Compositions and methods for treating selenoprotein-associated diseases

Abstract

The present invention features compositions and methods for treating selenoprotein-associated diseases. Non-limiting examples of selenoproteins include selenoprotein N (SELENON).

Description

[0001] COMPOSITIONS AND METHODS FOR TREATING SELENOPROTEIN¬

[0002] ASSOCIATED DISEASES

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] The present application claims priority to and the benefit of U.S. App. No. 63 / 632,819, filed April 11, 2024, the contents of which is hereby incorporated by reference in its entirety.

[0005] STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY

[0006] SPONSORED RESEARCH

[0007] This invention was made with government support under Grant No. AR044345 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] BACKGROUND OF THE INVENTION

[0009] SELENON (formerly SEPN1 or SelN) Related Myopathies (SELENO N-RM) are a group of rare congenital myopathies caused by pathogenic mutations of the SELENON gene that alter an individual’s ability to synthesize its encoded protein, Selenoprotein N. Selenoprotein N belongs to the group of selenoproteins, which thanks to several cis and trans elements, all have the ability to incorporate the amino acid selenocysteine (Sec) at a UGA codon. Anomalies at any of the cis and / or trans elements would result in a stop codon and premature termination of protein synthesis. Selenoprotein N is more prominent in fetal as compared to adult tissues and can be found throughout the body. Though not all biological roles of selenoprotein N are understood, it significantly impacts the calcium flux in the cells through redox regulation of the Sarco / Endoplasmic Reticulum Ca2+-ATPase (SERCA), which in turn seems to considerably affect skeletal muscles. Patients with mutations in the SELENON gene suffer from slowly progressive congenital muscular dystrophy with early onset rigidity of the spine and potentially life-threatening respiratory insufficiency. Presently, no specific palliative or curative therapies for SELONON- related myopathies exist. Accordingly, methods for treating such myopathies are urgently required.

[0010] SUMMARY OF THE INVENTION

[0011] As described below, the present invention features compositions and methods for treating related myopathies and other selenoprotein-associated diseases. In embodiments, the selenoprotein is selenoprotein N (SELENON) and / or a polypeptide encoded by the SELENON gene. In an aspect, the present disclosure provides a polynucleotide including a nucleic acid sequence encoding a selenoprotein polypeptide and a 3' untranslated region (UTR) including two or more selenocysteine insertion sequence (SECIS) elements.

[0012] In another aspect, the present disclosure provides a mammalian expression vector including the polynucleotide of any of the above aspects, or embodiments thereof.

[0013] In another aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) particle including the polynucleotide of any of the above aspects, or embodiments thereof.

[0014] In another aspect, the present disclosure provides a serotype 9 recombinant adeno- associated virus (rAAV9) particle including in order from 5’ to 3’ : a mammalian promoter, a SELENON polynucleotide, and a 3’ untranslated region (UTR) including two SELENON selenocysteine insertion sequence (SECIS) elements, where the 3’ untranslated region (UTR) is less than 800 bp in length.

[0015] In another aspect, the present disclosure provides a serotype myoAAV recombinant adeno-associated virus particle including in order from 5’ to 3’ : a mammalian promoter, a SELENON polynucleotide, and a 3’ untranslated region (UTR) including two SELENON selenocysteine insertion sequence (SECIS) elements, where the 3’ untranslated region (UTR) is less than 800 bp in length.

[0016] In another aspect, the present disclosure provides a cell including the expression vector or the particle of any of the above aspects, or embodiments thereof.

[0017] In another aspect, the present disclosure provides a pharmaceutical composition including the polynucleotide, the vector, the particle, and / or the cell, of any of the above aspects, or embodiments thereof, and a pharmaceutically acceptable excipient.

[0018] In another aspect the present disclosure provides a method for increasing selenoprotein expression in a cell. The method involves contacting the cell with the polynucleotide, the vector, and / or the particle of any of the above aspects, or embodiments thereof.

[0019] In another aspects, the present disclosure provides a method of treating a subject having a congenital selenoprotein-associated disease. The method involves administering to the subject an effective amount of the pharmaceutical composition of any of the above aspects, or embodiments thereof.

[0020] In another aspect, the present disclosure provides a method of treating a subject having a congenital selenoprotein-associated disease. The method involves administering to the subject an effective amount of a polynucleotide including a nucleic acid sequence encoding a selenoprotein polypeptide, and a 3' untranslated region (UTR) including two or more selenocysteine insertion sequence (SECIS) elements, thereby treating the congenital selenoprotein-associated disease.

[0021] In another aspect, the present disclosure provides a method of treating a subject having a congenital myopathy. The method involves administering to the subject an effective amount of a polynucleotide encoding, in order from 5' to 3': a regulatory element, a selenoprotein polypeptide, and a 3' untranslated region (UTR) including two or more selenocysteine insertion sequence (SECIS) elements, thereby treating the congenital myopathy.

[0022] In any of the above aspects, or embodiments thereof, the selenoprotein polypeptide is a

[0023] SELENON polypeptide.

[0024] In any of the above aspects, or embodiments thereof, the SELENON polypeptide or fragment thereof has at least about 85% sequence identity to the following amino acid sequence:

[0025] MGRARPGQRGPPSPGPAAQPPAPPRRRARSLALLGALLAAAAAAAVRVCARHAEAQAAARQELA

[0026] LKTLGTDGLFLFSSLDTDGDMYISPEEFKPIAEKLTGSTPAASCEEEELPPDPSEETLTIEARF

[0027] QPLLPETMTKSKDGFLGVSRLALSGLRNWTAAASPSAVFATRHFQPFLPPPGQELJGEPWWI I PS

[0028] ELSMFTGYLSNNRFYPPPPKGKEVI IHRLLSMFHPRPFVKTRFAPQGAVACLTAISDFYYTVMF

[0029] RIHAEFQLSEPPDFPFWFSPAQFTGHI ILSKDATHVRDFRLFVPNHRSLNVDMEWLYGASESSN

[0030] MEVDIGYIPQMELEATGPSVPSVILDEDGSMIDSHLPSGEPLQFVFEEIKWQQELSWEE7XARRL

[0031] EVAMYPFKKVSYLPFTEAFDRAKAENKLVHSILLWGALDDQSCUGSGRTLRETVLESSPILTLL

[0032] NESFISTWSLVKELEELQNNQENSSHQKLAGLHLEKYSFPVEMMICLPNGTWHHINANYFLDI

[0033] TSVKPEEIESNLFSFSSTFEDPSTATYMQFLKEGLRRGLPLLQP.

[0034] In any of the above aspects, or embodiments thereof, the nucleic acid sequence, or fragment thereof, has at least about 85% sequence identity to the following nucleic acid sequence:

[0035] ATGGGCCGGGCCCGGCCGGGCCAACGCGGGCCGCCCAGCCCCGGCCCCGCCGCGCAGCCTCCCG

[0036] CGCCACCGCGCCGCCGCGCCCGTTCCCTGGCGCTGCTCGGAGCCCTGCTGGCCGCCGCCGCTGC

[0037] CGCCGCCGTCCGGGTCTGCGCCCGCCACGCCGAGGCCCAGGCGGCCGCGCGGCAGGAACTGGCG

[0038] CTGAAGACCCTGGGGACAGATGGCCTTTTTCTCTTTTCCTCCTTGGACACTGACGGGGATATGT

[0039] ACATCAGCCCTGAGGAGTTCAAACCCATTGCTGAGAAGCTAACAGGGTCAACTCCCGCGGCCAG

[0040] CTGCGAGGAGGAGGAGTTGCCCCCTGACCCTAGCGAGGAGACGCTCACCATAGAAGCCCGATTC

[0041] CAGCCTCTGCTCCCGGAGACCATGACCAAGAGCAAAGATGGCTTCCTAGGGGTCTCCCGCCTCG

[0042] CCCTGTCCGGCCTCCGAAACTGGACAGCCGCCGCCTCACCAAGTGCAGTGTTTGCCACCCGCCA

[0043] CTTCCAGCCCTTCCTTCCCCCGCCAGGCCAGGAGCTGGGTGAGCCCTGGTGGATCATCCCCAGT

[0044] GAGCTGAGCATGTTCACTGGCTACCTGTCCAACAACCGCTTCTATCCACCGCCGCCCAAGGGCA

[0045] AGGAGGTCATCATCCACCGGCTCCTGAGCATGTTCCACCCTCGGCCCTTTGTGAAGACCCGCTT

[0046] TGCCCCTCAGGGAGCTGTGGCCTGCCTGACTGCCATCAGCGACTTCTACTACACTGTGATGTTC CGGATCCATGCCGAGTTCCAGCTCAGTGAGCCGCCCGACTTCCCCTTTTGGTTCTCCCCTGCTC

[0047] AGTTCACCGGCCACATCATCCTCTCCAAAGACGCCACCCACGTCCGCGACTTCCGGCTCTTCGT

[0048] GCCCAACCACAGGTCTCTGAATGTGGACATGGAGTGGCTTTACGGGGCCAGTGAAAGCAGCAAC

[0049] ATGGAGGTGGACATCGGCTACATACCCCAGATGGAGCTGGAGGCCACGGGCCCCTCTGTGCCCT

[0050] CCGTGATCCTGGATGAGGATGGCAGCATGATCGACAGCCACCTGCCTTCAGGGGAGCCCCTGCA

[0051] GTTTGTGTTTGAGGAGATCAAGTGGCAGCAGGAGCTGAGCTGGGAGGAGGCTGCCCGGCGCCTG

[0052] GAGGTGGCCATGTACCCCTTCAAGAAGGTCTCCTACTTGCCGTTCACTGAGGCCTTCGACCGAG

[0053] CCAAGGCTGAGAACAAGCTGGTGCACTCAATCCTGCTGTGGGGGGCCCTGGATGACCAGTCCTG

[0054] CTGAGGTTCAGGGCGGACTCTCCGGGAGACTGTCCTGGAAAGTTCGCCCATCCTCACCCTGCTC

[0055] AACGAGAGCTTCATCAGCACCTGGTCCCTGGTGAAGGAGCTGGAGGAACTGCAGAACAACCAGG

[0056] AGAACTCGTCCCACCAGAAGCTGGCTGGCCTGCACCTGGAGAAGTACAGCTTCCCCGTGGAGAT

[0057] GATGATCTGCCTGCCCAATGGCACCGTGGTCCATCACATCAATGCCAACTACTTCTTGGACATC

[0058] ACCTCCGTGAAGCCCGAGGAAATCGAGAGCAATCTCTTCAGCTTCTCATCCACCTTTGAAGACC

[0059] CGTCCACGGCCACCTACATGCAGTTCCTGAAGGAGGGACTCCGGCGTGGCCTGCCCCTCCTCCA

[0060] GCCCTAG.

[0061] In any of the above aspects, or embodiments thereof, one or more of the SECIS elements, or a fragment thereof, has at least about 85% sequence identity to a sequence listed in FIG. 3B, or to one of the following sequences:

[0062] SECIS element 1

[0063] CACGGACCCCATGGCAGGGGTGGCGTCTTCTGCATGATCCGCTCTGGTCAAACCCTTCCAGGCC

[0064] AGCCAGAGTGGGGATGGTCTGAGGGGCCAGCCCTTAGTGCAT;

[0065] SECIS element 2

[0066] CACGGACCCCATGGCAGGGGTGGCGTCTTCATGATCCGCTCTGGTCAAACCCTTCCAGGCCAGC

[0067] CAGAGTGGGGAGCCTGTCTGAGGGGCCAGCCCTTAGTGCAT;

[0068] SECIS element 3

[0069] CACGGACCCCATGGCAGGGGTGGCGTCTTCATGAGGGAGGGGCCCCAAACCCTTCCAGGCCAGG

[0070] ACCTCCCCTGAGCCTGTCTGAGGGGCCAGCCCTTAGTGCAT;

[0071] SECIS element 4

[0072] CCTGCCAGCCGCCCTGGCCCTGGTCACTGCATGATCCGCTCTGGTAAAGCCCTTGTGGGCGCCA

[0073] GAGTGGGGATGGTCTGTGACCTGCTGGGAAGGCAGGC;

[0074] SECIS element 5

[0075] CCTGCCAGCCGCCCTGGCCCTGGTCACTGCATGAGGGAGGGGCCCAAAGCCCTTGTGGGCGGAC

[0076] CTCCCCTGATGGTCTGTGACCTGCTGGGAAGGCAGGC;

[0077] SECIS element 6 CCTGCCAGCCGCCCTGGCCCTGGTCACATGAGGGAGGGGCCCAAAGCCCTTGTGGGCGGACCTC

[0078] CCCTGAGCCTGTCTGTGACCTGCTGGGAAGGCAGGC;

[0079] SECIS element 7

[0080] CCTGCCAGCCGCCCTGGCCCTGGTCACATGATCCGCTCTGGTCAAACCCTTCCAGGCCAGCCAG

[0081] AGTGGGGAGCCTGTCTGTGACCTGCTGGGAAGGCAGGC;

[0082] SECIS element 8

[0083] CACGGACCCCATGGCAGGGGTGGCGTCTTCTGCATGAGGGAGGGGCCCAAAGCCCTTGTGGGCG

[0084] GACCTCCCCTGATGGTCTGAGGGGCCAGCCCTTAGTGCAT;

[0085] SECIS element 9

[0086] CACGGACCCCATGGCAGGGGTGGCGTCTTCTGCATGATCCGCTCTGGTCAAACCCTTCCAGGCC

[0087] AGCCAGAGTGGGGATGGTCTGAGGGGCCAGCCCTTAGTGCAT;

[0088] SECIS element 10

[0089] CCTGCCAGCCGCCCTGGCCCTGGTCACATGAGGGAGGGGCCCAAAGCCCTTGTGGGCGGACCTC

[0090] CCCTGAGCCTGTCTGTGACCTGCTGGGAAGGCAGGC;

[0091] SECIS element 11

[0092] CACGGACCCCATGGCAGGGGTGGCGTCTTCATGATCCGCTCTGGTAAAGCCCTTGTGGGCGCCA

[0093] GAGTGGGGAGCCTGTCTGAGGGGCCAGCCCTTAGTGCAT; and

[0094] SECIS element 12

[0095] CCTGCCAGCCGCCCTGGCCCTGGTCACTGCATGAGGGAGGGGCCCCAAACCCTTCCAGGCCAGG

[0096] ACCTCCCCTGATGGTCTGTGACCTGCTGGGAAGGCAGGC

[0097] In any of the above aspects, or embodiments thereof, the SECIS elements are Type 1 and / or Type 2 SECIS elements. In any of the above aspects, or embodiments thereof, the SECIS elements are human SECIS elements.

[0098] In any of the above aspects, or embodiments thereof, one or more of the SECIS elements has at least about 85% sequence identity to the following sequence:

[0099] SELENON SECIS element

[0100] GGCAGCAGCCCCATGATGGCTGAATCCGAAATCCTCGATGGGTCCAGCTTGATGTCTTTGCAGC

[0101] TGCA .

[0102] In any of the above aspects, or embodiments thereof, the polynucleotide includes DNA or

[0103] RNA.

[0104] In any of the above aspects, or embodiments thereof, a polynucleotide sequence of the 3’

[0105] UTR includes a SELENON, SELENOW, SELENOM, or TXRD2 3’ UTR polynucleotide sequence. In any of the above aspects, or embodiments thereof, the 3’ UTR is less than about 1500 bp, 1250 bp, 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, or 300 bp in length. In any of the above aspects, or embodiments thereof, the 3’ UTR is less than about 800 bp in length.

[0106] In any of the above aspects, or embodiments thereof, the polynucleotide includes a promoter. In any of the above aspects, or embodiments thereof, the promoter is a constitutive promoter. In any of the above aspects, or embodiments thereof, the promoter is an inducible promoter. In any of the above aspects, or embodiments thereof, the promoter is a CMV promoter. In any of the above aspects, or embodiments thereof, the promoter is a muscle specific promoter. In any of the above aspects, or embodiments thereof, the promoter is a MHCK7 promoter.

[0107] In any of the above aspects, or embodiments thereof, the polynucleotide further includes adeno-associated virus (AAV) inverted terminal repeats (ITR).

[0108] In any of the above aspects, or embodiments thereof, the 3’ untranslated region (UTR) includes about 355 bp from the 3’ UTR of a TXRND2 gene, about 411 bp from the 3’ UTR of a SelW gene , about 198 bp from the 3’ UTR of a SelM gene, about 544 bp from the 3’ UTR of a Selenon gene, or about 775 bp from the 3’ UTR of a Selenon gene.

[0109] In any of the above aspects, or embodiments thereof, the vector is a viral particle. In any of the above aspects, or embodiments thereof, the viral particle is an adeno-associated viral (AAV) particle.

[0110] In any of the above aspects, or embodiments thereof, the rAAV particle is an rAAV9 virus particle. In any of the above aspects, or embodiments thereof, the rAAV particle includes a muscle-trophic viral capsid. In any of the above aspects, or embodiments thereof, the muscle- trophic viral capsid is a myoAAV viral capsid. In any of the above aspects, or embodiments thereof, the muscle-trophic viral capsid is myoAAV4A.

[0111] In any of the above aspects, or embodiments thereof, the selenoprotein includes one or more of: GPX1, GPX2, GPX3, GPX4, GPX6, TXNRD1 (TRI, TRXR1), TXNRD2 (TRXR2, TR3), TXNRD3 (TGR, TRXR3, TR2), DIO1 (DI), DIO2 (D2), DIOS (D3), MSRB1 (SELR, SELX, SEPX1), SEPHS2 (SPS2) SELENOF (SEP 15), SELENOH (SELH), SELENOI (SELI, EPT1), SELENOK (SELK), SELENOM (SELM, SEPM), SELENON (SEPN1, SELN), SELENOO (SELO), SELENOP (SEPPI, SEP, SELF, SEPP), SELENO S (SELS, SEPS1, PIMP), SELENOT (SELT) , SELENO V (SELF), or SELENOW ( SELW, SEPWP).

[0112] In any of the above aspects, or embodiments thereof, the mammalian promoter is a cytomegalovirus, MHCK7, or CK8 promoter. In any of the above aspects, or embodiments thereof, the cell is a cell in vitro or in vivo. In any of the above aspects, or embodiments thereof, the cell is a mammalian cell. In any of the above aspects, or embodiments thereof, the cell is a muscle cell.

[0113] In any of the above aspects, or embodiments thereof, the method increases levels of calcium ions in the sarcoplasmic reticulum of a muscle cell.

[0114] In any of the above aspects, or embodiments thereof, the polynucleotide is administered systemically or locally.

[0115] In any of the above aspects, or embodiments thereof, the subject is a mammal.

[0116] In any of the above aspects, or embodiments thereof, the selenoprotein-associated disease includes one or more of: congenital fiber type disproportion, congenital nondystrophic myopathy, congenital muscular dystrophy, Desmin-Related Myopathy with Mallory Body-like Inclusions, minicore myopathy, multicore myopathy, multiminicore myopathy, rigid spine muscular dystrophy, sedaghatian-type spondylometaphyseal dysplasia (SSDM), epilepsy, dilated cardiomyopathy, familial glucocorticoid deficiency, or spastic paraplegia.

[0117] In any of the above aspects, or embodiments thereof, the method reduces hypotonia, proximal muscle weakness, morphologic abnormalities in skeletal muscle, restrictive respiratory syndrome, respiratory weakness, spinal rigidity, neuronal degeneration, ataxia, seizures, skeletal disorder, brain atrophy, decreases in respiratory function, and / or scoliosis.

[0118] In any of the above aspects, or embodiments thereof, the selenoprotein includes one or more of: GPX1, GPX2, GPX3, GPX4, GPX6, TXNRD1, TXNRD2 (TXRD2), TXNRD3, DIO1, DIO2, DIO3, SEPHS2, SEPS1, SEPPI, SEP15, SEPN1 (SELENON), SEPX1, SEPW1 (SELENOW), SEPTI, SEEK, SELI, SELK, SEEM (SELENOM), SELO, or SELV.

[0119] In any of the above aspects, or embodiments thereof, the selenoprotein is one or more of: GPX4, TXNRD1, TXNRD2 (TXRD2), SEPN1 (SELENON), or SELI.

[0120] The invention provides methods for treating selenoprotein-associated diseases. Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

[0121] Definitions

[0122] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, Sth Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The

[0123] Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0124] By “selenoprotein N (SELENON) polypeptide, SEPN1 polypeptide,” or“SelN polypeptide” is meant a protein having at least about 85% amino acid sequence identity to

[0125] UniProtKB Accession No. Q9NZV5-1 or Q9NZV5-2, both provided below, or a fragment thereof, that has a function associated with transport of calcium ions across a membrane of the

[0126] Sarcoplasmic reticulum, that functions in oxidation-reduction reactions, or that otherwise is required for normal muscle function.

[0127] ▻Q9NZV5-1

[0128] MGRARPGQRGPPSPGPAAQPPAPPRRRARSLALLGALLAAAAAAAVRVCARHAEAQAAARQELA

[0129] LKTLGTDGLFLFSSLDTDGDMYISPEEFKPIAEKLTGSCSVTQTGVQWCSHSSLQPQLPWLNUS

[0130] SCLSLLRSTP7XASCEEEELPPDPSEETLTIEARFQPLLPETMTKSKDGFLGVSRLALSGLRNWT

[0131] 7VXASPSAVFATRHFQPFLPPPGQELGEPWWIIPSELSMFTGYLSNNRFYPPPPKGKEVIIHRLL

[0132] SMFHPRPFVKTRFAPQGAVACLTAISDFYYTVMFRIHAEFQLSEPPDFPFWFSPAQFTGHIILS

[0133] KDATHVRDFRLFVPNHRSLNVDMEWLYGASESSNMEVDIGYIPQMELEATGPSVPSVILDEDGS

[0134] MIDSHLPSGEPLQFVFEEIKWQQELSWEE7XARRLEVAMYPFKKVSYLPFTEAFDRAKAENKLVH

[0135] SILLWGALDDQSCUGSGRTLRETVLESSPILTLLNESFISTWSLVKELEELQNNQENSSHQKLA

[0136] GLHLEKYSFPVEMMICLPNGTWHHINANYFLDITSVKPEEIESNLFSFSSTFEDPSTATYMQF

[0137] LKEGLRRGLPLLQP .

[0138] ▻Q9NZV5-2

[0139] MGRARPGQRGPPSPGPAAQPPAPPRRRARSLALLGALLAAAAAAAVRVCARHAEAQAAARQELA

[0140] LKTLGTDGLFLFSSLDTDGDMYISPEEFKPIAEKLTGSTPAASCEEEELPPDPSEETLTIEARF

[0141] QPLLPETMTKSKDGFLGVSRLALSGLRNWT7VXASPSAVFATRHFQPFLPPPGQELGEPWWIIPS

[0142] ELSMFTGYLSNNRFYPPPPKGKEVIIHRLLSMFHPRPFVKTRFAPQGAVACLTAISDFYYTVMF

[0143] RIHAEFQLSEPPDFPFWFSPAQFTGHIILSKDATHVRDFRLFVPNHRSLNVDMEWLYGASESSN

[0144] MEVDIGYIPQMELEATGPSVPSVILDEDGSMIDSHLPSGEPLQFVFEEIKWQQELSWEE7XARRL

[0145] EVAMYPFKKVSYLPFTEAFDRAKAENKLVHSILLWGALDDQSCUGSGRTLRETVLESSPILTLL

[0146] NESFISTWSLVKELEELQNNQENSSHQKLAGLHLEKYSFPVEMMICLPNGTWHHINANYFLDI

[0147] TSVKPEEIESNLFSFSSTFEDPSTATYMQFLKEGLRRGLPLLQP .

[0148] In the above sequences “U” represents selenocysteine. By SEPN1 polynucleotide,” or “SelN polynucleotide” is meant a nucleic acid molecule that encodes a SELENON polypeptide and / or the flanking sequences (e.g., 3’ UnTranslated Region (UTR)) that modulate expression of the

[0149] SELENON polypeptide. An exemplary human SELENON polynucleotide sequences are provided below (SelenoDB Accession No. SPT00000073_2.0)

[0150] CCCCGCCCCGCTCTTTCGCTTCCCGGGCCGCCGGCAGCCGCCGCCAGCCGCAGCCATGGGCCGG

[0151] GCCCGGCCGGGCCAACGCGGGCCGCCCAGCCCCGGCCCCGCCGCGCAGCCTCCCGCGCCACCGC

[0152] GCCGCCGCGCCCGTTCCCTGGCGCTGCTCGGAGCCCTGCTGGCCGCCGCCGCTGCCGCCGCCGT

[0153] CCGGGTCTGCGCCCGCCACGCCGAGGCCCAGGCGGCCGCGCGGCAGGTCCGGGCCCGAGCTGGC

[0154] TGGGGCGGGAGCGCGGGAGCGGGGACTGAAGGACCCAGCCTCGGCAGCAGGGGGGACATGGGCA

[0155] TGGACGAGTCGGGGGAGGCGGAGGGCGGAGGGGGACTGGACGGGAGGGAGCACGGGAAGGAGGG

[0156] GACACGGGAGGCGGGTGGGGGGTGCCTGGGGCCGGGCTGATGGGGGGAAGGCGAACGGAGGCCA

[0157] GTGTGGGGCTGGGACAGACTGGAGGGGTCTCTATCCAGAGGGGTGACCTGGACACAGTGAAGCT

[0158] GGTTCCCGCAGAGTCTTAAAGGGTTACTGGGGAGCCCTGGGACAGTGAGGAGGGGACACAGGAG

[0159] AGGACAGAAGGTGCAGGAGGAACAGGGCCAAGCAGCCAGGGCCAGGCGGAAGGCGGAGTGGGGA

[0160] CAAACTGGGAGGGGGCAGTCGGACCAGACAGTGGATGAGCAGGGACCAGGAGGGACTGGAGGCT

[0161] CTGGGGCAGTTCAGAGGCAACATTTGCACAGATGGACCGGGAGGCACTGGGAGAGCTCAGGAAG

[0162] ATGGTGGGAGAAGGGGCTCCATGCGGAAGCAACTGCCTGTTGGGGACCAGGAGACCAGGCCGCC

[0163] AAATGGTGCCCAGCCCAGTCTCTCCTCCCAAGCAGGAACTGGCGCTGAAGACCCTGGGGACAGA

[0164] TGGCCTTTTTCTCTTTTCCTCCTTGGACACTGACGGGGATATGTACATCAGCCCTGAGGAGTTC

[0165] AAACCCATTGCTGAGAAGCTAACAGGTACCAGGAGAGACTGGCGGCTGGGGAGGAGGGCGCCTT

[0166] GGCCAACGGTGTCTTCACTGAGCAGGAGCGGCCGTCTGGAGTGGAGGGAAGCTCGTGGGATCTC

[0167] AGAGGCCCTGGACTCCTCCCAGCTCTGACACTTTGTAGCTGGTGGCTTGGGCGAGCTGCTCCAG

[0168] CAACCCCTGAGCCTGTTTTTCTCAGACTTGTGAGCAATAATACCAGCTAACATTCTTTGGTCTG

[0169] CGAGGTAGCTCACGCCTGTAATCCCAACACTTTGGGAGATGGAAGGATCGCTTGAGCCCAGGAG

[0170] TTCAAGACCAGCCTGGTCAACATGGCAAAACCCCATCCCTACAAAAATACAAAAATTAGCCGGG

[0171] TGTGGTGGTGCACACCTGTAGTCCCAGCTACTTGGGAGGCTGAGGTGGGAGGATCACTAGAGGC

[0172] CATGAGGCAGAGGTTGCAGTGAGTTGACATCACACCACTGCACTCCAGCCTGGGCGACAGAGTG

[0173] AGACCCTGTCTCAAAACAAACAAACAAAGCAAAACCAAAAAAACAAAAAAACCATTCCTTGGCC

[0174] ACCTCCTCCGTGCCAGGTATTTAAGTATTTCATGTGGGTTCTCTCTTTAAACCTCACAGTAGCC

[0175] CTGGGAGATGGACACTGTCATGATCCCCATGTTTAATAGTCTGAGAGACTAAGGCACAGGCAGG

[0176] CTCCCCAGTTTCCCAAGCTAGAAAGGTGCGGAGCCAGGGCTAGAACTGGGGCCATCTGGCTTCA

[0177] GGGTCCTGCACCAGCTCACAGGTTTCAGTAGAGAAAAGAAATGCACAGTGACGAATATGCAGTA

[0178] GATTTCCAATAGACAGCAGCTATAACTTTTTTTTTTTTTTTGAGACAGGGTCTTGTTCTGTCAC

[0179] CCAGACTGGAGTGCAGTGGTGCAGTCACAGCTCACTGCAGCCTCAACTTCCCTGGCTCAATTGA TCCTCCTGCCTCAGCCTCCTGAGTAAGTGGGACCACAGGCATGTGCTACCACGCCCAGCTGATT

[0180] TTTCTATTTTTGTAGAGATGCAGTCTCACTACGTTGCCTAGGCTGGTCTCAAATTCCTGGGCTC

[0181] AAACGATCCTCTTACCTTGGCCTCCCAAAGTGCTGGAATTACAGGTGTGAACCACCGCACCCAG

[0182] CCAGGAGCTATAACTATCATTACTAAATCTGAAACTTTGCTTAGGTGCTTTCAGATTTCTATTC

[0183] CATTCTTTGTTGTTGTTGTTGTTGAGATGGAGTCTTCCTCTGTCACCTAGACTGGAGTGCAGTG

[0184] ACAT7XATCTCAGCTCACTGCAGCTTCTGCCTCCCTGGTTC7XAGC7XATTCCCCTGCCTCAGCCTT

[0185] CCCAGGAGCTGGGATTACAGGCACCTACCACCATGCCGGGCTGATTTTGTATTTTTAGTAGAGA

[0186] CGGGGTTTTGCCTTGTTAGCTAGGCTGGTCTCGAACTCCTGACCTCAAGAGATCTGCCCGCCTC

[0187] AGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACTGTGCCCGGCCTCCTATTCCATTCTTG

[0188] CTATCATTTTTGCCTAGTGCAGTCAGTGATTATTTATTTATTTATTTGGTTAAAGATGAGGGGC

[0189] TGAAACATTCTAATGAATTATAAAACGCTTCTCTCGCTGCCCCACCACCACCCTAACCTCTGTG

[0190] CCCATTTTCTTCCAACTTCTTGTGACGTTTCTTGAACTTAGGATCTTGGACACCTACTTATTAC

[0191] CCAGATTGCCTGGTGTAAGTCAGTCATTGATGGGAAAGGCAAGAGAGACATGCTGGCTGTTTCG

[0192] GGTGCATAAGCTTCAAAGCGCTGTCACTAGCCTGCTGTTTTTCAGGGGTGTATTGTGTAGGTTG

[0193] GTTCCGTGCTGGCCCGGTGACCTGTGATCACTGCTAGAGGAGGAAGGTTGGATGAAAAATGCTA

[0194] ATGAGCTGGAGTGGATGTGTATCAATGTCTCTCAGTGGCAGGCAGTTCTGTCATGAAAGCAGAT

[0195] GGGCAGATAAACAGGTCGTCCCCTTGTGGCCTTGCCTGTACTTCTTGTTTTGCATACACTTCTG

[0196] CGCATTTGCACAGCTACCTCTTTGTCCTTTTTTGTGGCCAGAGTGAGAGTTCTCTCATAATGAC

[0197] ATGTTAGCAAAGCTTGAGGTGCACTTTTAATTTTTTGCACAGAAATCTTTTTCTCCATCAGGGT

[0198] AAAAGAATTCCCCCATTACCTCTAGGCTGTGAATGCAAAAATGGACCAAGTATTGAGAAATCAA

[0199] AGGGCAGGGGGAATCAAATCCAAGCAGGAAATAGAGCCAACTAGTTCTATTTGGTAGAGTTATG

[0200] AGAGTGTTTAATTTTCTGCCTTATCCCTTAAAATTTTTACAAATTACCTATAATGGGCAAATGT

[0201] TACTCAT7XAGTCAGG7VXAT7WXACTTTTTATT7VWXAGCC7VXAGGGCTGT7WXATGCTGAGTAG

[0202] TAGTATTATAATTAATAACATTATTACCAACTCCATTTGACAAATGAGAAAGCAAAGGCTCAGA

[0203] GAGGTGAAGTCCCTTCCCCAGATCATACAGCTAGGTGGTGGGGGCATCATCATTATTATTGGTT

[0204] TTTGTTTTTTTTGTTTTTTTGTTTTTTTTTTTGAGATGGAGTCTTGCTCTGTCACCCAGGCTGG

[0205] AGTGCTCGGCTCACTGCAACCTCTGCCTCCCAGGTTCAAGCAATTCTCCTGTCTCAGCCTCCAG

[0206] AGTAGCTGGGCTGGGATTACAGGGGTGCACCACCATGCCCAGCTAATTTTGGCATTTTTAGTAG

[0207] AGACGGGGTTTCATCATGTTGGCCAAGCTGGTCTCAAACTCCTAACCTCAGGTGATCCGCCTGC

[0208] TTCGGCTTCCCAAAGTGCTGGGATTACAGGCATGAGCTGCCTTGGGAATCATTATTATTAATAT

[0209] TTATCTGTGATATCAGTAAAATAATTGAATAAGCAAGGCTTACTAATAACCCTGCATATTCCTG

[0210] TTGCTCCCTTCTTCTTGGGTTTCTAGATTGCGTTTTGGAAAGTTTTACCTCATTGACTTACAAA

[0211] TACAG7VXAT7XAGTGATTCTTCAG7XATAGGGATTTGGATCAGGAGCAGCAGGGAGTGCTCATT7XA

[0212] AAGGGCAGATTCCAGACTCTCACCCCTGCAGATTCTCATTCAGTAGGTCTGAGGTGGGTTGTGA

[0213] GCATCTGTAGTTTAAAGGCATCCTGGGTGATTTTGAGGACTTCTGGTTGAGAACTGCTTTAATG CATCATTCAGCCTGCTAGCCAGTCAGTCAGCAAATGCCAGGCATCATCTCTGTACCACTACCCT

[0214] GCCCTGAGAGGGCAGACAGGGCAAGACCCACCCCCGCCCTCCGCTGGCTACACTTTGTTACTGG

[0215] TGACTATTACTCACCCCTTTTGAAATAGGCGTCATTTGTCCCTTAGCCTTTCAAAATGAATCCT

[0216] TCTATACTGGGAAGGTTATAGACTTGCCAGATCTCTGAGCAGGAAAAGATCCAAGAAATCAAAT

[0217] CCAGCCACACTGCCAGTCCAGCCCCTCCTACAGCTGGGGCAATGCCCCCCCCGCCCCCCCCCCC

[0218] CCGCCGCAAAAAAAAAAAAAGCAGGGGAAAGTGGGTCTCCTCACTCTCAAACCAGTGCTCTCCC

[0219] TATGACACCAAAGAGCCTGTTTCTGTTTCTTGCTTCATTCATTACTCCGGTTCCCTTGACTGAG

[0220] TTCCTCTCAGTCCCAGAAGACCTAGAAAAGGGTCGTGGTTGGTCATGGGTTTCGAGTAAACAGG

[0221] CCTGGGTTTCAATCCCGATTCCCCCACCATCTAGCTATGTGACCTGGGGAAGTGACTTTACCTC

[0222] TCTGAACACTTTCTCATTTGTCAAATGGAGTTGGTAATGTTATTATCTGCCTCCCTGGGCTATC

[0223] CTGAAACTAAAATGCGATACACTGGTTGTTACTTTGGTGACTGTTAACACCCCAGCTACCCCCA

[0224] CCCCCTGCCTGGCTCCGTTAATCACCAGCTAGTGTGGATGATGAGGGAGAGGTGAGCCCTGTAG

[0225] CATAAGGGCAGTGCCTCTCCGATGTCTGTGTCTCATAGGGTCAACTCCCGCGGCCAGCTGCGAG

[0226] GAGGAGGAGTTGCCCCCTGACCCTAGCGAGGAGACGCTCACCATAGAAGCCCGATTCCAGCCTC

[0227] TGCTCCCGGAGACCATGACCAAGAGCAAAGATGGCTTCCTAGGGGTGAGTTGGGGACCACAGGC

[0228] AGTGGGGTCATCTGTGTGTATCCCGAAGATGAGTTTCTGCTCCATTCATCCATCTTTCCTCTGC

[0229] CCTCTGTGTGCCCACTAGAGCCAGGCCCTGGAGGTCCATGTGCGGTGATGTTGTCCCTGCTGTC

[0230] CAGGCATACAGCCTGCTGGGGGAGCAGGAGAGAGAAGCATTAGCTTCTGCCCCAGACTGGGGAA

[0231] TCTGGGATAGCTTCCTAGAGGAAGTGACATCTGGTAGACTGCAGGATGAGGAGGGCTCAGCAGG

[0232] GTGACTGAGCACAGGTTGGGGGGGTCTCGGAGGAAGCAGCAGTGCAGTAGAGACCCTGTTAGTG

[0233] CAGCCGCCCCACATACTATGATGATTATTTCCCACACACTATGATGATTACTTCCCACACATCC

[0234] ATCTCTTCCCCACGGGGCATAGCATGATGCCTGGCTCCTGATAGGTACTCTGTTGAACTCTGAT

[0235] GATCTAGACCTGAGGTTTGGTCCCCAGCAGAGGGCTGTGGGGTGGGGAGGGCACCTGAGAAGGT

[0236] GGAGGTAGGAGGTGGACAGATGCTCTCCTTGCCAGCCCCTCCCCAACCCAAGGCCCTTTGGACA

[0237] GGGTCCTGAGCGCATTGCAGGATGAAGCTGCCTCAGCCCCTGCCTGCCCTTTTGCACTCTCTCC

[0238] CACTCTCTCCTCCCCTCTGTGGGCTTCCTCTGTTCCAACCACACCAGACTTCCTTCCTTCCAAA

[0239] TCCCCAGGCTTTCTTCCAACTGGGAGCCTTCTCACATGTGGTTCCCTCTGCCAGGAACACTGCC

[0240] TCTCTGTGCCCAGCCCACACAGCCCGACCCAGCCTTTTCATCTCAGCCAATGCCACTTCAGCAC

[0241] GAACAGAGGCCTGAAGGCAGGAGTGCCAGGCTGGTCACTGAGGCCAGGCAGTGGGTGTGTCTGG

[0242] GCCCAGGGGGCATGCAGGACCCACACAGGGGCATCCCAATCAGTGGGGCCAGGAGAGAAGGTGC

[0243] CAGACCCCACCAAGGGCTTTGTTGCCTGCCTTTGGCCTTCATTCTATGGTGGCTGAATATCGTG

[0244] CAGATTTTAAATAAGACAGCTTTCTCATCAGACCTGGGCTCTCCAGGCCAGGGACGGGGCATGT

[0245] GTAG7XATGGGGAG7XAGTGGGAGGC7XAG7XAGACCAGTTCTGGAGGGGAGACGGAGGAGATGGAGA

[0246] GTAAGGGCAGCCTGGAGGATGCTGGGGAAGGAAGGGCAGGGAGCGGCTCCTCATGGACAGTGGG

[0247] TGGGCAGGAGTTGGATGACTCAGGCTTCTGGCTGTGACCAGGGGCATGGTGGGCTGCTTGCCCA GATGGGGACTGTAGGCGGGGAGGGAGTAGGGGATAGAGAATGAGGTCAGTCTGCACATGTTGAA

[0248] GTTGAGGTCCCCAGGGGATGTTGGATGTCTGCTGCCCAGCAGCAGCCCCTTGGCAGTGCCCAAC

[0249] TCTAGAGCCTGGCAGCCACATGGCAAAAGAAACTTGGGAATGACCTTGAGGCTGGCACCTGTGG

[0250] CGGAATTGCCACCTGGCCCAGCCCAGGGAAGCAGTGCTGGGGAGCCCCTTTCTTTTTTTTTTTT

[0251] TTTTTTTTGGGACGGAGTCTCGCTCTGTCGCCCAGGCTGGAGTGCAGTGGCGCGATCTCTGCTC

[0252] ACTGCAAGCTCCGCCTCCCGGGTTCAAGCTATTCTCCTGCTTCAGCCTCCCGAGTAGCTGGGAC

[0253] TACAGGTGCCCACCACCACACCCGGCTAATTTCTTTGTATTTTTAGTAGAGACGGGGTTTCACC

[0254] ATGTTGACCAGGATGGTTTCAATCTCCTGACCTCGTGATCCACCTGCCTCGGCCTCCCGAATTA

[0255] TAGGCGTACAGGGATTATAGGGATTACAGGTGTGAGCCACCATGCCCGGCCGGGAGCCCCTTTC

[0256] TTAACCTCATCGCTGCAGGTTCAGGGAGGGACAGAAATTCCAAGGAGGAGCTCACAGGGAAGGG

[0257] CATTGCCCGTTTTGGGTCCTGTTTACTTGCCGTCTCCCACTATAAGCTCCAAGAAGACAGGGTG

[0258] CACAGTGTACCCTGATAGAAACTTCCTATACAGTAGGTGCATAAAACATTTTGTGGAATGAACA

[0259] AATGAATAAACGTGTTCTTCTCAGCCAAGGAGACAGTGCAGCAGATAGGCAGGAGTCCAGGAAG

[0260] TGGTCAAAACCGGTCTGACCTTCCACAAAAGGAGATGGGGAACCCATCTCCATGGATTTGGCAG

[0261] TGATGGGCTGGTCTGAGCAGATAAGTCCGCACAGACAACAAGAACTGGGGTGAGGCAGGAGCAG

[0262] CACCCCTAGCCCTGGCCCCCGAGCAGAGCCCCAGCAAGCCCAGCACCCATCGCTGGCAGGTCCC

[0263] CTGCAGGGGGTGGCAAGCCTACAAGCAGGACCCAGGAAGGCGGGCTCGGCCTGGCCCGGCACAG

[0264] CCAGCTCTGCCTTGAGAACCGCCTGTTAAATCTGTCTCTGTGGGAGCTCCCTGCCTGCTTTCCT

[0265] CCTACCTCCCACCAAGCCTGAGCTCCTGCTGCCAGGGAGGTGGCGACAGAAGAGGGAATCTGCT

[0266] GTTCCCCAAATGCTGGCCGACGTGTAGACAGCTCTGTGGTCTCTGAGCGTCCTCTCCGAGCCAT

[0267] CAGCTTGTCAGACAGAGGCGAGGCAGGTCACCGATGAGGACTCGGGCTTAGGGAAGGCCTTTTG

[0268] CCCAGGGTGGGGCAGGCTGTACTTAAGGCAGGGCCCTTCTGGGGGTGGCCTTCCTTCCCCTTCC

[0269] CTGTGGTCTCATCCTGCTCTTGACCCTAGAGATTTCACCTCAAACATGATTGGCCACCACCCTC

[0270] ATCGGACTTAGCTCCAAACCGAAACCCTGAGCTGGCCAGTGGGACATTAGTCCCTGGGAGGAGA

[0271] CTTGGAGAGGAGACTGGGGAGGGAGAGTAGGGCTGCTGGAGGGCCAGGGAAGCCAGGCAGGGTG

[0272] GTGGCACCCAAGGCTGGCCCATGGGCTTATGCATAGGATGGAGGTGGGACTATGTGAGGCACAG

[0273] AGCCCCACCATCCCCATTCCAACCTTGCCCTCGAGCGAGCTGCTCTGGCCTGGGCTGCGCCTGG

[0274] GTGGGCTGTGCCTGGGTGGGCTGTGCCTGGGTGGGCTGTGCCTGGGTGGGCTGTGCCTGGGTGG

[0275] GCTGCAGAGGCACTGCAGCAGGGAGAGGAGCCTCTCTCTGGCGGGATATTGCTAAGCTTGTGGC

[0276] CATCATAAGGGCAGGAACCTCCCTGGGGTCCATCTGCTCCCTTGGTGTGGGCTGGCTCGGGGCT

[0277] TGAGGGAGACCTCCACCCACATGGTACAGGAGACCCCGGAGTCAGGTTCTCAGATTCCTGGAGC

[0278] TTTGCTTTCCCCCGCCCCAGGTCTCCCGCCTCGCCCTGTCCGGCCTCCGAAACTGGACAGCCGC

[0279] CGCCTCACCAAGTGCAGTGTTTGCCACCCGCCACTTCCAGCCCTTCCTTCCCCCGCCAGGCCAG

[0280] GAGCTGGGTGAGCCCTGGTGGATCATCCCCAGTGAGCTGAGCATGTTCACTGGCTACCTGTCCA

[0281] ACAACCGCTTCTATCCACCGCCGCCCAAGGGCAAGGAGGTGAGGACAGCTGGGGTGCGACGTGG GGCCCCTCCGCCCGAGCCCAGGAGTGGCCACCCCTCTGTCTGCGCCTGGACCCCAGTGCCAGGC

[0282] CTGCTCCACCTGCTCTCCTGCCTTCCTCTGGACCTGCCCCCTCCCCATGGGGCCCCTGGGCCGT

[0283] CGGGTCTGGGCTGACCCCCACCCCAGCGGATCCAGGCCCAGCGGGACCCAGCAAGCCAGTACGT

[0284] GCCTCCCGCCGCCCCCAGGTCATCATCCACCGGCTCCTGAGCATGTTCCACCCTCGGCCCTTTG

[0285] TGAAGACCCGCTTTGCCCCTCAGGGAGCTGTGGCCTGCCTGACTGCCATCAGCGACTTCTACTA

[0286] CACTGTGATGTTCCGGTGAGTGGGCCACACTGGCTGGCCTGGAGCACCGGGGAGGCATGACGGT

[0287] ACAGCGCCCAGAGGGGAGGGCCCAGCTTGAGCCCCCCAGCTCCACCTCTCCTCCCACTGCCGTC

[0288] TTGGGCAAGCAGCTTTGGCTCTCTGAGCCTCAGTTTTCTCACCTGTGGATCCGGGTGCTGATGA

[0289] CTTCCTCGTAAGGCACGTGAGGGTTCAGAAAGAAAAGTTGTGGAACTTGCTTGGCACGGGGCTG

[0290] GTAGCCATGGTTGTGCCATCCACACAGAAGTGGGAAGGACACCCAGTCACACATGCCCTCCAGA

[0291] AGCTGTGAAGGCTGGGGGAGGGGGTCCAGGCAGGGCTTCCAGAGGAGGGGCATGTGACCCGGGC

[0292] CTCGAAGGATGAGCAGCATTTGGAGAGCCTCGCTGCTGCCCACTGGCCCATCCACCTCCACCCC

[0293] CACACTCAGCCTCCTCTCCCTGATGATTCTGGCCAAGGCTTCCCGGGCTCCTGGGGAGAAGGTG

[0294] GGCAGCTCTGGTGCAGCAGATCCCCTTCCCCACAGGATCCATGCCGAGTTCCAGCTCAGTGAGC

[0295] CGCCCGACTTCCCCTTTTGGTTCTCCCCTGCTCAGTTCACCGGCCACATCATCCTCTCCAAAGA

[0296] CGCCACCCACGTCCGCGACTTCCGGCTCTTCGTGCCCAACCACAGGTGGGAGCTTGACCCTGGC

[0297] CCAGCCTTGGCTCCCTCCTACAGCTTGTCCTGCTCCCCAGCTCCAGGAGCCTAGGGGCCTCTTC

[0298] TGTCTCTGCCCCTTCCTTTGCTCCCCAGAGCCCATCTCATGGGGCTGACGTGGCAGGAGGCGGC

[0299] ATGAGGCAGGCAAGAGAATGAGCTGATCACCCACCAGCTCTTCCCAGGCACCCGTGGCCTGGCC

[0300] CTCCACCTCTGTGCCTGGGACCCAGTGCAGCAGTCCGCAGACCCTTCCTGAAGCTGGGCCTCCC

[0301] CGCCCTCATACTGCTGACAGGCCCCAGGCAGAGCTGTAACCGCAGCCTGTGCACGCCACCAAGA

[0302] GGAAGGCCAGCAGCTAGGCAGGGACCCAGCTGTGGGGTCCAGGAAGGCTTCCCAGAAGAAAAAG

[0303] GTCATCTCAGCAGAGATCAGGGCTCTGAGGGTTTTGCTGGGCAGAGGAACAGCACATCCAAAGG

[0304] CCAAGGGCAGGCCCTCCACTGCTGCAGAGCCCCAGAGGGGCTTCCGGGACGGTGCTACACCAGC

[0305] CACCTGCTTGCCTCACCTCCTCAGGGCTGGCCACCTGTCAGCAGAGTAGAGGACCCCGGAGGCC

[0306] CTGGCTTCAAGTTTGTCCAGATTCCTAACTGATAGCAAGTGGCCACCGAGTGGGCCTGAGTGAC

[0307] CGCTCTACAGGGATGAGACCTGGCTGGGAGCCAGGAGGTCTGCAGTTAAGTCTCTGCTGTGGAA

[0308] CCCATTGGCTGTGTGGCTGTGGGCAAGGCCCTGGCCCTCTGTAGGCCTGTTTCCTCATCTGCCG

[0309] GATGGGAGACTGACTCCTGGCCTACTTCACCCACAGGGCAGGCGTGAGAGGATGGTCGGGATAA

[0310] CATGGGAAGAGAAAATTGCCCCTAGGAGTGGGGGTGGCTTCCCTCCCTCCCCAGAGTGGGAAGG

[0311] GCAGGGGCTTCCCGGTCAAAGGGAAAAGGAAGAGGAGGGCACAAGCCCTCCATCTGCTCACCTA

[0312] GGGCTGGGCACACCTGCCTCCATCGCAGGGTTGTTGAGGAGGGGGCCTGGACTGTATGGGACAG

[0313] GAGGAGAGAGGCAGTGGCCAGCTCCAGGACCAGCTGTTCCTGGGCTCATGTCTGAGGACCAGGC

[0314] ACCAGGGACTGCTGACCCCCGGGGGTTACCTAGCTCTGCCCTCTGTGCCCCCTGCTGCGGAGGT

[0315] GGTGGTTTGCCAGTGCTGGGGCTCCCTCGTGCGGCGGATCTGCAGATTGCACTGCTCGGCTCAG ATTTCCTGCTGCCCGCCAGAGGCCTGTGCAGGGACCACGGTGCTCGTTTTCATTCCTGCCCCAC

[0316] CCTGTGACCCGCCTTGAGGCACTGGCCATCTCTCTGCTTCAAAGGGTCCTGGAGAGTACACGGG

[0317] CCATGGCTGTGCAGGCCCTTTGTGAATTGCGAGGGGCAGGGCTGGCGGATGGTGAAGTGGCTCC

[0318] AGTGCCTTAGGAAGGGGCAGCCTGGTGGGGAAGAGGGCCAGGGCTCTGCAGCCAGAATCCCAGT

[0319] GGCTCTGAGAGCAGGTGTTCACCTTGAGAAACCTCAGTGTCCTCATCTGGAAAGTGGAGACAGT

[0320] TGCAGGTATTATGTGAGAT7VXAGTGTGACACAGAT7XATGGGCTTTGATGATGGTGTCACTCTGC

[0321] CCTGGCCATCCCAGGTCTCTGAATGTGGACATGGAGTGGCTTTACGGGGCCAGTGAAAGCAGCA

[0322] ACATGGAGGTGGACATCGGCTACATACCCCAGGTGAGCGCACAGGAGGCTCCCATCCAGGTGGG

[0323] CTCGGCTGCAGGGCCCCGCCCTCCCTCTGCAATGAGTGGAGCATTTTGGAGGGTCTCTAGGGGA

[0324] GCCCCTGAGGATTCTTGCCCATCTCTGAGCCTTCCCCCTACCACTGACCTCTGGCCCAGATGGA

[0325] GCTGGAGGCCACGGGCCCCTCTGTGCCCTCCGTGATCCTGGATGAGGATGGCAGCATGATCGAC

[0326] AGCCACCTGCCTTCAGGGGAGCCCCTGCAGTTTGTGTTTGAGGAGATCAAGTGGCAGCAGGAGC

[0327] TGAGCTGGGAGGAGGCTGCCCGGCGCCTGGAGGTGGCCATGTACCCCTTCAAGAAGGTGAGGCT

[0328] GGGCAGGGGTGAAGGCCAGGGTCAGGCTGCATGGGCAGAGGCTGGGAGCTGTGGAGCAGCAGCT

[0329] GGGCACAGACGCTGGTATGTGTGCAGGGCTTGCTACTGAGGCTGTCTCACTGTTGGGCCCAGCT

[0330] GTGCCAGCGGGGCCTCCCCGCTGCCATTCAGAACTTGGAGGAAGAGGCCAGGTGCAATGGCTCA

[0331] TGCCTATAATCCAAAACTTTGGGAGGCTGAGGCAGAGAATCACTTGTGGCCAGGAGTTCAAGAG

[0332] CAATCTGGGCAACATAGCGAGACCCCATATCTACAAAAGAATTTTTTTAATTACCTGGGGGTGG

[0333] TGGTAGCACGCACCTGTAGTACCAAAGCTTTGGGGAGGCTGAGGCAGGAGGACTGTTTGAGCCC

[0334] AGGAGTTCAAGGCTGCAATGAGCTATGATTGCGCCACTGCACTCCAGCCTGGGCAACAGAGTGA

[0335] GACTCCATCTCAAAATAAAAAATAAATGAAAATAATAATAATAAATAGAACTTGGAGGAGACAT

[0336] CCATCCCCTGGCTTAGTAGCACCCACTTCACACACATACAAACACACACACTAACATATATATA

[0337] CACACACACATACACACATATACAAACATACACAAACATATATACACATACAAATGTACATATA

[0338] CACAGACATACACACAAATATATATGCCTACACACAAACACACACACACACACACACACACACA

[0339] CACACACACACACACTTGCACACACTACAGACTCAGCCCGAGTGGGACCCTGGCCGCTTTGATG

[0340] ATGGCTTCGCTCTGTCTCGGTGTGGCCCCAGGTCTCCTACTTGCCGTTCACTGAGGCCTTCGAC

[0341] CGAGCCAAGGCTGAGAACAAGCTGGTGCACTCAATCCTGCTGTGGGGGGCCCTGGATGACCAGT

[0342] CCTGCTGAGGTGAGGGGCCCGGCTGGATCTAAGGGGAGCAGTGGGAAAGTCCACACCTTGTGGG

[0343] GTACCAGAGGCTCTGAGACCAATGGGACTCTTCTGTTGAGTTGTGAGGGCCTCAGGGACTGCCC

[0344] ATGGTAGGGGCGTGGGGTGAGGGCTTGGGGGTTTCTGTGCTGAGCCCAGAGGGAGCTACCAGAT

[0345] GCTGAAGAAAGGGCCCTGGCAGACTGGGTTCAAACTCAGCCATTGTCAGCTTGGTAACCTTGAC

[0346] CAAGTGTCTTCCCCTCTGTGAGCCTCAGTTTTCTCAATAGTAAGAGGGGATAACACACTTACCT

[0347] CTCATAGCTGTGGACATGGAGGTG7VXAGTGCCGCATACACTGT7VXAGTGTTATATACGTGT7XAG

[0348] AGAAAAAATCGGGCCAGAGGCTGGGCTTGTGTTAATTGATTCAGGAAATTCACCAGAGGCCCCC

[0349] TAGATGCAACGTCCTTTGGGTGTCTGGCAGTGGGCACAAAGATGAACAAAACAGTGCCCCACCC TCACCCCGTCAACCGTCAGTGCAGCAGTGGGCTGGGTGCTTGCGTCCCACAGTGAGGAAGGCAG

[0350] AAGGGGTCCCTGCCCTCAAAGGGGGAGACACAGTGCAAAGGCAGACACCAAATGAGTCAGTACA

[0351] AGCACGGTGAGTGTTCTACAGGGGACAGTCCAGAGTGGCACCAGAGTGTATAACTGGGGGTCAG

[0352] GGAAGGCTTTGTGAAGACAGTGATATTTACGCTGAGACTTTGAAGGATGAGTAGGAGTTTCTCA

[0353] AGTGACCGGATGGAGTGTTCCAGGTAGAGGAACAGCCTGTGTGAAGGCCCTGGGACACAGAGCG

[0354] TTCATTGGATTT7XAG7XAGCTGTCACTGTCTGC7XAGTCCCCAGGGCCAGGTAG7XACCCCT7XACCC

[0355] TAATCTCAGCCCAGGATGCTGGTATATTATTGATGTTAATTCCAACTATCATTTATTCAGCACC

[0356] TACTCTACCTCAGGCTCTTTGAATCTGTTATTTCACTTGGGTGCAGAGACTGTCCTGCAGCCCG

[0357] TGAGGTCTCCCCAAAGCAAGATTGCACCCCAGCAAGATGTGGGGGCGCCTCACCCTTCTGTCTT

[0358] CCTGAACAGGTTCAGGGCGGACTCTCCGGGAGACTGTCCTGGAAAGTTCGCCCATCCTCACCCT

[0359] GCTCAACGAGAGCTTCATCAGCACCTGGTCCCTGGTGAAGGAGCTGGAGGAACTGCAGGTGAGC

[0360] GGGCAGGTGGCAGGAACAGGAGCGTCCGGAACAGTGGTGGGGGCCGCGGCATCAGGAGTGTGCA

[0361] ACTGTCCCCACAGAACAACCAGGAGAACTCGTCCCACCAGAAGCTGGCTGGCCTGCACCTGGAG

[0362] AAGTACAGCTTCCCCGTGGAGATGATGATCTGCCTGCCCAATGGCACCGTGGTAGGCACCCCCA

[0363] CTCAGACCCCACAGGGCCCAGGCACCTCGGGGCCCCGGGAGCAACCAGCAGGAGGCGTGGATGT

[0364] GCAGACTTCATCAGGCTTCGGGACTGTCCCTGCCACTTCCTGGCTGC7VXAGCCTC7XAGCTGGTC

[0365] TCTTACCCAGTCTAAGCCTCCGTTGTCCCCGTGTAGAAACAGGAATCGTGATGCAGGTGTTGGT

[0366] CATCGGGGGCTGCGCAGCAGTGAGCATAGTCCTGAGGCAGAGTCAGCGCCTCCCTAGAAACACA

[0367] GCCTGGAAGGGAAGGCTCACCTTGGACCATGGCGGCCAGTCCTGGGATGGAAGGGGAGGGAGGA

[0368] GCCAGGCCTTGTGACAGAGACCAGGAGAGGGCCCTGAGCCAAGATTCAAGACCCCACCTCTCCC

[0369] T7XAGGGCCCCTTACCTAGCACTCAGC7XAGTCTTCTCACTGGGGCCTCACAGCAGCTTTGGGAGA

[0370] TGGATGGTCATGAATACCCATTTTATAAACAAGGAGGCCCAGAAAGTTGAGAGACTGGACAAAT

[0371] GTTGCAGTC7XATT7XAGTGCAGCTGGCTTTG7XACCGAGGCCTGCCCTTGAGCCTCAGATCTCCAG

[0372] CTCTTCTGATCAAAGCGACCCCTCATCCTTTAAGTCCCAAGGCATGGTTAGTGTGGGACCCAGG

[0373] GCTGTAGGAGGGGGCCGGCTTCCTCAGGATCTTAGGATGGGAGCAGTTCAGGAGCAGGGGCTGT

[0374] CACGGGAGAGGTGTCCAGTGAGGGTTTGGTGT7VXAGG7VXAGGGCACCAGGCGG7XAGGCATCTGC

[0375] TGTCCCAGGCGCAAGGGTGATGAGATGGTGTCGGGGAACTGGATGAGGCTGGGCCTGGTTGGGA

[0376] G7XACCAGGCCTCCCATTCATATG7XATGTTGATTG7XATGCTGATTCATTCATTTGTTCATCTACT

[0377] GGGCACCTACTGTGTGCCAGGCACAAAGATAAAGACACAGCCCCGTGCTGAAGGAGCTCAGTCT

[0378] CTGGAGCGAGAGGCCAAAGGAGAGGCCCATTGAATGTGAGGCTGCAGTGAGAGAAGGGAGCACG

[0379] GAGTTCAGGATGGGAGTCCGGGGTCACAGGAACAGAGGAGAGGGGTCATTTCGGGGGGCGGGGG

[0380] CAGAGGGCTAGTAGAAGAGGTGATGTTGGAAGGTGTTGGGTTTTCACTTGGTAGGCAAGGCAGG

[0381] GAGGAGCCCCCTCGTCAGACACAGGGTGATGAGATGGTGTCGGGTGCTGGCGAGGGCTTACGGC

[0382] AGCACTGCCTGCCCACCATCCACCTCAGACAAGGACAGTCAAGGCCTGATAGCATCCCTGCTTC

[0383] TCCCCATAGAAGAGAGGGCACAGGGAGCCTGGGGGCCCCCCTCCGCCCCACTTGCCTCACCCGG CCCTTCTCCCAGGTCCATCACATCAATGCCAACTACTTCTTGGACATCACCTCCGTGAAGCCCG

[0384] AGGAAATCGAGAGCAATCTCTTCAGCTTCTCATCCACCTTTGAAGACCCGTCCACGGCCACCTA

[0385] CATGCAGTTCCTGAAGGAGGGACTCCGGCGTGGCCTGCCCCTCCTCCAGCCCTAGAGTGCCTGG

[0386] ACGGGATCTGATGCACAGGCCCCCACGCCTCAGAGCCAGAGTGGTCCTCAGCCCATTTCAGACT

[0387] GCAGATGCCGCCCACTCCCACCCCACTCCTAGGCTGCCTTGGAGGGTACAAGATCCACTGAGGG

[0388] TGGCCACCACAGCCTTGGCTCCATGGTGGCGGGTAGACAAGGGATGCCTGGGCTGACTGGGCAG

[0389] AGGAACCTCTAGCTCTGACTGTCACTCGGCTCTCCCTACCCATTTGGCTCTGGAAGCTGCTTGG

[0390] CCCCCCCAGATCAGGGCCTGGGTGAACTCCCTGGACCTTTCCTAGCCAGCCGCACAGTCTAGGC

[0391] CCTTGTGGGGTGAAGAATGGAGGGAGGAGCAGGCTAGGAAGACGGGGCCACCACCCTCTCCTTG

[0392] CTTTCAGCCCTTCCCACAGGAAACATCAAGAAGCCCCAGCCAGGAGGGGCCAGGCTGCCAAGGC

[0393] GGCTCCCCTGTTTATCTAGAGCCTTCGTTCCTGGCCATACCCCGGACTGCCCTCCTGTGCCTGA

[0394] TGTCCCCAGCTGGGGTCAGTCTCAACAGGAGCCAGTCTTCTGGAGCCTCTGGGCAGAACCCTCC

[0395] ATCAGAGTGGAAATCAGACGGGACCCCCTGCAGCTTCCCTGACCACGCCACTGACCAGCTATCT

[0396] GGGGAAGTTTACTGTGAAGGGGTTTCTGCCTTTAGCAATGGGGTTCACTAAGGGGGTTCCCGAG

[0397] GCCCAGGGCCAAGGCACTCCCACCGCCTACCTTAGCACAGGGTCTCTGCAGGACTGCGGGAGCC

[0398] AGCGCTCCTGCCGCCCCTCTTGCCCCTCAGACCTTGCATCCACAGAAGCACAACCCAGCCAAAC

[0399] ACCACAGCCTTCTCCAGAGCCGGCACTGTCCCGGCAACCAGGGGTGCCCCAGGCTAGCTCTTCT

[0400] ACCTCTGGGGCACCACGGACTCCCCTTGGCCACTCTTGGGACTTTGGTCCACGTCCTGAGCCAC

[0401] TGACCACGGCCAGTCTCTCTTTTTATATGTGCAGAAAAGTGTTTTTACACAAACTTTCTCATGG

[0402] TTTGTAGGTATTTTTTTATAACCCCAGTGCTGAGGAGAAAGGAGGGGCAGTGGCTTCCCCGGCA

[0403] GCAGCCCCATGATGGCTGAATCCGAAATCCTCGATGGGTCCAGCTTGATGTCTTTGCAGCTGCA

[0404] CCTATGGGAAGAAGTAGTCCTCTCTTCCTTCTCCTCTTCAGCTTTTTAAAAACAGTCCTCAGAG

[0405] GATCCATGATCCCCAGCACTGTCCCATCCTCCACAAAGGCCCACAGGCATGCCTGTACTCTCTT

[0406] TCATTAAGGTCTTGAAGTCAGGCTGCCCCCTCCCCAGCCCCCAGTTCTCTCCCCACCCCCTCAC

[0407] CCCACCCGGGGCTCACTCAGCCTGGCAGAGGAAGAAGGAAGGCAGACATCTCCGCAGCCACTCC

[0408] TGGGCCTTTTATGTGCCGAGTTACCCCACTTGCCTTGGGCGTGTCCACTGAGCCTTCCCCAGCC

[0409] AGTCTTGTTCTCAATTTTGTTTTGTTTTGTTTTGAGACGGAGTCTTGCTCTGTCACCCAGGCTG

[0410] GAGTGCTATGGCTCGATCTTGGCTCACTGCAACCTCCACCTCCCAGGTTCAAGCAATTCTCTTG

[0411] CCTCAGCCTCCCGAGTAGCTGGGATTACAGGTGCATGCCACCATGGCTGGCTAATTTTTGTATT

[0412] TTTAGTAGAGATGGGGTTTCACCATATTGGTCAGGCTGATCTGGAACTTCTGACCTCAGGTGAT

[0413] CCACCTGCCTCAGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCAATCGTGCCCAGCCTTGTT

[0414] CTTAATTTTGTATCATCCAGTCATCGCTAATATTACACGCACCTTCTCACTTAATCCTCACGAC

[0415] AAGCCTGTGAGGCAGATGCTCATTGTTCCCATCTTGATGAAACTTGAGTCTCAGGGAAGTGAAG

[0416] TGACTTGCCCAGGGTCACTCAGGTAGAGTTGAGATTCAAACCCACATGTGGCTCCAAAGTCTGC

[0417] ATCTGGATTTGGGGGTGTTTTTTGGCATGGCACCCTCACCTCTCTCCCTGCCTGTTTTCCCCAA AGTGGAAAGGAAGGCCTTTCAAACCAGAGTGTCTCACTCCCCTCTGACCTCCAGACCAGATGGG

[0418] GCATGAGCCAGCCAGCTCAGCCAGGCTCCCTGTGTCCTGGGAGGAAGTGTCCCCATCCCCCATG

[0419] CCCCTTATGGGGAGGGAGGGCGTCTGATGCTCTCTCTCTGCCTCCCCCCCCATCCTGTCAGGCA

[0420] CAGGTGACGGGGGCAGCCCATGCGAGCCCTTCTCCTGCTGCTCTGGGAGGGCCAGTTCCACATT

[0421] GAGCCAGCCTGGTCCCATGGAAAATGATGGCCTGGGCTTTCTGAGGCCTTATCTGATGCCTCTG

[0422] CAGTTCATGTCCCCCACCAGGCCTCGAGGCTCAGGGTGGGAGAGGGCCCCGGGCTGCCCTGTCA

[0423] CTCCTCTAACACTTCCCTCCCCTGTCCCCAACATGCCCTGTAATAAAATTAGAGAAGACTAAC.

[0424] In the above sequence, untranslated regions (UTRs) of exons are indicated by italicized text, translated regions of exons are indicated by bold text, Exon 3 is indicated by bold-italic text, a stop codon within Exon 3 is indicated by bold-italic-underlined text, introns are indicated by plain text, the Sec codon is indicated by bold underlined text, and a sequence encoding a SECIS element is indicated by italic, double -underlined text. In some embodiments, a SELENON polynucleotide includes Exon 3. In some embodiments, a SELENON polynucleotide does not include Exon 3. In some embodiments, the stop codon within Exon 3 is used as a Sec codon. In some embodiments, the SELENON polynucleotide comprises one or more sequences corresponding to one or more modified or engineered selenocysteine insertion (SECIS) elements.

[0425] By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof. In embodiments, the agent is a polynucleotide encoding a SELENON polypeptide or a viral particle comprising such a polynucleotide.

[0426] By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

[0427] By "alteration" is meant a change in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. The alteration can be an increase or a decrease. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels. "

[0428] By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

[0429] In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of' or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. Any embodiments specified as “comprising” a particular component s) or element(s) are also contemplated as “consisting of’ or “consisting essentially of’ the particular component(s) or element(s) in some embodiments.

[0430] The term “congenital” indicates a condition present in a subject from birth. In an embodiment, a congenital disease or disorder is caused by a pathogenic mutation in the genome of a subject or present in a cell (e.g., cell in vitro or in vivo).

[0431] By “cysteine” is meant an amino acid compound with the formula o

[0432] HS OH

[0433] NH2and / or corresponding to CAS no. 52-90-4, and pharmaceutically acceptable salts thereof.

[0434] “Detect” refers to identifying the presence, absence or amount of the analyte to be detected.

[0435] By "detectable label" is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.

[0436] By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Non-limiting examples of diseases suitable for treatment using the compositions and methods disclosed herein are selenoprotein-associated diseases and SELENON-related myopathies (e.g., Rigid Spine Muscular Dystrophy, Rigid Spine Syndrome, Multiminicore myopathy, Multiminicore Disease, Minicore Disease / Myopathy, Multicore Disease / Myopathy, Congenital Fiber Type Disproportion, Desmin-Related Myopathy with Mallory Body-like Inclusions, Sedaghatian-type spondylometaphyseal dysplasia (SSDM), Epilepsy, Dilated Cardiomyopathy, Familial Glucocorticoid Deficiency, Spastic Paraplegia). Non-limiting examples of SELENON-related myopathies include congenital fiber type disproportion, congenital (e.g., nondystrophic) myopathies, congenital muscular dystrophy, Desmin-Related Myopathy with Mallory Body-like Inclusions, minicore myopathy, multicore myopathy, multiminicore myopathy / disease (MmD), and rigid spine muscular dystrophy. Symptoms and / or pathological features associated with selenoprotein-associated diseases and SELENON-related myopathies include primary hypotonia and weakness (e.g., proximal weakness), insulin resistance, abnormal lung development, morphologic abnormalities in skeletal muscle (e.g., scoliosis or evolving scoliosis), restrictive respiratory syndrome or respiratory weakness (e.g., evolving respiratory weakness), and spinal rigidity. In embodiments the disease or disorder is a “selenoprotein-associated disease or disorder” that is associated with reduced levels of selenoprotein expression and / or with a mutation in a selenoprotein polynucleotide. In embodiments, the disease is associated with a mutation in one or more of GPX1, GPX2, GPX3, GPX4, GPX6, TXNRD1 (TRI, TRXR1), TXNRD2 (TRXR2, TR3), TXNRD3 (TGR, TRXR3, TR2), DIO1 (DI), DIO2 (D2), DIOS (D3), MSRB1 (SELR, SELX, SEPX1), SEPHS2 (SPS2) SELENOF (SEP 15), SELENOH (SETH), SELENOI (SELI, EPT1), SELENOK (SELK) , SELENOM (SEEM, SEPM), SELENON (SEPN1, SELN), SELENOO (SELO), SELENOP (SEPPI, SEP, SELF, SEPP), SELENOS (SELS, SEPS1, PIMP), SELENOT (SELT), SELENO V (SELF), SELENOW ( SELW, SEPWT).

[0437] By "effective amount" is meant the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

[0438] An "expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, containing a regulatory element(s) operably linked to a polypeptide-encoding polynucleotide sequence, where the regulatory element(s) enable transcription of the polynucleotide sequence in a host cell. Non-limiting examples of regulatory elements include inducible promoters, tissue-preferred regulatory elements, and enhancers.

[0439] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0440] By “gene” is meant a polynucleotide sequence that is transcribed as a single unit.

[0441] "Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

[0442] By “increase” is meant to alter positively by at least 5% relative to a reference. An increase may be by 5%, 10%, 25%, 30%, 50%, 75%, or even by 100%.

[0443] The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0444] By "isolated polynucleotide" is meant a nucleic acid that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

[0445] By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0446] By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a developmental state, condition, disease, or disorder. Exemplary markers include selenoproteins, such as SELENON.

[0447] As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.

[0448] By "polypeptide" or “amino acid sequence” is meant any chain of amino acids, regardless of length or post-translational modification. In various embodiments, the post-translational modification is glycosylation or phosphorylation. In various embodiments, conservative amino acid substitutions may be made to a polypeptide to provide functionally equivalent variants, or homologs of the polypeptide. In some aspects the invention embraces sequence alterations that result in conservative amino acid substitutions. In some embodiments, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the conservative amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references that compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Non-limiting examples of conservative substitutions of amino acids include substitutions made among amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. In various embodiments, conservative amino acid substitutions can be made to the amino acid sequence of the proteins and polypeptides disclosed herein.

[0449] By “reduce” is meant to alter negatively by at least 5% relative to a reference. A reduction may be by 5%, 10%, 25%, 30%, 50%, 75%, or even by 100%.

[0450] By “reference” is meant a standard or control condition. In embodiments, a reference is a subject or cell not containing a pathogenic mutation in a selenoprotein polynucleotide. In some embodiments, the reference is a subject or cell expressing a functional selenoprotein. In some instances, the reference is a subject or a cell from a subject that does not have a congenital myopathy.

[0451] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

[0452] By “selenocysteine insertion sequence (SECIS) element polynucleotide” is meant a polynucleotide molecule that binds to a SECIS binding protein. Representative SECIS element sequences include nucleotide sequences having at least about 85% sequence identity to a sequence listed in FIG. 3B or a fragment thereof. Further non-limiting examples of SECIS elements are described herein below. In embodiments, a SECIS element polynucleotide is an RNA molecule.

[0453] By “selenocysteine (Sec)” is meant an amino acid compound with the formula and / or corresponding to CAS no. 10236-58-5, or pharmaceutically acceptable salts thereof.

[0454] Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a doublestranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a doublestranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507). For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg / ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg / ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

[0455] For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. The terms “pathogenic mutation”, “pathogenic variant”, “disease casing mutation”, disease causing variant”, “deleterious mutation”, or “predisposing mutation” refers to a genetic alteration or mutation that is associated with a disease or disorder or that increases an individual’s susceptibility or predisposition to a certain disease or disorder. In some embodiments, the pathogenic mutation comprises at least one wild-type amino acid substituted by at least one pathogenic amino acid in a protein encoded by a gene. In some embodiments, the pathogenic mutation is in a terminating region (e.g., stop codon). In some embodiments, the pathogenic mutation is in a non-coding region (e.g., intron, promoter, etc.).

[0456] By “selenoprotein-associated disease” is meant any pathology associated with a deficiency in a selenoprotein. For example, the deficiency may be associated with a defect in the sequence or expression of a selenoprotein polypeptide. In some embodiments, a selenoprotein- associated disease is a SELENON-related myopathy. In some embodiments, the selenoprotein- associated disease is a congenital selenoprotein-associated disease.

[0457] By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0458] Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e"3and e"100indicating a closely related sequence.

[0459] By "subject" is meant an animal. The animal can be a mammal. The mammal can be a human or non-human mammal, such as a bovine, equine, canine, ovine, rodent, or feline.

[0460] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0461] As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and / or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.

[0462] As used herein, a “vector” refers to any vehicle or polynucleotide that may be used to deliver a desired polynucleotide sequence into a cell. In an embodiment the vehicle is a viral particle (e.g., an rAAV viral particle). A vector (e.g., a plasmid) can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. An insertional vector is capable of inserting itself into a host nucleic acid. A vector can also include one or more selectable marker genes and other genetic elements. An expression vector is a vector containing an expression cassette that contains the necessary regulatory sequences to allow transcription and translation of inserted polynucleotide(s) in a host cell. In some embodiments, the vector contains a SELENON polynucleotide. In some embodiments, the vector is a viral vector. Exemplary viral vectors include, but are not limited to, retroviral, including lentiviral, adenoviral, baculoviral and avian viral vectors, and including such vectors allowing for stable, single-copy genomic integrations. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is an adeno-associated virus (AAV) vector.

[0463] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

[0464] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art.

[0465] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0466] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0467] BRIEF DESCRIPTION OF THE DRAWINGS

[0468] FIG. 1 provides a schematic providing an overview of selenoprotein expression and characterizing features of selenoprotein family polypeptides. FIG. 2 provides a schematic illustrating a physiological function of the SEPN1 (SELENON) polypeptide in a muscle cell.

[0469] FIGs. 3 A and 3B provide an illustration of the secondary structure of Type 1 and Type 2 eukaryotic SECIS elements and a multiple sequence alignment of human SECIS elements. Not intending to be bound by theory, the secondary structure of SECIS is conserved, but the primary structure is different from one selenoprotein to another. In FIG. 3B, Human Type 1 SECIS elements include Gpxl, Gpx2, SelN, Diol, and SelV; whereas the other human SECIS elements listed in FIG. 3B are Type 2 SECIS elements. As shown in FIG. 3 A, Type 2 SECIS elements contain an additional bulge in the apical loop. The illustration of FIG. 3 A shows the four main structural motifs of SECIS elements, which are identified by arrows. A bracket indicates the position of the SECIS core that contains a quartet of non-Watson-Crick base pairs. Highly conserved nucleotides are represented in letters. The dots symbolize the two sheared tandem GA base pairs that are important for kink-turn structures. The asterisks indicate the variations from the consensus sequence (AA) that are present as CC in SelM, SelO, and Trxr3 SECIS elements. In FIG. 3B, a sequence alignment of 26 human SECIS elements is provided (Latreche et al., 2009, Nucleic Acids Research 37(17):5868-5880). Highly conserved nucleotides are represented in bold. Nucleotides involved in helix 2 are underlined. Gpx, glutathione peroxidase; Trxr, thioredoxin reductase; Dio, iodothyronine deiodinase; Sei, selenoprotein; Sell5, 15 kDa selenoprotein; Sps2, selenophosphate synthetase.

[0470] FIG. 4 provides a genetic map for the adeno-associated virus (AAV) genome. Not intending to be bound by theory, the ~5-kb AAV genome contains three open reading frames (orfs) that code for functional proteins. The rep orf (dark grey) codes for four Rep proteins (Rep78, Rep68, Rep52, and Rep40) that are synthesized from mRNAs initiated from the p5 and pl9 promoters, each of which is either spliced or left intact. The two larger proteins (Rep78 and Rep68) have site-specific, single-strand endonuclease, DNA helicase, and ATPase activities that are required for AAV DNA replication. The two smaller Rep proteins (Rep52 and Rep40) are required for packaging DNA into capsids and retain only the helicase domain that is present in the larger Rep proteins. The p40 promoter initiates an mRNA that is alternatively spliced to make three capsid proteins from the cap orf (light grey). The two minor capsid proteins, VP2 and VP1, contain the same amino acid sequence that is present in VP3 but contain additional N- terminal sequences that are required for infection. The ratio of VP1, VP2, and VP3 in the capsid is approximately 1 : 1 : 10. The additional N-terminal sequences present in VP1 and VP2 contain nuclear localization signals and a phospholipase A2 activity, both of which are required for infection. The spliced mRNA that codes for VP3 from a conventional AUG start codon also codes for the minor VP2 protein, which has additional N-terminal residues, from a weak upstream ACG start codon (asterisk). In addition, the VP2 / VP3 mRNA codes for an assemblyactivating protein (AAP) from a weak CTG start codon (asterisk) but in a different reading frame. AAP facilitates nuclear import of the major VP3 capsid protein and promotes assembly and maturation of the capsid, but AAP is not present in the mature capsid. Also shown are the 145-base (not to scale) T-shaped AAV inverted terminal repeats (ITRs).

[0471] FIG. 5 provides a schematic of an expression cassette flanked by two inverted terminal repeats (ITRs), which function in facilitating insertion of the expression cassette into an AAV capsid. In the embodiment of FIG. 5, the expression cassette contains a promoter as a regulatory element controlling transcription from a polynucleotide encoding a polynucleotide of interest (“gene of interest”).

[0472] FIG. 6 provides a plasmid map for the AAV vector pAAV-MCS.

[0473] FIG. 7 provides a schematic comparing the size of the human SEPN1 transcript to size restrains on the capacity for an AAV vector to encapsidate a polynucleotide sequence.

[0474] FIG. 8 provides an illustration showing that an AAV vector cannot properly encapsidate a full wild-type human SEPN1 transcript.

[0475] FIG. 9 provides a schematic annotating features of two polynucleotide constructs encoding a SEPN1 (SELENON) polypeptide without Exon 3 and one construct encoding a polynucleotide encoding a SEPN1 polypeptide with Exon 3, which includes an inframe TGA that could either encode a selenocysteine or a stop codon. The constructs were each cloned into pAAV-MCS. Throughout the figures, “myc” indicates a cMyc tag, Acc65I indicates a restriction site, and EcoRI indicates a restriction site. “Native codon” indicates a wild-type human SELENON polynucleotide sequence and “optimized codon” indicates a codon-optimized sequence.

[0476] FIG. 10 presents the same constructs as shown in FIG. 9, but includes information indicating the size of full-length and truncated versions of the SELENON polypeptides encoded by each construct depending on whether the UGA codon encodes a selenocysteine or stop.

[0477] FIG. 11 provides a Western blot image and a gel image. The three constructs shown in FIG. 9 were expressed in HEK 293T cells and the gel image and Western blot were prepared using these cells. The strongest expression of the full length selenoprotein N is observed with construct- 1 that contains the native sequence of SELENON gene without exon-3.

[0478] FIG. 12 presents a schematic annotating features of additional versions of polynucleotide construct- 1 (native SELENON sequence and excluding exon-3), prepared to evaluate the influence of features of the 3' untranslated region on expression of the selenoprotein encoded by each construct in cells. Throughout the figures, “Flag” indicates a FLAG tag-encoding polynucleotide sequence. Each construct encoded a SELENON polypeptide containing an N- terminal cMyc tag and a C -terminal FLAG tag. Construct 1 -my c-flag TXNDR2-UTR / TXNRD2 contained 355 bp from the 3' UTR of the TXNRD2 gene, which included the SECIS element from the TXNRD2 gene. Constructl -my c-flag SelW-UTR / SelW contained 411 bp from the 3' UTR of the SelW gene, which included the SECIS element of the SelW gene. Constructl -my c- flag SelM-UTR / SelM contained 198 bp from the 3' UTR of the SelM gene, which included the SECIS element of the SelM gene. Constructl -my c-FLAG SepnUTR544 / UTR544 / 544 contained 544 bp from the 3' UTR of the SELENON gene, which included the SECIS element of the SELENON gene. Constructl -my c-flag SepnUTR775 / UTR775 / 775 was 775 bp in length and contained two copies of the SECIS element of the SELENON gene and part of its flanking regions.

[0479] FIGs. 13A and 13B presents a Western blot image and a bar graph showing expression of the constructs of FIG. 12 in HEK293T cells transfected with plasmids encoding the constructs using lipofectamine. The “Native” sample was Construct- 1 from FIG. 9 including the native full size 3’ UTR sequence of human SELENON gene. FIG. 13 A presents a Western blot. FIG. 13 A and the top panel of FIG. 13B show portions of the same Western blot. The lower panel of FIG. 13B shows the ratio of truncated to full-length polypeptide detected in the Western blot of FIG. 13A and in the top panel of FIG. 13B.

[0480] FIG. 14 provides an image of an immunofluorescent gel run to detect expression of SEPN1 in HEK293T cells. The HEK293T cells were transduced using different volumes of AAV-SEPN1-UTR544 and AAV-SEPN1-UTR775. Little expression of the truncated SEPN1 polypeptide was detected. The SEPN1 polypeptides were detected using an anti-myc antibody.

[0481] FIG. 15 provides a bar graph showing transcript levels of endogenous and exogenous Sei enon! SELENON measured using qRT-PCR. Transcript levels were normalized to expression levels of PPIA and RPS18. Transcript levels were measured in liver and quadriceps of mice injected with AAV9-CMV-Myc-Selenon-Sec-Flag-UTR775 viral vectors. Throughout the figures, “WT” indicates a mouse with an intact SELENON gene, and “Selenon-KO” indicates a mouse with a deletion in exon 9 of the SELENON gene / polynucleotide. The numbers below the X-axis of FIG. 15 are identifiers for the mice corresponding to each measurement.

[0482] FIGs. 16A and 16B provide a Western blot image and a graph presenting polypeptide expression levels measured from the Western blot. The Western blot was used to detect transduced human SELENON protein in quadriceps and liver of injected mice using an anti-myc antibody. The mice were injected at 4 weeks and were sacrificed at 4-weeks post-injection to evaluate SELENON expression. FIG. 16A presents Western blot gel images. FIG. 16B presents a bar graph providing quantitation of the Western blot bands of FIG. 16A normalized to tubulin levels. The numbers above the gel images of FIG. 16A and below the X-axis of the bar graph of FIG. 16B are identifiers for the mice corresponding to each measurement.

[0483] FIG. 17 provides a western blot image showing long-term expression levels of transduced SELENON protein in quadriceps muscles of mice injected with 1E+14 Vg / kg of AAV9-CMV-Myc-Selenon-Sec-Flag-UTR775 at 4 weeks and sacrificed at 7 months post injection. The numbers above the gel images of FIG. 17 and below the X-axis of the bar graph of FIG. 17 are identifiers for the mice corresponding to each measurement. The positive control lane corresponds to protein extracted from HEK293T cells transfected with the plasmid pAAV9- CMV-Myc-Selenon-Sec-Flag-UTR775.This plasmid was also encapsidated in AAV9 particles that were injected into the mice. The negative control lane corresponds to untransfected HEK293T cells

[0484] FIG. 18 provides a Western blot image showing expression levels of transduced SELENON protein in various tissues (brain, lung, liver, kidney, spleen, heart, tibialis anterior (TA), paraspinal muscle, triceps, and diaphragm) of 3 mice (BL3077, BL3112, BL2990). These three mice were chosen because their quadriceps showed low, medium and high expression of SELENON respectively, as shown in FIG. 17, and allowed the assessment of the transgene expression across different muscle groups and tissues. For the most part the expression of the transgene in different muscles of these three mice matched the quadriceps. The only deviation was the diaphragm of BL3077 that showed a higher expression compared to the other 2 mice, while the quadriceps of this mouse had the lowest transgene expression.

[0485] FIG. 19 provides a Western blot image and a graph showing expression levels of SELENON protein in HEK293T or C2C12 cells transfected with plasmid pAAV-CMV-Myc- Selenon-Flag-UTR775 or pAAV-MHCK7-selenon-UTR775 using lipofectamine. Lane 1 : HEK293T cells transfected with pAAV-CMV-Myc-Selenon-Flag-UTR775, lane 2: HEK293T cells transfected with pAAV-MHCK7-selenon-UTR775, lane 3: untransfected HEK293T cells, lane 4: C2C12 cells transfected with pAAV-CMV-Myc-Selenon-Flag-UTR775, lane 5: C2C12 cells transfected with pAAV-MHCK7-selenon-UTR775, lane 6: untransfected C2C12 cells. The goal of this experiment was to compare the promotors CMV and MHCK7 in HEK293T (Human embryonic kidney) and C2C12 cells (mouse muscle cell line). FIG. 19 shows that both CMV and MHCK7 derive a strong expression of the full length SELENON protein in HEK293T cells, with CMV leading to a greater expression. However, in the mouse muscle cell line C2C12 the CMV promotor shows a very low expression (undisguisable from the endogenous SELENON expression). MHCK7 which is synthetic muscle specific promoter shows a stronger expression than CMV in C2C12 cells. FIG. 20 provides a plasmid map for the vector pAAV-CMV-myc-HU.SELENON-flag- UTR775.

[0486] FIG. 21 provides a plasmid map for the vector pAAV-MHCK7-HU.SELENON- UTR775.

[0487] FIG. 22 provides an immunoblot showing the expression of selenoprotein N in mice treated at 4 weeks with either saline or AAV9-MHCK7-Hu.SELENON-UTR775 (lot#4965) at 1E+14 and sacrificed at 13 weeks. In each lane, 30 microgram of protein extracted from the gastrocnemius muscle were loaded and detected with a custom made anti-SELENON antibody (Rabbit #3882) at 1 :500 dilution. The secondary antibody starBright™ Blue 700 Goat Antirabbit IgG was used at 1 :5000. hFAB™ Rhodamine labeled anti-Tubulin primary antibody (Biorad# 12004165) was used at 1 : 10,000 to detect tubulin and was used to normalize and compare the expression of SELENON from different lanes. The lower panel, shows the quantification of western blot bands’ intensity. For each lane, the intensity of the SELENON band was normalized to the intensity of the tubulin band.

[0488] FIG. 23 provides an immunoblot showing the expression of selenoprotein N in mice treated at 4 weeks with either saline or AAV9-MHCK7-Hu.SELENON-UTR775 (lot#4965) 1E+14 Vg / kg or myoAAV4A-MHCK7-Hu.SELENON-UTR775 (lot#5545) 1E+13 Vg / kg and sacrificed at 6 weeks (2 -weeks treatment). Protein from gastrocnemius, tibialis anterior, quadriceps, diaphragm, heart, liver and brain were extracted. In each lane 30 micrograms of protein were loaded and detected with a custom made anti-SELENON antibody (Rabbit #3882) at 1 :500 dilution. The secondary antibody starBright™ Blue 700 Goat Anti-rabbit IgG was used at 1 :5000. hFAB™ Rhodamine labeled anti-Tubulin primary antibody (Biorad# 12004165) was used at 1 : 10,000 to detect tubulin and was used to normalize and compare the expression of SELENON from different lanes. The lower panel, shows the quantification of western blot bands’ intensity. For each lane, the intensity of the SELENON band was normalized to the intensity of the tubulin band.

[0489] DETAILED DESCRIPTION OF THE INVENTION

[0490] The invention features compositions and methods that are useful for treatment of SELENON-related myopathies and other selenoprotein-associated diseases. In embodiments, the selenoprotein is selenoprotein N (SELENON / SEPN1) or a SELENON polynucleotide.

[0491] The invention is based, at least in part, upon the discovery that an expression vector (e.g., an Adeno- Associated Virus (AAV) vector) comprising at least two selenocysteine insertion sequence (SECIS) elements can be used to direct exogenous SELENON polypeptide expression in a mammalian cell. In embodiments, the 3' UTR of a selenoprotein polynucleotide is a SELENON, SELENOW, SELENOM, or TXRD2 3’ UTR. In some instances, the 3' UTR of the selenoprotein polynucleotide comprises two or more selenocysteine insertion sequence (SECIS) elements. In embodiments, the 3' UTR is less than about 1500 bp, 1250 bp, 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, or 300 bp in length. SELENON expression vectors containing two SECIS elements are useful to treat SELENON -related myopathies and other selenoprotein- associated diseases.

[0492] Diseases Associated with Selenoproteins

[0493] Some congenital (e.g., nondystrophic) myopathies are associated with selenoprotein deficiencies. Such myopathies may be associated with primary hypotonia and weakness. Onset of a selenoprotein related myopathy is typically early in the life of a human subject (e.g., prior to 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years of age) and relatively nonprogressive; however, later, and adult-onset forms (e.g., onset after about or at least about 18, 19, 20, 25, 30, 35, 40, 45, or 50 years of age) do exist. In an embodiment, the onset of a congenital myopathy is about or less than about 2 or 3 years of age. These congenital myopathies are rare genetic diseases with an incidence that can be as low as 0.06 per 1,000 live births (i.e., about 1 per 16,667 live births) and represent about 10% of all cases of neuromuscular disorders. The congenital myopathies are associated with distinctive and specific morphologic abnormalities in skeletal muscle, which are a main pathological feature of these myopathies. In some instances, a subject with a congenital myopathy can walk but has significant proximal weakness (e.g., as much as 63% of subjects develop Achilles, elbow, or other contractures). Non-limiting examples of symptoms associated with congenital myopathies include insulin resistance, abnormal lung development, spinal rigidity, scoliosis (e.g., evolving scoliosis), and respiratory weakness (e.g., evolving respiratory weakness).

[0494] The evolving respiratory weakness may be treated using nocturnal or full-time non- invasive ventilation. In embodiments, respiratory weakness has an onset that occurs at about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 33, or 35 years of age. In embodiments, the respiratory weakness has an onset that occurs at about 13.9 years of age. Death from a congenital myopathy occurring within the first two decades of life is typically associated with respiratory weakness.

[0495] In embodiments, the disease is a disease associated with a mutation in one or more of GPX1, GPX2, GPX3, GPX4, GPX6, TXNRD1, TXNRD2 (TXRD2), TXNRD3, DIO1, DIO2, DIOS, SEPHS2, SEPS1, SEPPI, SEP15, SEPN1 (SELENON), SEPX1, SEPW1 (SELENOW), SEPTI, SETH, SELI, SELK, SEEM (SELENOM), SELO, and SELV. In embodiments, the congenital myopathies are associated with a mutation in a selenoprotein N (SELENON SEPN 1) polynucleotide or polypeptide (e.g., in a cell of a subject). In embodiments, the congenital myopathy is congenital muscular dystrophy, optionally with spinal rigidity and / or restrictive respiratory syndrome. In some instances, the congenital myopathy is multiminocore disease. Further non-limiting examples of congenital myopathies include congenital fiber type disproportion (CFTD), multicore myopathy, multiminicore myopathy / disease (MmD), minicore myopathy, and rigid spine muscular dystrophy (RSMD). In embodiments, the congenital myopathy is associated with loss-of-function (LOF) mutations (e.g., an autosomal recessive LOF mutation) in a SEPN H SELENON polynucleotide. About 10% of all congenital myopathy cases (e.g., congenital muscular dystrophy cases) are related to a mutation in a SELENON polynucleotide. An analysis of the Genome Aggregation Database (gnomAD) revealed the pathogenic allele frequency for diseases associated with a mutation in a SELENON polynucleotide as being about 2.2 cases (95% confidence interval: 1.4-3.1) per million subjects. The prevalence of the pathogenic allele was determined in three regions to be the following: 1) in North America was 1,274 (95% confidence interval: 810-1,795), 2) in Europe was 1,631 (95% confidence interval: 1,038-2,297), and 3) in Asia was 9,819 (95% confidence interval: 6,248-13,835). In some instances, the congenital myopathy is associated with a pathogenic mutation in & RYR1 polynucleotide.

[0496] Multiminicore myopathy and rigid spine muscular dystrophy are associated with as much as 10% of congenital myopathy cases with an incidence as high as one or two per 300,000. In multiminicore myopathies, microscopic multiple “minicores” are present in affected skeletal fibers.

[0497] In an embodiment, the disease is a disease associated with a mutation in a glutathione peroxidase 4 (GPX4) polynucleotide or polypeptide (e.g., in a cell of a subject). In embodiments, the disease associated with a mutation in GPX4 is characterized by neuronal degeneration, ataxia, seizures, skeletal disorder, brain atrophy, and / or respiratory failure. In embodiments, the disease associated with a mutation in GPX4 is Sedaghatian-type spondylometaphyseal dysplasia (SSDM). In embodiments, the mutation in a GPX4 polynucleotide or polypeptide is g.H05253_1105260del, g. H05455OA, g. H05813G>A, g.l 106233J 106237del, or g.1106406J 106407del.

[0498] In embodiments, the disease is a disease associated with a thioredoxin reductase (e.g., TXNRD1, TXNRD2, or TXNRD3).

[0499] In embodiments, the disease is a disease associated with a mutation in a thioredoxin reductase 1 (TXNRD1) polynucleotide or polypeptide (e.g., in a cell of a subject). In embodiments, the disease associated with a mutation in TXNRD1 is epilepsy. In an embodiment, the mutation in a TXNRD1 polynucleotide or polypeptide is g, 104714898C>T or p.Prol90Leu.

[0500] In an embodiment, the disease is a disease associated with a mutation in a thioredoxin reductase 2 (TXNRD2) polynucleotide or polypeptide (e.g., in a cell of a subject). In embodiments, the disease associated with a mutation in TXNRD2 is dilated cardiomyopathy or familial glucocorticoid deficiency. In embodiments, the mutation in a TXNRD2 polynucleotide or polypeptide is g, 19907120C>T, g,19868204C>T, g, 19865895A>C, p.Ala59Thr, p.Gly375Arg, or p.Y447Ter.

[0501] In an embodiment, the disease is a disease associated with a mutation in a selenoprotein I (SELENOI or SELI) polynucleotide or polypeptide (e.g., in a cell of a subject). In embodiments, the disease associated with a mutation in SELENOI is spastic paraplegia. In embodiments, the mutation in a SELENOI polynucleotide or polypeptide is g.26596259G>C, g.26607825A>G, or p.Argl l2Pro.

[0502] Selenoproteins

[0503] In various aspects, the invention features polynucleotides that encode a selenoprotein described herein and a 3' untranslated region containing one, two, or more of the selenocysteine insertion sequence (SECIS) elements described herein. It can be advantageous for the polynucleotide to comprise at least two SECIS elements. In an embodiment, the polynucleotide is of a size (e.g., less than 2.6 kb or less than 4.6 kb) that provides for its delivery by a recombinant adeno-associated virus particle. In embodiments the polynucleotide contains a regulatory element(s) operably linked to the portions of the polynucleotide encoding the selenoprotein and the 3’ untranslated region.

[0504] Selenoproteins are polypeptides that contain a selenocysteine (Sec) (FIG. 1). The selenocysteine amino acid residue is typically encoded by a TGA codon which encodes a stop codon in non-selenoproteins genes). In an embodiment, a polynucleotide sequence encoding the selenoprotein contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Sec codons and the encoded selenoprotein contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 selenocysteins, respectively. Not intending to be bound by theory, FIG. 1 provides a schematic overview of a process by which a polynucleotide (e.g., mRNA) encoding a selenoprotein codon (e.g., UGA) is translated in a cell. In particular, translation of the Sec codon / sel enocysteine incorporation site (e.g., UGA) involves the binding of a selenocysteine insertion sequence (SECIS) binding protein to a selenocysteine insertion sequence (SECIS) element in the 3’ UTR of a polynucleotide (e.g., an mRNA transcript) encoding the selenoprotein. Binding of the SECIS binding protein to the SECIS element is associated with the recruitment of elongation factors, and a selenocysteine-containing tRNA (sec-tRNA) and incorporation of selenocysteine into a selenoprotein polypeptide translated from the selenoprotein-encoding polynucleotide.

[0505] Non-limiting examples of selenoproteins include those described in Moghadaszadeh and Beggs, “Selenoproteins and Their Impact on Human Health Through Diverse Physiological Pathways,” Physiology (Bethesda), 21 :307-315 doi: 10.1152 / physiol.00021.2006, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Non-limiting examples of selenoproteins include GPX1, GPX2, GPX3, GPX4, GPX6, TXNRD1, TXNRD2 (TXRD2), TXNRD3, DIO1, DIO2, DIO3, SEPHS2, SEPS1, SEPPI, SEP 15, SEPN1 (SELENON), SEPX1, SEPW1 (SEI. ENOW), SEPTI, SELH, SELI, SELK, SELM (SELENOM), SELO, and SELV. In embodiments, the selenoprotein has a biochemical activity such as oxi doreduction, selenocysteine synthesis, and / or selenium transport. In embodiments, the selenoprotein has a physiological role in cancer prevention or development, male fertility, thyroid metabolism, immune function, central nervous system function, and / or muscle function. In some instances, the selenoprotein is a glutathione peroxidase (e.g., cytosolic GPx (cGPx; GPx-1; GPX1), gastrointestinal GPx (GI-GPx; GPx-2; GPX2), plasma GPx (pGPx; GPx-3; GPX3), phospholipid hydroperoxide GPx (PHGPx, GPx-4; GPX4), sperm nuclei GPx (snGPx; GPX4), or GPx-6 (GPX6)), a thioredoxin reductase (e.g., thioredoxin reductase 1 (TrxRl; TXNRD1), thioredoxin reductase 2 (TrxR2; TXRD2; SelZfl; SelZf2; TXRD2; TXNRD2), and thioredoxin reductase 3 (TrxR3; TGR; TXNRD3)), an iodothyronine deiodinase (e.g., Type 1 deiodinase (DIO1; IOD1; DI; DIO1), Type 2 deiodinase (DIO2; IOD2; D2; DIO 2), or Type 3 deiodinase (DIO3; IOD3; D3; DIO3)), a selenophosphate synthetase (SPS2; SEPHS2), a selenoprotein S (SelS; VIMP; SEPS1), a selenoprotein P (SEPPI; SelP; SEPPI), a selenoprotein 15 kDa (Sell 5; SEP 15), a selenoprotein N (SelN; SEPN1; SEPN1; SELENON), a selenoprotein X (SelX; SelR; SEPX1), a selenoprotein W (SelW; SELENOW; SEPW1; SELENOW), a selenoprotein T (SelT; SEPTI), a selenoprotein H (SELH), a selenoprotein I (SELI), a selenoprotein K (SELK), a selenoprotein M (SELENOM; SELENOM; SELM), a selenoprotein O (SELO), or a selenoprotein V (SELV). In embodiments, the polypeptide is expressed predominantly (e.g., about or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all expression of the polypeptide and / or transcription from a polynucleotide encoding the polypeptide in a subject) in muscle. In other embodiments, the selenoprotein polynucleotide is expressed throughout the body, in the gastrointestinal tract, in the kidney, in the plasma, in the brain, in the testis, in the liver, in the heart, in the thyroid, in the pituitary gland, in skeletal muscle, in the prostate, in leukocytes, or various combinations thereof.

[0506] Not intending to be bound by theory, SELENON / SEPN1 is associated with transport of calcium into the lumen of the sarcoplasmic reticulum (SR) through an active sarco / endoplasmic reticulum Ca2+-ATPase (SERCA) (see FIG. 2). This flux of calcium into the SR is associated with muscle relaxation. Selenoprotein N deficiency in mice is associated with core lesions in muscle, altered lung development, and / or rigidity of the trunk and limbs associated with physical stress (e.g., endurance running or swimming). Not intending to be bound by theory, lowering levels of SEPN1 in a subject / cell is associated with reduced levels of calcium in the sarcoplasmic reticulum. For example, mice modified to delete exon 9 of SEPN1 / SELENON (i.e., SELENON / SEPN1 KO mice) express little-to-no SEPN1 polypeptide and, thus, have muscle fibers with lower SR calcium levels relative to non-SEPNl KO mice. Also, selenon-KO fish (i.e., fish modified to delete exon 9 of SEPNE) raised in an increased-viscosity fluid (e.g., 0.1% methyl cellulose) show reduced growth relative to selenon-KO fish grown in a lower viscosity fluid (e.g., no methyl cellulose).

[0507] Selenocysteine Insertion Sequence (SECIS) Elements

[0508] In various aspects, the invention features polynucleotides containing 1) a sequence encoding a selenoprotein and 2) a sequence corresponding to a selenoprotein insertion sequence (SECIS) element.

[0509] A selenocysteine insertion sequence (SECIS) element is a polynucleotide molecule (e.g., an mRNA molecule) that forms a stem-loop structure (FIG. 3 A). In embodiments, a SECIS element is about or at least about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length. In embodiments, a SECIS element is less than about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length. In embodiments, a SECIS element contains a helix 2 sequence (see FIG. 3 A), a highly conserved “AAA” sequence, and / or a SECIS core sequence (see FIG. 3 A) containing the sequences “UGAN” and “NGAN.” In some instances, the SECIS element has a Type 1 structure or a Type 2 structure (see FIG. 3 A). In embodiments, a SECIS element contains a sequence(s) with about or at least about 85%, 90%, 95%, 99%, or 100% sequence identity to one or more of the sequences underlined in the sequences shown in FIG. 3B, or fragments thereof. In some instances the SECIS element contains two or more sequences with about or at least about 85%, 90%, 95%, 99%, or 100% sequence identity to two or more sequences underlined in the sequences shown in FIG. 3B, where two of the sequences are from the same SECIS element polynucleotide sequence depicted in FIG. 3B. In embodiments, a loop of the SECIS element is about or at least about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. In embodiments, the loop of the SECIS element is no more than about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. In embodiments, the SECIS element contains a Helix 2 portion (see FIG. 3 A) that is about or at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 nucleotides in length. In embodiments, the SECIS element contains a Helix 2 portion that is no more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 nucleotides in length. In some embodiments, the SECIS element comprises elements (e.g., Apical loop, Helix 2, Internal loop, or Helix 1) selected from one or more different SECIS elements (e.g., those listed in FIG. 3B). The SECIS element can be eukaryotic (e.g., human) or non-eukaryotic (e.g., bacterial, or archaeal).

[0510] As described above and shown in FIG. 1, SECIS elements are an important feature of polynucleotides (e.g., mRNA molecules) encoding selenoproteins and are typically disposed in a 3’ untranslated region (UTR) of a polynucleotide sequence encoding a selenoprotein. Not intending to be bound by theory, the secondary structure of SECIS elements is conserved in humans, but the primary sequence varies between selenoproteins. In embodiments, a selenoprotein polynucleotide sequence (e.g., a SELENON polynucleotide sequence) contains a SECIS element derived from a SELENON, SELENOW, SELENOM, or TXRD2 polynucleotide. Non-limiting examples of SECIS element sequences are provided in FIG. 3B. In embodiments, a SECIS element suitable for use in the methods provided herein contains a sequence with about or at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a sequence provided in FIG. 3B, one of the following sequences, or fragments thereof. Although the sequences of FIG. 3B and the SECIS element sequences provided below are provided as DNA sequences, it will be understood by one of ordinary skill in the art that a SECIS element in an RNA molecule (e.g., an mRNA transcript) will contain an RNA equivalent of the DNA sequences (e.g., a sequence where all of the T’s of a DNA sequence provided herein for a SECIS element are substituted with U’s). In some embodiments, the SECIS elements comprise polynucleotide sequences having at least 85%, 90%, 95%, 99%, or 100% sequence identity to polynucleotide sequences in naturally occurring SECIS elements. In some embodiments, a SECIS element of the present disclosure comprises polynucleotide sequences having at least 85%, 90%, 95%, 99%, or 100% sequence identity to polynucleotide sequences in more than one naturally occurring SECIS element. Exemplary chimeric SECIS elements (i.e., elements comprising polynucleotide sequences from more than one naturally occurring SECIS element) are provided below:

[0511] SECIS element 1

[0512] CACGGACCCCATGGCAGGGGTGGCGTCTTCTGCATGATCCGCTCTGGTCAAACCCTTCCAGGCC

[0513] AGCCAGAGTGGGGATGGTCTGAGGGGCCAGCCCTTAGTGCAT.

[0514] SECIS element 2 CACGGACCCCATGGCAGGGGTGGCGTCTTCATGATCCGCTCTGGTCAAACCCTTCCAGGCCAGC

[0515] CAGAGTGGGGAGCCTGTCTGAGGGGCCAGCCCTTAGTGCAT.

[0516] SECIS element 3

[0517] CACGGACCCCATGGCAGGGGTGGCGTCTTCATGAGGGAGGGGCCCCAAACCCTTCCAGGCCAGG

[0518] ACCTCCCCTGAGCCTGTCTGAGGGGCCAGCCCTTAGTGCAT.

[0519] SECIS element 4

[0520] CCTGCCAGCCGCCCTGGCCCTGGTCACTGCATGATCCGCTCTGGTAAAGCCCTTGTGGGCGCCA

[0521] GAGTGGGGATGGTCTGTGACCTGCTGGGAAGGCAGGC.

[0522] SECIS element 5

[0523] CCTGCCAGCCGCCCTGGCCCTGGTCACTGCATGAGGGAGGGGCCCAAAGCCCTTGTGGGCGGAC

[0524] CTCCCCTGATGGTCTGTGACCTGCTGGGAAGGCAGGC.

[0525] SECIS element 6

[0526] CCTGCCAGCCGCCCTGGCCCTGGTCACATGAGGGAGGGGCCCAAAGCCCTTGTGGGCGGACCTC

[0527] CCCTGAGCCTGTCTGTGACCTGCTGGGAAGGCAGGC.

[0528] SECIS element 7

[0529] CCTGCCAGCCGCCCTGGCCCTGGTCACATGATCCGCTCTGGTCAAACCCTTCCAGGCCAGCCAG

[0530] AGTGGGGAGCCTGTCTGTGACCTGCTGGGAAGGCAGGC.

[0531] SECIS element 8

[0532] CACGGACCCCATGGCAGGGGTGGCGTCTTCTGCATGAGGGAGGGGCCCAAAGCCCTTGTGGGCG

[0533] GACCTCCCCTGATGGTCTGAGGGGCCAGCCCTTAGTGCAT.

[0534] SECIS element 9

[0535] CACGGACCCCATGGCAGGGGTGGCGTCTTCTGCATGATCCGCTCTGGTCAAACCCTTCCAGGCC

[0536] AGCCAGAGTGGGGATGGTCTGAGGGGCCAGCCCTTAGTGCAT.

[0537] SECIS element 10

[0538] CCTGCCAGCCGCCCTGGCCCTGGTCACATGAGGGAGGGGCCCAAAGCCCTTGTGGGCGGACCTC

[0539] CCCTGAGCCTGTCTGTGACCTGCTGGGAAGGCAGGC.

[0540] SECIS element 11

[0541] CACGGACCCCATGGCAGGGGTGGCGTCTTCATGATCCGCTCTGGTAAAGCCCTTGTGGGCGCCA

[0542] GAGTGGGGAGCCTGTCTGAGGGGCCAGCCCTTAGTGCAT.

[0543] SECIS element 12

[0544] CCTGCCAGCCGCCCTGGCCCTGGTCACTGCATGAGGGAGGGGCCCCAAACCCTTCCAGGCCAGG

[0545] ACCTCCCCTGATGGTCTGTGACCTGCTGGGAAGGCAGGC.

[0546] Further non-limiting examples of SECIS elements are provided in Latreche, et al.,

[0547] “Novel structural determinants in human SECIS elements modulate the translational recoding of

[0548] UGA as selenocysteine,” Nucleic Acids Research, 37:6868-5880 (2009) doi: 10.1093 / nar / gkp635, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0549] Adeno-Associated Virus (AAV) Vectors

[0550] Adeno-associated virus is a small (20-26 nm), icosahedral, and nonenveloped virus.

[0551] AAV particles contain a single-stranded DNA genome consisting of approximately 4.7 kb (see FIG. 4). The genome contains three open reading frames (ORFs) encoding replication proteins (Reps), capsid proteins (Caps), and the assembly activating protein (AAP), and is flanked by two inverted terminal repeats (ITRs) (see FIG. 4). Interestingly, adeno-associated virus (rAAV) particles have tissue-specific targeting capabilities, such that a heterologous gene of the rAAV will be delivered specifically to one or more predetermined tissue(s) or cell(s). A capsid protein encoded by the rAAV facilitates the tissue-specific targeting. In various embodiments, the recombinant adeno-associated virus (rAAV) particles disclosed herein are encoded by or encode any one of the vectors and / or polynucleotides described herein or are produced by any one of the methods described herein.

[0552] More than 30 naturally occurring serotypes of AAV are available and are useful in the particles, vectors, nucleotide molecules, and methods described herein. Many natural variants in the adeno-associated virus (AAV) capsid exist, allowing identification and use of an AAV with properties specifically suited for neural cells as well as other cell types. AAV viruses (i.e., AAV particles) can be engineered by conventional molecular biology techniques, making it possible to optimize these particles for cell specific delivery of the desired nucleic acid sequences, for minimizing immunogenicity, for tuning stability and particle lifetime, for efficient degradation, for accurate delivery to the nucleus, etc.’

[0553] The use of recombinant adeno-associated viruses (rAAVs) is a mode of exogenous delivery of polynucleotides (e.g., DNA) because AAVs are relatively non-toxic, provide efficient gene transfer, and can be easily optimized for specific purposes. Further advantages of AAV vectors include 1) low immunogenicity, 2) good biosafety rating, 3) infect both dividing and quiescent cells in vivo, and 4) can mediate long-term gene delivery in vivo. Methods for the design and use of adeno-associated viruses (AAVs) for delivery of polynucleotides to a cell are described in Adeno- Associate Virus, Methods and Protocols, R.S. Snyder and P Moullier, Editors., Methods in Molecular Biology, vol 807, Humana Press, DOI 10.1007 / 978-1-61779- 370-7, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Among the serotypes of AAVs isolated from human or non-human primates (NHP) and well characterized, human serotype 2 is the first AAV that was developed as a gene transfer vector. This serotype has been widely used for efficient gene transfer experiments in different target tissues and animal models. Other AAV serotypes useful in the vectors and methods of this disclosure include, but are not limited to, Anc80, AAV1, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, DJ, retro, rh.10, rh.39, rh,43, CSP3, SHH10, 7m8, PHP.b, PHP. eb, Myo-AAV, AAV-Myo and the like (see, e.g., WO 2005 / 033321, U.S. Pat. No. 7,198,951, and U.S. Patent Application Pub No. 2022 / 0228173 Al for a discussion of various AAV serotypes, the contents of each of which are hereby incorporated by reference in their entirety). In certain embodiments the serotype is selected to optimize a desired mode of delivery. In embodiments, an AAV vector provided herein has a specific viral capsid (e.g., an AAV9 capsid).

[0554] In some embodiments, the viral capsid used is a muscle-trophic viral capsid. For example, the myoAAV family of viral capsids is a family of muscle-trophic viral capsids useful for encapsidating polynucleotides and / or constructs of the present disclosure. The MyoAAV family of viral capsids includes MyoAAV 1A-1F, MyoAAV2A-2F, MyoAAV 3A-3F, and Myo AAV 4A-4F. The myoAAV family of viral capsids is discussed in further detail in, for example, Tabebordbar et al., Cell (2021) 184:4919-4938, the contents of which are hereby incorporated by reference.

[0555] Adeno-associated virus components suitable for inclusion in particles and vectors of the present invention include the capsid proteins, including the virion particle (VPs) proteins VP1, VP2, VP3, and hypervariable regions, the replication proteins (rep), including rep 78, rep 68, rep 52, and rep 40, and the sequences encoding these proteins. These components may be readily utilized in a variety of vector systems and cells.

[0556] In some embodiments, the recombinant adeno-associated virus (rAAV) particle comprises or is a vector of the present invention. In some embodiments, the viral particle is a recombinant AAV particle containing a polynucleotide sequence (e.g., an expression cassette) containing a heterologous polynucleotide and / or a regulatory element (e.g., a promoter) flanked by one or two AAV inverted terminal repeats (ITRs) (see FIG. 5). Typically, the polynucleotide sequence is less than about 4.3 kb, 4.4 kb, 4.5 kb, 4.6 kb, 4.7 kb, or 4.8 kb in length. In various embodiments, the heterologous gene is encapsidated in the AAV particle. The AAV particle comprises capsid proteins. In some embodiments, the vector comprises a heterologous gene operatively linked to regulatory elements including promoters and transcription initiation and termination sequences, thereby forming an expression cassette.

[0557] Regulatory Elements

[0558] Non-limiting examples of regulatory elements include transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and sequences that enhance secretion of the encoded product. A great number of regulatory elements, including promoters which are native, constitutive, inducible and / or tissue-specific, are known in the art and are suitable for use in embodiments of the present invention.

[0559] In some embodiments of the present invention a polyadenylation sequence can be inserted following a heterologous gene sequence. In various embodiments, the polyadenylation sequence is inserted before a 3' adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence. A rAAV vector useful in the present invention may also comprise an intron sequence. A non-limiting example of an intron sequence is an intron derived from SV-40, and is referred to as the SV-40 T intron sequence. Vectors of the present invention in various embodiments comprise an internal ribosome entry site (IRES). An IRES sequence is used to produce more than one polypeptide from a single gene transcript. An IRES sequence may be used to produce a protein that includes more than one polypeptide chain.

[0560] The precise nature of sequences needed for gene expression in host cells may vary between species, tissues or cell types. In some embodiments, vectors of the present invention comprise 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively of a heterologous gene, such as, to provide non-limiting examples, a TATA box, a capping sequence, a CAAT sequence, an enhancer elements, and the like. In various embodiments, a 5' non-transcribed sequences can include a promoter region that includes a promoter sequence for transcriptional control of an operably joined polynucleotide. In some embodiments, vectors of the present invention include enhancer sequences or upstream activator sequences as desired. The vectors of the invention may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.

[0561] Examples of suitable promoters useful for expressing a selenoprotein polypeptide include, but are not limited to the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al (1985) Cell, 41 :521-530), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter (e.g., chicken β-actin promoter), the phosphoglycerol kinase (PGK) promoter, the EFla promoter, the CBA promoter, UBC promoter, GUSB promoter, NSE promoter, Synapsin promoter, MeCP2 (methyl-CPG binding protein 2) promoter, GFAP; CBh promoter and the like. Exemplary promoters include, but are not limited to, the MoMLV LTR, a CK6 promoter, a transthyretin promoter (TTR), a TK promoter, a tetracycline responsive promoter (TRE), an HB V promoter, an hAAT promoter, a LSP promoter, chimeric liver-specific promoters (LSPs), the E2F promoter, the telomerase (hTERT) promoter; the cytomegalovirus enhancer / chicken beta-actin / Rabbit β-globin promoter (CAG promoter; Niwa et al., Gene, 1991, 108(2): 193-9), the elongation factor 1 -alpha promoter (EFl -alpha) promoter (Kim et al., Gene, 1990, 91(2):217-23 and Guo et al., Gene Ther., 1996, 3(9):802-10). In some embodiments, the promoter comprises a human β-glucuronidase promoter or a cytomegalovirus enhancer linked to a chicken β-actin (CBA) promoter. The promoter can be a constitutive, inducible, or repressible promoter.

[0562] Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41 :521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFlα promoter [Invitrogen],

[0563] Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Non-limiting examples of inducible promoters regulated by exogenously supplied promoters include the zinc- inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (see, e.g., WO 98 / 10088); the ecdysone insect promoter (see, e.g., No et al, Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline-repressible system (see, e.g., Gossen et al, Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (see, e.g., Gossen et al, Science, 268: 1766-1769 (1995), and Harvey et al, Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486-inducible system (see, e.g., Wang et al, Nat. Biotech., 15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)) and the rapamycin-inducible system (see, e.g., Magari et al, J. Clin. Invest., 100:2865-2872 (1997)). Still other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, blood glucose level, acute phase, a particular differentiation state of the cell, or in replicating cells only.

[0564] In another embodiment, the native promoter for a heterologous polynucleotide comprised by the vector will be used. The native promoter may be preferred when it is desired that expression of the heterologous gene should mimic the native expression. The native promoter may be used when expression of the heterologous gene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.

[0565] In some embodiments, the promoter expresses the heterologous gene in a muscle cell. In some embodiments, the heterologous gene is exclusively expressed in muscle cells (e.g., expressed in a muscle tissue and not expressed in other tissues of the body).

[0566] In some embodiments, vectors of the present invention comprise expression control sequences imparting tissue-specific gene expression capabilities. In some cases, the tissuespecific expression control sequences bind tissue-specific transcription factors that induce transcription in a tissue specific manner. Exemplary tissue-specific regulatory sequences include, but are not limited to, the following tissue specific promoters: a liver-specific thyroxin binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, a a-myosin heavy chain (a-MHC) promoter, or a cardiac Troponin T (cTnT) promoter. Other exemplary promoters include Beta-actin promoter, hepatitis B virus core promoter; alpha-fetoprotein (AFP) promoter, bone osteocalcin promoter; bone sialoprotein promoter, CD2 promoter; immunoglobulin heavy chain promoter; T cell receptor a-chain promoter, neuronal such as neuron-specific enolase (NSE) promoter, neurofilament light-chain gene promoter, the neuron-specific vgf gene promoter, and the synthetic promoter MHCK7 that derives gene expression in skeletal and cardiac muscle. In some embodiments, the expression control sequence allows for specific expression in a muscle cell.

[0567] In some embodiments, the promoter is a muscle-specific promoter. Exemplary musclespecific promoters include, but are not limited to, the following promoters: the SPc5-12 promoter, the SP-031 promoter, the MH promoter, the Sk-CRM4 / DES promoter, the CK6 promoter, the MHCK7 promoter, the dMCK promoter, the tMCK promoter, the CK8 promoter, and the CK8e promoter. Muscle-specific promoters are described in, for example, Skopenkova et al., Acta Naturae (2021) 13(l)(48):47-58.

[0568] Modifications

[0569] In embodiments, the polynucleotides provided herein contain one or more modifications or analogs.

[0570] For example, in some embodiments a polynucleotide contains one or more analogs (e.g., altered backbone, sugar, or nucleobase). Some non-limiting examples of analogs include 5- bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine.

[0571] In embodiments, the polynucleotide contains a modified backbone and / or linkages (e.g., between adjacent nucleosides). Non-limiting examples of modified backbones include those that contain a phosphorus atom in the backbone and those that do not contain a phosphorus atom in the backbone. Non-limiting examples of modified backbones include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonate such as 3' -alkylene phosphonates, 5 '-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkyl phosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3 '-5' linkages, 2'-5' linked analogs, and those having inverted polarity wherein one or more intemucleotide linkages is a 3' to 3', a 5' to 5' or a 2' to 2' linkage.

[0572] In embodiments, a polynucleotide contains short chain alkyl or cycloalkyl linkages (e.g., between adjacent nucleosides), mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. In embodiments, a polynucleotide includes one or more of the following: morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

[0573] In embodiments, a polynucleotide contains a nucleic acid mimetic. The term “mimetic can be intended to include polynucleotides wherein only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with non-furanose groups, replacement of only the furanose ring can also be referred as being a sugar surrogate. The heterocyclic base moiety or a modified heterocyclic base moiety can be maintained for hybridization with an appropriate target nucleic acid. One such nucleic acid can be a peptide nucleic acid (PNA). In a PNA, the sugar-b ackbone of a polynucleotide can be replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleotides can be retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. In embodiments, the backbone in PNA compounds contains two or more linked aminoethylglycine units that give PNA an amide containing backbone. Heterocyclic base moieties can be bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.

[0574] In embodiments, a polynucleotide contains a morpholino backbone structure. For example, a nucleic acid can contain a 6-membered morpholino ring in place of a ribose ring. In some of these embodiments, a phosphorodiamidate or other non-phosphodiester internucleoside linkage can replace a phosphodiester linkage.

[0575] A polynucleotide can contain linked morpholino units having heterocyclic bases attached to the morpholino ring. Linking groups can link morpholino monomeric units. Non-ionic morpholino-based oligomeric compounds can have less undesired interactions with cellular proteins. Morpholino-based polynucleotides can be nonionic mimics of nucleic acids. A variety of compounds within the morpholino class can be joined using different linking groups. A further class of polynucleotide mimetic can be referred to as cyclohexenyl nucleic acids (CeNA). In some instances, the furanose ring normally present in a nucleic acid molecule is replaced with a cyclohexenyl ring. CeNA DMT protected phosphoramidite monomers can be prepared and used for oligomeric compound synthesis using phosphoramidite chemistry. In some cases, incorporation of CeNA monomers into a nucleic acid chain increases the stability of a DNA / RNA hybrid. CeNA oligoadenylates can form complexes with nucleic acid complements with similar stability to the native complexes. In embodiments, a polynucleotide contains Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring thereby forming a 2'-C, 4'-C-oxymethylene linkage, thereby forming a bicyclic sugar moiety. The linkage can be a methylene ( — CH2), group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2. LNA and LNA analogs can display very high duplex thermal stabilities with complementary nucleic acid (Tm=+3 to +100C ), stability towards 3'- exonucleolytic degradation and good solubility properties.

[0576] In embodiments, a polynucleotide contains nucleobase modifications (often referred to simply as “base modifications”) or substitutions. In embodiments, unmodified nucleobases include one or more of the purine bases, (e.g., adenine (A) and guanine (G)), and / or the pyrimidine bases, (e.g., thymine (T), cytosine (C) and uracil (U)). Non-limiting examples of modified nucleobases include nucleobases such as 5 -methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2 -propyl and other alkyl derivatives of adenine and guanine, 2 -thiouracil, 2-thiothymine and 2 -thiocytosine, 5-halouracil and cytosine, 5-propynyl ( — C=C — CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5 -trifluoromethyl and other 5 -substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F- adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3 -deazaguanine and 3 -deazaadenine. Further non-limiting examples of modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido(5,4-b)(l,4)benzoxazin- 2(3H)-one), phenothiazine cytidine (lH-pyrimido(5,4-b)(l,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (l,4)benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido(5,4-b)(l,4)benzothiazin- 2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H- pyrimido(5,4-(b) (l,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4, -b)indol-2- one), pyridoindole cytidine (H-pyrido(3',2':4, 5)pyrrolo[2,3-d]pyrimidin-2-one).

[0577] Recombinant Adeno- Associated Virus (rAAV) Particle Preparation

[0578] Numerous methods are known in the art for the production of recombinant adeno- associated virus (rAAV) particles and / or vectors, including transfection, stable cell line production, and infectious hybrid virus production systems which include adenovirus- AAV hybrids, herpesvirus- AAV hybrids and baculovirus-AAV hybrids. In various embodiments, rAAV production cultures for the production of rAAV virus particles all include: 1) suitable host cells, including, for example, human-derived cell lines such as HeLa, A549, or 293 cells, or insect-derived cell lines such as SF-9, in the case of baculovirus production systems; 2) suitable helper virus function, provided by wild-type or mutant adenovirus (such as temperature sensitive adenovirus), herpes virus, baculovirus, or a plasmid construct providing helper functions; 3) AAV rep and cap genes and gene products; 4) a heterologous gene (such as a therapeutic gene) flanked by at least one adeno-associated virus (AAV) inverted terminal repeat (ITR) sequences; and 5) suitable media and media components to support recombinant adeno-associated virus (rAAV) particle production. Suitable media known in the art may be used for the production of rAAV particles. These media include, without limitation, media produced by Hyclone Laboratories and JRH including Modified Eagle Medium (MEM), Dulbecco’s Modified Eagle Medium (DMEM), custom formulations such as those described in U.S. Pat. No. 6,566,118, and Sf-900 II SFM media as described in U.S. Pat. No. 6,723,551, each of which is incorporated herein by reference in its entirety, particularly with respect to custom media formulations for use in production of recombinant adeno-associated virus (AAV) particles.

[0579] Methods for preparing recombinant adeno-associated virus particles can involve culturing a cell which contains a nucleic acid sequence encoding a recombinant adeno-associated virus rAAV particle. In various embodiments, the nucleic acid sequence can comprise a sequence encoding a capsid protein or a fragment thereof and a functional replication open reading frame (Rep). In various embodiments, the cell also comprises nucleotide sequences encoding sufficient helper functions to permit packaging of a recombinant adeno-associated virus vector comprising a nucleotide sequence encoding a heterologous gene sequence into the AAV capsid proteins. In various embodiments, the adeno-associated virus (AAV) vector comprises a nucleotide sequence comprising inverted terminal repeats (ITRs) and a sequence encoding a heterologous gene. In various embodiments, the cell comprises the AAV vector.

[0580] In some aspects, a method is provided for producing any recombinant adeno-associated virus (rAAV) particle as described herein comprising (a) culturing a host cell under a condition that rAAV particles are produced, wherein the host cell comprises (i) one or more AAV package genes, wherein each said AAV packaging gene encodes an AAV replication and / or encapsidation protein; (ii) a rAAV vector comprising a heterologous gene encoding a therapeutic polypeptide and / or nucleic acid as described herein flanked by at least one AAV inverted terminal repeat (ITR), and (iii) an AAV helper function; and (b) recovering the rAAV particles produced by the host cell.

[0581] In some embodiments, components to be cultured in the host cell to package a rAAV vector in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the components (e.g., recombinant adeno-associated virus vector, Rep open reading frame, Cap open reading frame, and / or helper functions) may be provided by cell that has been engineered to contain one or more of the components using methods known to those of skill in the art. In some embodiments, the cell will contain the component(s) under the control of a promoter. Non-limiting examples of the promoter include all promoters described herein. The promoter can be an inducible promoter or a constitutive promoter. In still another alternative, a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. The cell can be derived from 293 cells (which contain El helper functions under the control of a constitutive promoter), but which comprise Rep and / or Cap open reading frames under the control of inducible promoters.

[0582] A recombinant adeno-associated virus (AAV) vector, a Rep open reading frame, a Cap open reading frame, and helper functions useful for producing the recombinant adeno-associated virus (rAAV) particles of the invention may be delivered to a cell using any appropriate genetic element (e.g., a vector). The selected genetic element may be delivered by any suitable method, including those described herein. Methods used to construct any embodiment of this invention are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. In various embodiments, a method for generating rAAV particles is not a limitation on the present invention.

[0583] In some embodiments, recombinant adeno-associated virus particles may be produced using the triple transfection method, an embodiment of which is described in U.S. Pat. No. 6,001,650. Briefly, a plasmid containing a replication open reading frame (Rep) and a capsid open reading frame (Cap), along with a helper adenoviral plasmid, may be transfected (e.g., using the calcium phosphate method) into a cell line (e.g., HEK-293 cells), and recombinant adeno-associated virus (rAAV) particles may be collected and optionally purified. In some embodiments, recombinant adeno-associated virus (rAAV) particles are produced by transfecting a cell with a vector or nucleotide molecule describe herein to be packaged into the rAAV particles, a vector encoding adeno-associated virus (AAV) helper function genes (i.e., an AAV helper function vector), and / or a vector encoding accessory function genes (i.e., an accessory functions vector). The AAV helper function genes can include Rep and Cap open reading frames, which can function in trans for productive adeno-associated virus replication and encapsidation. The accessory function vector encodes nucleotide sequences for non- AAV derived viral and / or cellular functions upon which AAV is dependent for replication (i.e., “accessory functions”). The accessory functions include those functions useful for adeno- associated virus replication, including, without limitation, those moieties involved in activation of adeno-associated virus (AAV) gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus, and vaccinia virus.

[0584] Recombinant adeno-associated virus (rAAV) particles can be purified and formulated using standard techniques known in the art. For example, rAAV particles can be filter sterilized using a filter (e.g., a 22 pm filter). In embodiments, rAAV particles are formulated in 0.001%F68+DPBS.

[0585] In various embodiments, a cell comprising a nucleotide sequence or vector of the present invention is a 293 cell or a cell derived from a 293 cell. A non-limiting example of a 293 cell is available from ATCC, catalog number CRL-1573. In some embodiments, the cell is a HeLa, A549, 293, or insect-derived cell (e.g., SF-9). The cell can be a mammalian cell, insect cell, plant cell, bacterial cell, archaeal cell, or a fungal cell (e.g., a yeast cell).

[0586] Suitable recombinant adeno-associated virus (rAAV) particle production culture media of the present invention may be supplemented with serum or serum-derived recombinant proteins at a level of 0.5%-20% (v / v or w / v). Alternatively, as is known in the art, recombinant adeno- associated virus (rAAV) particles may be produced in serum-free conditions which may also be referred to as media with no animal-derived products. One of ordinary skill in the art may appreciate that commercial or custom media designed to support production of recombinant adeno-associated virus (rAAV) particles may also be supplemented with one or more cell culture components know in the art, including without limitation glucose, vitamins, amino acids, and or growth factors, in order to increase the titer of rAAV in production cultures.

[0587] Recombinant adeno-associated virus (rAAV) particle production cultures can be grown under a variety of conditions (over a wide temperature range, for varying lengths of time, and the like) suitable to the particular host cell being utilized. As is known in the art, rAAV particle production cultures can include attachment-dependent cultures which can be cultured in suitable attachment-dependent vessels such as, for example, roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors. Recombinant adeno-associated virus (rAAV) particle production cultures may also include suspension-adapted host cells such as HeLa, 293, and SF-9 cells which can be cultured in a variety of ways including, for example, spinner flasks, stirred tank bioreactors, and disposable systems such as the Wave bag system.

[0588] Recombinant adeno-associated virus (rAAV) particles of the invention may be harvested from rAAV production cultures by lysis of the host cells of the production culture or by harvest of the spent media from the production culture, provided the cells are cultured under conditions known in the art to cause release of rAAV particles into the media from intact cells. Suitable methods of lysing cells are also known in the art and include for example multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals, such as detergents and / or proteases.

[0589] In some embodiments, a production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtration through a series of depth filters including, for example, a grade DOHC Millipore Millistak+ HC Pod Filter, a grade A1HC Millipore Millistak+ HC Pod Filter, and a 0.2 pm Filter Opticap XL 10 Millipore Express SHC Hydrophilic Membrane filter. Clarification can also be achieved by a variety of other standard techniques known in the art, such as, centrifugation or filtration through any cellulose acetate filter of 0.2 pm or greater pore size known in the art.

[0590] In some embodiments, the rAAV production culture harvest is further treated with Benzonase® to digest any high molecular weight DNA present in the production culture. In some embodiments, the Benzonase® digestion is performed under standard conditions known in the art including, for example, a final concentration of 1-2.5 units / ml of Benzonase® at a temperature ranging from ambient to 37° C. for a period of 30 minutes to several hours. Recombinant adeno-associated virus (rAAV) particles may be isolated or purified in various embodiments using one or more of the following purification steps: equilibrium centrifugation; flow-through anionic exchange filtration; tangential flow filtration (TFF) for concentrating the rAAV particles; rAAV capture by apatite chromatography; heat inactivation of helper virus; rAAV capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and rAAV capture by anionic exchange chromatography, cationic exchange chromatography, or affinity chromatography. These steps may be used alone, in various combinations, or in different orders. Methods to purify rAAV particles are found, for example, in Xiao et al., (1998) Journal of Virology 72:2224-2232; U.S. Pat. Nos. 6,989,264 and 8,137,948; and WO 2010 / 148143.

[0591] Expression Level Analysis

[0592] Aspects of the disclosure relate to methods that include or measure an expression level of a selenoprotein polypeptide (e.g., SELENON) or polynucleotide. Any method for expression level analysis known in the art is contemplated for use herein. Such assays may be used for diagnostic purposes. Levels of the selenoprotein may be variably reduced in biological samples (e.g., muscle tissue, muscle cells, blood, serum, plasma) obtained from a subject with a disease (e.g., a congenital myopathy) that may be treated by the methods provide herein. While not all patients having a selenoprotein-associated disease or disorder (e.g., a congenital myopathy) have a significant decrease in selenoprotein expression, some subjects may have selenoprotein expression decreased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% relative to a normal control. Increased levels of the selenoprotein (e.g., SELENON) transcript (e.g., mRNA) and / or polypeptide can be an indication of presence of a healthy cell (e.g., a cell in a muscle tissue). Exemplary assays are described below. mRNA assays

[0593] One of ordinary skill in the art is familiar with various methods for analyzing mRNA levels. Examples of mRNA-based assays include but are not limited to oligonucleotide microarray assays, quantitative RT-PCR, Northern analysis, and multiplex bead-based assays. Other mRNA detection and quantitation methods include multiplex detection assays known in the art, e.g., xMAP® bead capture and detection (Luminex Corp., Austin, TX).

[0594] An exemplary method is a quantitative RT-PCR assay which may be carried out as follows: mRNA is extracted from cells in a biological sample (e.g., muscle cells) using the Rneasy kit (Qiagen). Total mRNA is used for subsequent reverse transcription using the SuperScript III First-Strand Synthesis SuperMix (Invitrogen) or the SuperScript VILO cDNA synthesis kit (Invitrogen). A small volume (e.g., 5 pl) of the RT reaction is used for quantitative PCR using SYBR™ Green PCR Master Mix and gene-specific primers, in triplicate, using an ABI 7300 Real Time PCR System. mRNA detection binding partners include oligonucleotide or modified oligonucleotide (e.g. locked nucleic acid) probes that hybridize to a target mRNA. mRNA-specific binding partners can be generated using the sequences provided herein or known in the art. Methods for designing and producing oligonucleotide probes are well known in the art (see, e.g., US Patent No. 8036835; Rimour et al. GoArrays: highly dynamic and efficient microarray probe design. Bioinformatics (2005) 21 (7): 1094-1103; and Wernersson et al. Probe selection for DNA microarrays using OligoWiz. Nat Protoc. 2007;2(l 1):2677-91).

[0595] Protein assays

[0596] One of ordinary skill in the art is familiar with various methods for measuring protein levels. Protein levels may be measured using protein-based assays such as but not limited to immunoassays (e.g., Western blots, enzyme-linked immunosorbent assay (ELISA), or immunofluorescence or colorimetric cell staining), multiplex bead-based assays, and assays involving aptamers (such as SOMAmer™ technology) and related affinity agents.

[0597] A brief description of an exemplary immunoassay, an ELISA, is provided here. A Biological sample is applied to a substrate having bound to its surface protein-specific binding partners (i.e., immobilized protein-specific binding partners). The protein-specific binding partner (which may be referred to as a “capture ligand” because it functions to capture and immobilize the protein on the substrate) may be an antibody or an antigen-binding antibody fragment such as Fab, F(ab)2, Fv, single chain antibody, Fab and sFab fragment, F(ab’)2, Fd fragments, scFv, and dAb fragments, although it is not so limited. Other binding partners are described herein. Protein present in the biological sample binds to the capture ligands, and the substrate is washed to remove unbound material. The substrate is then exposed to soluble protein-specific binding partners (which may be identical to the binding partners used to immobilize the protein). The soluble protein-specific binding partners are allowed to bind to their respective proteins immobilized on the substrate, and then unbound material is washed away. The substrate is then exposed to a detectable binding partner of the soluble proteinspecific binding partner. In one embodiment, the soluble protein-specific binding partner is an antibody having some or all of its Fc domain. Its detectable binding partner may be an anti-Fc domain antibody. As will be appreciated by those in the art, if more than one protein is being detected, the assay may be configured so that the soluble protein-specific binding partners are all antibodies of the same isotype. In this way, a single detectable binding partner, such as an antibody specific for the common isotype, may be used to bind to all the soluble protein-specific binding partners bound to the substrate.

[0598] Other examples of protein detection and quantitation methods include multiplexed immunoassays as described for example in US Patent Nos. 6939720 and 8148171, and published US Patent Application No. 2008 / 0255766, and protein microarrays as described for example in published US Patent Application No. 2009 / 0088329.

[0599] Protein detection binding partners include protein-specific binding partners. Proteinspecific binding partners can be generated using the sequences provided herein or known in the art. In some embodiments, binding partners may be antibodies. As used herein, the term “antibody” refers to a protein that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term “antibody” encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab’)2, Fd fragments, Fv fragments, scFv, and dAb fragments) as well as complete antibodies. Methods for making antibodies and antigen-binding fragments are well known in the art (see, e.g. Sambrook et al, “Molecular Cloning: A Laboratory Manual” (2ndEd.), Cold Spring Harbor Laboratory Press (1989); Lewin, “Genes IV”, Oxford University Press, New York, (1990), and Roitt et al., “Immunology” (2ndEd.), Gower Medical Publishing, London, New York (1989), W02006 / 040153, WO2006 / 122786, and W02003 / 002609).

[0600] Binding partners also include non-antibody proteins or peptides that bind to or interact with a target protein, e.g., through non-covalent bonding. For example, if the protein is a ligand, a binding partner may be a receptor for that ligand. In another example, if the protein is a receptor, a binding partner may be a ligand for that receptor. In yet another example, a binding partner may be a protein or peptide known to interact with a protein. Methods for producing proteins are well known in the art (see, e.g. Sambrook et al, “Molecular Cloning: A Laboratory Manual” (2ndEd.), Cold Spring Harbor Laboratory Press (1989) and Lewin, “Genes IV”, Oxford University Press, New York, (1990)) and can be used to produce binding partners such as ligands or receptors.

[0601] Binding partners also include aptamers and other related affinity agents. Aptamers include oligonucleic acid or peptide molecules that bind to a specific target. Methods for producing aptamers to a target are known in the art (see, e.g., published US Patent Application No. 2009 / 0075834, US Patent Nos. 7435542, 7807351, and 7239742). Other examples of affinity agents include SOMAmer™ (Slow Off-rate Modified Aptamer, SomaLogic, Boulder, CO) modified nucleic acid-based protein binding reagents.

[0602] Sequence Analysis

[0603] Aspects of the disclosure relate to methods that include sequencing a selenoproteinencoding polynucleotide (e.g., a selenoprotein gene sequence) in a cell to identify a pathogenic mutation associated with a selenoprotein disease or disorder. In embodiments, it is advantageous to administer vectors, polynucleotides, and / or compositions provided herein to a subject and / or cell harboring such a pathogenic mutation in a selenoprotein gene sequence (e.g., a mutation that results in reduced expression levels or functionality of a selenoprotein, such as SELENON, in a cell). In embodiments, a subject having a congenital myopathy is selected for treatment based upon the identification of a pathogenic mutation in a selenoprotein-encoding polynucleotide sequence in the subject’s genome. In embodiments, administration of vectors, polynucleotides, and / or compositions provided herein to a subject and / or cell harboring such a pathogenic mutation in a selenoprotein gene sequence (e.g., a mutation that results in reduced expression levels or functionality of a selenoprotein, such as SELENON, in a cell) is effective to correct haploinsufficiency of the selenoprotein gene sequence in the subject and / or cell.

[0604] The sequencing of a polynucleotide can be carried out using any suitable commercially available sequencing technology. In another embodiment, the sequencing of a polynucleotide is carried out using chain termination method of DNA sequencing (e.g., Sanger sequencing). In yet another embodiment, commercially available sequencing technology is a next-generation sequencing technology, including as non-limiting examples combinatorial probe anchor synthesis (cPAS), DNA nanoball sequencing, droplet-based or digital microfluidics, heliscope single molecule sequencing, nanopore sequencing, GeneGap sequencing, massively parallel signature sequencing (MPSS), microfluidic Sanger sequencing, microscopy-based techniques (e.g., transmission electronic microscopy DNA sequencing), RNA polymerase (RNAP) sequencing, single-molecule real-time (SMRT) sequencing, SOLiD sequencing, ion semiconductor sequencing, polony sequencing, Pyrosequencing (454), sequencing by hybridization, sequencing by synthesis (e.g., Illumina™ sequencing), sequencing with mass spectrometry, and tunneling currents DNA sequencing.

[0605] Pharmaceutical Compositions

[0606] In some aspects, the present invention provides pharmaceutical compositions. To prepare the pharmaceutical compositions of this invention, an effective amount of the recombinant adeno-associated virus (rAAV) particles, nucleotide molecules, and / or vectors of the present invention are combined with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration. These pharmaceutical compositions are desirable in unitary dosage form suitable, particularly, for administration percutaneously, or by parenteral injection. Any of the usual pharmaceutical media may be employed such as, for example, water, glycols, oils, alcohols, and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs, and solutions; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents, and the like in the case of powders, pills, capsules, and tablets. For parenteral compositions, the carrier will usually comprise sterile water, at least in large part, though other ingredients, for example, to aid solubility and cell viability, may be included. Other ingredients may include antioxidants, viscosity stabilizers, chelating agents, buffers, preservatives. If desired, further ingredients may be incorporated in the compositions, e.g. anti-inflammatory agents, antibacterials, antifungals, disinfectants, vitamins, antibiotics.

[0607] One suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present invention. As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. Supplementary active ingredients can also be incorporated into the compositions.

[0608] In some embodiments, the pharmaceutical composition comprises preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0609] In some embodiments, recombinant adeno-associated virus particle compositions are formulated to reduce aggregation of recombinant adeno-associated virus (rAAV) particles in the composition, particularly where high rAAV concentrations are present (e.g., about 1013viral genomes (Vg) / ml or more). Methods for reducing aggregation of rAAVs are known in the art and include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc.

[0610] In some embodiments, the pharmaceutical compositions of the present invention may contain at least about 0.1% of an additional active compound. A concentration of the additional active compound may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation. The amount of active compound in a pharmaceutical composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the pharmaceutical composition. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical compositions, and as such, a variety of dosages and treatment regimens may be desirable.

[0611] In some embodiments, the pharmaceutical compositions of the present invention comprise an exosome. In some embodiments, the adeno-associated virus (AAV) particles are exosome-associated AAV vectors, as described in Gene. Ther. 23:380-392 (2016), the entirety of the disclosure of which is incorporated herein by reference for all purposes.

[0612] The pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is sterile and fluid to the extent that easy syringability exists. In various embodiments, compositions of the present invention are stable under conditions of manufacture and storage and are preserved against the contaminating action of microorganisms, such as bacteria and fungi. A carrier suitable for use in the pharmaceutical composition can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In some embodiments, the pharmaceutical composition may comprise isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0613] The pharmaceutical composition may be buffered, if necessary, and a liquid diluent first rendered isotonic with sufficient saline or glucose. For example, a recombinant adeno- associated virus (rAAV) particle, vector, or plasmid may be dissolved or dispersed in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at a proposed site of administration on a patient. Sterile injectable solutions can be prepared by incorporating a recombinant adeno- associated virus (rAAV) particle, vector, polynucleotide sequence, and / or plasmid in a solvent with various of the other ingredients enumerated herein, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0614] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present invention into target cells. In particular, a recombinant adeno-associated virus (rAAV) particle, vector, or plasmid may be formulated for delivery encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like. Liposomes have been developed with improved serum stability and circulation half-times (see, e.g., U.S. Pat. No. 5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers are known to one of skill in the art.

[0615] Nanocapsule formulations of the recombinant adeno-associated virus (rAAV) particle, vector, or plasmid may be used. Nanocapsules can generally entrap substances in a stable and reproducible way.

[0616] Methods of Treatment

[0617] The present invention provides methods of treating SELENON-r elated myopathies and / or other selenoprotein-associated diseases, where the methods involve administering a therapeutically effective amount of a pharmaceutical composition comprising a vector and / or polynucleotide of the invention to a subject (e.g., a mammal such as a human). The method includes the step of administering to a subject a therapeutic amount of a composition described herein sufficient to treat the disease and / or or disorder or symptom thereof, under conditions such that the disease or disorder is treated. In some embodiments, the composition is a pharmaceutical composition described herein.

[0618] The subject method has wide applicability to the treatment of selenoprotein-associated diseases and / or disorders (e.g., myopathies). In this regard, the subject method is useful for, but not limited to, treatment of a disease or disorder such as multiminicore myopathy / disease (MmD), and rigid spine muscular dystrophy. In embodiments the disease or disorder is a selenoprotein-associated disease or disorder, optionally wherein the disease or disorder is associated with reduced levels of selenoprotein expression and / or with a pathological mutation to a polynucleotide (e.g., a gene) encoding a selenoprotein in a cell.

[0619] The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of an agent described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).

[0620] The therapeutic methods of the invention in general comprise administration of a therapeutically effective amount of the compositions described herein, such as a composition comprising a recombinant adeno-associated virus (rAAV) particle or vector, to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease and / or disorder, or symptom thereof. Determination of those subjects “at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like). The compounds herein may be also used in the treatment of any disorder and / or disease resulting in abnormal development of a subject and / or abnormal and / or reduced muscle function.

[0621] The pharmaceutical compositions of this invention can be administered by any suitable routes including, by way of illustration, oral, topical, rectal, transdermal, subcutaneous, intravenous, intramuscular, intranasal, intracranial, intracerebral, intraventricular, intrathecal, and the like. In some embodiments, the administration modalities as described in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 may be used to deliver compositions of the present invention. In some embodiments, a preferred mode of administration is by portal vein injection. For therapeutic uses, the compositions and agents disclosed herein may be administered by any convenient method; for example, parenterally, conveniently in a pharmaceutically or physiologically acceptable carrier, e.g., phosphate buffered saline, saline, deionized water, or the like. The compositions may be added to a retained physiological fluid such as blood or synovial fluid.

[0622] Generally, the amount of a composition administered will be empirically determined. In various embodiments, a dosage of the compositions of the present invention administered to a subject will generally be in the range of about or at least about 1E+9 viral genomes (Vg), 1E+10 Vg, 1E+11 Vg, 1E+12 Vg, 1E+13 Vg, 1E+14 Vg, 1E+15 Vg, 1E+16 Vg per kg or total dose. In some embodiments, a dosage of the compositions of the present invention administered to a subject will generally be in the range of about 1E+9 to about 1E+14 Vg per kg. Other additives may be included, such as stabilizers, bactericides, etc. In various embodiments, these additives can be present in conventional amounts.

[0623] In various embodiments, the adeno-associated virus (rAAV) particles, nucleotide molecules, and / or vectors of the present invention are administered in sufficient amounts to transfect a cell of a desired tissue (e.g., a muscle tissue) and to provide sufficient levels of gene transfer and expression without undue adverse effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to a selected organ or tissue (e.g., a muscle), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parental routes of administration. Routes of administration may be combined, if desired.

[0624] The dose of adeno-associated virus (rAAV) particles, nucleotide molecules, and / or vectors used to achieve a particular “therapeutic effect,” e.g., the units of dose in viral genome (Vg) copies / per kilogram of body weight (Vg / kg), will vary based on several factors including, but not limited to: the route of administration, the level of gene or RNA expression used to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. One of skill in the art can readily determine a dose range to treat a patient having a particular disease and / or disorder based on the aforementioned factors, as well as other factors that are well known in the art. In some embodiments, the therapeutic effect is a restoration or increase in muscle function. In some embodiments, the therapeutic effect is an increase in transport of calcium ions across a membrane of the sarcoplasmic reticulum and / or an increase in the concentration of calcium ions (e.g., a time-averaged and / or homeostatic concentration) in the sarcoplasmic reticulum of a muscle cell(s).

[0625] Administration of recombinant adeno-associated virus (rAAV) particles, nucleotide molecules, and / or vectors of the present invention to a subject may be by, for example, intramuscular injection or by administration into the bloodstream of the subject. Administration into the bloodstream may be by injection into a vein, an artery, or any other vascular conduit. In some embodiments, the recombinant adeno-associated virus (rAAV) particles, nucleotide molecules, and / or vectors are administered into the bloodstream by way of isolated limb perfusion, a technique well known in the surgical arts, the method essentially enabling the artisan to isolate a limb from the systemic circulation prior to administration. A variant of the isolated limb perfusion technique, described in U.S. Pat. No. 6,177,403, can also be employed by the skilled artisan to administer the recombinant adeno-associated virus (rAAV) particles, nucleotide molecules, and / or vectors into the vasculature of an isolated limb to potentially enhance transduction into muscle cells or tissue. Moreover, in certain instances, it may be desirable to deliver the virions to a muscle of the subject. The compositions of the invention may comprise a recombinant adeno-associated virus (rAAV) particle, nucleotide molecule, and / or vector, either alone or in combination with one or more other recombinant adeno-associated virus (rAAV) particles, nucleotide molecules, and / or vectors (e.g., a second recombinant adeno-associated virus (rAAV) particle, nucleotide molecule, and / or vector encoding one or more different heterologous genes). In some embodiments, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different recombinant adeno- associated virus (rAAV) particles, nucleotide molecules, and / or vectors each comprising one or more different heterologous genes. In embodiments, the heterologous gene encodes a selenocysteine-encoding polynucleotide described herein.

[0626] Recombinant adeno-associated virus particles, nucleotide molecules, and / or vectors of the present invention can be inserted into a delivery device which facilitates introduction by injection or implantation into a subject. Such delivery devices include tubes, e.g., catheters, for injecting cells and fluids into the body of a recipient subject. In a preferred embodiment, the tubes additionally have a needle, e.g., a syringe, through which the cells of the invention can be introduced into the subject at a desired location. Recombinant adeno-associated virus particles, nucleotide molecules, and / or vectors of the invention can be inserted into such a delivery device, e.g., a syringe, in different forms. For example, the recombinant adeno-associated virus particles, nucleotide molecules, and / or vectors can be suspended in a solution or embedded in a support matrix when contained in such a delivery device. As used herein, the term “solution” includes a pharmaceutically acceptable carrier or diluent in which the recombinant adeno-associated virus particles, nucleotide molecules, and / or vectors of the invention remain functional and / or viable. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and / or dispersion media. The use of such carriers and diluents is well known in the art. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. In some embodiments, the selection of the carrier is not a limitation of the present invention. The solution is preferably sterile and fluid. Preferably, the solution is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Solutions of the invention can be prepared by incorporating recombinant adeno-associated virus particles, nucleotide molecules, and / or vectors as described herein in a pharmaceutically acceptable carrier or diluent and, as other ingredients enumerated herein, followed by filtered sterilization. Optionally, recombinant adeno-associated virus particles, nucleotide molecules, and / or vectors may be administered on support matrices. Support matrices in which recombinant adeno-associated virus particles, nucleotide molecules, and / or vectors can be incorporated or embedded include matrices which are recipient-compatible and which degrade into products which are not harmful to the recipient. Natural and / or synthetic biodegradable matrices are examples of such matrices. Natural biodegradable matrices include plasma clots, e.g., derived from a mammal, and collagen matrices. Synthetic biodegradable matrices include synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acid. Other examples of synthetic polymers and methods of incorporating or embedding cells into these matrices are known in the art. These matrices provide support and protection for the cells in vivo.

[0627] Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a bioactive factor at a particular target site.

[0628] One feature of certain embodiments of an implant can be the linear release of the recombinant adeno-associated virus particles, nucleotide molecules, and / or vectors, which can be achieved through the manipulation of the polymer composition and form. By choice of monomer composition or polymerization technique, the amount of water, porosity and consequent permeability characteristics can be controlled. The selection of the shape, size, polymer, and method for implantation can be determined on an individual basis according to the disorder and / or disease to be treated and the individual patient response. The generation of such implants is generally known in the art.

[0629] In another embodiment of an implant recombinant adeno-associated virus particles, nucleotide molecules, and / or vectors are encapsulated in implantable hollow fibers or the like. Such fibers can be pre-spun and subsequently loaded with the recombinant adeno-associated virus particles, nucleotide molecules, and / or vectors, or can be co-extruded with a polymer which acts to form a polymeric coat about the recombinant adeno-associated virus particles, nucleotide molecules, and / or vectors. Such encapsulated cells can then be combined with a neural stimulant.

[0630] In addition to the methods of delivery described above, the following techniques are also contemplated as alternative methods of delivering the recombinant adeno-associated virus particles, nucleotide molecules, and / or vectors to a subject. Ultrasound has been used as a device for enhancing the rate and efficacy of drug permeation into and through a circulatory system. Other drug delivery alternatives contemplated are intraosseous injection (see, e.g., U.S. Pat. No. 5,779,708), microchip devices (see, e.g., U.S. Pat. No. 5,797,898), ophthalmic formulations, transdermal matrices (see, e.g., U.S. Pat. Nos. 5,770,219 and 5,783,208), and feedback- controlled delivery (see, e.g., U.S. Pat. No. 5,697,899).

[0631] In embodiments, a composition of the present disclosure is administered to a subject before or after the first onset of symptoms associated with a congenital myopathy in the subject and / or before or after first detection of a pathogenic mutation in the subject that is associated with a congenital myopathy.

[0632] Kits

[0633] The rAAV vector particles, polynucleotides, and / or compositions of the disclosure may be supplied along with additional reagents in a kit. The kits can include instructions for the treatment regime or assay, reagents, equipment (test tubes, reaction vessels, needles, syringes, etc.) and standards for calibrating or conducting the treatment or assay. The instructions provided in a kit according to the invention may be directed to suitable operational parameters in the form of a label or a separate insert. Optionally, the kit may further comprise a standard or control information so that the test sample can be compared with the control information standard to determine if whether a consistent result is achieved.

[0634] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[0635] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0636] EXAMPLES Example 1: Validation of Expression Constructs in Cell-Based Assays

[0637] Experiments were undertaken to develop an expression construct suitable for use in treatment of a SELENON-related myopathy or other selenoprotein-associated disease in a subject. Introduction of the SELENON polynucleotide to a subject would be associated with an increase in expression of functional SELENON polypeptides in the subject, thereby treating the SELENON-related myopathy disease or other selenoprotein-associated disease.

[0638] Recombinant adeno-associated virus (rAAV) vectors are suitable for use in delivering an expression construct containing a SELENON polynucleotide to a cell; however, the size of an expression construct that may be encapsidated by the rAAV capsid is limited to less than about 4.6 kb. Although the polypeptide-coding portion of the SELENON (SEPN1) transcript meets these size restrictions (see FIG. 7), the SELENON transcript 3’ untranslated region (UTR), which is required for proper incorporation of selenocysteine into the SELENON polypeptide, brings the total length of the SELENON transcript to about 4.2 kb, which together with the promotor and poly A signal sequences would exceed the 4.6kb limit and therefore is too long for encapsidation by an rAAV capsid (see FIG. 8). Thus, experiments were undertaken to determine the minimum portion of the 3’ UTR of the SELENON transcript, or the 3’ UTR of other selenoprotein transcripts, necessary to facilitate expression of a functional SELENON polypeptide in a cell from an expression construct. In particular, experiments were undertaken to determine how much sequence flanking the SECIS element in the 3’ UTR of a wild-type SELENON gene, or the 3’ UTR of other selenoprotein genes, was necessary for expression of a functional SELENON polypeptide from an expression construct.

[0639] First, a polynucleotide encoding the entire SELENON gene was synthesized by Genescript and cloned into a pAAV-MCS vector (FIGs. 6 and 9). Three constructs were synthesized (FIG. 9). Construct- 1 contained the wild-type SELENON gene sequence and a sequence encoding an N-terminal cMyc tag. Construct-2 contained a codon-optimized version of the SELENON gene sequence and a sequence encoding an N-terminal cMyc tag. Construct-3 was identical to construct- 1 (native sequence) in every way except that it also contained exon 3 of the SELENON gene, which carries an in-frame UGA codon. It had never been shown before whether the UGA codon in exon 3 encodes a selenocysteine or a translation termination.” All 3 constructs contained the full length native 3 ’UTR of human SELENON gene allowing a direct comparison of the expression of the different coding sequences.

[0640] The sequences of these 3 constructs are provided in Table 1, below.

[0641]

[0642]

[0643]

[0644]

[0645]

[0646]

[0647]

[0648]

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655]

[0656]

[0657] Table 1: Sequences of SELENON Comparison Constructs

[0658] For polynucleotide sequences: Inverted Terminal Repeats (ITRs) are shown in CAPITAL LETTERS; myc is shown in underlined italics;

[0659] Hu.Selenon is shown in bold italics; 3’ UTR is shown in bold underline; SECIS sequence is shown in BOLD UNDERLINE CAPITAL

[0660] LETTERS

[0661] For polypeptide sequences: myc is shown in underlined italics; selenocysteines are shown in bold underline.

[0662] As shown in FIG. 10, within each construct, failure to incorporate selenocysteine into a polypeptide translated from each respective construct would result in truncation at the Sec codon (UGA), which typically functions as a stop codon in polynucleotides encoding non-selenoprotein polynucleotides.

[0663] One microgram of each of the three pAAV-MCS vectors encoding the constructs were transfected into HEK 293T cells using lipofectamine. Polypeptide expression from each of the constructs was measured using Western blots and stained gels, as shown in FIG. 11. Construct- 1 showed the highest levels of expression of full-length polypeptide and was selected for subsequent experiments to characterize the shortest 3’ UTR sequence that would allow selenocteyine incorporation into SELENON polypeptide.

[0664] Five new constructs were synthesized, each containing a different 3’ UTR nucleotide sequence (see FIG. 12). Each construct was cloned into a pAAV-MCS vector. Each construct encoded a SELENON polypeptide containing an N-terminal cMyc tag and a C -terminal FLAG tag. One construct (Constructl-myc-flag TXNDR2-UTR / TXNRD2) contained 355 bp from the 3’ UTR of the TXNRD2 gene, which included the SECIS element from the TXNRD2 gene. One construct (Constructl-myc-flag SelW-UTR / SelW) contained 411 bp from the 3’ UTR of the SelW gene, which included the SECIS element of the SelW gene. Another construct (Constructl- myc-flag SelM-UTR / SelM) contained 198 bp from the 3’ UTR of the SelM gene, which included the SECIS element of the SelM gene. Another construct (Construct 1 -my c-FL AG SepnUTR544 / UTR544 / 544) contained 544 bp from the 3’ UTR of the SELENON gene, which included the SECIS element of the SELENON gene. Another construct (Constructl-myc-flag SepnUTR775 / UTR775 / 775) was 775 bp in length and contained two copies of the SECIS element of the SELENON gene and its flanking regions. Sequences of these five new constructs is shown in Table 2, below.

[0665]

[0666] 00

[0667]

[0668]

[0669]

[0670]

[0671]

[0672]

[0673] 00

[0674] 00

[0675]

[0676]

[0677]

[0678]

[0679]

[0680]

[0681]

[0682]

[0683]

[0684]

[0685] Table 2: Sequences of Five New Constructs

[0686] Inverted Terminal Repeats (ITRs) are shown in CAPITAL LETTERS; myc is shown in underlined italics; Hu.Selenon is shown in bold italics; flag is shown in underline; 3’ UTR is shown in bold underline; SECIS sequence is shown in BOLD UNDERLINE CAPITAL LETTERS.

[0687] Each of the five pAAV-MCS vectors encoding the new rAAV vector constructs was transfected into HEK293T cells using lipofectamine. Polypeptide expression was measured using a Western blot (see FIG. 13 A). Expression levels in the cells transfected with Construct- 1 containing the native full length 3’UTR was used as a control. All the five tested constructs allowed the expression of the full length SELENON polypeptide. The construct containing two SECIS elements from SELENON (i.e., Constructl-myc-flag SepnUTR775) resulted in the strongest expression of the full length SELENON protein. It is well known that when selenoproteins are overexpressed in vitro, a significant amount of truncated polypeptide is produced due to the poor encoding of selenocysteine at the UGA codon. It is believed that this is caused by the exhaustion of some the elements necessary in the selenoprotein synthesis (i.e. SECIS binding protein, selenocysteine and its corresponding tRNA). As expected, transfection with all five constructs resulted in production of the truncated SELENON polypeptide. To characterize the most efficient construct (i.e. highest expression of the full length SELENON and lowest expression of the truncated SELENON), the ratio of expressed truncated polypeptide to expressed full-length polypeptide was determined for each construct (see FIG. 13B). The construct containing two SECIS elements from SELENON (i.e., Constructl-myc-flag SepnUTR775) was associated with the lowest ratio of truncated polypeptide expression to full- length polypeptide expression. The next lowest ratio of was observed for the UTR544 construct. UTR 775 and UTR544 derived the selenocysteine incorporation in the SELNON polypeptide at levels comparable to the full length native SELENON 3’UTR.

[0688] Next, having identified constructs associated with expression of the lowest ratios of truncated polypeptide to full-length polypeptide were incorporated into rAAV particles to produce the viral vectors AAV9-CMV-Myc-SelenonSec-Flag-UTR775 and AAV9-CMV-Myc- SelenonSec-Flag-UTR544, which were then used to infect HEK293T cells. The rAAV particles were AAV9 serotype rAAV particles. The constructs were packaged into the AAV9 particles using standard techniques. The particles were purified using iodixanol gradients and ultracentrifugation. The particles were titered with SYBR™-Green based qPCR assays. Purified rAAV particles were further filter sterilized by a 0.22 μm filter. The rAAV particles were suspended in a solution containing 0.001% Pluronic F-68 Non-ionic Surfactant in Dulbecco’s phosphate-buffered saline (DPBS). The constructs both were associated with a dose response and very little truncated SELENON / SEPN1 polypeptide was detected on western blot (see FIG. 14). The data of this example shows that the infection of HEK293T cells using the AAV9- CMV-Myc-SelenonSec-Flag-UTR775 vector resulted in the expression of the full length SELENON protein, demonstrating that although the designed UTR775 was shorter than the native SELENON 3’UTR, it was sufficient to derive selenocysteine insertion in the SELENON protein. Further, the reduced size of the 3’UTR met the limited cargo capacity of AAVs and could be successfully packaged in an AAV capsid.

[0689] Example 2: Administration of AAV-SELENON to Mice

[0690] Having identified the AAV9-CMV-Myc-SelenonSec-Flag-UTR775 viral vector as being associated with a low ratio of truncated polypeptide to full-length polypeptide and with high levels of total full-length SELENON expression, the in vivo efficacy of the vector in mice was then evaluated. These in vivo experiments demonstrated expression of full length human SELENON mRNA and protein in mice injected with the AAV9-CMV-Myc-Selenon-Sec-Flag- UTR775 vector (FIG. 20).

[0691] The term “AAV9-CMV-Myc-SelenonSec-Flag-UTR775” refers to an AAV9 serotype adeno-associated viral vector particle with a cargo containing a polynucleotide encoding a nucleotide sequence encoding a human SELENON polypeptide under the control of a CMV promoter. The nucleotide sequence encoding the human SELENON polypeptide excludes exon 3 and contains the selenocysteine codon (Sec) in exon 10 and further encodes an N-terminal cMyc tag and a C -terminal FLAG tag. The polynucleotide further encodes a 3’ UTR downstream of the sequence encoding the C -terminal FLAG tag. The 3’ untranslated region (UTR) contains a fragment of the wild-type SELENON 3’ UTR and contains two selenocysteine insertion sequence (SECIS) elements and its flanking region.

[0692] Twelve mice (5 WT and 7 Selenon-KO) were injected at 4 weeks of age with 1E+14 vg / kg of AAV9-CMV-Myc-SelenonSec-Flag-UTR775. Table 3 provides details relating to representative mice that received injections. The KO mice contained a deletion of Exon 9 of the Selenon gene and, therefore, expressed an mRNA transcript from the altered gene but no SELENON polypeptide. The mice were sacrificed at 8 weeks of age (4 weeks post injection) and the expression of the SELENON transgene in the liver and quadriceps was assessed by qRT- PCR and western blot using an anti-myc tag antibody (FIGs. 15, 16A, and 16B). A multiplex Taqman assay was used to detect the endogenous murine Selenon transcript and the transcript resulting from the human SELENON transgene delivered by AAV. The assay used to detect mRNA transcribed from the transduced SELENON construct (i.e., that introduced to the mice using AAV9-CMV-Myc-SelenonSec-Flag-UTR775) targeted a region of UTR775 that was not naturally present and was therefore unique and specific. The assay detecting the endogenous mouse Selenon mRNA transcript targeted mouse Selenon mRNA and did not detect mRNA associated with the AAV9-CMV-Myc-SelenonSec-Flag-UTR775 construct, which encoded a human SELENON polypeptide. The levels of transcript expressed from the endogenous Selenon gene or associated with the AAV9-CMV-Myc-SelenonSec-Flag-UTR775 vector (i.e., with the SELENON transgene) were normalized to PPI A and RPS18 housekeeping genes. As shown in FIG. 15, the mRNA levels of the endogenous murine Selenon were slightly reduced in Selenon- KO animals suggesting a possible decay of the mutant transcripts.

[0693]

[0694]

[0695] The human SELENON transgene showed robust expression in quadriceps of both WT and Selenon-KO injected mice. Interestingly, transgene expression was almost undetectable in the liver. The AAV9-derived expression of SELENON was also confirmed at the protein level by western blot (FIGs. 16A and 16B). An anti-myc antibody was used to detect the human SELENON protein, which included an N-terminal myc tag, because commercially available SELENON antibodies worked poorly. Expression of SELENON protein was undetectable in the liver, but robust expression was observed in the quadriceps muscle, thereby confirming the qRT- PCR results (FIG. 17).

[0696] Three mice (BL3077, BL3112, BL2990) having low, medium and high expression of SELENON in their quadriceps, were selected to assess transgene expression across different muscle groups and tissues. A Western Blot was performed to assay expression levels of transduced SELENON protein in various tissues (brain, lung, liver, kidney, spleen, heart, tibialis anterior (TA), paraspinal muscle, triceps, and diaphragm) of the 3 mice (FIG. 18). For the most part the expression of the transgene in different muscles of these three mice matched the quadriceps. The only deviation was the diaphragm of BL3077 that showed a higher expression compared to the other 2 mice, while the quadriceps of this mouse had the lowest transgene expression.

[0697] Next, expression levels of SELENON protein in HEK293T or C2C12 cells transfected with plasmid pAAV-CMV-Myc-Selenon-Flag-UTR775 (FIG. 20) or pAAV-MHCK7-selenon- UTR775 (FIG. 21) using lipofectamine was assayed (FIG. 19). Both CMV and MHCK7 were shown to drive a strong expression of the full length SELENON protein in HEK293T cells, with CMV leading to a greater expression. However, in the mouse muscle cell line C2C12 the CMV promotor showed a very low expression (undisguisable from the endogenous SELENON expression). MHCK7 which is synthetic muscle specific promoter showed a stronger expression than CMV in C2C12 cells, and was thus selected for further experimentation.

[0698] In conclusion, it was shown that the described construct (CMV-Myc-Selenon-Sec-Flag- UTR775) could be successfully packaged in an AAV9 capsid and that the latter could infect mice in vivo and drive robust expression of full length human SELENON mRNA and protein in skeletal muscle.

[0699] Example 3: Administration of SELENON with a MHCK7 Promoter to Mice

[0700] Sixteen mice (6 WT and 10 Selenon-KO) were injected at 4 weeks of age with 1E+14 vg / kg of AAV9-CMV-Myc-SelenonSec-Flag-UTR775. Table 4 provides details relating to representative mice that received injections. The KO mice contained a deletion of Exon 9 of the Selenon gene and, therefore, expressed an mRNA transcript from the altered gene but no SELENON polypeptide. The mice were sacrificed at 13 weeks of age (9 weeks post injection) and the expression of the SELENON transgene was assayed using immunoblot (FIG. 22). In each lane, 30 microgram of protein extracted from the gastrocnemius muscle was loaded and detected with a custom made anti-SELENON antibody (Rabbit #3882) at 1 :500 dilution. The secondary antibody starB right™ Blue 700 Goat Anti-rabbit IgG was used at 1 :5000. hFAB™ Rhodamine labeled anti-Tubulin primary antibody (Biorad# 12004165) was used at 1 : 10,000 to detect tubulin and was used to normalize and compare the expression of SELENON from different lanes. The assay indicated that the construct was well expressed in mice, and was indeed responsible for expression of SELENON at levels much higher than that found in WT mice.

[0701]

[0702] Next, 4 mice were injected with either saline, AAV9-MHCK7-Hu.SELENON-UTR775 (lot#4965) 1E+14 Vg / kg, or myoAAV4A-MHCK7-Hu.SELENON-UTR775 (lot#5545) 1E+13 Vg / kg and sacrificed at 6 weeks (2 -weeks treatment). Table 5 provides details relating to representative mice that received injections.

[0703] Table 5. Information relating to mice injected with either saline, AAV9-MHCK7- Hu.SELENON-UTR775 (lot#4965) 1E+14 Vg / kg, or myoAAV4A-MHCK7-Hu.SELENON- UTR775 (lot#5545) 1E+13 Vg / kg. The abbreviation “DOB” indicates “date of birth,” the abbreviation “DOD” indicates “date of death,” the abbreviation “WT” indicates “wild type SELENON polynucleotide sequence,” and the abbreviation “KO” indicates “ SELENON knockout,” and the abbreviations “M” and “F” indicate “male” and “female,” respectively. SELENON knockout mice contained a deletion of exon 9 of the SELENON gene / polynucleotide.

[0704] A Western Blot was next performed to assay protein expression in the mice (FIG. 23).

[0705] Protein from gastrocnemius, tibialis anterior, quadriceps, diaphragm, heart, liver and brain were extracted from each mouse. In each lane 30 micrograms of protein were loaded and detected with a custom made anti-SELENON antibody (Rabbit #3882) at 1 :500 dilution. The secondary antibody starB right™ Blue 700 Goat Anti-rabbit IgG was used at 1 :5000. hFAB™ Rhodamine labeled anti-Tubulin primary antibody (Biorad# 12004165) was used at 1 : 10,000 to detect tubulin and was used to normalize and compare the expression of SELENON from different lanes. The results indicated that the myoAAV4A was able to induce expression of SELENON in the mice at levels at least equivalent to that of the AAV9 encapsidated construct, despite a magnitude lower level of administration. MyoAAV capsids thus represent a promising group of capsids for use in expressing SELENON. Materials and Methods

[0706] The following constructs and capsids were used in the preceding Examples.

[0707] >pAAV-MHCK7-Hu.SELENON-UTR775 (gtgtccac was added before KOZAK) cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccgg cctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttctagacggaagagaaggtgacccttacc cagttgttcaactcacccttcagattaaaaataactaaggtaagggcctgggtaggggaggtggtgtgagacggtcctgtctctcctctatctg cccatcggccctttggggaggaggaatgtgcccaaggactaaaaaaaggccctggagccagaggggcgagggcagcagacctttcatg ggcaaacctcagggctgctggccactacgggtctaggctgcccatgtaaggaggcaaggcctggggacacccgagatgcctggttataat taacccagacatgtggctgcccccccccccccaacacctgctgcctgctaaaaataaccctgtccctggtggatatcaaggctgtggggga ctgagggcaggctgtaacaggcttgggggccagggcttatacgtgcctgggactcccaaagtattactgttccatgttcccggcgaagggc cagctgtcccccgccagctagactcagcacttagtttaggaaccagtgagcaagtcagcccttggggcagcccatacaaggccatggggc tgggcaagctgcacgcctgggtccggggtgggcacggtgcccgggcaacgagctgaaagctcatctactctcaggggcccctccctgg ggacagcccctcctggctagtcacaccctgtaggctcctctatataacccaggggcacaggggctgccctcattctaccaccacctccaca gcacagacagacactcaggagccagccagcccagtgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactg ggcttgtcgagacagagaagactcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccacgcc accATGGGCCGGGCCCGGCCGGGCCAACGCGGGCCGCCCAGCCCCGGCCCCGCCGCG

[0708] CAGCCTCCCGCGCCACCGCGCCGCCGCGCCCGTTCCCTGGCGCTGCTCGGAGCCCTG

[0709] CTGGCCGCCGCCGCTGCCGCCGCCGTCCGGGTCTGCGCCCGCCACGCCGAGGCCCA

[0710] GGCGGCCGCGCGGCAGGAACTGGCGCTGAAGACCCTGGGGACAGATGGCCTTTTTC

[0711] TCTTTTCCTCCTTGGACACTGACGGGGATATGTACATCAGCCCTGAGGAGTTCAAAC

[0712] CCATTGCTGAGAAGCTAACAGGGTCAACTCCCGCGGCCAGCTGCGAGGAGGAGGAG

[0713] TTGCCCCCTGACCCTAGCGAGGAGACGCTCACCATAGAAGCCCGATTCCAGCCTCTG

[0714] CTCCCGGAGACCATGACCAAGAGCAAAGATGGCTTCCTAGGGGTCTCCCGCCTCGC

[0715] CCTGTCCGGCCTCCGAAACTGGACAGCCGCCGCCTCACCAAGTGCAGTGTTTGCCAC

[0716] CCGCCACTTCCAGCCCTTCCTTCCCCCGCCAGGCCAGGAGCTGGGTGAGCCCTGGTG

[0717] GATCATCCCCAGTGAGCTGAGCATGTTCACTGGCTACCTGTCCAACAACCGCTTCTA

[0718] TCCACCGCCGCCCAAGGGCAAGGAGGTCATCATCCACCGGCTCCTGAGCATGTTCCA

[0719] CCCTCGGCCCTTTGTGAAGACCCGCTTTGCCCCTCAGGGAGCTGTGGCCTGCCTGAC

[0720] TGCCATCAGCGACTTCTACTACACTGTGATGTTCCGGATCCATGCCGAGTTCCAGCT

[0721] CAGTGAGCCGCCCGACTTCCCCTTTTGGTTCTCCCCTGCTCAGTTCACCGGCCACATC

[0722] ATCCTCTCCAAAGACGCCACCCACGTCCGCGACTTCCGGCTCTTCGTGCCCAACCAC

[0723] AGGTCTCTGAATGTGGACATGGAGTGGCTTTACGGGGCCAGTGAAAGCAGCAACAT

[0724] GGAGGTGGACATCGGCTACATACCCCAGATGGAGCTGGAGGCCACGGGCCCCTCTG

[0725] TGCCCTCCGTGATCCTGGATGAGGATGGCAGCATGATCGACAGCCACCTGCCTTCAG

[0726] GGGAGCCCCTGCAGTTTGTGTTTGAGGAGATCAAGTGGCAGCAGGAGCTGAGCTGG GAGGAGGCTGCCCGGCGCCTGGAGGTGGCCATGTACCCCTTCAAGAAGGTCTCCTA

[0727] CTTGCCGTTCACTGAGGCCTTCGACCGAGCCAAGGCTGAGAACAAGCTGGTGCACTC

[0728] AATCCTGCTGTGGGGGGCCCTGGATGACCAGTCCTGCTGAGGTTCAGGGCGGACTCT

[0729] CCGGGAGACTGTCCTGGAAAGTTCGCCCATCCTCACCCTGCTCAACGAGAGCTTCAT

[0730] CAGCACCTGGTCCCTGGTGAAGGAGCTGGAGGAACTGCAGAACAACCAGGAGAACT

[0731] CGTCCCACCAGAAGCTGGCTGGCCTGCACCTGGAGAAGTACAGCTTCCCCGTGGAG

[0732] ATGATGATCTGCCTGCCCAATGGCACCGTGGTCCATCACATCAATGCCAACTACTTC

[0733] TTGGACATCACCTCCGTGAAGCCCGAGGAAATCGAGAGCAATCTCTTCAGCTTCTCA

[0734] TCCACCTTTGAAGACCCGTCCACGGCCACCTACATGCAGTTCctgaaggagggactccggcgtgg cctgcccctcctccagccctagggtaccgcctaccttagcacagggtctctgcaggactgcgggagccagcgctcctgccgcccctcttgc ccctcagaccttgcatccacagaagcacaacccagccaaacaccacagccttctccagagccggcactgtcccggcaaccaggggtgcc ccaggctagctcttctacctctggggcaccacggactccccttggccactcttgggactttggtccacgtcctgagccactgaccacggcca gtctctctttttatatgtgcagaaaagtgtttttacacaaactttctcatggtttgtaggtatttttttataaccccagtgctgaggagaaaggaggg gcagtggcttccccggcagcagccccatgatggctgaatccgaaatcctcgatgggtccagcttgatgtctttgcagctgcacctatgggaa gaagtagtcctctcttccttctcctcttcagctttttaaaaacagtcctcagaggatccatgatccccagcactgtcccatcctccacaaaggcc cacaggcatgcctgtttggtccacgtcctgagccactgaccacggccagtctctctttttatatgtggggcagtggcttccccggcagcagcc ccatgatggctgaatccgaaatcctcgatgggtccagcttgatgtctttgcagctgcacctatgggaagaagtagtcctctcttccttctcctctt cagctttttaaaaacagtcctcagaggatccatgatccccagcactgtcccatcctcgaattcggccgcttcgagcagacatgataagataca ttgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagc tgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggagatgtgggaggttttttaaagcaagtaaaacctctac aaatgtggtaaaatcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggt cgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggtattttctcc ttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtg gttacgcgcagcgtgaccgctacacttgccagcgccttagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttcc ccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcac gtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaaca ctcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgc gaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgcc aacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcag aggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttctta gacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaata accctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttc ctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaaca gcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattg acgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggat ggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccga aggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgac gagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaatt aatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccg gtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggc aactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatacttt agattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttcca ctgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccg ctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttc ttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgcc agtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgc acacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaaggga gaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatcttt atagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaac gcggcctttttacggttcctggccttttgctggccttttgctcacatgt

[0735] In the above sequence, the SELENON polypeptide is encoded by the CAPTIAL

[0736] LETTERS, and the gtgtccac insertion is underlined.

[0737] >MyoAAV-4A modified from pAAV2 / 9n (Addgene #112865) tcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatg tgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactca ctatagggcgaattgggtaccgggccccccctcgatcgaggtcgacggtatcgggggagctcgcagggtctccattttgaagcgggaggt ttgaacgcgcagccgccatgccggggttttacgagattgtgattaaggtccccagcgaccttgacgagcatctgcccggcatttctgacagc tttgtgaactgggtggccgagaaggaatgggagttgccgccagattctgacatggatctgaatctgattgagcaggcacccctgaccgtgg ccgagaagctgcagcgcgactttctgacggaatggcgccgtgtgagtaaggccccggaggctcttttctttgtgcaatttgagaagggaga gagctacttccacatgcacgtgctcgtggaaaccaccggggtgaaatccatggttttgggacgtttcctgagtcagattcgcgaaaaactgat tcagagaatttaccgcgggatcgagccgactttgccaaactggttcgcggtcacaaagaccagaaatggcgccggaggcgggaacaagg tggtggatgagtgctacatccccaattacttgctccccaaaacccagcctgagctccagtgggcgtggactaatatggaacagtatttaagcg cctgtttgaatctcacggagcgtaaacggttggtggcgcagcatctgacgcacgtgtcgcagacgcaggagcagaacaaagagaatcag aatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggagctggtcgggtggctcgtggacaaggggattacct cggagaagcagtggatccaggaggaccaggcctcatacatctccttcaatgcggcctccaactcgcggtcccaaatcaaggctgccttgg acaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagcagcccgtggaggacatttccagcaatcgg atttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctgggatgggccacgaaaaagttcggcaagaggaac accatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatagcccacactgtgcccttctacgggtgcgtaaact ggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggaggggaagatgaccgccaaggtcgtggagt cggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggcccagatagacccgactcccgtgatcgtc acctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagcagccgttgcaagaccggatgttcaaatttg aactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagactttttccggtgggcaaaggatcacgtggttga ggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtgagcccaaacgggtg cgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacaggtaccaaaacaaatgttctcgtcacgtg ggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaaatatctgcttcactcacggacagaaagactgtttag agtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgtgctacattcatcatatcatgggaaaggtgcca gacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataaatgatttaaatcaggtATGGCTGCCG

[0738] ATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGT

[0739] GGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAAC

[0740] GCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGAC

[0741] AAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCT

[0742] ACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGAC

[0743] GCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCG

[0744] AGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGC

[0745] GGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGG

[0746] ACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAAT

[0747] TTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACC

[0748] TCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACC

[0749] AGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGC

[0750] ATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGG

[0751] GCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGA

[0752] GGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGAC

[0753] TTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAAC

[0754] AACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAA

[0755] GAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGT

[0756] CCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGA

[0757] GGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCT

[0758] GACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATA

[0759] TTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGA

[0760] GAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGA

[0761] ATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGAC

[0762] AGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAG

[0763] GGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGT

[0764] GACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAA TGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAG

[0765] AGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAA

[0766] GAGACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAAC

[0767] TACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACAAACCACCAGTCAA

[0768] ACTCCAGAGGAGACTACAACTCCTTGGCACAGGCGCAGACCGGTTGGGTTCAAAAC

[0769] CAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACC

[0770] CATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGG

[0771] AGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACC

[0772] TGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTA

[0773] TTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCA

[0774] AGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTG

[0775] AATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGAT

[0776] ACCTGACTCGTAATCTGTAAttgcttgttaatcaataaaccgtttaattcgtttcagttgaactttggtctctgcgaagggcga attcgtttaaacctgcaggactagaggtcctgtattagaggtcacgtgagtgttttgcgacattttgcgacaccatgtggtcacgctgggtattta agcccgagtgagcacgcagggtctccattttgaagcgggaggtttgaacgcgcagccgccaagccgaattctgcagatatccatcacact ggcggccgctcgactagagcggccgccaccgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcat ggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgccta atgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaa cgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcgg tatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaag gccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcaga ggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttacc ggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaa gctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgact tatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaacta cggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaaca aaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacgg ggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaa tgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatt tcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcg agacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgc ctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgt ggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcg gttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtc atgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccgg cgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatct taccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaa caggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcattta tcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccac ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttat aaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagg gcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatc ggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaagga gcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgt cccat

[0777] In the above sequence, the MyoAAV-4A capsid is encoded by the CAPTIAL LETTERS, and the differences from pAAV2 / 9n are additionally underlined.

[0778] Other Embodiments

[0779] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adapt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0780] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0781] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:

1. A polynucleotide comprising a nucleic acid sequence encoding a selenoprotein polypeptide and a 3' untranslated region (UTR) comprising two or more selenocysteine insertion sequence(SECIS) elements.

2. The polynucleotide of claim 1, wherein the selenoprotein polypeptide is a SELENON polypeptide.

3. The polynucleotide of claim 2, wherein the SELENON polypeptide or fragment thereof has at least about 85% sequence identity to the following amino acid sequence:MGRARPGQRGPPSPGPAAQPPAPPRRRARSLALLGALLAAAAAAAVRVCARHAEAQAAARQELALKTLGTDGLFLFSSLDTDGDMYISPEEFKPIAEKLTGSTPAASCEEEELPPDPSEETLTIEARFQPLLPETMTKSKDGFLGVSRLALSGLRNWT7VXASPSAVFATRHFQPFLPPPGQELGEPWWI IPSELSMFTGYLSNNRFYPPPPKGKEVI IHRLLSMFHPRPFVKTRFAPQGAVACLTAISDFYYTVMFRIHAEFQLSEPPDFPFWFSPAQFTGHI ILSKDATHVRDFRLFVPNHRSLNVDMEWLYGASESSNMEVDIGYIPQMELEATGPSVPSVILDEDGSMIDSHLPSGEPLQFVFEEIKWQQELSWEE7XARRLEVAMYPFKKVSYLPFTEAFDRAKAENKLVHSILLWGALDDQSCUGSGRTLRETVLESSPILTLLNESFISTWSLVKELEELQNNQENSSHQKLAGLHLEKYSFPVEMMICLPNGTWHHINANYFLDITSVKPEEIESNLFSFSSTFEDPSTATYMQFLKEGLRRGLPLLQP.

4. The polynucleotide of any one of claims 1-3, wherein the nucleic acid sequence, or fragment thereof, has at least about 85% sequence identity to the following nucleic acid sequence:ATGGGCCGGGCCCGGCCGGGCCAACGCGGGCCGCCCAGCCCCGGCCCCGCCGCGCAGCCTCCCGCGCCACCGCGCCGCCGCGCCCGTTCCCTGGCGCTGCTCGGAGCCCTGCTGGCCGCCGCCGCTGCCGCCGCCGTCCGGGTCTGCGCCCGCCACGCCGAGGCCCAGGCGGCCGCGCGGCAGGAACTGGCGCTGAAGACCCTGGGGACAGATGGCCTTTTTCTCTTTTCCTCCTTGGACACTGACGGGGATATGTACATCAGCCCTGAGGAGTTCAAACCCATTGCTGAGAAGCTAACAGGGTCAACTCCCGCGGCCAGCTGCGAGGAGGAGGAGTTGCCCCCTGACCCTAGCGAGGAGACGCTCACCATAGAAGCCCGATTCCAGCCTCTGCTCCCGGAGACCATGACCAAGAGCAAAGATGGCTTCCTAGGGGTCTCCCGCCTCGCCCTGTCCGGCCTCCGAAACTGGACAGCCGCCGCCTCACCAAGTGCAGTGTTTGCCACCCGCCACTTCCAGCCCTTCCTTCCCCCGCCAGGCCAGGAGCTGGGTGAGCCCTGGTGGATCATCCCCAGTGAGCTGAGCATGTTCACTGGCTACCTGTCCAACAACCGCTTCTATCCACCGCCGCCCAAGGGCAAGGAGGTCATCATCCACCGGCTCCTGAGCATGTTCCACCCTCGGCCCTTTGTGAAGACCCGCTTTGCCCCTCAGGGAGCTGTGGCCTGCCTGACTGCCATCAGCGACTTCTACTACACTGTGATGTTCCGGATCCATGCCGAGTTCCAGCTCAGTGAGCCGCCCGACTTCCCCTTTTGGTTCTCCCCTGCTCAGTTCACCGGCCACATCATCCTCTCCAAAGACGCCACCCACGTCCGCGACTTCCGGCTCTTCGTGCCCAACCACAGGTCTCTGAATGTGGACATGGAGTGGCTTTACGGGGCCAGTGAAAGCAGCAACATGGAGGTGGACATCGGCTACATACCCCAGATGGAGCTGGAGGCCACGGGCCCCTCTGTGCCCTCCGTGATCCTGGATGAGGATGGCAGCATGATCGACAGCCACCTGCCTTCAGGGGAGCCCCTGCAGTTTGTGTTTGAGGAGATCAAGTGGCAGCAGGAGCTGAGCTGGGAGGAGGCTGCCCGGCGCCTGGAGGTGGCCATGTACCCCTTCAAGAAGGTCTCCTACTTGCCGTTCACTGAGGCCTTCGACCGAGCCAAGGCTGAGAACAAGCTGGTGCACTCAATCCTGCTGTGGGGGGCCCTGGATGACCAGTCCTGCTGAGGTTCAGGGCGGACTCTCCGGGAGACTGTCCTGGAAAGTTCGCCCATCCTCACCCTGCTCAACGAGAGCTTCATCAGCACCTGGTCCCTGGTGAAGGAGCTGGAGGAACTGCAGAACAACCAGGAGAACTCGTCCCACCAGAAGCTGGCTGGCCTGCACCTGGAGAAGTACAGCTTCCCCGTGGAGATGATGATCTGCCTGCCCAATGGCACCGTGGTCCATCACATCAATGCCAACTACTTCTTGGACATCACCTCCGTGAAGCCCGAGGAAATCGAGAGCAATCTCTTCAGCTTCTCATCCACCTTTGAAGACCCGTCCACGGCCACCTACATGCAGTTCCTGAAGGAGGGACTCCGGCGTGGCCTGCCCCTCCTCCAGCCCTAG.

5. The polynucleotide of any one of claims 1-4, wherein one or more of the SECIS elements, or a fragment thereof, has at least about 85% sequence identity to a sequence listed in FIG. 3B, or to one of the following sequences:SECIS element 1CACGGACCCCATGGCAGGGGTGGCGTCTTCTGCATGATCCGCTCTGGTCAAACCCTTCCAGGCCAGCCAGAGTGGGGATGGTCTGAGGGGCCAGCCCTTAGTGCAT;SECIS element 2CACGGACCCCATGGCAGGGGTGGCGTCTTCATGATCCGCTCTGGTCAAACCCTTCCAGGCCAGCCAGAGTGGGGAGCCTGTCTGAGGGGCCAGCCCTTAGTGCAT;SECIS element 3CACGGACCCCATGGCAGGGGTGGCGTCTTCATGAGGGAGGGGCCCCAAACCCTTCCAGGCCAGGACCTCCCCTGAGCCTGTCTGAGGGGCCAGCCCTTAGTGCAT;SECIS element 4CCTGCCAGCCGCCCTGGCCCTGGTCACTGCATGATCCGCTCTGGTAAAGCCCTTGTGGGCGCCAGAGTGGGGATGGTCTGTGACCTGCTGGGAAGGCAGGC;SECIS element 5CCTGCCAGCCGCCCTGGCCCTGGTCACTGCATGAGGGAGGGGCCCAAAGCCCTTGTGGGCGGACCTCCCCTGATGGTCTGTGACCTGCTGGGAAGGCAGGC;SECIS element 6CCTGCCAGCCGCCCTGGCCCTGGTCACATGAGGGAGGGGCCCAAAGCCCTTGTGGGCGGACCTCCCCTGAGCCTGTCTGTGACCTGCTGGGAAGGCAGGC;SECIS element 7CCTGCCAGCCGCCCTGGCCCTGGTCACATGATCCGCTCTGGTCAAACCCTTCCAGGCCAGCCAGAGTGGGGAGCCTGTCTGTGACCTGCTGGGAAGGCAGGC;SECIS element 8CACGGACCCCATGGCAGGGGTGGCGTCTTCTGCATGAGGGAGGGGCCCAAAGCCCTTGTGGGCGGACCTCCCCTGATGGTCTGAGGGGCCAGCCCTTAGTGCAT;SECIS element 9CACGGACCCCATGGCAGGGGTGGCGTCTTCTGCATGATCCGCTCTGGTCAAACCCTTCCAGGCCAGCCAGAGTGGGGATGGTCTGAGGGGCCAGCCCTTAGTGCAT;SECIS element 10CCTGCCAGCCGCCCTGGCCCTGGTCACATGAGGGAGGGGCCCAAAGCCCTTGTGGGCGGACCTCCCCTGAGCCTGTCTGTGACCTGCTGGGAAGGCAGGC;SECIS element 11CACGGACCCCATGGCAGGGGTGGCGTCTTCATGATCCGCTCTGGTAAAGCCCTTGTGGGCGCCAGAGTGGGGAGCCTGTCTGAGGGGCCAGCCCTTAGTGCAT; andSECIS element 12CCTGCCAGCCGCCCTGGCCCTGGTCACTGCATGAGGGAGGGGCCCCAAACCCTTCCAGGCCAGGACCTCCCCTGATGGTCTGTGACCTGCTGGGAAGGCAGGC6. The polynucleotide of any one of claims 1-5, wherein the SECIS elements are Type 1 and / orType 2 SECIS elements.

7. The polynucleotide of any one of claims 1-6, wherein the SECIS elements are human SECIS elements.

8. The polynucleotide of any one of claims 1-7, wherein one or more of the SECIS elements has at least about 85% sequence identity to the following sequence:SELENON SECIS elementGGCAGCAGCCCCATGATGGCTGAATCCGAAATCCTCGATGGGTCCAGCTTGATGTCTTTGCAGCTGCA .

9. The polynucleotide of any one of claims 1-8, wherein the polynucleotide comprises DNA orRNA.

10. The polynucleotide of any one of claims 1-9, wherein a polynucleotide sequence of the 3’ UTR comprises a SELENON, SELENOW, SELENOM, or TXRD2 3’ UTR polynucleotide sequence.

11. The polynucleotide of any one of claims 1-10, wherein the 3’ UTR is less than about 1500 bp, 1250 bp, 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, or 300 bp in length.

12. The polynucleotide of any one of claims 1-11, wherein the 3’ UTR is less than about 800 bp in length.

13. The polynucleotide of any one of claims 1-12, wherein the polynucleotide comprises a promoter.

14. The polynucleotide of claim 13, wherein the promoter is a constitutive promoter.

15. The polynucleotide of claim 13, wherein the promoter is an inducible promoter.

16. The polynucleotide of claim 14, wherein the promoter is a CMV promoter.

17. The polynucleotide of claim 13, wherein the promoter is a muscle specific promoter.

18. The polynucleotide of claim 17, wherein the promoter is a MHCK7 promoter.

19. The polynucleotide of any one of claims 1-18, wherein the polynucleotide further comprises adeno-associated virus (AAV) inverted terminal repeats (ITR).

20. The polynucleotide of any one of claims 1-19, wherein the 3’ untranslated region (UTR) comprises about 355 bp from the 3’ UTR of a TXRND2 gene, about 411 bp from the 3’ UTR of a SelW gene , about 198 bp from the 3’ UTR of a SelM gene, about 544 bp from the 3’ UTR of a Selenon gene, or about 775 bp from the 3’ UTR of a Selenon gene.

21. A mammalian expression vector comprising the polynucleotide of any one of claims 1-20.

22. The mammalian expression vector of claim 21, wherein the vector is a viral particle.

23. The mammalian expression vector of claim 22, wherein the viral particle is an adeno- associated viral (AAV) particle.

24. A recombinant adeno-associated virus (rAAV) particle comprising the polynucleotide of any one of claims 1-20.

25. The rAAV particle of claim 24, wherein the rAAV particle is an rAAV9 virus particle.

26. The rAAV particle of claim 24, wherein the rAAV particle comprises a muscle-trophic viral capsid.

27. The rAAV particle of claim 26, wherein the muscle-trophic viral capsid is a myoAAV viral capsid.

28. The rAAV particle of claim 26 or 27, wherein the muscle-trophic viral capsid is myoAAV4A.

29. The polynucleotide of any one of claims 1-20, the vector of any one of claims 21-23, or the particle of any one of claims 24-28, wherein the selenoprotein is selected from the group consisting of: GPX1, GPX2, GPX3, GPX4, GPX6, TXNRD1 (TRI, TRXR1), TXNRD2 (TRXR2, TR3), TXNRD3 (TGR, TRXR3, TR2), DIO1 (D1), DIO2 (D2), DIO3 (D3), MSRB1 (SELR, SELX, SEPX1), SEPHS2 (SPS2) SELENOF (SEP 15), SELENOH (SELH), SELENOI (SELI, EPT1), SELENOK (SELK), SELENOM (SEEM, SEPM), SELENON (SEPN1, SELN), SELENOO (SELO), SELENOP (SEPPI, SEP, SELF, SEPP), SELENO S (SELS, SEPS1, VIMP), SELENOT (SELT) , SELENO V (SELV), and SELENOW ( SELW, SEPWP).

30. A serotype 9 recombinant adeno-associated virus (rAAV9) particle comprising in order from 5’ to 3’ : a mammalian promoter, a SELENON polynucleotide, and a 3’ untranslated region (UTR) comprising two SELENON selenocysteine insertion sequence (SECIS) elements, wherein the 3’ untranslated region (UTR) is less than 800 bp in length.

31. A serotype myoAAV recombinant adeno-associated virus particle comprising in order from 5’ to 3’ : a mammalian promoter, a SELENON polynucleotide, and a 3’ untranslated region(UTR) comprising two SELENON selenocysteine insertion sequence (SECIS) elements, wherein the 3’ untranslated region (UTR) is less than 800 bp in length.

32. The recombinant adeno-associated virus particle of claim 30 or 31, wherein the mammalian promoter is a cytomegalovirus, MHCK7, or CK8 promoter.

33. A cell comprising the expression vector of any one of claims 21-23 or 29 or the particle of any one of claims 24-32.

34. A pharmaceutical composition comprising the polynucleotide of any one of claims 1-20 or 29, the vector of any one of claims 21-23 or 29, the particle of any one of claims 24-32, and / or the cell of claim 33, and a pharmaceutically acceptable excipient.

35. A method for increasing selenoprotein expression in a cell, the method comprising contacting the cell with the polynucleotide of any one of claims 1-20 or 29, the vector of any one of claims 21-23 or 29, and / or the particle of any one of claims 24-32.

36. The method of claim 35, wherein the cell is a cell in vitro or in vivo.

37. The method of claim 35 or claim 36, wherein the cell is a mammalian cell.

38. The method of any one of claims 35-37, wherein the cell is a muscle cell.

39. The method of any one of claims 35-38, wherein the method increases levels of calcium ions in the sarcoplasmic reticulum of a muscle cell.

40. A method of treating a subject having a congenital selenoprotein-associated disease, the method comprising administering to the subject an effective amount of the pharmaceutical composition of claim 34.

41. A method of treating a subject having a congenital selenoprotein-associated disease, the method comprising administering to the subject an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a selenoprotein polypeptide, and a 3' untranslated region (UTR) comprising two or more selenocysteine insertion sequence (SECIS) elements, thereby treating the congenital selenoprotein-associated disease.

42. A method of treating a subject having a congenital myopathy, the method comprising administering to the subject an effective amount of a polynucleotide encoding, in order from 5' to 3': a regulatory element, a selenoprotein polypeptide, and a 3' untranslated region (UTR) comprising two or more selenocysteine insertion sequence (SECIS) elements, thereby treating the congenital myopathy.

43. The method of any one of claims 40-42, wherein the polynucleotide is administered systemically or locally.

44. The method of any one of claims 40-43, wherein the subject is a mammal.

45. The method of any one of claims 40-44, wherein the selenoprotein-associated disease is selected from the group consisting of: congenital fiber type disproportion, congenital nondystrophic myopathy, congenital muscular dystrophy, Desmin-Related Myopathy with Mallory Body-like Inclusions, minicore myopathy, multicore myopathy, multiminicore myopathy, rigid spine muscular dystrophy, sedaghatian-type spondylometaphyseal dysplasia (SSDM), epilepsy, dilated cardiomyopathy, familial glucocorticoid deficiency, and spastic paraplegia.

46. The method of any one of claims 40-45, wherein the method reduces hypotonia, proximal muscle weakness, morphologic abnormalities in skeletal muscle, restrictive respiratory syndrome, respiratory weakness, spinal rigidity, neuronal degeneration, ataxia, seizures, skeletal disorder, brain atrophy, decreases in respiratory function, and / or scoliosis.

47. The method of any one of claims 40-46, wherein the selenoprotein is selected from the group consisting of: GPX1, GPX2, GPX3, GPX4, GPX6, TXNRD1, TXNRD2 (TXRD2), TXNRD3, DIO1, DIO2, DIO3, SEPHS2, SEPS1, SEPPI, SEP15, SEPN1 (SELENON), SEPX1, SEPW1 (SELENO W), SEPTI, SELH, SELI, SELK, SELM (SELENOM), SELO, and SELV.

48. The method of claim 47, wherein the selenoprotein is selected from the group consisting of: GPX4, TXNRD1, TXNRD2 (TXRD2), SEPN1 (SELENON), and SELI.

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