG GSRITGYWDFLTEENKWQRVMKSLSLQYSTKDLKEGKEYTFRVSAENENGEGTPSEIVWA KDDWAPDLDLKDLPDLCYLAKENSNFRLKIPIKGKPAPSVSWKKGEDPLATDTRVSVESTA VNTTLWYDCQKSDAGKYTITLKNVAGTKEGTLSIKWGKPGIPTGPIKFDEVTAEAMTLKW GPPKDDGGSEITNYVLEKRDSVNNKWVTCASAVQKTTFRVTRLHEGIEYTFRVSAENKYGVG EGLKSEPIVAKHPFDVPDAPPPPNIVDVRHDSVSLTWTDPKKTGGSPITGYHIEFKERNSLL WKRANKTPIRMKDFKVTGLTEGLEYEFRVMAINLAGVGKPSLPSEPWALDPIDPPGKPEVI SVTRNSVTLIWTEPKYDGGHKLTGYIVEKRDLPSKSWMKANHVNVPDCAFTVTDLVEGGKYE FRIRAKNTAGAI SAPSES TGT 11 CEDE YEAPT I VLDPT IKDGLTVKAGDS I VLSAI S I LGKP LPKSSWSRAGKDIRPSDIAQITSTPTSSMLTVKYATRKDAGEYTITATNPFGTKEEHVKVSV LDVPGPPGPIEISNVSAEKATLTWTPPLEDGGSPIKAYVLEKRETSRLLWTWSEDIQACRH WTKLIQGNEYLFRVSAVNHYGKGEPVQSEPVKMVDRFGPPGPPGKPEISNVTKNTATVSWK RPTDDGGSEITGYHVERREKKGLRWVRATKTPVSDLRCKVTGLQEGNTYEFRVSAENRAGIG PPSDASNPVLMKDVAYPPGPPSNAHVTDTTKKSASLAWGKPHYDGGLEITGYWEHQKVGDD AWIKDTTGTALRITQFWPDLQTKEKYNFRISAINDAGVGEPAVIPNVEIVEKEVAPDFELD AELRRTLWRAGLSIRIFVPIKGRPAPEVTWTKDNINLKHRANIENTESFTLLIIPECNRYD TGKFVMTIENPAGKKSGFVNVRVLDTPGPVLNLRPTDITKDSVTLHWDLPLIDGGSRITNYI VEKREATRKSYSTVTTKCHKCTYKVTGLTEGCEYFFRVMAENEYGVGEPTETTEPVRASEAP LPPDSLNIMDITKNTVSLAWPKPRHDGGSKITGYVIEAQRKGSDQWTHISTVKGLECWRNL TEGEEYTFQVMAVNSAGRSAPRESRPVIVKEQTMLPELDLRGIYQKLVIARAGDNIKVEIPV LGRPKPTVTWKKGDQILKQTQRVNVENTATSTILNINECVRSDSGPYPLTAKNTVGEVGDVI TIQVHDIPGPPTGPIKFDEVSSDFVTFSWEPPENDGGVPISNYWEMRQTDSTTWVELATTVAttorney Docket No. TVD-013WO2 IRTTYKATRLTTGVEYQFRVRAQNRYGVGPGITSASWANYPFKVPGPPGTPQVTAVTKDSM TISWHEPLSDGGSPILGYHIERKERNGILWQTVSKALVPGNIFKSTGLTDGIAYEFRVIAEN MAGKSKPSKPSEPMFALDPIDPPGKPVPLNITRHTVALKWAKPEYTGGFKITSYWEKRDLP NGRWLKANFSNILENEFTVSGLTEDAAYEFRVIAKNAAGAISPPSEPSDAITCRDDLEAPRI MVDVRFKDTITLKAGEAFKLEADVSGRPPPTMEWTKDGKELEGTGKLEIKIADFSTHLINKD SSRTDSGAYILTATNPGGFAKHI FNVKVLDRPGPPEGPLAVSDVTSEKCVLSWLPPLDDGGA KIDHYIVQKRETSRLAWTNVATEVQVTKLKVTKLLKGNEYI FRVMAVNKYGVGEPLESEPVL AVDPYGPPDPPKNPEVTTITKDSMWCWGHPDSDGGSEI INYIVERRDKAGQRWVKCNKKAL TDLRFKVSGLTEGHEYEFRIMAENAAGISAPSATSPFYKACDTVFKPGPPGNPRVLDTSRSS ISIAWNKPIYDGGSEITGYMVEIALPEEDEWQWTPPAGLKATSYTITSLIENQEYKIRIYA MNSEGLGEPALVPGTPKAEERMLPPEIELDADLRKWTIRACCTLRLFVPIKGRPAPEVKWA REHGESLDKAS IESTSSYTLLWGNVNRFDSGKYILTVENSSGSKSAFVNVRVLDTPGPPQN LKIKEVTKTSVTLTWEPPLLDGGSKIKNYIVEKRESTRKAYSTVATNCHKTSWKVDQLQEGC SYYFRVLAENEYGIGLPAETAESVKASERPLPPGKITLTDVTRNSVSLSWEKPEHDGGSRIL GYIVEMQSKGSDRWATCATVKVTEATITGLIQGEEYSFRVSAQNEKGISDPRQLSVPVIAKD LVIPPAFKLLFNTFTVLAGEDLKIDVPFIGRPPPAVTWHKDDIPLKQTTRVNAESTENNSLL TIKEACREDVGHYTVKLTNSAGEATETLNVIVLDKPGPPTGPVKMDEVTADSVTLSWEPPKY DGGSSINNYIVEKRDTSTTAWQIVSATVARTTIKACRLKTGCEYQFRIAAENRYGKSTYLNS EPWAQYPFKVPGPPGTPFVTLASKDSMEVQWHEPVSDGGSKVIGYHLERKERNS ILWVKLN KTPIPQTKFKTTGLEEGIEYEFRVSAENIVGIGKPSKPSECYAAHDPCDPPGRPEAI IVTRN SVTLQWKKPTYDGGSKITGYIVEKKELPDGRWMKASFTNI IDTQFEVTGLLEDHRYEFRVIA RNAAGVFSEPSESTGAITARDEVEPPRISMDPKYRDTVWQAGESFKIDADIYGKPIPTTQW VKGDQELSSTARLEIKSTDFATSLSVKDAVRVDSGNYILKAKNVAGEKSVTINVKVLDRPGP PEGPVAISGVTAEKCTLAWKPPLQDGGSDITNYIVERRETSRLVWTLVDANVQTLSCKVLKL LEGNEYI FRIMAVNKYGVGEPLESESLIAKNPFWPDAPKAPEVTAVTKDSMIWWERPASD GGSEILGYVLEKRDKEGIRWTRCHKRLIGELRLRVTGLLENHNYEFRVSAENAAGLSEPSPP SAYQKACDPIYKPGPPNNPKVMDVTRSSVFLSWTKPIYDGGCEIQGYIVEKCDVSVGEWTMC TPPTGINKTNLEVEKLLEKHEYNFRICAINKAGVGEHADVPGPVMVEEKLEAPDIDLDLELR KVI N I RAGGS LRL EVP I KGRP T PE VKWGKVDGD I RDAAI I DVT S S FT S LVLDNVNRYDS GKY TLTLENSSGTKSAFVTVRVLDTPSPPVNLKVTEITKDSVSITWEPPLLDGGSKIKNYIVEKR EATRKSYAAWTNCHKNSWKIDQLQEGCSYYFRVTAENEYGIGLPARTADPIKVAEVPQPPG KITVDDVTRNSVSLSWTKPEHDGGSKI IQYIVEMQAKNTDKWSECARVKSLDAVITNLTQGE EYLFRVIAVNEKGRSDPRSLAVPI IAKDLVIEPDVRPAFSSYSVQVGQDLKIEVPISGRPKP SISWTKDGMPLKQTTRINVTDSLDLTTLSIKETHKDDGGQYGITVSNWGQKTAAIEIITLDAttorney Docket No. TVD-013WO2 KPDPPKGPVKFDEISAESITLSWNPPLYTGGCQITNYIVQKRDTTTTVWDWSATVARTTLK VTKLKTGTEYQFRIFAENRYGQSFALESEPWAQYPYKEPGPPGTPFVTAISKESMWQWHE PINNGGSPVIGYHLERKERNSILWTKVNKTIIHDTQFKALNLEEGIEYEFRVYAENIVGVGK ASKNSECYVARDPCDPPGTPEAI IVKRNEITLQWTKPVYDGGSMITGYIVEKRDLPEGRWMK ASFTNVIETQFTVSGLTEDQRYEFRVIAKNAAGAISKPSDSTGPITAKDEVELPRISMDPKF RDTIWNAGETFRLEADVHGKPLPTIEWLRGDKEIEESARCEIKNTDFKALLIVKDAIRIDG GQYILRASNVAGSKSFPVNVKVLDRPGPPEGPVQVTGVTAEKCTLAWSPPLQDGGSDISHYV VEKRETSRLAWTWASEWTNSLKVTKLLEGNKYI FRIMAVNKYGVGEPLESAPVLMKNPFV LPGPPKSLEVTNIAKDSMTVCWNRPDSDGGSEI IGYIVEKRDRSGIRWIKCNKRRITDLRLR VTGLTEDHEYEFRVSAENAAGVGEPSPATVYYKACDPVFKPGPPTNAHWDTTKNSITLAWS KPIYDGGSEILGYWEICKADEEEWQIVTPQTGLRVTRFEIAKLIEHQEYKIRVCALNKVGL GEAASVPGTVKPEDKLEAPELDLDSELRKGIWRAGGSARIHIPFKGRPTPEITWSKEEGEF TDKVQIEKGINFTQLSIDNCDRNDAGKYILKLENSSGSKSAFVTVKVLDTPGPPQNLAVKEV RKDSVLLVWEPPIIDGGAKVKNYVIDKRESTRKAYANVSSKCNKTSFRVENLTEGAIYYFRV MAENEFGVGVPTETSDAVKASEPPSPPGKVTLTDVSQTSASLMWEKPEHDGGSRILGYWEM QPKGTEKWSWAESKVCNAWSGLSSGQEYQFRVKAYNEKGKSDPRVLGIPVIAKDLTIQPS FKLPFNTYSVQAGEDLKIEIPVIGRPRPKISWVKDGEPLKQTTRVNVEETATSTILHIKESS KDDFGKYSVTATNSAGTATENLSVIVLEKPGPPVGPVKFDEVSADFWISWEPPAYTGGCQI SNYIVEKRDTTTTNWQMVSATVARTTIKISKLKTGTEYQFRIFAENRYGKSTPLDSKPVWQ YPFKEPGPPGTPFVTSISKDQMLVQWHEPVNDGGSKVTGYHLEQKEKNSILWVKLNKIPIQD TKFKTTGLDEGLEYEFRVSAENIVGIGKPSKVSECYVARDPCDPPGRPEAIVITRNSVTLKW KKPVYDGGSKITGYIVEKKDLPDGRWMKASFTNWETEFTVTGLVEDQRYEFRVIARNAADN FSEPSESSGAITARDEIDAPNASLDPKYRDVI IVHAGETFVLEADIRGKPIPDI IWSKDGNE LEETAARMEIKSTLQKTTLIVKDCIRTDGGQYTLKLSNVGGTKTIPITVKVLDRPGPPEGPL KVTGVTAEKCYLAWNPPLQDGGASISHYIIEKRETSRLSWTQVSNEVQALNYKVTKLLPGNE YIFRVMAVNKYGIGEALESEPVIACNPYKRPGPPSTPEASAITKDSMVLTWTRPVDDGGAEI EGYILEKRDKEGIRWTKCNKKTLTDLRFRVTGLTEGHSYEFRVAAENAAGVGEPSEPSVFYR ACDALYPPGPPSNPKVTDTSRSSVSLAWNKPI YDGGAPVRGYVIELKKAAADEWTTCTPPSG LQGKQFTVTKLKENTEYNFRICAFNTEGVGEPATIPGSWAQERMEAPEIELDADLRKWTL RASATLRLFVTIKGRPEPEVKWEKAEGILTERAQIEVTSSYTMLVIDNVTRFDSGRYNLTLE NNSGSKTAFVNVRVLDSPSAPVNLTIREVKKDSVTLSWEPPLIDGGAKITNYIVEKRETTRK AYATITNNCTKNTFKIENLQEGCSYYFRVLASNEYGIGLPAETAEPVKVSEPPLPPGRVTLV DVTRNTATIKWEKPESDGGSKITGYWEMQTKGSEKWSACTQVKTLETTISGLTAGEEYVFR VAAVNEKGRSDPRQLGVPVIAKDIEIKPSVELPFNTFNVKANDQLKIDIPFKGRPQATVAWKAttorney Docket No. TVD-013WO2 KDGQVLRETTRVNVASSKTVTTLSIKEASREDVGTYELCVSNTAGSITVPITVIVLDRPGPP GPIRIDEVSCDNVSISWNPPEYDGGCQISNYIVEKRETTSTTWQWSQAVARTSIKIVRLTT GSEYQFRVCAENRYGKSSYSESSAWAEYPFSPPGPPGTPKWHATKSTMWSWQVPVNDGG SQVIGYHLEYKERSS ILWSKANKVLIADTQMKVSGLDEGLMYEYRVYAENIAGIGKCSKACE PVPARDPCDPPGQPEVTNITRKSVSLKWSKPRYDGGAKITGYIVERRELPDGRWLKCNFTNV QETYFEVTELTEDQRYEFRVFARNAADSVSEPSESTGPITVKDDVEAPRIMMDVKFRDVIIV KAGEVLKINADIAGRPLPVISWAKDGVEIEERAKTEIVSTDYTTTLTVKDCVRRDTGQYVLT LKNVAGTRTMAVNCKVLDKPGPPAGPLEINGLTAEKCSLSWGRPQEDGGADIDYYIVEKRET SRLAWTICEAELRTTSCKVTKLLKGNEYIFRVTGVNKYGVGEPLESMAVKALDPFTTPSPPT SLEITSVTKDSMTLCWSRPETDGGSDISGYI IERREKNSLRWMRVNKKPVYDLRVKSTGLRE GCEYEYRVFAENAAGLSLPSETSPLVRAEDPVFLPSPPSKPKIVDSGKTTITIGWVKPLFDG GAPITGYTVEYKKSEETDWKVAIQSFRGTEYTMSGLTTGDEYVFRVRSLNKMGASDPSDSSD PQVAKEREEEPVFDVDSEMRKTLIVKAGSSFTMTVPFRGRPIPNVSWSKPDTDLRTRAYIDS TDSRTLLTIENANRNDSGKYTLTIQNVLSAASMTFWKVLDSPGPPANITVREVTKETAMLS WDVPENDGGAPVKNYHIEKREASKKAWVSVTNNCNRLSYKVTNLQEGAI YYFRVSGENEFGV GVPAETKEGVKITEKPSPPEKLGVTSVSKDSVSLSWLKPEHDGGSRI IHYWEALEKGQKTW VKCAWKTTHHWSGLRESHEYFFRVFAENQAGLSDPRELLLPVLIKDQLEPPEIDMKNFPS HTVYVRAGSNLKVDI P I SGKPLPKVTLSRDGVPLKATMRFNTE I TAENLT INLKESVTTDAG RYEITAANSSGTTKTFINIIVLDRPGPPTGPVAISDITEESVTLKWEPPKYDGGSHVTNYIV LKRETSTAVWSEVSATVARTMIKVMKLTTGEEYQFRIKAENRFGISDHIDSVCVWKLPYTT PGPPSTPWVSNVTRESITVGWHEPVSNGGSAVTGYHLEMKDRNSILWQKANKMIIRTTHFKV TTISAGLIYEFRVYAENAAGIGKPSHPSEPVLAIDACEPPRNVRITDISKNSVNLSWQQPAF DGGSKITGYIVERRDLPDGRWTKASFTNVIETQFTVSGLTQNSQYEFRVFARNAVGSVSNPS EWGPITCIDSYGGPVIDLPLEYTEWKYRAGTSVKLRAGISGKPEPTIEWYKDDKELQTNA LVCVENSTDLASILIKDANRLNSGSYELKLRNAMGSASATIRVQILDKPGPPGGPIEFKTVT AEKITLLWRPPADDGGAKITHYIVEKRETSRWWSMVAENLEECIVTTTKI IKGNEYVFRVR AVNKYGIGEPLESEPWAKNAFVTPGPPSIPEVTKITKNSMTWWDRPTVDGGSEINGYFLE RRDKKSLAWLKVLKETIRDTRQKVTGLTENSDYQYRVCAVNAAGVGPFSEPSDFYKAADPID PPGPPAKIRIADSTKSSITLGWSKPVYDGGSDVTGYWEMKQGDEEEWTIVSTRGEVRTTEY WSNLKPGVNYYFQVSAVNCAGQGEPITMTEPAQAKDVLEEPEIDLDVALRTSVIAKAGEDV QLLIPFKGRPPPTVTWRKDEKNLGSDTRYSIQNTDSSSLLVIPQVTRNDTGKYILTIENGVG QPKSSTVSVKVLDTPAACQKLQVKHVSLGTVTLLWDPPLIDGGSPI INYVIEKRDATKRTWS WSHKCSGTSFKVTDLSEKTPFFFRVLAENEIGIGEPCETTEPVKAAEVPAPIRDLSMKDST KTSWLSWTKPDFDGGSIITDYLVERKGKGEQAWSHAGISKTCEIEIGQLKEQSVLEFRVSAAttorney Docket No. TVD-013WO2 RNEKGQSDPVT I GPLTVKELVI TPEVDLSE I PGAQI SVRI GHNVHLELPYKGKPKPS I SWLK DGLPLKESEYVRFSKTENKITLSIKNSKKEHGGKYTVILDNAVCRNSFPITIITLGPPSKPK GPIRFDEIKADSAIMSWDIPEDDGGGEITCYSIEKREASQTNWKMVCSSVARTTFKVSNLVK DSEYQFRVRAENRYGVSEPLASNI IVAKHQFRIPGPPGKPVIYNVTSDGMSLTWDAPVYDGG SEVTGFHVEKKERNSILWQRVNTSPISGREYRATGLIEGLDYQFRVYAENSAGLSSPSDPSK FTLAVSPVDPPGTPDYIDVTRETITLKWNPPLRDGGSKIVAYSIEKRQGSDRWVRCNFTDVS ECQYTVTGLSPGDRYEFRI lARNAVGTISPPSQSSGLIMTRDENVPPTVEFGPEYFDGLVIK SGDSLRIKALVQGRPVPRVTWFKDGVEIERRMNMEITDVLGSTSLFVRDATRDHRGVYTVEA KNVSGSTKAEVTVKVQDTPGKWGPIRFTNITGEKMTLWWEAPLNDGCAPVTHYI IEKRETS RLAWALIEDNCEALSYTAIKLITGNEYQFRISAVNKFGVGRPLESDPWAQIQYTIPDAPGV PEPSNVTGNSITLTWTRPESDGGSEIQHYILERREKKSTRWVKVISKRPISETRFKVTGLVE GNEYEFHVMAENAAGIGPASGISRLIKCREPVNPPSAPSWKVTDTSKTTVSLEWARPVFDG GMEI IGYI IEMCKADLGDWHKVNTEPCVKTRYTVTDLQAGEEYKFRVSAVNGAGKGDSCEVT GTIKAVDRLSAPELDIDANFKQTHIVRAGVSIRLFIAYQGRPTPTAVWSKPDSNLSIRADIH TTDSFSTLTVENCNRNDAGKYTLTVENNSGKKSITFTVKVLDSPGPPGPITFKDVTRGSATL MWDAPLLDGGARIHHYVIEKREASRRSWQWSEKCTRQILKVSELTEGVPYYFRVSAENEYG VGEPYEMPEPIVATEQPAPPRRLDWDTSKSSAVLAWLKPDHDGGSRITSYLLEMRQKGSDF WVEAGHTKQLTFTVERLVENTEYEFRVKAKNDAGYSEPREAFSSVI IKEPQIEPTADLTGIT NQL I TCKAGS T FT I DVP I S GRPAPKVTWKLEEMRLKE TDRMS I AT TKDRT TLTVKDSMRGDS GRYFLTLENTAGVKTFTITVWIGRPGPVTGPIEVSSVSAESCVLSWTEPKDDGGTEITNYI VEKRESGTTAWQLINSSVKRTQIKVTHLTKYKEYCFRVSSENRFGVSKPLESVPIVAEHPFV PPSAPTRPEVYYVSANAMSIRWEEPYHDGGSKIVGYWVEKKERNTILWVKENKVPCLECNYK VTGLVEGLEYQFRTYALNAAGVSKASEASRPIMAQNPVDPPGRPEVTDVTRSTVSLIWSAPV YDGGSKWGYI lERKPVSEVGDGRWLKCNYTIVSDNFFTVTALSEGDTYEFRVLAKNAAGVI SKGSESTGPVTCRDEYAPPKAELDARLQGDLVTIRAGSDLVLDAAVGGKPEPKI IWTKGDKE LDLCEKISLQYTGKRATAVIKYCDRSDSGKYTLTVKNASGTKSVSVMVKVLDSPGPCGKLTV SRVTEEKCTLAWSLPQEDGGAEITHYIVERRETSRLNWVIVEGECLTASYWTRLIKNNEYT FRVRAVNKYGLGVPVESEPIVARNSFTIPSQPGIPEEVGAGKEHI I IQWTKPESDGGNEISN YLVDKREKKSLRWTRVNKDYWYDTRLKVTSLMEGCDYQFRVTAVNAAGNSEPSEASNFISC REPSYTPGPPSAPRWDTTKRSISLAWTKPMYDGGTDIIGYVLEMQEKDTDQWCRVHTNATI RNNEFTVPDLKMGQKYSFRVAAVNAKGMSDYSETTAEIEPVERLEIPDLELADDLKKTVIVR AGASLRLMVSVSGRPSPVITWSKKGIDLANRAI IDNTESYSLLIVDKVNRYDAGKYTIEAEN QSGKKSATVLVKVYDTPGPCPSVSVKEVSRDSVT I TWE I PT I DGGAPVNNYI IEKREAAMRA FKTVTTKCSKTLYRISGLVEGTMYYFRVLPENIYGIGEPCETSDAVLVSEVPLVPTKLEWDAttorney Docket No. TVD-013WO2 VTKSTVTLAWEKPLYDGGSRLTGYVLEACKAGTERWMKWTLKPTVLEHTVISLNEGEQYLF RVRAQNEKGVSEPRE I VTPVTVQDLRVLPT I DLS TMPQKT IHVPAGRP IELVI P I TGRPPPT ASWFFAGSKLRESERVTVETHTKVTKLTIRETTIRDTGEYTLELKNVTGTTSETIKVI ILDK PGPPTGPIKIDEIDATSVTISWEPPELDGGAPLSGYWEQRDAHRPGWLPVSESVTRPTFKF TRLTEGNEYVFRVAATNRFGIGSYLQSEVIECRSSISIPGPPETLQIFDVSRDGMTLTWYPP EDDGGSQVTGYIVERKEVRADRWVRVNKVPVTMTRYRSTGLIEGLEYEHRVTAINARGTGKP SRPSKPTVAMDPIAPPGKPQNPRVTDTTRTSVSLAWSVPEDEGGSKVTGYLIEMQKVDQREW TKCNTTPTKIREYTLTHLPQGAEYRFRVLACNAGGPGEPAEVPGTVKVTEMLEYPDYELDER YQEGVFVRQGGVIRLTIPIKGKPFPVCKWTKEGQDISKRAMIATSETHTELVIKEADRNDSG TYDLVLENKCGKKTVYIKVKVIGSPNTPEGPLEYDDIQARSVRVSWRPPADDGGADILGYIL ERREVPKAAWYTIDSRVRGTSLWKGLKENVEYHFRVSAENQFGISKPLKSEEPVIPKTPLN PPEPPSNPPEVLDVTKSSVSLSWSRPKDDGGSRVTGYYIERKETSTDKWVRHNKTQITTTMY TVTGLVPDAEYQFRIIAQNDVGLSETSPASEPWCKDPFDKPSQPGELEILSISKDSVTLQW EKPECDGGKEILGYWVEYRQSGDSAWKKSNKERIKDRQFTIGGLLEATEYEFRVFAENETGL SRPRRTAMSVKTKLTSGEAPGVRKEMADVTTKLGEAAQLSCQIVGRPLPDIKWYRFGKELIQ SRKYKMSSDGRTHTLTVMTDEQEDEGVYTCVATNEVGEVESSSKLLLQAAPQFHPGYPLKEK YYGAVGSTLRLHVMYIGRPVPAMTWFHGQKLLQNSEKITIENTEHYTHLVMKNVQRKTHAGK YKVQLSNAFGTVDATLDVEIQDKPDKPTGPIVIEALLKNSWISWKPPADDGGSWITNYWE KCEAKEGAEWQLVSSAISVTTCRIVNLTENAGYYFRVSAQNTFGISEPLEVASIVIIKSPFE KPGVPGKPTITAVTKDSCWAWKPPASDGGAKIRNYYLERREKKQNKWIAVTTEEIRETVFS VQNLIEGLEYEFRVKCENLGGESEWSEISEPVTPKSDVPIQAPHFKEELRNLNVRYQSNATL VCKVTGHPKPIVKWYRQGKEI IADGLKYRIQEFKGGYHQLI IASVTDDDATVYQVRATNQGG SVSGTASLEVEVPAKIHLPKTLEGMGAVHALRGEWSIKIPFSGKPDPVITWQKGQDLIDNN GHYQVIVTRSFTSLVFSNGVERKDAGFYWCAKNRFGIDQKTVELDVADVPDPPRGVKVSDV SRDSVNLTWTEPASDGGSKVTNYIVEKCATTAERWLRVGQARETRYTVINLFGKTSYQFRVI AENKFGLSKPSEPSEPTVTKEDKTRAMNYDDEVDETREVTTTKASHSKTKELYEKYMIAEDL GRGE EG I VHRCVE T S S KRT FMAKFVKVKGT DQVLVKKE I S I LN I ARHRN I L YLHE S EE SMEE LVMIFEFISGLDIFERINTSAFELNEREIVSYVRQVCEALEFLHSQNIGHFDIRPENIIYQT RKNSTIKI lEFGQARQLKPGDNFRLLFTAPEYYAPEVHQHDWSTATDMWSLGTLVYVLLSG INPFLAETNQQMIENIMNAEYTFDEEAFKEISLEAMDFVDRLLVKERKSRMTASEALQHPWL KQR I DRVS TKVI RT LKHRRY YHT L I KKDLNMWS AAR ISCGGAIRS QRGVS VAKVKVAS I E I GPVSGQIMHAIGEEGGYVKYVCKIENYDQSTQVTWYFGVRQLENSEKYEITYEDGVATMYVK DITKFDDGTYRCKWNDYGEDSSYAELFVKGVREVYDYYCRRTKKVKRRTDAMRLLERPPEF TLPLYNKTAYVGENVRFGVTITVHPEPRVTWYKSGQKIKPGDDEKKYTFESDKGLYQLTINSAttorney Docket No. TVD-013WO2 VTTDDDAEYTWARNKHGEDSCKAKLTVTLHPPPTETTLRPMFKRLLANAECHEGQSVCFEI RVSGIPAPTLKWEKDGQPLSLGPHIEIVHEGLDYYALHIRDTLPEDTGYYRVTATNTAGSTS CQAHLQVERLRYVKQEFQSKEERERHVQKQIDKTLRMAEILSGTETVPLTPVAQEALREAAI LYKPAVSTKTVKGEYRLQTEEKKEERKLRMPYEVPEPRRFKQATVEEDQRIKQFVPMSDMKW YKKIRDQYEMPGKLDRWQKRPKRIRLSRWEQFYVMPLPRITDQYRPKWRIPKLTQDDLEMV RPARRRTPSPDYDLYYYRRRRRSLGDMSDEELLLPIDDYLAMKRTEEERLRLEEELELGFSA SPPSRSPPRFELSSLRYSSPPAHVKVEDRRRDFRYSTYHVPTKEETSTSYAELRERHAQASY RQPKLRQRIMAEKEEEELLRPVTTTQRLSEYKSELDYMSKEEKSKKKSKRQRQVTEITEIEE EYEISRRAQRESSSSVSRLLRRRRSLSPTYIELMRPVSELIRSHPRPAEEYEDDAERRSPTP ERTRPRSPSPVSSERSLSRFERSARFDI FSRYESMKAALKTQKTSERKYEVLSQQPFTLDHA PRITLRMRSHRVPCGQNTRFILNVQSKPTAEVKWYHNGVELQESSKIHYTNTSGVLTLEILD CQTEDGGTYRAVCTNYKGEASDYATLDVTGGAYTTYASQRRDEEVPKSVFPELTKTEAYAVS SFKRTSELEAASSVREVKSQMTETRESLSTYEHYASAEMKSATSEEKSLEEKATVRKIKTTL AARILTKPRSITVHEGESARFSCDTDGEPVPTVTWLREGQWSTSARHQVTTTKYKSTFEIS SVQASDEGNYSVWENSDGKQEAQFTLTVQKARVIEKAVTSPPRVKSPEPRVKSPETVKSPK RVKSPEPVTSHPKAVSPTETKPTEKGQHLPVSAPPKITQSLKAEASKDIAKLTCAVESSALC AKEVAWYKDGKKLKENGHFQFHYSADGTYELKIHNLSESDCGEYVCEVSGEGGTSKTSFQFT GQSFKSIHEQVSSISETTKSVQKTAESPEAKKQEPIAPESISSKPVIVTGLRDTTVSSDSVA KFTIKVTGEPQPTITWTKDGKAIAQGSKYKLSSNKEEFILEILKTETSDGGLYACTVTNSAG SVSSSCKLTIKAVKDTEAQKVSTQKTSEVTSQKKASAQEEISQKALTSEEIKMSEVKSHETL AIKEEASKVLIAEEVKRSAAASLEKSIVHEEVTKTSQASEEVKTHAEIKTLSTQMNITKGQR ATLKANIAGATDVKWVLNGTELPNSEEYRYGVSGSDQTLTIKQASHREEGILSCIGKTSQGV VKCQFDLTLSEELSDAPSFITQPRSQNINEGQNVLFSCEVSGEPSPEIEWFKNNLPISISSN ISVSRSRNVYTLEIRNAAVSDSGKYTIKAKNFHGQCSATASLTVLPLVEEPPREWLKTSSD VS LHGS VS S QS VQMSASKQEAS FS S FS S S SAS SMTEMKFASMSAQSMS SMQE S FVEMS S S S F MGKSSMTQLESSTSRMLKAGGRGIPPKIEALPSDISIDEGKVLTVACAFTGEPTPEITWSCG GRKIQNQEQQGRFHIENTDDLTTLI IMDVQKQDGGLYTLSLGNEFGSDSATVNINIRSM(SEQ IDNO: 307).

[0196] The disclosure also provides a method of treating Duchenne muscular dystrophy in a subject in need thereof, wherein the subject has a DMD gene with a PTC. In some embodiments, the method comprises administering to the subject an effective amount of a suppressor tRNA disclosed herein (e.g., a Gin-suppressor tRNA, e.g, an Gin suppressor tRNA, such as tr0904 or tr0524), or a nucleic acid or expression vector disclosed hereinAttorney Docket No. TVD-013WO2 encoding the same. Exemplary Gin-suppressor tRNAs include tRNAs described in TABLE 1, TABLE 2, TABLE 3, TABLE 7, TABLE 8, TABLE 9, and TABLE 15 In certain embodiments, the suppressor tRNA is selected from any one of SEQ ID NOs: 175, 176, 177, 178, 236, 238, 240, 241, 242, 249, 250, 251, and 252. In certain embodiments, the suppressor tRNA is selected from any one of SEQ ID NOs: 176, 179, 180, 181, 182, 183, 184, 185, 186, 239, and 241. In certain embodiments, the suppressor tRNA is selected from any one of SEQ ID NOs: 187, 188, 189, 190, 191, 192, 193, 194, 195, 915, and 916. In some embodiments, the suppressor tRNA is selected from any one of SEQ ID NOs: 254-274, 918, 925, 1149, and 1150. In other embodiments, the suppressor tRNA includes a nucleic acid sequence selected from any one of SEQ ID NOs: 175-237, 901-909, 915-916, 921-924, 1000-1119, 1122-1123, and 1125-1131, wherein each thymine in any one of SEQ ID NOs: 175-237, 901-909, 915-916, 921-924, 1000-1119, 1122-1123, and 1125-1131 is replaced by uracil. In other embodiments, the suppressor tRNA includes a nucleic acid sequence selected from any one of SEQ ID NOs: 238-252, 910-914, 1120, 1121, and 1124 or the nucleic acid encoding the suppressor tRNA includes the nucleic acid sequence of any one of SEQ ID NOs: 238-253, 910-914, 1120, 1121, and 1124. In certain embodiments, the suppressor tRNA is tr0904 (SEQ ID NO: 176). In certain embodiments, the suppressor tRNA is tr0524 (SEQ ID NO: 241). In certain embodiments, the suppressor tRNA is trl344 (SEQ ID NO: 236). In certain embodiments, the suppressor tRNA is trl519 (SEQ ID NO: 191). In certain embodiments, the suppressor tRNA is tri 517 (SEQ ID NO: 190). In certain embodiments, the suppressor tRNA is tri 511 (SEQ ID NO: 187). In certain embodiments, the suppressor tRNA is trl622 (SEQ ID NO: 916). In certain embodiments, the suppressor tRNA is trl723 (SEQ ID NO: 1088). In certain embodiments, the suppressor tRNA is trl773 (SEQ ID NO: 1091). In certain embodiments, the suppressor tRNA is trl794 (SEQ ID NO: 1104). In certain embodiments, the suppressor tRNA is trl806 (SEQ ID NO: 1106). In certain embodiments, the suppressor tRNA is tri 821 (SEQ ID NO: 1112). In certain embodiments, the suppressor tRNA is trl624 (SEQ ID NO: 1122). In certain embodiments, the suppressor tRNA is trl625 (SEQ ID NO: 1123). In certain embodiments, the suppressor tRNA is trl626 (SEQ ID NO: 1125). In certain embodiments, the suppressor tRNA is trl627 (SEQ ID NO: 1126). In certain embodiments, the suppressor tRNA is tri 853 (SEQ ID NO: 1127). In certain embodiments, the suppressor tRNA is tri 854 (SEQ ID NO: 1128). In certain embodiments, the suppressor tRNA is tri 857 (SEQ ID NO: 1129). In certain embodiments, the suppressor tRNA is tri 858 (SEQ ID NO: 1130).Attorney Docket No. TVD-013WO2

[0197] For example, in some embodiments, the subject has a DMD gene with a PTC and the method comprises administering to the subject an effective amount of a suppressor tRNA comprising a nucleotide sequence selected from any one of SEQ ID NOs: 175-274, 901-916, 918, 921-925, 1000-1119, 1122-1123, 1125-1131, 1149, and 1150 or a nucleic acid or expression vector encoding the same.

[0198] In certain embodiments, the functional DMD 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 entries Pl 1532 (human) or Pl 1531 (mouse), which sequences are hereby incorporated by reference in their entirety. In certain embodiments, the functional DMD gene product is the DMD Pl 1532-1 or Pl 1531-1 isoform (as set forth in UniProt # Pl 1532 (human) or #P11531 (mouse)), 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 Pl 1532-1 or Pl 1531-1 isoform thereof, provided below as SEQ ID NOs: 304 and 305, respectively:MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKL PKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNI ILHWQVKNVM KNIMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFDWNSV VCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQQVSIEAI QEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVTTS DPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDV EWKDQFHTHEGYMMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECL RVASMEKQSNLHRVLMDLQNQKLKELNDWLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVL QEDLEQEQVRVNSLTHMWWDESSGDHATAALEEQLKVLGDRWANICRWTEDRWVLLQDIL LKWQRLTEEQCLFSAWLSEKEDAVNKIHTTGFKDQNEMLSSLQKLAVLKADLEKKKQSMGKL YSLKQDLLSTLKNKSVTQKTEAWLDNFARCWDNLVQKLEKSTAQISQAVTTTQPSLTQTTVM ETVTTVTTREQILVKHAQEELPPPPPQKKRQITVDSEIRKRLDVDITELHSWITRSEAVLQS PEFAI FRKEGNFSDLKEKVNAIEREKAEKFRKLQDASRSAQALVEQMVNEGVNADS IKQASE QLNSRWIEFCQLLSERLNWLEYQNNI IAFYNQLQQLEQMTTTAENWLKIQPTTPSEPTAIKS QLKICKDEVNRLSDLQPQIERLKIQS lALKEKGQGPMFLDADFVAFTNHFKQVFSDVQAREK ELQTIFDTLPPMRYQETMSAIRTWVQQSETKLSIPQLSVTDYEIMEQRLGELQALQSSLQEQ QSGLYYLSTTVKEMSKKAPSEISRKYQSEFEEIEGRWKKLSSQLVEHCQKLEEQMNKLRKIQ NHIQTLKKWMAEVDVFLKEEWPALGDSEILKKQLKQCRLLVSDIQTIQPSLNSVNEGGQKIK NEAEPEFASRLETELKELNTQWDHMCQQVYARKEALKGGLEKTVSLQKDLSEMHEWMTQAEEAttorney Docket No. TVD-013WO2 EYLERDFEYKTPDELQKAVEEMKRAKEEAQQKEAKVKLLTESVNSVIAQAPPVAQEALKKEL ETLTTNYQWLCTRLNGKCKTLEEVWACWHELLSYLEKANKWLNEVEFKLKTTENIPGGAEEI SEVLDSLENLMRHSEDNPNQIRILAQTLTDGGVMDELINEELETFNSRWRELHEEAVRRQKL LEQSIQSAQETEKSLHLIQESLTFIDKQLAAYIADKVDAAQMPQEAQKIQSDLTSHEISLEE MKKHNQGKEAAQRVLSQIDVAQKKLQDVSMKFRLFQKPANFEQRLQESKMILDEVKMHLPAL ETKSVEQEWQSQLNHCVNLYKSLSEVKSEVEMVIKTGRQIVQKKQTENPKELDERVTALKL HYNELGAKVTERKQQLEKCLKLSRKMRKEMNVLTEWLAATDMELTKRSAVEGMPSNLDSEVA WGKATQKEIEKQKVHLKSITEVGEALKTVLGKKETLVEDKLSLLNSNWIAVTSRAEEWLNLL LEYQKHMETFDQNVDHITKWI IQADTLLDESEKKKPQQKEDVLKRLKAELNDIRPKVDSTRD QAANLMANRGDHCRKLVEPQISELNHRFAAISHRIKTGKASIPLKELEQFNSDIQKLLEPLE AEIQQGVNLKEEDFNKDMNEDNEGTVKELLQRGDNLQQRITDERKREEIKIKQQLLQTKHNA LKDLRSQRRKKALEISHQWYQYKRQADDLLKCLDDIEKKLASLPEPRDERKIKEIDRELQKK KE E LNAVRRQAE GL S E DGAAMAVE P T Q I QL S KRWRE I E S K FAQ FRRLN FAQ I H T VRE E TMMV MTEDMPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQ QSSGRIDI IHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRR FHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTWRTLNATGEEII QQSSKTDAS ILQEKLGSLNLRWQEVCKQLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADN IASIPLEPGKEQQLKEKLEQVKLLVEELPLRQGILKQLNETGGPVLVSAPISPEEQDKLENK LKQTNLQWIKVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNHLLLWLSPIRNQLEIYNQ PNQEGPFDVKETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRLLQ ELRAKQPDLAPGLTTIGASPTQTVTLVTQPWTKETAISKLEMPSSLMLEVPALADFNRAWT ELTDWLSLLDQVIKSQRVMVGDLEDINEMI IKQKATMQDLEQRRPQLEELITAAQNLKNKTS NQEARTI ITDRIERIQNQWDEVQEHLQNRRQQLNEMLKDSTQWLEAKEEAEQVLGQARAKLE SWKEGPYTVDAIQKKITETKQLAKDLRQWQTNVDVANDLALKLLRDYSADDTRKVHMITENI NASWRS IHKRVSEREAALEETHRLLQQFPLDLEKFLAWLTEAETTANVLQDATRKERLLEDS KGVKELMKQWQDLQGEIEAHTDVYHNLDENSQKILRSLEGSDDAVLLQRRLDNMNFKWSELR KKSLNIRSHLEASSDQWKRLHLSLQELLVWLQLKDDELSRQAPIGGDFPAVQKQNDVHRAFK RELKTKEPVIMSTLETVRI FLTEQPLEGLEKLYQEPRELPPEERAQNVTRLLRKQAEEVNTE WEKLNLHSADWQRKIDETLERLRELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLE KVKALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQL HEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNN VRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQI INCLTTIYDRLE QEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGI ISLCKAHLEDKYRYLFKQVAS STGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRAttorney Docket No. TVD-013WO2 LEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPI IGFRYRSLKHFNYDICQSCFFSGRVAK GHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPV TLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLI QHYCQSLNQDSPLSQPRSPAQILISLESEERGELERILADLEEENRNLQAEYDRLKQQHEHK GLSPLPSPPEMMPTSPQSPRDAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQL LEQPQAEAKVNGTTVSSPSTSLQRSDSSQPMLLRWGSQTSDSMGEEDLLSPPQDTSTGLEE VMEQLNNSFPSSRGRNTPGKPMREDTM (SEQ ID NO: 304);MLWWEEVEDCYEREDVQKKTFTKWINAQFSKFGKQHIDNLFSDLQDGKRLLDLLEGLTGQKL PKEKGSTRVHALNNVNKALRVLQKNNVDLVNIGSTDIVDGNHKLTLGLIWNI ILHWQVKNVM KTIMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFTSSWSDGLALNALIHSHRPDLFDWNSV VSQHSATQRLEHAFNIAKCQLGIEKLLDPEDVATTYPDKKSILMYITSLFQVLPQQVSIEAI QEVEMLPRTSSKVTREEHFQLHHQMHYSQQITVSLAQGYEQTSSSPKPRFKSYAFTQAAYVA TSDSTQSPYPSQHLEAPRDKSLDSSLMETEVNLDSYQTALEEVLSWLLSAEDTLRAQGEISN DVEEVKEQFHAHEGFMMDLTSHQGLVGNVLQLGSQLVGKGKLSEDEEAEVQEQMNLLNSRWE CLRVASMEKQSKLHKVLMDLQNQKLKELDDWLTKTEERTKKMEEEPFGPDLEDLKCQVQQHK VLQEDLEQEQVRVNSLTHMWWDESSGDHATAALEEQLKVLGDRWANICRWTEDRWIVLQD ILLKWQHFTEEQCLFSTWLSEKEDAMKNIQTSGFKDQNEMMSSLHKISTLKIDLEKKKPTME KLSSLNQDLLSALKNKSVTQKMEIWMENFAQRWDNLTQKLEKSSAQISQAVTTTQPSLTQTT VMETVTMVTTREQIMVKHAQEELPPPPPQKKRQITVDSELRKRLDVDITELHSWITRSEAVL QSSEFAVYRKEGNISDLQEKVNAIAREKAEKFRKLQDASRSAQALVEQMANEGVNAESIRQA SEQLNSRWTEFCQLLSERVNWLEYQTNI ITFYNQLQQLEQMTTTAENLLKTQSTTLSEPTAI KSQLKICKDEVNRLSALQPQIEQLKIQSLQLKEKGQGPMFLDADFVAFTNHFNHI FDGVRAK EKELQTIFDTLPPMRYQETMSSIRTWIQQSESKLSVPYLSVTEYEIMEERLGKLQALQSSLK EQQNGFNYLSDTVKEMAKKAPSEICQKYLSEFEEIEGHWKKLSSQLVESCQKLEEHMNKLRK FQNHIKTLQKWMAEVDVFLKEEWPALGDAEILKKQLKQCRLLVGDIQTIQPSLNSVNEGGQK IKSEAELEFASRLETELRELNTQWDHICRQVYTRKEALKAGLDKTVSLQKDLSEMHEWMTQA EEEYLERDFEYKTPDELQTAVEEMKRAKEEALQKETKVKLLTETVNSVIAHAPPSAQEALKK ELETLTTNYQWLCTRLNGKCKTLEEVWACWHELLSYLEKANKWLNEVELKLKTMENVPAGPE EITEVLESLENLMHHSEENPNQIRLLAQTLTDGGVMDELINEELETFNSRWRELHEEAVRKQ KLLEQSIQSAQEIEKSLHLIQESLEFIDKQLAAYITDKVDAAQMPQEAQKIQSDLTSHEISL EEMKKHNQGKDANQRVLSQIDVAQKKLQDVSMKFRLFQKPANFEQRLEESKMILDEVKMHLP ALETKSVEQEVIQSQLSHCVNLYKSLSEVKSEVEMVIKTGRQIVQKKQTENPKELDERVTAL KLHYNELGAKVTERKQQLEKCLKLSRKMRKEMNVLTEWLAATDTELTKRSAVEGMPSNLDSEAttorney Docket No. TVD-013WO2 VAWGKATQKEIEKQKAHLKSVTELGESLKMVLGKKETLVEDKLSLLNSNWIAVTSRVEEWLN LLLEYQKHMETFDQNIEQITKWI IHADELLDESEKKKPQQKEDILKRLKAEMNDMRPKVDST RDQAAKLMANRGDHCRKWEPQISELNRRFAAISHRIKTGKASIPLKELEQFNSDIQKLLEP LEAEIQQGVNLKEEDFNKDMSEDNEGTVNELLQRGDNLQQRITDERKREEIKIKQQLLQTKH NALKDLRSQRRKKALEISHQWYQYKRQADDLLKCLDEIEKKLASLPEPRDERKLKEIDRELQ KKKEELNAVRRQAEGLSENGAAMAVEPTQIQLSKRWRQIESNFAQFRRLNFAQIHTLHEETM WTTEDMPLDVSYVPSTYLTEISHILQALSEVDHLLNTPELCAKDFEDLFKQEESLKNIKDN LQQISGRIDI IHKKKTAALQSATSMEKVKVQEAVAQMDFQGEKLHRMYKERQGRFDRSVEKW RHFHYDMKVFNQWLNEVEQFFKKTQNPENWEHAKYKWYLKELQDGIGQRQAWRTLNATGEE I IQQSSKTDVNILQEKLGSLSLRWHDICKELAERRKRIEEQKNVLSEFQRDLNEFVLWLEEA DNIAITPLGDEQQLKEQLEQVKLLAEELPLRQGILKQLNETGGAVLVSAPIRPEEQDKLEKK LKQTNLQWIKVSRALPEKQGELEVHLKDFRQLEEQLDHLLLWLSPIRNQLEIYNQPSQAGPF DIKEIEVTVHGKQADVERLLSKGQHLYKEKPSTQPVKRKLEDLRSEWEAVNHLLRELRTKQP DRAPGLSTTGASASQTVTLVTQSWTKETVISKLEMPSSLLLEVPALADFNRAWTELTDWLS LLDRVIKSQRVMVGDLEDINEMI IKQKATLQDLEQRRPQLEELITAAQNLKNKTSNQEARTI ITDRIERIQIQWDEVQEQLQNRRQQLNEMLKDSTQWLEAKEEAEQVIGQVRGKLDSWKEGPH TVDAIQKKITETKQLAKDLRQRQISVDVANDLALKLLRDYSADDTRKVHMITENINTSWGNI HKRVSEQEAALEETHRLLQQFPLDLEKFLSWITEAETTANVLQDASRKEKLLEDSRGVRELM KPWQDLQGEIETHTDIYHNLDENGQKILRSLEGSDEAPLLQRRLDNMNFKWSELQKKSLNIR SHLEASSDQWKRLHLSLQELLVWLQLKDDELSRQAPIGGDFPAVQKQNDIHRAFKRELKTKE PVIMSTLETVRI FLTEQPLEGLEKLYQEPRELPPEERAQNVTRLLRKQAEEVNAEWDKLNLR SADWQRKIDEALERLQELQEAADELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRG EIAPLKENVNRVNDLAHQLTTLGIQLSPYNLSTLEDLNTRWRLLQVAVEDRVRQLHEAHRDF GPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYR TAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLV NVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGI ISLCKAHLEDKYRYLFKQVASSTGFCDQ RRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMV WLPVLHRVAAAETAKHQAKCNICKECPI IGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYP MVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWP VDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSL NQDSPLSQPRSPAQILISLESEERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPS PPEMMPTSPQSPRDAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQPQAE AKVNGTTVSSPSTSLQRSDSSQPMLLRWGSQTSESMGEEDLLSPPQDTSTGLEEVMEQLNN SFPSSRGRNAPGKPMREDTM (SEQ ID NO: 305).Attorney Docket No. TVD-013WO2

[0199] 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, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0200] 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 a dilated cardiomyopathy, a method or composition described herein is administered in combination with one or more additional therapeutic agents, e.g., ACE inhibitors, beta blockers, cardiac glycosides, diuretics, vasodilators, aldosterone antagonists, anti -arrhythmia medications, blood thinners, and neprilysin inhibitors. During the treatment of Duchenne muscular dystrophy, a method or composition described herein is administered in combination with one or more additional therapeutic agents, e.g., a corticosteroid, a histone deacetylase inhibitor, Translama® (ataluren), Duvyzat™ (givinostat), AGAMREE® (vamorolone), Elevidys® (delandistrogene moxeparvovec-rokl), deflazacort, casimersen, eteplirson, viltolarsen, or golodirsen.

[0201] 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.Attorney Docket No. TVD-013WO2EXAMPLES

[0202] 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

[0203] This Example describes using parental and engineered suppressor tRNAs thereof to increase dystrophin (DMD) protein expression in a cell line wherein the endogenous DMD gene contained a premature stop codon (PTC). Suppressor tRNAs solely with the anticodon modified are referred to as ‘parental suppressor tRNAs,’ while suppressor tRNAs with one or more mutations relative to a parental suppressor tRNA are referred to as ‘engineered suppressor tRNAs’ or ‘engineered suppressor tRNAs thereof.’ Together, parental and engineered suppressor tRNAs are referred to as ‘suppressor tRNAs.’ The example demonstrated that the increase in expression was due, at least in part, to readthrough of a PTC by the parental and / or engineered suppressor tRNA.Materials and MethodsA A V Production

[0204] 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 derived from publicly 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 plasmids used this Example included a single-stranded (ssAAV) transfer plasmid encoding three copies of a suppressor tRNA of the disclosure, functionally linked to U6 promoters (e.g., SEQ ID NO: 917) located between two inverted terminal repeats (ITRs) (FIG. 2).

[0205] Suppressor tRNA vectors were packaged into the myotropic capsid MyoAAV 2A. Triple-plasmid transfection was conducted using polyethylenimine (PEI, Polyscience) and optionally included other transfection reagents to produce rAAV particles. The transfected plasmids included:Attorney Docket No. TVD-013WO2 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.

[0206] The three plasmids were co-transfected into HEK293T cells. Briefly, the HEK293T 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.Cell Culture

[0207] Dystrophin-deficient H2K myoblasts (H2K-mdxamp;) were derived from a mouse model of Duchenne muscular dystrophy (DMD) containing an inactivating glutamine (Q) to a STOP (X) nonsense mutation in the dystrophin gene. This mutation arose in 1981 in a C57BL / 10ScSn colony at University of Leicester. A C-to-T substitution in the CAA codon in exon 23 (ENSMUST00000114000 chrX:g.83803333C> T; c.2983C> T; p. Q995*) resulted in a termination codon (TAA) in place of a glutamine (Q) codon. H2K-mt / x mutant and H2K-wild-type (H2K-WT) control cells were plated on Day 0 and allowed to differentiate into myotubes until Day 3, at which time they were transduced by MyoAAV 2 A expressing suppressor tRNAs from Batch 1, Batch 2, or Batch 3 (TABLES 19-21, respectively) at a multiplicity of infection (MOI) of 3E5 vector genomes (VG). Batch 1 included 13 Gln(Q)> TAA, Ser(S)> TAA, and Tyr(Y)> TAA suppressor tRNAs (tr0157, tr0523, tr0524, tr0525, tr0903, tr0904, tr0905, tr0906, trO538, trl233, trl344, tr0211, and tr0212). Batch 2 included 11 Gln(Q)> TAA suppressor tRNAs (trl499, trl503, trl505, trl502, tr0524, trl500,Attorney Docket No. TVD-013WO2 tri 504, trl506, tr0163, tri 501, and tr0904). Batch 3 included eleven Gln(Q)> TAA suppressor tRNAs (tri 511, trl513, tri 515, trl517, tri 519, trl521, trl523, trl525, and trl531, tri 621, and tri 622). Myotubes were harvested seven days after transduction (Day 10) and homogenized in RIPA lysis buffer. Total protein lysates were quantified using a BCA protein assay and normalized to 1 pg / pL for analysis by Western blot (FIG. 4).TABLE 19: Batch 1SEQ ID Suppr. Parent. Amino Anticodon Class16Sequence17NO: ID Suppr. AcidID175 tr0903 tr0521 Gin TTA E GGTTCCATGGTGTAA TGGTAAGCACTCTGG ACTttaAATCCAGCGA TCCGAGTTCGAGTCT CGGTGGAGCCT176 tr0904 tr0163 Gin TTA E GGCCCCATGGTGTAA TGGTTAGCACTCTGG TCTttaAAACCAGCGA TCCGAGTTCAAATCT CGGTGGGACCT177 tr0905 tr0156 Gin TTA E GGTTCCATGGTGTAA TGGTTAGCACTCTGG ACTttaAATCCAGCGA TACGAGTTCAAATCT CGTTGGAACCT178 tr0906 trO155 Gin TTA E GGTTCCGTGGTGTAA TGGTTAGCACTCTGG ACTttaAATCCAGCGA CCCGAGTTCAAATCT CGGCGGGACCT236 tr1344 trO538 Ser TTA E GTAGTCGTGGCCGAG TGGTTAAGGCGGCAG GCTttaAACCTGTTGG GGTTTCCCCGCACGG GTTCGAATCCCGTCG ACTACG238 tr0157 tr0157 Gin TTA P GGTTCCATGGTGTAA TGGTTAGCACTCTGG ACTttaAATCCAGCGA TCCGAGTTCAAATCTCGGTGGGACCT16In TABLE 19, P denotes parental suppressors and E denotes engineered suppressors.17For sequence entries in TABLE 19, the 3-nucleotide anticodon is denoted via lowercase lettersAttorney Docket No. TVD-013WO2 SEQ ID Suppr. Parent. Amino Anticodon Class16Sequence17NO: ID Suppr. AcidID240 tr0523 tr0523 Gin TTA P GGTCCCATGGTGTAA TGGTtAGCACTCTGG ACTttaAATCCAGCAaT CCGAGTTCGAATCTC GGTGGGACCT241 tr0524 tr0524 Gin TTA P GGCCCCATGGTGTAA TGGTcAGCACTCTGG ACTttaAATCCAGCGaT CCGAGTTCAAATCTC GGTGGGACCC242 tr0525 tr0525 Gin TTA P GGTTCCATGGTGTAA TGGTtAGCACTCTGG ACTttaAATCCAGCGaT CCGAGTTCAAGTCTC GGTGGAACCT249 tri 233 tri 233 Ser TTA P GTAGTCGTGGCCGAG TGGTtAAGGCGATGG ACTttaAATCCATTGG GGTCTCCCCGCGCAG GTTCGAATCCTGCCG ACTACG250 trO538 trO538 Ser TTA P GTAGTCGTGGCCGAG TGGTtAAGGCGATGG ACTttaAATCCATTGG GGTTTCCCCGCGCAG GTTCGAATCCTGCCG ACTACG251 tr0211 tr0211 Tyr TTA P CCTTCGATAGCTCAG CTGGTAGAGCGGAG GACTttaGATCCTTAG GtCGCTGGTTCGATTC CGGCTCGAAGGA252 tr0212 tr0212 Tyr TTA P CCTTCGATAGCTCAG TTGGTAGAGCGGAG GACTttaGATCCTTAG GtCGCTGGTTCGAATCCGGCTCGAAGGAAttorney Docket No. TVD-013WO2TABLE 20: Batch 2SEQ Suppr. Variant Variant Amino Anticodon Class Sequence19ID ID Site (see Acid 18NO: FIG. 3)176 tr0904 N / A N / A Gin TTA E GGCCCCATGGTGT AATGGTTAGCACT CTGGTCTttaAAACC AGCGATCCGAGTT CAAATCTCGGTGG GACCT179 trl499 tr0524 3 Gin TTA E GGTCCCATGGTGT AATGGTcAGCACTC TGGACTttaAATCCA GCGaTCCGAGTTCA AATCTCGGTGGGA CCC180 tri 500 tr0904 3 Gin TTA E GGTCCCATGGTGT AATGGTTAGCACT CTGGTCTttaAAACC AGCGATCCGAGTT CAAATCTCGGTGG GACCT181 trl501 tr0904 4,31,39 Gin TTA E GGCTCCATGGTGT AATGGTTAGCACT CTGGACTttaAATCC AGCGATCCGAGTT CAAATCTCGGTGG GACCT182 tri 502 tr0524 31,39 Gin TTA E GGCCCCATGGTGT AATGGTcAGCACTC TGGTCTttaAAACCA GCGaTCCGAGTTCA AATCTCGGTGGGA CCC183 tri 503 tr0524 73 Gin TTA E GGCCCCATGGTGT AATGGTcAGCACTC TGGACTttaAATCCA GCGaTCCGAGTTCA AATCTCGGTGGGACCTIn TABLE 20, P denotes parental suppressors, and E denotes engineered suppressors.For sequence entries in TABLE 20, the 3-nucleotide anticodon is denoted via lowercase letters.Attorney Docket No. TVD-013WO2 SEQ Suppr. Variant Variant Amino Anticodon Class Sequence19ID ID Site (see Acid 18NO: FIG. 3)184 tri 504 tr0904 73 Gin TTA E GGCCCCATGGTGT AATGGTTAGCACT CTGGTCTttaAAACC AGCGATCCGAGTT CAAATCTCGGTGG GACCC185 tri 505 tr0524 20a Gin TTA E GGCCCCATGGTGT AATGGTTAGCACT CTGGACTttaAATCC AGCGaTCCGAGTTC AAATCTCGGTGGG ACCC186 tri 506 tr0904 20a Gin TTA E GGCCCCATGGTGT AATGGTCAGCACT CTGGTCTttaAAACC AGCGATCCGAGTT CAAATCTCGGTGG GACCT239 tr0163 tr0904 31,39 Gin TTA E GGCCCCATGGTGT AATGGTTAGCACT CTGGACTttaAATCC AGCGATCCGAGTT CAAATCTCGGTGG GACCT241 tr0524 N / A N / A Gin TTA P GGCCCCATGGTGT AATGGTcAGCACTC TGGACTttaAATCCA GCGaTCCGAGTTCA AATCTCGGTGGGACCCTABLE 21: Batch 3SEQ Suppr. ID Parental Amino Anticodon Sequence20ID Suppressor AcidNO: ID187 trl511 tr0520 Gin TTA GGTTCCATGGTGTAATG GTGAGCACTCTGGACTtta AATCCAGCGATCCGAGT TCGAGTCTCGGTGGAACCCFor sequence entries in TABLE 21, the 3 -nucleotide anticodon is denoted via lowercase letters.Attorney Docket No. TVD-013WO2 SEQ Suppr. ID Parental Amino Anticodon Sequence20ID Suppressor AcidNO: ID188 trl513 tr0162 Gin TTA GGTCCCATGGTGTAATG GTTAGCACTCTGGACTtta AATCCAGCAATCTGAGT TCAAATCTCGGTGGGAC TT189 trl515 tr0520 Gin TTA GGTTCCATGGTGTAATG GTGAGCACTCTGGACTtta AATCCAGCGATCCGAGT TCGAGCCTCGGTGGAAC CT190 trl517 tr0520 Gin TTA GGTTCCATGGTGTAATG GTGAGCACTCTGGACTtta AATCCAGCGACCCGAGT TCGAGTCTCGGTGGAAC CT191 trl519 tr0521 Gin TTA GGTTCCATGGTGTAATG GTAAGCACTCTGGACTtta AATCCAGCGACCCGAGT TCGAGTCTCGGTGGAAC CT192 tri 521 tr0523 Gin TTA GGTCCCATGGTGTAATG GTTAGCACTCTGGACTtta AATCCAGTAATCCGAGT TCGAATCTCGGTGGGAC CT193 tri 523 tr0520 Gin TTA GGTTCCATGGTGTAATG GTGAGCACTCTGGACTtta AATCCAGTGATCCGAGT TCGAGTCTCGGTGGAAC CT194 tri 525 tr0523 Gin TTA GGTCCCATGGTGTAATG GTTAGCACTCTGGACTtta AATCCAGCAACCCGAGT TCGAATCTCGGTGGGAC CT195 tri 531 tr0523 Gin TTA GGTCCCATGGTGTAATG GTAAGCACTCTGGACTtta AATCCAGCAATCCGAGT TCGAATCTCGGTGGGAC CT915 tri 621 tr0163 Gin TTA GGCCCCATGGTGTAATG GTTAGCACTCTGGACTtta AATCCAGCGATCCGAGT TCGAGTCTCGGTGGGACCTAttorney Docket No. TVD-013WO2 SEQ Suppr. ID Parental Amino Anticodon Sequence20ID Suppressor AcidNO: ID916 tri 622 tr0524 Gin TTA GGCCCCATGGTGTAATG GTcAGCACTCTGGACTtta AATCCAGCGaTCCGAGT TCGAGTCTCGGTGGGACCCDystrophin Quantification by Jess Western Blot (Jess WB)

[0208] Full-length dystrophin was detected by capillary Jess Automated Western Blot (Bio-Techne), as per manufacturer’s instructions, using an anti-dystrophin antibody (Abeam; ab 154168, 1 / 250 dilution). Dystrophin signal intensity was normalized to total protein signal intensity. For a precise measure of dystrophin protein expression levels, a standard curve was established using pooled untreated wild-type (“D2-WT”) and pooled untreated mutant (“D2-mt&”) lysates at increasing concentrations (1%, 5%, 10%, 25%, 50%, 75%, and 100% wild-type levels, R2> 0.98). The accuracy of the standard curve was then ensured by comparing expected and experimentally determined levels for 15% and 60% of wild-type dystrophin levels (CV<30%). Resulting dystrophin signals from samples were then quantified to the standard curve.Results

[0209] Dystrophin-deficient H2K-mdxamp; cells were transduced with MyoAAV 2A expressing suppressor tRNAs from Batch 1. Batch 1 included both parental suppressor tRNAs and engineered suppressor tRNAs with one or more mutations relative to a parental suppressor tRNA (FIGs. 3A-3B). Twelve suppressor tRNAs were tested: seven Gln(Q)> TAA suppressors (tr0157, tr0523, tr0524, tr0525, tr0904, tr0905, and tr0906), three Ser(S)> TAA suppressors (trO538, trl233, and trl344), and two Tyr(Y)> TAA suppressors (tr0211 and tr0212) (see TABLE 19). Cells were lysed seven days after transduction and dystrophin protein levels were measured by capillary Jess Automated Western Blot.

[0210] Full-length dystrophin protein was detected in untreated H2K wild-type (“H2K-WT”) cells and in all dystrophin-deficient H2K-mdxamp; cells transduced with Batch 1 suppressor tRNAs, but not in untreated H2K-mdx cells (FIG. 5A). Rescue of full-length dystrophin varied by suppressor tRNA, ranging from a high of 28.11% (± 2.49) of wild-type dystrophin level for tr0212 to a low of 5.25% (± 0.74) for tr0157.Attorney Docket No. TVD-013WO2

[0211] Dystrophin-deficient H2K-mdxamp; cells were transduced with MyoAAV 2A expressing suppressor tRNAs from Batch 2 (FIG. 3A). Batch 2 included 11 suppressor tRNAs, all of which were Gln(Q)> TAA suppressors (TABLE 20). These suppressors derived from tr0525 and tr0904, the top performing suppressors from Batch 1 in a mouse model of Duchenne Muscular Dystrophy (FIG. 7). Sequence analysis identified nucleotide motifs present in tr0524 and / or tr0904 that deviated from Gln-tRNA consensus features. These included C3 in the acceptor stem, C20a in the D-loop, the T31, A39 nucleotide pair in the anticodon stem, and C73 in the acceptor stem (FIG. 3B). Sequence variants in Batch 2 were designed to explore the functional importance of the unique nucleotide motifs identified in tr0524 and / or tr0904 from Batch 1 (FIG. 3C). Full-length dystrophin protein was detected in all dystrophin-deficient H2K-mdxamp; cells transduced with Batch 2 suppressor tRNAs (FIG.5B) Rescue of full-length dystrophin varied by suppressor tRNA, ranging from a high of 24.66% (± 1.99) of wild-type dystrophin level for tri 505 to a low of 0% for tri 501. Multiple sequence variants from Batch 2 resulted in increased rescue of full-length dystrophin relative to the starting suppressors tr0524 and tr0904.

[0212] Dystrophin-deficient H2K-mdxamp; cells were transduced with MyoAAV 2A expressing suppressor tRNAs from Batch 3 (FIG. 3A). Batch 3 included 11 suppressor tRNAs, all of which were Gln(Q)> TAA suppressors (TABLE 21). These suppressor tRNAs were identified by large-scale screening for functional suppression of an inactivating glutamine (Q) to a STOP (X) nonsense mutation in a reporter gene. Full-length dystrophin protein was detected in all dystrophin-deficient H2K-mdxamp; cells transduced with Batch 3 suppressor tRNAs (FIG. 5C). Rescue of full-length Dystrophin varied by suppressor tRNA, ranging from a high of 69.03% (± 5.24) for tri 519 to a low of 22.09% (± 2.79) for trl531.EXAMPLE 2

[0213] This Example describes using parental and engineered suppressor tRNAs thereof to rescue full-length dystrophin via intramuscular injection in a mouse model of Duchenne Muscular Dystrophy. Suppressor tRNAs solely with the anticodon modified are referred to as ‘parental suppressor tRNAs,’ while suppressor tRNAs with one or more mutations relative to a parental suppressor tRNA are referred to as ‘engineered suppressor tRNAs’ or ‘engineered suppressor tRNAs thereof.’ Together, parental and engineered suppressor tRNAs are referred to as ‘suppressor tRNAs.’ The example demonstrated that the increase in expression was due, at least in part, to readthrough of a PTC by the parental and / orAttorney Docket No. TVD-013WO2 engineered suppressor tRNA.Materials and MethodsA A V Production

[0214] 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 derived from publicly 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 plasmids used this Example included a single-stranded (ssAAV) transfer plasmid encoding three copies of a suppressor tRNA of the disclosure, functionally linked to U6 promoters (e.g., SEQ ID NO: 917) located between two inverted terminal repeats (ITRs) (FIG. 2).

[0215] Suppressor tRNA vectors were packaged into the myotropic capsid MyoAAV 2A. 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.

[0216] The three plasmids were co-transfected into HEK293T cells. Briefly, the HEK293T 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 gAttorney Docket No. TVD-013WO2 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.Animals

[0217] D2. B 10 (DBA / 2-congenic) Dmdmdxmice (also referred to as DBA / 2J-mdx or D2-mdx mice) were used for the study. This mouse model of Duchenne muscular dystrophy (DMD) contains an inactivating glutamine (Q) to a STOP (X) nonsense mutation in the dystrophin gene. This mutation arose in 1981 in a C57BL / 10ScSn colony at University of Leicester. A C-to-T substitution in the CAA codon in exon 23 (ENSMUST00000114000 chrX:g.83803333C> T; C.2983OT; p. Q995*) resulted in a termination codon (TAA) in place of a glutamine (Q) codon. The Jackson Laboratory Repository created the DBA / 2J-congenic D2- / 77t / x mouse model (Stock No. 013141) by backcrossing C57BL / 10-mdx mice to DBA / 2J inbred mice for several generations using a marker-assisted, speed congenic approach.

[0218] The Dmdmdxmutant allele is on the X chromosome. Hemizygous D2-mt& male mice were used for this study. These mice are viable but developed myopathic features of DMD that recapitulated several of the human characteristics of DMD myopathology (lower hind limb muscle weight, fewer myofibers, increased fibrosis and fat accumulation, and muscle weakness).Intramuscular (IM) Injection of AAVs

[0219] AAV virus stocks were provided by the manufacturer in phosphate buffered saline (PBS) 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. Twelve-week-old D2-mdx were dosed via intramuscular administration (tibialis anterior; TA) at 1E11 VG / muscle. Injections were performed on both TA muscles of mice under isoflurane anesthesia. Ten animals were dosed per experimental condition to reduce the biological and technical variability in downstream experiments. Untreated wild-type (“D2-WT”) and D2-mdx hemizygous male mice (“D2-mdx”) were used as control groups. Animals wereAttorney Docket No. TVD-013WO2 euthanized at 18 weeks of age (6 weeks post-treatment) (FIG. 6). Mice were euthanized through CO2 exposure until cessation of respiration followed by cervical dislocation to confirm euthanasia prior to tissue collection. Both the left and right TA muscles were removed. The right TA was flash frozen in liquid nitrogen, stored at -70 °C, and utilized for protein analysis. The left TA was cut transversely into two pieces, with the lower-half, including tendon, placed into 10% Neutral Buffered Formalin (NBF) and the upper-half cryo-embedded with optimal cutting temperature (OCT) compound.Protein Extraction

[0220] Tissue from right TA muscles was homogenized in a 10% SDS lysis buffer. Total protein lysates were quantified using a BCA protein assay and normalized to 5 pg / pL for capillary Western blot (Jess WB) and Liquid Chromatography Mass Spectrometry (LC-MS) analysis.Dystrophin Quantification by Jess Western Blot

[0221] Full-length Dystrophin was detected by capillary Jess Automated Western Blot (Bio-Techne), as per manufacturer’s instructions, using an anti-dystrophin antibody (Abeam; ab 154168, 1 / 250 dilution). Dystrophin signal intensity was normalized to total protein signal intensity. For a precise measure of dystrophin protein expression levels, a standard curve was established using pooled untreated wild-type (“D2-WT”) and pooled untreated mutant (“D2-mt&”) lysates at increasing concentrations (1%, 5%, 10%, 25%, 50%, 75%, and 100% wild-type levels, R2> 0.98). The accuracy of the standard curve was then ensured by comparing expected and experimentally determined levels for 15% and 60% of wild-type dystrophin levels (CV < 30%). Resulting dystrophin signals from samples were then quantified to the standard curve.Dystrophin Quantification by Immunohistochemistry

[0222] Left TA muscles were transversally bisected and embedded in OCT compound. Cryo-embedded tissues were sectioned into -7-10 pm thick cross-sections using a microtome cryostat. TA muscle cross-sections were permeabilized using 1% Triton X-100 and blocked in PBS-3% BSA-2% NGS. They were then stained for dystrophin (Abeam; MA5-32565) and laminin-2 (muscle fiber marker; Abeam; L0663) to determine the number of muscleAttorney Docket No. TVD-013WO2 fibers positive for dystrophin expression. Cross sections were mounted for visualization and pictures were acquired using a FV3000 Olympus confocal microscope (20x objective). Analysis was done using Cell Profiler with a custom analysis pipeline.Liquid Chromatography-Mass Spectrometry (LC-MS / MS)

[0223] Total protein concentration was confirmed by a microBCA assay and 40 pg of protein per sample was used. Proteins were reduced for 10 minutes at 65 °C with 10 mM DTT and alkylated for 30 minutes at room temperature in the dark with 15 mM iodoacetamide. Proteins were precipitated overnight at -80 °C with 8 volumes of precipitation solution (8:1 acetone / methanol). The following day, pellets were washed 3 times with 250 pL cold methanol. Protein pellets were resolubilized in 50 pl of 50 mM Tris pH 8.0 + 0.75M + 0.1% NaDOC and predigested with 0.66 pg of Trypsin / LysC for 2 hours at 37 °C with agitation. Another 0.66 pg of Trypsin / LysC was added and the digestion was continued overnight at 37 °C with agitation. The following day, samples were spiked with 20 pL of a Master mix of heavy peptides (1 pg / mL) to each sample mix. Samples were acidified with 2% formic acid and the peptides were purified by reversed phase SPE.Peptides were resuspended in 40 pL of H2O, 0.1% FA. Once resuspended, 4 pL of each sample were pooled to be injected as GPF samples to be used for spectral library generation. Samples and pools were transferred in HPLC vials with insert and injected on a 60 minute LC-MS / MS gradient.

[0224] Acquisition was performed with a ABSciex TripleTOF 6600 (ABSciex, Foster City, CA, USA) equipped with an electrospray interface with a 25 pm iD capillary and coupled to an Eksigent UHPLC (Eksigent, Redwood City, CA, USA). Analyst TF 1.8 software was used to control the instrument and for data processing and acquisition. The pool for the ion library was acquired in SWATH (DIA) acquisition mode using gas phase fractionation (GPF). The samples were analyzed in SWATH acquisition mode. The source voltage was set to 5.35 kV and maintained at 325 °C, curtain gas was set at 50 psi, gas one at 40 psi and gas two at 35 psi. Separation was performed on a reversed phase Kinetex XB column 0.3 pm i.d., 2.6 pm particles, 150 mm long (Phenomenex) which was maintained at 60 °C. Samples were injected by loop overfilling into a 5 pL loop. For the 60 minutes LC gradient, the mobile phase consisted of the following solvent A (0.2% v / v formic acid and 3% DMSO v / v in water) and solvent B (0.2% v / v formic acid and 3% DMSO in EtOH) at a flow rate of 3 pL / min.Attorney Docket No. TVD-013WO2

[0225] GPF acquisitions of pooled library-building samples and study samples were processed with DIA-NN (v. 1.8.1) against a customized proteomic database that included sequences resulting from UAA-to-glutamine substitutions at native stop codons. FDR was set to 0.01; maximum missed cleavages: 1; fragment m / z 100-1800; precursor m / z 300-1250. Mass accuracy: 20 ppm; MBR; quant strategy: high accuracy; RT-dependent cross-run normalization. After validation of the run using the generated DIA-NN stats report (MSI signal; MS2 signal; MS1 / MS2 signal ratios, number of precursor and identified proteins were checked), the report was used with notebook to explore the protein quantification data. A protein centric approach was used to analyze and filter the raw DIA-NN output (maximum q-value for precursors & proteins: 0.01, sum top 5 precursors to generate protein data).Missing values were imputed using a K-nearest neighbor approach and normalized using RT-LOESS prior to summing to protein level data. Following verification of homoscedasticity and normal distribution within the data, protein quantitation was analyzed using a one-way ANOVA comparison between un-injected D2-mt& and all injected D2-mt / x groups with Benjamini -Hochberg procedure for controlling FDR.Results

[0226] Twelve-week-old D2-mt / x hemizygous male mice were intramuscularly injected in the TA with AAVs expressing suppressor tRNAs from Batch 1. Thirteen suppressor tRNAs were tested: eight Gln(Q)> TAA suppressors (tr0157, tr0523, tr0524, tr0525, tr0903, tr0904, tr0905, and tr0906), three Ser(S)> TAA suppressors (trO538, trl233, and tr1344), and two Tyr(Y)> TAA suppressors (tr0211 and tr0212) (TABLE 19). Animals were euthanized at 18 weeks of age (6 weeks post-treatment) and dystrophin protein levels were measured in TA muscle by capillary Jess Automated Western Blot.

[0227] Full-length dystrophin protein was detected in TA muscle from untreated wildtype (“D2-WT”) mice and in all cohorts of T)2-mdx hemizygous male mice dosed with suppressor tRNAs, but not in untreated D2- / ??t / x hemizygous male mice (FIG. 7). Rescue of full-length dystrophin varied by suppressor tRNA, ranging from a high of 23.01% (± 10.32) of wild-type level for tr0904 to a low of 1.99% (± 2.03) for trl233. All Batch 1 suppressor tRNAs that rescued full-length dystrophin protein in D2- / ??t / x mice also rescued full-length dystrophin in dystrophin-deficient H2I<- / ??t / x cells (FIG. 5A). There were, however, differences in suppressor activity between in vivo mouse and in vitro cell models. In mice,Attorney Docket No. TVD-013WO2 Gln(Q)> TAA suppressors produced the highest levels of dystrophin rescue, whereas in H2K-mdx cells both Ser(S)> TAA and Tyr(Y)> TAA suppressors outperformed the best Gln(Q)> TAA suppressors.

[0228] TA muscles from the three top performing suppressor tRNA cohorts (tr0524, tr0904, and tr1344) were further analyzed by immunohistochemistry (IHC) to measure the number of dystrophin-positive muscle fibers compared to untreated wild-type and untreated mutant controls. TA muscles from untreated D2-WT and D2-mt& mice showed 95.0% and 0.7% dystrophin-positive muscle fibers, respectively. The detection of a small number of positive fibers in mutant mice was likely due to the presence of sporadic dystrophin-positive muscle fibers, called revertant fibers (RFs), which are observed in otherwise dystrophinnegative backgrounds in DMD patie...

Claims

Attorney Docket No. TVD-013WO2 What is claimed is:

1. A nucleic acid encoding a suppressor tRNA, wherein the nucleic acid comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 254-274, 918, 925, 1149, and 1150, wherein Ni is a thymine (T) or cytosine (C) and N2 is thymine (T) or cytosine (C), provided that when Ni is a cytosine (C) then N2 is a thymine (T).

2. A nucleic acid encoding a suppressor tRNA, wherein the suppressor tRNA comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 175-237, 901-909, 915-916, 921-924, 1088-1119, 1122-1123, and 1125-1131, wherein each thymine in any one of SEQ ID NOs: 175-237, 901-909, 915-916, 921-924, 1088-1119, 1122-1123, and 1125-1131 is replaced by uracil.

3. The nucleic acid of claim 2, wherein the tRNA is capable of being aminoacylated with glutamine, tyrosine, serine, or leucine.

4. The nucleic acid of any one of claims 1-3, wherein the tRNA is capable of being aminoacylated with glutamine.

5. The nucleic acid of any one of claims 1-4, wherein the tRNA comprises a trinucleotide anticodon, wherein the anticodon is 5'-UCA-3' and recognizes an UGA stop codon.

6. The nucleic acid of any one of claims 1-4, wherein the tRNA comprises a trinucleotide anticodon, wherein the anticodon is 5'-UUA-3' and recognizes an UAA stop codons.

7. The nucleic acid of any one of claims 1-4, wherein the tRNA comprises a trinucleotide anticodon, wherein the anticodon is 5'-CUA-3' and recognizes an UAG stop codons.

8. The nucleic acid of any one of claims 1-7, wherein the nucleic acid comprises a 5' flanking sequence, a 3' flanking sequence, or both 5' and 3' flanking sequences.

9. The nucleic acid of claim 8, wherein the 5' flanking sequence or the 3' flanking sequence comprises a regulatory element.Attorney Docket No. TVD-013WO2 10. The nucleic acid of claim 8 or 9, wherein the 5' flanking sequence comprises a leader sequence, a promoter element, or a secondary structure.

11. The nucleic acid of any one of claims 8-10, wherein the 3' flanking sequence comprises a terminator element or a poly-T element.

12. The nucleic acid of any one of claims 8-11, wherein the 5' flanking region comprises a nucleotide sequence set forth in TABLE 16.

13. The nucleic acid of any one of claims 8-12, wherein the 3' flanking region comprises a nucleotide sequence set forth in TABLE 16.

14. The nucleic acid of any one of claims 1-13, wherein the nucleic acid comprises an internal tRNA promoter.

15. A nucleic acid comprising: (a) a U6 promoter comprising a nucleic acid sequence selected from any one of SEQ ID NOs: 917, 1132-1134, and 1151-1152; and (b) a nucleic acid encoding a suppressor tRNA.

16. The nucleic acid of claim 15, wherein the U6 promoter comprises a U6-8 promoter comprising the nucleic acid sequence of SEQ ID NO: 1132.

17. A nucleic acid comprising: (a) a 5' flanking sequence comprising a nucleic acid sequence selected from any one of SEQ ID NOs: 1153-1156 and 1159; and (b) a nucleic acid encoding a suppressor tRNA.

18. The nucleic acid of any one of claims 15-17, wherein the nucleic acid encoding the suppressor tRNA comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 254-274, 918, 925, 1149, and 1150, wherein Ni is a thymine (T) or cytosine (C) and N2 is thymine (T) or cytosine (C), provided that when Ni is a cytosine (C) then N2 is a thymine (T).

19. The nucleic acid of any one of claims 15-17, wherein the suppressor tRNA comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 175-237, 901-909, 915-916, 921-924, 1088-1119, 1122-1123, and 1125-1131, wherein each thymine in any one of SEQAttorney Docket No. TVD-013WO2 ID NOs: 175-237, 901-909, 915-916, 921-924, 1088-1119, 1122-1123, and 1125-1131 is replaced by uracil.

20. The nucleic acid of any one of claims 15-17, wherein the suppressor tRNA comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 238-252, 910-914, 1120, 1121, and 1124.

21. A nucleic acid comprising: (a) a 5' flanking sequence comprising a nucleic acid sequence selected from any one of SEQ ID NO: 1157 and SEQ ID NO: 1158; and (b) a nucleic acid encoding a suppressor tRNA, wherein the nucleic acid encoding the suppressor tRNA comprises:(a) a nucleic acid sequence selected from any one of SEQ ID NOs: 254-274, 918, 925, 1149, and 1150, wherein Ni is a thymine (T) or cytosine (C) and N2 is thymine (T) or cytosine (C), provided that when Ni is a cytosine (C) then N2 is a thymine (T);(b) a nucleic acid sequence selected from any one of SEQ ID NOs: 175-237, 901-909, 915-916, 921-924, 1088-1119, 1122-1123, and 1125-1131, wherein each thymine in any one of SEQ ID NOs: 175-237, 901-909, 915-916, 921-924, 1088-1119, 1122-1123, and 1125-1131 is replaced by uracil; or(c) a nucleic acid sequence selected from any one of SEQ ID NOs: 238-252, 910-914, 1120, 1121, and 1124.

22. The nucleic acid of claim 20 or 21, wherein the nucleic acid encoding the suppressor tRNA comprises the nucleic acid sequence of any one of SEQ ID NOs: 238-253, 910-914, 1120, 1121, and 1124.

23. A vector comprising the nucleic acid of any one of claims 1-22.

24. The vector of claim 23, wherein the vector comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copy numbers of the nucleic acid encoding the suppressor tRNA.

25. The vector of claim 23 or 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 adeno-associated virus (AAV) vector or a lentiviral vector.Attorney Docket No. TVD-013WO2 28. The vector of claim 27, wherein the viral vector is an AAV.

29. The vector of claim 28, wherein the AAV comprises an AAV inverted terminal repeat (ITR) derived from an AAV serotype selected from the group consisting of: AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16, or a derivative thereof.

30. The vector of claim 29, wherein the AAV ITR is derived from AAV-9 or AAV-rh74.

31. The vector of any one of claims 28-30, wherein the AAV is a single-stranded AAV or a self-complementary AAV.

32. A tRNA encoded by the nucleic acid of any one of claims 1-22.

33. The tRNA of claim 32, wherein the tRNA is aminoacylated with glutamine, serine, tyrosine, or leucine.

34. The tRNA of claim 32 or 33, wherein the tRNA is aminoacylated with glutamine.

35. The tRNA of any one of claims 32-34, wherein the tRNA comprises a tri -nucleotide anticodon, wherein the anticodon is 5'-UCA-3' and recognizes an UGA stop codon.

36. The tRNA of any one of claims 32-34, wherein the tRNA comprises a tri -nucleotide anticodon, wherein the anticodon is 5'-UUA-3' and recognizes an UAA stop codons.

37. The tRNA of any one of claims 32-34, wherein the tRNA comprises a tri -nucleotide anticodon, wherein the anticodon is 5'-CUA-3' and recognizes an UAG stop codons.

38. A pharmaceutical composition comprising the nucleic acid of any one of claims 1-22 or the vector of any one of claims 23-31 and a pharmaceutically acceptable excipient.Attorney Docket No. TVD-013WO2 39. 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 1-22, the vector of any one of claims 23-31, or the pharmaceutical composition of claim 38.

40. A method of treating a 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 1-22, the vector of any one of claims 23-31, or the pharmaceutical composition of claim 38, thereby to treat the disorder, wherein the disorder is a muscular dystrophy or a cardiology disorder.

41. A method of treating a disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of a nucleic acid encoding a suppressor tRNA, a vector comprising the nucleic acid, or a pharmaceutical composition comprising the nucleic acid or the vector and a pharmaceutically acceptable excipient, wherein the suppressor tRNA comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 238-252, 910-914, 1120, 1121, and 1124, thereby to treat the disorder, wherein the disorder is a muscular dystrophy or a cardiology disorder.

42. The method of claim 41, wherein the nucleic acid encoding the suppressor tRNA comprises the nucleic acid sequence of any one of SEQ ID NOs: 238-253, 910-914, 1120, 1121, and 1124.

43. The method of any one of claims 40-42, wherein the disorder is a muscular dystrophy.

44. The method of claim 43, wherein the muscular dystrophy comprises a disorder listed in TABLE 1745. The method of claim 43 or 44, wherein the muscular dystrophy is Duchenne muscular dystrophy, limb girdle syndrome, congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy, McArdle disease, Glycogen storage disease type V, a desminopathy, hypokalemic periodic paralysis, malignant hyperthermia, actin-accumulation myopathy, congenital myopathy, congenital myasthenic syndrome, dystroglycanopathy, Muscular dystrophy, limb-girdle, autosomal dominant 4, autosomal recessive limb-girdle muscular dystrophy, autosomal dominant limb-girdle muscular dystrophy type ID, muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A14, autosomalAttorney Docket No. TVD-013WO2 recessive limb-girdle muscular dystrophy type 2P, autosomal recessive limb-girdle muscular dystrophy type 2N, autosomal recessive limb-girdle muscular dystrophy type 2B, muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type Al, muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 4, emery-Dreifuss muscular dystrophy 3, autosomal recessive, X-linked myopathy with postural muscle atrophy, emery-Dreifuss muscular dystrophy 4, autosomal dominant, X-linked Emery-Dreifuss muscular dystrophy, Emery-Dreifuss muscular dystrophy 5, autosomal dominant, glycogen storage disease, type V, McArdle disease, desmin-related myofibrillar myopathy, congenital myopathy 18, congenital myopathy with fiber type disproportion, actin accumulation myopathy, congenital myopathy 11, myopathy, congenital, with tremor, congenital myopathy with fiber type disproportion, Bailey-Bloch congenital myopathy, myopathy, proximal, and ophthalmoplegia, congenital myasthenic syndrome 5, congenital myasthenic syndrome 10, congenital myasthenic syndrome 4 A, congenital myasthenic syndrome 11, congenital myasthenic syndrome 9, congenital myasthenic syndrome 2 A, congenital myasthenic syndrome 13, muscular dystrophy-dystroglycanopathy (congenital with brain and eye anomalies), type A, 7, autosomal recessive limb-girdle muscular dystrophy type 2D, autosomal recessive limb-girdle muscular dystrophy type 2E, autosomal recessive limb-girdle muscular dystrophy type 2F, myofibrillar myopathy 3, autosomal dominant limb-girdle muscular dystrophy type IF, autosomal recessive limb-girdle muscular dystrophy type 2 J, autosomal dominant limb-girdle muscular dystrophy type 1G, autosomal recessive limb-girdle muscular dystrophy type 2Q, autosomal recessive limb-girdle muscular dystrophy type R18, autosomal recessive limb-girdle muscular dystrophy type 2G, autosomal recessive limb-girdle muscular dystrophy type 2C, autosomal recessive limb-girdle muscular dystrophy type 2L, limb-girdle muscular dystrophy due to POMK deficiency, limb-girdle muscular dystrophy type 2A, or merosin deficient congenital muscular dystrophy.

46. The method of claim 45, wherein the muscular dystrophy is Duchenne muscular dystrophy.

47. The method of any one of claims 43-46, wherein the muscular dystrophy is associated with a premature termination codon (PTC) in a DMD, CAPN3, FKRP, DNAJB6, GMPPB, DYSF, DAG1, POMT2, POMT1, FKTN, LMNA, FHL1, SYNE1, EMD, SYNE2, PYGM, DES, CACNA1S, RYR1, ACTA1, MYBPC1, TPM3, STAC3, MYH2, COLQ, D0K7, CHRNE, RAPSN, MUSK, CHRNB1, DPAGT1, CRPPA, SGCA, SGCB, SGCD, SGCG,Attorney Docket No. TVD-013WO2 MYOT, TNPO3, HNRNPDL, PLEC, TRAPPCI 1, TCAP, ANO5, POMK, LAMA2, CAPN3, FKRP, DNAJB6, GMPPB, DAG1, POMT2, DYSF, POMT1, FKTN, LMNA, FHL1, SYNE1, EMD, SYNE2, PYGM, DES, CACNA1S, RYR1, ACTA1, HDAC1, MYBPC1, TPM3, STAC3, MYH2, COLQ, DOK7, CHRNE, RAPSN, MUSK, CHRNB1, DPAGT1, CRPPA, SGCA, SGCB, SGCD, MYOT, TNPO3, TTN, HNRNPDL, PLEC, TRAPPCI 1, TCAP, SGCG, ANO5, POMK, and / or LAMA2 gene.

48. The method of claim 47, wherein the muscular dystrophy is associated with a PTC in a DMD gene.

49. The method of any one of claims 43-48, wherein a DMD gene in the subject comprises a PTC.

50. The method of claim 49, wherein production of the tRNA in the subject increases expression of the DMD gene, and the increased expression ameliorates at least one symptom of DMD.

51. The method of claim 49 or 50, wherein production of the tRNA in the subject increases expression of the DMD gene to at least 5% of a DMD gene expression level in a subject not having DMD or in the subject being treated prior to the start of treatment.

52. The method of any one of claims 49-51, wherein production of the tRNA in the subject increases expression of full-length dystrophin encoded by the DMD gene, and the increased expression ameliorates at least one symptom of DMD.

53. The method of claim 52, wherein production of the tRNA in the subject increases expression of the full-length dystrophin in at least one muscle of the subject to at least 3% of a full-length dystrophin expression level in a subject not having DMD or in the subject being treated prior to the start of treatment.

54. The method of claim 53, wherein the at least one muscle comprises gastrocnemius, quadriceps, diaphragm, triceps, heart, and tibialis anterior.

55. The method of claim 54, wherein the at least one muscle is the gastrocnemius, quadriceps, diaphragm, and / or triceps.Attorney Docket No. TVD-013WO2 56. The method of any one of claims 48-55, wherein production of the tRNA in the subject improves proteomic dysregulation arising from the loss of full-length dystrophin (e.g., as measured by LC-MS / MS).

57. The method of claim 56, wherein production of the tRNA in the subject decreases expression of one or more proteins selected from the group consisting of: Utm, Anxal, Anxa2, Anxa5, Fnl, Tubb2A, Tubb5, Tubb6, TubalC, Vim, Serpinhl, Clicl, Coll4Al, Dtna, Sgcb, Sgcd, Sgcg, Dagl, Sspn, Sgca, Calr, and Cacnbl relative to the expression of the one or more proteins before production of the tRNA.

58. The method of claim 56 or 57, wherein production of the tRNA in the subject increases expression of one or more proteins selected from the group consisting of: Plec, Synm, SntAl, SntB2, SlOOal 1, and S100al3 relative to the expression of the one or more proteins before production of the tRNA.

59. The method of any one of claims 48-58, wherein production of the tRNA in the subject improves histopathology of dystrophin (e.g, as measured by immunohistochemistry).

60. The method of any one of claims 40-42, wherein the disorder is a cardiology disorder.

61. The method of claim 60, wherein the cardiology disorder comprises a disorder listed in TABLE 1862. The method of claim 60 or 61, wherein the cardiology disorder is a titinopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, Brugada syndrome, long QT syndrome, Friderich’s ataxia, or cholesterol emia.

63. The method of claim 62, wherein the cardiology disorder is a titinopathy.

64. The method of claim 62 or 63, wherein the cardiology disorder is dilated cardiomyopathy.

65. The method of any one of claims 60-64, wherein the cardiology disorder is associated with a PTC in a TTN, LMNA, FLNC, MYBPC3, BAG3, RBM20, LAMP2, SCN5A, KCNH2, KCNQ1, FXN, and / or LDLR gene.Attorney Docket No. TVD-013WO2 66. The method of claim 65, wherein the cardiology disorder is associated with a PTC in a TTN gene.

67. The method of any one of claims 60-66, wherein a TTN gene in the subject comprises a PTC.

68. The method of claim 67, wherein production of the tRNA in the subject increases expression of the TTN gene, and the increased expression ameliorates at least one symptom of the cardiology disorder.

69. The method of claim 67 or 68, wherein production of the tRNA in the subject increases expression of the TTN gene by at least 5%, as compared to expression of the TTN in the subject prior to administration.

70. The method of any one of claims 66-69, wherein production of the tRNA in the subject increases expression of full-length titin encoded by the TTN gene, and the increased expression ameliorates at least one symptom of the cardiology disorder.

Images