POLYNUCLEOTIDE COMPOSITIONS FOR THE TREATMENT OF PREMATURE TERMINATION DISEASES

FIELD OF THE INVENTION

[0001] The present disclosure relates to polynucleotide compositions comprising (a) a complementary oligonucleotide comprising at least 10 consecutive nucleobases having at least 80% complementarity to a target gene having a premature UGA stop codon; (b) an optional linker selected from the group consisting of (i) an oligonucleotide and (ii) a chemical linker; and (c) an oligonucleotide comprising a SECIS element (selenocysteine insertion sequence), or pharmaceutically acceptable salts thereof. Methods of treatment and uses of the polynucleotide compositions are also provided herein.

BACKGROUND

[0002] Nonsense mutations occur in DNA when a sequence change gives rise to a stop codon rather than a codon specifying an amino acid. The presence of this new, premature, stop codon leads to production of a shortened protein that is either less active or, more likely, non-functional. A number of conditions are associated with such premature stop codons. Treatments for conditions associated with premature stop codons are required.

SUMMARY OF THE DISCLOSURE

[0003] Polynucleotide compositions comprising: (a) a complementary oligonucleotide comprising at least 10 consecutive nucleobases having at least 80% complementarity to a target gene having a premature UGA stop codon; (b) an optional linker, selected from the group consisting of: (i) an oligonucleotide and (ii) a chemical linker; and (c) an oligonucleotide comprising a SECIS element, or pharmaceutically acceptable salts thereof, are provided herein.

[0004] In some aspects, the target gene is selected from the group consisting of genes listed in Table 1. In some aspects, the target gene is DNA that encodes a pre-mRNA. In some aspects, the target gene is RNA corresponding to any one of the DNA of Table 1. In some aspects, the target gene is pre-mRNA. In some aspects, the target gene is mature RNA. [0005] In some aspects, the complementary oligonucleotide comprises at least 10 consecutive nucleobases having at least 90% complementarity to the target gene. In some aspects, the complementary oligonucleotide comprises at least 15 consecutive nucleobases.

[0006] In some aspects, the complementary oligonucleotide is single-stranded. In some aspects, the complementary oligonucleotide comprises at least one duplex region. In some aspects, the complementary oligonucleotide contains at least one hairpin loop. In some aspects, the complementary oligonucleotide hybridizes to a region within 15,000 nucleotides of a premature stop codon within the target gene. In some aspects, the complementary oligonucleotide hybridizes 3' to the premature stop codon of the target gene. In some aspects, the complementary oligonucleotide hybridizes to a 3' untranslated region of the target gene. In some aspects, the complementary oligonucleotide hybridizes 5' to the premature stop codon of the target gene. In some aspects, the complementary oligonucleotide hybridizes to a 5' untranslated region of the target gene.

[0007] In some aspects, the polynucleotide composition comprises a linker. In some aspects, the linker is an oligonucleotide. In some aspects, the linker is 1-50 consecutive linked nucleotides. In some aspects, the linker is single-stranded. In some aspects, the linker comprises at least one duplex region. In some aspects, the linker comprises at least one hairpin. In some aspects, the linker of the polynucleotide compositions described herein comprise a linker comprising a nucleotide sequence selected from the group consisting of UAU, UGUUAA, GCUGAUAGA, TAT, TGTTAA, SEQ ID NOs: 171-173, and SEQ ID NOs: 184-186. In some aspects, the linker of the polynucleotide compositions described herein comprise a linker consisting of a nucleotide sequence selected from the group consisting of UAU, UGUUAA, GCUGAUAGA, TAT, TGTTAA, SEQ ID NOs: 171-173, and SEQ ID NOs: 184-186.

[0008] In some aspects, the polynucleotide composition comprises a linker that is a chemical linker. In some aspects, the chemical linker is cleavable. In some aspects, the cleavable linker is an enzymatically-cleavable peptide linker. In some aspects, the enzymatically-cleavable peptide linker comprises val-cit linkage. In some aspects, the cleavable linker is an acid sensitive hydrazone linker. In some aspects, the cleavable linker is a glutathione-sensitive disulfide linker.

[0009] In some aspects, the polynucleotide composition comprises a linker that is a chemical linker that is non-cleavable. In some aspects, the non-cleavable linker is succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1-1 carboxylate (SMCC). [0010] In some aspects, the polynucleotide composition comprises a linker that is a chemical linker that is a polyethylene glycol linker. In some aspects, the polyethylene glycol linker is monodispersed. In some aspects, the polyethylene glycol linker is polydispersed. In some aspects, the polyethylene glycol is tetraethylene glycol.

[0011] In some aspects, the polynucleotide composition comprises a linker that is a chemical linker that comprises glycerol or a glycerol homolog of the formula HO — (CH2)o — CH(OH) — (CH ₂ ) _P — OH, wherein o and p independently are integers from 1 to 6, from 1 to 4 or from 1 to 3.

[0012] In some aspects, the polynucleotide composition comprises a linker that is a chemical linker that is l,3-diamino-2-hydroxypropane.

[0013] In some aspects, the polynucleotide composition comprises a linker that is a chemical linker that has a formula of HO — (CH2)m — C(O)NH — CH2 — CH(OH) — CH2 — NHC(O) — (CH2)m — OH, wherein m is an integer from 0 to 10, from 0 to 6, from 2 to 6 or from 2 to 4.

[0014] In some aspects, the polynucleotide composition comprises a linker that is a chemical linker that is selected from a group consisting of Azido-PEG12-NHS ester, 4- hydroxy-thalidomide, thalidomide-acid, thalidomide-O-PEG-acid, thalidomide-O-PEG- Amine, thalidomide-O-PEG-azide, thalidomide-O-PEG-NHS ester, thalidomide-O-PEG- propargyl, dBETl, thalidomide-O-PEG-t-butyl ester, thalimide-O-PEG-tosyl, 4-fluoro- thalidomide, pomalidomide, thalidomide-O-acetamido-C4-amine, thalidomide-O-amido- PEG4-azide, thalidomide-O-amido-PEG4-propargyl, pomalideomide-PEG-NH-Boc, pomalidomide-PEG-Ph-NH2, P131, ARV-825, pomalidomide-PEG-azide, pomalidomide 4'- PEG-azide, D-biotin-PEG-thalidomide, D-amino-PEG-thalidomide, VHL ligand 1, and EGFR PROTAC.

[0015] In some aspects, the polynucleotide composition does not include a linker.

[0016] In some aspects, the polynucleotide composition comprises an oligonucleotide comprising a SECIS element comprises at least one ribonucleotide. In some aspects, the polynucleotide composition comprises an oligonucleotide comprising a SECIS element comprises at least one deoxyribonucleotide. In some aspects, the oligonucleotide comprising a SECIS element comprises at least one duplex region. In some aspects, the oligonucleotide comprising a SECIS element comprises at least one hairpin. In some aspects, the oligonucleotide comprising a SECIS element has a Kd for SBP2 of less than 100 nM when measured by RNA electromobility shift assay. [0017] In some aspects, the polynucleotide composition comprises an oligonucleotide comprising the SECIS element comprises the sequence of TnnATGATGnCnnnnnCnnAAA, wherein: T is thymine; A is adenine; G is guanine; C is cytosine; and n is independently selected from thymine, adenine, guanine, cytosine, and uracil.

[0018] In some aspects, the polynucleotide composition comprises an oligonucleotide comprising the SECIS element comprises the sequence of ATGAnGnCnnnnnCCnAAAnCCTC, wherein: T is thymine; A is adenine; G is guanine; C is cytosine; and n is independently selected from thymine, adenine, guanine, cytosine, and uracil.

[0019] In some aspects, the polynucleotide composition comprises an oligonucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementarity to SEQ ID NOs: 5-31. In some aspects, the polynucleotide composition comprises an oligonucleotide comprising the SECIS element comprises one or more sequences selected from the group consisting of SEQ ID NOs: 5-31. In some aspects, the polynucleotide composition comprises an oligonucleotide comprising the SECIS element consisting one or more sequences selected from the group consisting of SEQ ID NOs: 5-31.

[0020] In some aspects, the polynucleotide composition comprises an oligonucleotide comprising the SECIS element comprises the sequence of UnnAUGAUGnCnnnnnCnnAAA, wherein: U is uracil; A is adenine; G is guanine; C is cytosine; and n is independent selected from thymine, adenine, guanine, cytosine, and uracil.

[0021] In some aspects, the polynucleotide composition comprises an oligonucleotide comprising the SECIS element comprises the sequence of AUGAnGnCnnnnnCCnAAAnCCUC, wherein: U is uracil; A is adenine; G is guanine; C is cytosine; and n is independent selected from thymine, adenine, guanine, cytosine, and uracil.

[0022] In some aspects, the polynucleotide composition comprises an oligonucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementarity to SEQ ID NOs. 32-58. In some aspects, the polynucleotide composition comprises an oligonucleotide comprising the SECIS element comprises one or more sequences selected from the group consisting of SEQ ID NOs. 32-58. In some aspects, the polynucleotide composition comprises an oligonucleotide comprising the SECIS element consisting one or more sequences selected from the group consisting of SEQ ID NOs. 32-58. [0023] In some aspects, the polynucleotide composition comprises an oligonucleotide comprising the SECIS element comprises 20-200, 30-190, 40-180, 50-170, 60-160, 70-150, 80-140, 90-130, 100-120, or 110 nucleotides.

[0024] In some aspects, one or more of the complementary oligonucleotide, linker that is an oligonucleotide, and oligonucleotide comprising a SECIS element of the polynucleotide composition comprises at least one alternative nucleobase. In some aspects, the alternative nucleobase is 5-methylcytosine, pseudouridine, 5-methoxyuridine, or combinations thereof.

[0025] In some aspects, one or more of the complementary oligonucleotide, linker that is an oligonucleotide, and oligonucleotide comprising a SECIS element of the polynucleotide composition comprises at least one alternative sugar moiety and/or internucleoside linkage. In some aspects, the alternative sugar moiety and/or internucleoside linkage is 2'-O-methyl, a bicyclic nucleic acid, morpholino, phosphorothioate, phosphorodiamidate morpholino, peptide nucleic acid, locked nucleic acid, 2'-fluoro, 2'0,4'C-ethylene-bridged nucleic acid, tricycle-DNA, tricycle-DNA phosphorothioate, 2'O-[2-(N-methylcarbamoyl)ethyl], 2'-O- methyl-phosphorothioate, 2'-O-methoxy-ethyl, or combinations thereof.

[0026] In some aspects, the polynucleotide composition comprises a total of 30-1000 nucleobases.

[0027] In some aspects, the polynucleotide composition further comprises a targeting moiety. In some aspects, the targeting moiety is selected from a group consisting of an antibody, cholesterol, peptide, aptamer, and combinations thereof. In some aspects, the targeting moiety is attached to the 5' end of the polynucleotide composition. In some aspects, the targeting moiety is attached to the 3' end of the polynucleotide composition. In some aspects, the targeting moiety is attached to the polynucleotide composition using a linker that is an oligonucleotide linker or a chemical linker. In some aspects, the linker is the same or different as the linker attaching the complementary oligonucleotide to the oligonucleotide comprising a SECIS element.

[0028] In some aspects, the polynucleotide composition is in a pharmaceutically acceptable salt form. In some aspects, the polynucleotide composition is in a free form.

[0029] In some aspects the polynucleotide composition is circular.

[0030] Vectors comprising a polynucleotide composition described herein are also provided. In some aspects, the vector is a viral vector. In some aspects, the vector is a retrovirus vector. In some aspects, the vector is an AAV vector. [0031] Pharmaceutical compositions comprising one or more of the polynucleotide compositions and/or vector described herein, and a pharmaceutically acceptable carrier or excipient are also provided. In some aspects, the pharmaceutical composition further comprises a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, a liposome, an exosome, or extracelluar vesicles.

[0032] Methods of increasing expression of a protein encoded by a gene containing a premature stop codon in a subject in need thereof, comprising administering to the subject one or more of the polynucleotide compositions, vectors, or pharmaceutical compositions described herein are also provided. In some aspects, there is an increase of at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, of functional expression of the protein when compared with a control.

[0033] Methods of treating, preventing, or delaying the progression of a condition caused by one or more premature stop mutations in a subject in need thereof, comprising administering to the subject one or more of the polynucleotide compositions, vectors, or pharmaceutical compositions described herein are also provided. In some aspects, the condition is a cancer. In some aspects, the condition is Rett syndrome, cystic fibrosis, autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, Duchenne muscular dystrophy, Becker's muscular dystrophy, cystinosis, tuberous sclerosis, colorectal cancer, choroideremia, laminopathies, retinitis pigmentosa, Stargardt disease, phenylketonuria, ataxia telangiectasia, fabry, niemann-Pick A/B, gangliosidosis type 1, MPS I-H, Hunter syndrome (MPS IIIB), Maroteaux-Lamy syndrome (MPS VI), neuronal ceroid lipofuscinoses (NCLs), hemophilia A, hemophilia B, spinal muscular atrophy, Leber congenital amaurosis (LCA) type 2, ocular coloboma, usher syndrome type 1, congenital aniridia, cancer, Greig cephalopolysyndactyly syndrome, achalasia, adrenocortical insufficiency, or alacrimia.

[0034] Methods of reducing the level of premature termination in a cell comprising contacting the cell with one or more of the polynucleotide compositions, vectors, or pharmaceutical compositions described herein are also provided. In some aspects, the cell is in a subject. In some aspects, the subject is a human. In some aspects, the subject is identified as having premature termination.

[0035] In some aspects, the polynucleotide composition, vector, or the pharmaceutical composition described herein is administered by oral, parenteral, intrathecal, intracerebroventricular, intraparenchymal, buccal, sublingual, intraocular, intravenous, intraperitoneal, intra-articular, subcutaneous, intramuscular, transepithelial, intranasal, intrapulmonary, intraparenchymal, topical, or inhalation route.

[0036] Polynucleotide compositions, vectors, or pharmaceutical compositions described herein for use in preventing or delaying the progression of a premature termination disease in a subject are also provided.

[0037] Use of one or more of the polynucleotide compositions, vectors, or pharmaceutical compositions provided herein in the preparation for a medicament for use in preventing or delaying the progression of a premature termination disease in a subject are also provided.

Definitions

[0038] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular aspects, and are not intended to limit the claimed technology, because the scope of the technology is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. For the purposes of the present invention, the following terms are defined below.

[0039] In this application, unless otherwise clear from context, (i) the term “a” can be understood to mean “at least one”; (ii) the term “or” can be understood to mean “and/or”; and (iii) the terms “including” and “comprising” can be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps.

[0040] The term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term "at least", and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21- nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers in the series or range. [0041] As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, an oligonucleotide with “no more than 3 mismatches to a target sequence” has 3, 2, 1, or 0 mismatches to a target sequence. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range.

[0042] As used herein, the term “administration” refers to the administration of a composition (e.g., a polynucleotide composition or a pharmaceutical composition comprising a polynucleotide composition as described herein) to a subject. Administration to a subject (e.g., to a human) can be by any appropriate route.

[0043] The term "target gene" as used herein refers to a gene which contains a premature stop codon mutation of UGA. The target gene can be DNA that encodes a pre-mRNA, pre- mRNA, or mature RNA. Non-limiting examples of target genes known to include premature stop codon mutations are MECP2, CFTR, PKD1, PKD2, PKHD1, DMD, CTNS, TSC1, TSC2, APC, Repl, LAMA2, LAMA3, LAMB1, LAMB2, LAMB3, LAMC2, RPGR, PAH, ATM, GLA, SMPD1, GLB1, IDUA, IDS, NAGLU, ARSB, CLN1, CLN2, FVIII, FIX, SMN2, RPE65, PAX2, LAMB1, RHO, RP2, ABCA4, PCDH15, USH1C, Pax6, APC, ATM, BRCA1, BRCA2, CDH1, CDKN2A, NF1, NF2, PTCH, TP53, VHL, GLI3, KRIT1, AAAS, SOX10, FOXC2, PRKAR1A, SMARCB1, PDCD10, TREM2.

[0044] As used herein, "SECIS element" refers to a cv.s-acting mRNA structure that is required for expression of selenocysteine (Sec)-containing proteins.

[0045] “G,” “C,” “A,” “T,” and “U” each generally stand for a naturally-occurring nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a base, respectively. However, it will be understood that the term "nucleotide" can refer to an alternative nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of oligonucleotides by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured herein.

[0046] The terms “nucleobase” and “base” include the purine (e.g. adenine and guanine) and pyrimidine (e.g. uracil, thymine, and cytosine) moiety present in nucleosides and nucleotides which form hydrogen bonds in nucleic acid hybridization. The term nucleobase also encompasses alternative nucleobases which can differ from naturally-occurring nucleobases, but are functional during nucleic acid hybridization. In this context, “nucleobase” refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as alternative nucleobases.

[0047] The term “nucleoside” refers to a monomeric unit of an oligonucleotide or a polynucleotide having a nucleobase and a sugar moiety. A nucleoside can include those that are naturally-occurring as well as alternative nucleosides, such as those described herein. The nucleobase of a nucleoside can be a naturally-occurring nucleobase or an alternative nucleobase. Similarly, the sugar moiety of a nucleoside can be a naturally-occurring sugar or an alternative sugar.

[0048] The term “alternative nucleoside” refers to a nucleoside having an alternative sugar or an alternative nucleobase, such as those described herein.

[0049] In some aspects the nucleobase moiety is modified by changing the purine or pyrimidine into a modified purine or pyrimidine, such as substituted purine or substituted pyrimidine, such as an “alternative nucleobase” selected from N6-methyladenosine, isocytosine, pseudoisocytosine, 5-methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uridine, 5-bromouridine 5-thiazolo-uridine, 2-thio-uridine, pseudouridine, 1- methylpseudouridine, 5-methoxyuridine, 2'-thio-thymine, inosine, diaminopurine, 6- aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0050] The nucleobase moieties can be indicated by the letter code for each corresponding nucleobase, e.g. A, T, G, C, or U, wherein each letter can include alternative nucleobases of equivalent function. In some aspects, e.g., for gapmers, 5-methyl cytosine LNA nucleosides can be used.

[0051] A “sugar” or “sugar moiety,” includes naturally occurring sugars having a furanose ring. A sugar also includes an “alternative sugar,” defined as a structure that is capable of replacing the furanose ring of a nucleoside. In some aspects, alternative sugars are non-furanose (or 4'-substituted furanose) rings or ring systems or open systems. Such structures include simple changes relative to the natural furanose ring, such as a six- membered ring, or can be more complicated as is the case with the non-ring system used in peptide nucleic acid. Alternative sugars can include sugar surrogates wherein the furanose ring has been replaced with another ring system such as, for example, a morpholino or hexitol ring system. Sugar moieties useful in the preparation of oligonucleotides having motifs include, without limitation, P-D-ribose, P-D-2'-deoxyribose, substituted sugars (such as 2', 5' and bis substituted sugars), 4'-S-sugars (such as 4'-S-ribose, 4'-S-2'-deoxyribose and 4'-S-2'- substituted ribose), bicyclic alternative sugars (such as the 2'-0 — CH2-4' or 2'-0 — (CH2)2-4' bridged ribose derived bicyclic sugars) and sugar surrogates (such as when the ribose ring has been replaced with a morpholino or a hexitol ring system). The type of heterocyclic base and internucleoside linkage used at each position is variable and is not a factor in determining the motif. In most nucleosides having an alternative sugar moiety, the heterocyclic nucleobase is generally maintained to permit hybridization.

[0052] A “nucleotide,” as used herein, refers to a monomeric unit of an oligonucleotide or polynucleotide that comprises a nucleoside and an internucleosidic linkage. The internucleosidic linkage can include a phosphate linkage. Similarly, “linked nucleosides” can be linked by phosphate linkages. Many “alternative internucleosidic linkages” are known in the art, including, but not limited to, phosphate, phosphorothioate, and boronophosphate linkages. Alternative nucleosides include bicyclic nucleosides (BNAs) (e.g., locked nucleosides (LNAs (e.g., A-LNA, 5mC L-NA, G-LNA, T-LNA) and constrained ethyl (cEt) nucleosides), peptide nucleosides (PNAs), phosphotriesters, phosphorothionates, phosphoramidates, and other variants of the phosphate backbone of native nucleoside, including those described herein.

[0053] An “alternative nucleotide,” as used herein, refers to a nucleotide having an alternative nucleoside or an alternative sugar, and an internucleoside linkage, which can include alternative nucleoside linkages.

[0054] The terms “oligonucleotide” and “polynucleotide,” as used herein, are defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides can be referred to as nucleic acid molecules or oligomers. Oligonucleotides are commonly made in the laboratory by solid-phase chemical synthesis followed by purification. When referring to a sequence of the oligonucleotide, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides. The oligonucleotide can be man-made. For example, the oligonucleotide can be chemically synthesized, and be purified or isolated. Oligonucleotide is also intended to include (i) compounds that have one or more furanose moieties that are replaced by furanose derivatives or by any structure, cyclic or acyclic, that can be used as a point of covalent attachment for the base moiety, (ii) compounds that have one or more phosphodiester linkages that are either modified, as in the case of phosphoramidate or phosphorothioate linkages, or completely replaced by a suitable linking moiety as in the case of formacetal or riboacetal linkages, and/or (iii) compounds that have one or more linked furanose-phosphodiester linkage moieties replaced by any structure, cyclic or acyclic, that can be used as a point of covalent attachment for the base moiety. The oligonucleotide can comprise one or more alternative nucleosides or nucleotides (e.g., including those described herein). It is also understood that oligonucleotide includes compositions lacking a sugar moiety or nucleobase but are still capable of forming a pairing with or hybridizing to a target sequence. “Oligonucleotide” refers to a short polynucleotide (e.g., of 100 or fewer linked nucleosides).

[0055] A "linker" or "linking group" is a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds. The polynucleotides disclosed herein can comprise one or more linkers capable of linking one or more oligonucleotides disclosed herein to one or more other oligonucleotides disclosed herein, and/or to any other oligonucleotide, and/or to any conjugate moiety. For example, a linker could be used to link an oligonucleotide disclosed herein to a polynucleotide that targets the gene of interest.

[0056] Linkers may be susceptible to cleavage ("cleavable linker") thereby facilitating release of the different oligonucleotides and/or different conjugate moieties disclosed herein. Such cleavable linkers may be susceptible, for example, to nuclease-induced cleavage, acid- induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at suitable conditions. Suitable cleavable linking groups for use in cleavable linkers include those which are sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together.

[0057] Alternatively, linkers may be substantially resistant to cleavage ("non-cleavable linker"). Such non-cleavable linkers can be any chemical moiety capable of linking one or more different oligonucleotides disclosed herein to one or more other oligonucleotides disclosed herein, and/or to any conjugate moiety in a stable, covalent manner and does not fall off under the categories listed above for cleavable linkers. Thus, non-cleavable linkers are substantially resistant to acid-induced cleavage, nuclease-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage and disulfide bond cleavage. Furthermore, non-cleavable refers to the ability of the chemical bond in the linker or adjoining to the linker to withstand cleavage induced by an acid, a nuclease, photolabile- cleaving agent, a peptidase, an esterase, or a chemical or physiological compound that cleaves a disulfide bond, at conditions under which the oligonucleotides disclosed herein do not lose their activity or intended purpose.

[0058] Conjugate moieties can be attached to the oligonucleotide directly or through a linking moiety (e.g. linker or tether). Linkers serve to covalently connect a third region, e.g. a conjugate moiety to an oligonucleotide (e.g. the termini of region A or C). In some aspects, the conjugate or oligonucleotide conjugate can, comprise a linker region which is positioned between the oligonucleotide and the conjugate moiety. In some aspects, the linker between the conjugate and oligonucleotide is biocleavable. Phosphodiester containing biocleavable linkers are described in more detail in WO 2014/076195 (herein incorporated by reference).

[0059] In some aspects, two or more linkers can be linked in tandem. When multiple linkers connect one or more oligonucleotides disclosed herein to one or more other oligonucleotides disclosed herein, and/or to any conjugate moiety, each of the linkers can be the same or different.

[0060] As used herein, and unless otherwise indicated, the term "complementary," when used to describe a first nucleotide or nucleoside sequence in relation to a second nucleotide or nucleoside sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide or nucleoside sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides or nucleosides.

[0061] “Complementary” sequences, as used herein, can include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and alternative nucleotides or nucleosides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing. Complementary sequences between an oligonucleotide and a target sequence as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide or nucleoside sequence to an oligonucleotide or polynucleotide comprising a second nucleotide or nucleoside sequence over the entire length of one or both nucleotide or nucleoside sequences. Such sequences can be referred to as "fully complementary" with respect to each other herein. However, where a first sequence is referred to as "substantially complementary" with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via an RNase H- mediated pathway. “Substantially complementary” can refer to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest.

[0062] The term “complementarity” refers to two or more oligomers (i.e., each comprising a nucleobase sequence) that are related with one another by Watson-Crick basepairing rules. Complementarity may be “partial,” in which less than all of the nucleobases of a given nucleobase sequence are matched to the other nucleobase sequence according to base pairing rules. For example, in some aspects, complementarity between a given nucleobase sequence and the other nucleobase sequence may be about 70%, about 75%, about 80%, about 85%, about 90% or about 95%. Or, there may be “complete” or “perfect” (100%) complementarity between a given nucleobase sequence and the other nucleobase sequence to continue the example. The degree of complementarity between nucleobase sequences can have significant effects on the efficiency and strength of hybridization between the sequences.

[0063] By “reducing the level of premature termination” is meant decreasing the level of premature termination caused by premature termination mutations, in particular, mutations that lead to a premature UGA termination codon within the coding region of a gene, in a cell or subject, e.g., by administering a polynucleotide composition, or pharmaceutically acceptable salt thereof, to the cell or subject.

[0064] The phrase "contacting a cell with a polynucleotide composition," as used herein, includes contacting a cell by any possible means. Contacting a cell with a polynucleotide composition includes contacting a cell in vitro with the polynucleotide composition or contacting a cell in vivo with the polynucleotide composition. The contacting can be done directly or indirectly. Thus, for example, the polynucleotide composition can be put into physical contact with the cell by the individual performing the method, or alternatively, the polynucleotide composition can be put into a situation that will permit or cause it to subsequently come into contact with the cell.

[0065] Contacting a cell in vitro can be done, for example, by incubating the cell with the polynucleotide composition. Contacting a cell in vivo can be done, for example, by injecting the polynucleotide composition into or near the tissue where the cell is located, or by injecting the polynucleotide composition into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located.

[0066] As used herein, "lipid nanoparticle" or "LNP" is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., a polynucleotide. LNP refers to a stable nucleic acid-lipid particle. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate).

[0067] As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the polynucleotide composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the polynucleotide composition, although in some examples, it can. Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids.

[0068] As used herein, the terms “effective amount,” “therapeutically effective amount,” and “a “sufficient amount” of an agent that reduces the level premature termination (e.g., in a cell or a subject) described herein refer to a quantity sufficient to, when administered to the subject, including a human, effect beneficial or desired results, including clinical results, and, as such, an “effective amount” or synonym thereto depends on the context in which it is being applied. For example, in the context of treating a disorder or disease caused by premature termination, it is an amount of the agent that reduces the level of premature termination sufficiently to achieve a treatment response as compared to the response obtained without administration of the agent that reduces premature termination. The amount of a given agent that reduces the level of premature termination described herein that will correspond to such an amount will vary depending upon various factors, such as the given agent, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, and/or weight) or host being treated, and the like, but can nevertheless be routinely determined by one of skill in the art. Also, as used herein, a “therapeutically effective amount” of an agent that reduces the level and/or activity of premature termination of the present disclosure is an amount which results in a beneficial or desired result in a subject as compared to a control. As defined herein, a therapeutically effective amount of an agent that reduces the level of premature termination of the present disclosure can be readily determined by one of ordinary skill by routine methods known in the art. Dosage regimen can be adjusted to provide the optimum therapeutic response.

[0069] A “therapeutically-effective amount” or “prophylactically effective amount” also includes an amount (either administered in a single or in multiple doses) of a polynucleotide composition that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. Polynucleotide compositions employed in the methods herein can be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

[0070] The term “pharmaceutical composition,” as used herein, represents a composition containing a polynucleotide composition described herein formulated with a pharmaceutically acceptable excipient. Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, capsule, caplet, gelcap, or syrup); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); for intrathecal injection; for intracerebroventricular injections; for intraparenchymal injection; for intraocular administration (e.g., for intravitreal or subretinal administration); or in any other pharmaceutically acceptable formulation.

[0071] A “pharmaceutically acceptable excipient,” as used herein, refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the polynucleotide composition) and having the properties of being substantially nontoxic and non-inflammatory in a patient. Excipients can include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0072] As used herein, the term “pharmaceutically acceptable salt” means any pharmaceutically acceptable salt of the compound of any of the compounds described herein. For example, pharmaceutically acceptable salts of any of the compounds described herein include those that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66: 1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting a free base group with a suitable organic acid.

[0073] The compounds described herein can have ionizable groups so as to be capable of preparation as pharmaceutically acceptable salts. These salts can be acid addition salts involving inorganic or organic acids or the salts can, in the case of acidic forms of the compounds described herein, be prepared from inorganic or organic bases. Frequently, the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases and methods for preparation of the appropriate salts are well-known in the art. Salts can be prepared from pharmaceutically acceptable non-toxic acids and bases including inorganic and organic acids and bases. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemi sulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

[0074] By a “reference” is meant any useful reference used to compare protein or mRNA levels or activity. The reference can be any sample, standard, standard curve, or level that is used for comparison purposes. The reference can be a normal reference sample or a reference standard or level. A “reference sample” can be, for example, a control, e.g., a predetermined negative control value such as a “normal control” or a prior sample taken from the same subject; a sample from a normal healthy subject, such as a normal cell or normal tissue; a sample (e.g., a cell or tissue) from a subject not having a disease; a sample from a subject that is diagnosed with a disease, but not yet treated with a compound described herein; a sample from a subject that has been treated by a compound described herein; or a sample of a purified protein (e.g., any described herein) at a known normal concentration. By “reference standard or level” is meant a value or number derived from a reference sample. A “normal control value” is a pre-determined value indicative of non-disease state, e.g., a value expected in a healthy control subject. Typically, a normal control value is expressed as a range (“between X and Y”), a high threshold (“no higher than X”), or a low threshold (“no lower than X”). A subject having a measured value within the normal control value for a particular biomarker is typically referred to as “within normal limits” for that biomarker. A normal reference standard or level can be a value or number derived from a normal subject not having a disease or disorder (e.g., a nucleotide or trinucleotide repeat expansion disorder); a subject that has been treated with a compound described herein. In some aspects, the reference sample, standard, or level is matched to the sample subject sample by at least one of the following criteria: age, weight, sex, disease stage, and overall health. A standard curve of levels of a purified protein, e.g., any described herein, within the normal reference range can be used as a reference. [0075] As used herein, the term “subject” refers to any organism to which a composition can be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). A subject can seek or be in need of treatment, require treatment, be receiving treatment, be receiving treatment in the future, or be a human or animal who is under care by a trained professional for a particular disease or condition.

[0076] As used herein, the terms "treat," "treated," and "treating" mean both therapeutic treatment and prophylactic or preventative measures wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder, or disease, or obtain beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of a condition, disorder, or disease; stabilized (i.e., not worsening) state of condition, disorder, or disease; delay in onset or slowing of condition, disorder, or disease progression; amelioration of the condition, disorder, or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder, or disease.

Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0077] As used herein, the terms “variant” and “derivative” are used interchangeably and refer to naturally-occurring, synthetic, and semi-synthetic analogues of a compound, peptide, protein, or other substance described herein. A variant or derivative of a compound, peptide, protein, or other substance described herein can retain or improve upon the biological activity of the original material.

[0078] The details of one or more aspects are set forth in the description below. Other features, objects, and advantages will be apparent from the description and from the claims.

OVERVIEW OF DRAWINGS

[0079] FIG. 1 A exemplifies the luciferase gene containing the 5' UTR, ORF, and 3' UTR. FIG. IB shows exemplifies the luciferase gene containing L258X which introduces a premature UGA stop codon. The dashed lines in FIG. IB exemplify potential target gene regions for a complementary oligonucleotide to hybridize to the luciferase gene. [0080] FIG. 2 exemplifies various configurations of the polynucleotide compositions disclosed herein hybridizing to a target gene of interest, wherein the optional linker can be an oligonucleotide. The mRNA exemplified as the target gene of interest can be pre-mRNA or mature mRNA. Although mRNA is exemplified as the target gene in FIG. 2, the target gene could also be DNA.

[0081] FIG. 3 exemplifies various configurations of the polynucleotide compositions disclosed herein hybridizing to a target gene of interest, wherein the optional linker can be an oligonucleotide. The mRNA exemplified as the target gene of interest can be pre-mRNA or mature mRNA. Although mRNA is exemplified as the target gene in FIG. 3, the target gene could also be DNA.

[0082] FIG. 4A exemplifies a polynucleotide composition hybridizing to a target gene of interest, wherein the optional linker can be a chemical linker. FIG. 4B exemplifies a circular polynucleotide composition (e.g., RNA) hybridizing to a target gene of interest. The mRNA exemplified as the target gene of interest can be pre-mRNA or mature mRNA. Although mRNA is exemplified as the target gene in FIG. 4, the target gene could also be DNA.

[0083] FIG. 5A is a diagram depicting the dual luciferase reporter construct with the CMV promoter is driving the cassette composed of wild-type Renilla luciferase, a T2A selfcleaving element, followed by firefly luciferase that contains a mutation resulting in a UGA stop codon at position 258. The transcription of the constructs results in an mRNA with a beta-globin 3’ untranslated region where many of the bifunctional oligonucleotides described are targeted. FIG. 5B is a diagram of an alternative reporter construct that is composed of two fluorescent protein cassettes. The first is mCherry driven by the PGK promoter and the second is wildtype GFP or mutated version with individual UGA stop codons at positions 48, 57, or 70 driven by the CMV promoter. As with the FIG. 5A construct, the GFP cassette results in a beta-globin 3’UTR mRNA.

[0084] FIG. 5 exemplifies a luciferase-based reporter construct. FIG. 5A exemplifies a luciferase-based reporter construct comprising Renilla reniformis luciferase followed by a sequence encoding a 2A self-cleaving peptide (T2A) followed by firefly luciferase containing UGA ²⁵⁸ followed by the BGH 3’UTR, where many of the bifunctional oligonucleotides described are targeted. FIG. 5B exemplifies a luciferase-based reporter construct comprising a first cassette comprising a mCherry coding sequence driven by the PGK promoter and a second cassette comprising an eGFP coding sequence driven by the CMV promoter. A UGA stop codon is incorporated into three potential sites Cys ⁴⁸ , Trp ⁵⁷ , and Cys70. [0085] FIG. 6 represents a comparison of stop codon readthrough of firefly luciferase with a premature stop codon mutation (L258X) using various in frame 3’ UTR SECIS elements as described in Study 1. The firefly luciferase was normalized to Renilla luciferase signal (controlling for variations in HEK293 transfection efficiency) and data is expressed as the ratio of raw firefly to Renilla luminescence (arbitrary units). The “no oligo control” condition, which is the dual luciferase reporter construct containing the L258X mutation without treatment, serves as the negative control.

[0086] FIG. 7 represents a dose response comparison (250-1500 nM) of stop codon readthrough of firefly luciferase with a premature stop codon mutation (L258X) using a complementary oligonucleotide against various regions of the beta-globin 3’ UTR with either a GPX4 or Dio3 SECIS element as described in Study 2. The firefly luciferase was normalized to Renilla luciferase signal (controlling for variations in HEK293 transfection efficiency) and data is expressed as the ratio of raw firefly to Renilla luminescence (arbitrary units). The firefly luciferase alone signal (i.e. no oligonucleotide) was subtracted from each condition as background noise.

[0087] FIG. 8 represents a dose response comparison (500-1500 nM) of stop codon readthrough of firefly luciferase with a premature stop codon mutation (L258X) using a complementary oligonucleotide against various regions of the beta-globin 3’ UTR with an attached SECIS element (in the SECIS-linker-complementary region 3’ orientation) as described in Study 3. The firefly luciferase was normalized to Renilla luciferase signal (controlling for variations in HEK293 transfection efficiency) and data is expressed as the ratio of raw firefly to Renilla luminescence (arbitrary units). The firefly luciferase alone signal (i.e. no oligonucleotide) was subtracted from each condition as background noise.

[0088] FIG. 9 represents a dose response comparison (500-15 OOnM) of stop codon readthrough of firefly luciferase with a premature stop codon mutation (L258X) using a complementary oligonucleotide against various regions of the beta-globin 3’ UTR with an attached SECIS element (in the complementary region 3 ’-linker- SECIS element orientation) as described in Study 4. The firefly luciferase was normalized to Renilla luciferase signal (controlling for variations in HEK293 transfection efficiency) and data is expressed as the ratio of raw firefly to Renilla luminescence (arbitrary units). The firefly luciferase alone signal (i.e. no oligonucleotide) was subtracted from each condition as background noise.

[0089] FIG. 10 represents a comparison of stop codon readthrough of firefly luciferase with a premature stop codon mutation (L258X) using plasmid-based delivery of the functional oligonucleotide as described in Study 5. Briefly, the plasmid-delivered DNA sequence can transcribed into the functional RNA oligonucleotide with a complementary oligonucleotide against the B-globin 3’ UTR with a SECIS element. The firefly luciferase was normalized to Renilla luciferase signal (controlling for variations in HEK293 transfection efficiency) and data is expressed as the ratio of raw firefly to Renilla luminescence (arbitrary units). The firefly luciferase alone signal (ie no oligonucleotide) was subtracted from each condition as background noise.

DETAILED DESCRIPTION

I. Polynucleotide Compositions

[0090] A bifunctional polynucleotide composition described herein requires one arm (i.e., a complementary oligonucleotide) that targets a gene-of-interest (GOI) having a premature UGA stop codon and a second arm that contains a SECIS element. In some aspects, these two arms are joined via an optional linker. Such bifunctional oligonucleotides can result in selenocysteine incorporation into the UGA stop codon (for example, in a premature UGA stop codon mutation) and, hence, readthrough and restoration of translation, resulting in the generation of a full-length protein. While replacement of the original amino acid with a selenocysteine may result in a non-functional protein, in many cases selenocysteine incorporation and the generation of a full-length protein can restore sufficient levels of functional activity, thereby resulting in the correction of the loss of function. SECIS elements are found in the 3’ UTR of proteins containing selenocysteine proteins and are approximately 75 nucleotides in length. The SECIS elements across the endogenous selenoprotein transcript 3’UTRs are variable in sequence, but their secondary structures are conserved in a consensus stem-loop-stem-loop folding.

[0091] Therefore, polynucleotide compositions comprising: (a) a complementary oligonucleotide comprising at least 10 consecutive nucleobases having at least 80% complementarity to a target gene having a premature UGA stop codon; (b) an optional linker, selected from the group consisting of: (i) an oligonucleotide and (ii) a chemical linker; and (c) an oligonucleotide comprising a SECIS element, or pharmaceutically acceptable salts thereof, are provided herein.

[0092] In some aspects, the polynucleotide compositions described herein comprises a total of 30-1000 nucleobases. In some aspects the polynucleotide compositions described herein comprises a total of 30-950, 30-900, 30-850, 30-800, 30-750, 30-700, 30-650, 30-600, 30-650, 30-600, 30-550, 30-500, 30-450, 30-400, 30-350, 30-300, 30-250, 30-200, 40-1000,

40-950, 40-900, 40-850, 40-800, 40-750, 40-700, 40-650, 40-600, 40-650, 40-600, 40-550,

40-500, 40-450, 40-400, 40-350, 40-300, 40-250, 40-200, 40-175, 50-1000, 50-950, 50-900,

50-850, 50-800, 50-750, 50-700, 50-650, 50-600, 50-650, 50-600, 50-550, 50-500, 50-450,

50-400, 50-350, 50-300, 50-250, or 50-200.

[0093] In some aspects, the polynucleotide composition is in a pharmaceutically acceptable salt form. In some aspects, the polynucleotide composition is in a free form.

[0094] In some aspects, the polynucleotide composition is circular.

[0095] In some aspects, the complementary oligonucleotide and the oligonucleotide comprising a SECIS element are on the same oligonucleotide strand. In some aspects, the complementary oligonucleotide and the oligonucleotide comprising a SECIS element are on the same oligonucleotide strand connected by a linker. In some aspects, the complementary oligonucleotide and the oligonucleotide comprising a SECIS element are on the same oligonucleotide strand and are directly linked without a linker.

[0096] In some aspects, the complementary oligonucleotide and the oligonucleotide comprising a SECIS element are on different oligonucleotide strands. In some aspects, the complementary oligonucleotide and the oligonucleotide comprising a SECIS element are on different oligonucleotide strands and are connected by one or more duplex regions.

A. Target Gene

[0097] The target gene can be any DNA, pre-mRNA, or mature mRNA containing a premature stop codon (e.g., UGA premature stop codon) leading to a shortened protein that is less functional, especially non-functional. In some aspects, the target gene is selected from the group consisting of genes listed in Table 1.

Table 1

[0098] In some aspects, the target gene is DNA.

[0099] In some aspects, the target gene is RNA corresponding to any one of the DNA of

Table 1. In some aspects, the target gene is pre-mRNA. In some aspects, the target gene is mature RNA.

B. Complementary Oligonucleotide

[0100] In some aspects, the complementary oligonucleotide (also referred to as a “complementarity region”) comprises at least 10 consecutive nucleobases having at least 90% complementarity to the target gene. In some aspects, the complementary oligonucleotide comprises at least 10 consecutive nucleobases having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementarity to the target gene.

[0101] In some aspects, the complementary oligonucleotide comprises at least 15 consecutive nucleobases. In some aspects, the complementary oligonucleotide comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 consecutive nucleobases. [0102] In some aspects, the complementary oligonucleotide comprises 10-50 consecutive nucleobases. In some aspects, the complementary oligonucleotide comprises 10-45, 10-40, 15-50, 15-45, 15-40, 20-50, 20-45, 20-40, or 25-50 consecutive nucleobases.

[0103] In some aspects, the complementary oligonucleotide is single-stranded. In some aspects, the complementary oligonucleotide comprises at least one duplex region. In some aspects, the complementary oligonucleotide contains at least one hairpin loop.

[0104] In some aspects, the complementary oligonucleotide hybridizes to a region within 15,000 nucleotides of a premature stop codon within the target gene. In some aspects, the complementary oligonucleotide hybridizes to a region within 15,000, within 14,000, within 13,000, within 12,000, within 11,000, within 10,000, within 9500, within 9000, within 8500, within 8000, within 7500, within 7000, within 6500, within 6000, within 5500, within 5000, within 4500, within 4000, within 3500, within 3000, within 2500, within 2000, within 1900, within 1800, within 1700, within 1600, within 1500, within 1400, within 1300, within 1200, within 1100, within 1000, within 900, within 950, within 900, within 850, within 800, within

750, within 700, within 650, within 600, within 550, within 500, within 450, within 400, within 350, within 300, within 250, within 200, within 150, within 100, or within 50 nucleotides of a premature stop codon within the target gene.

[0105] In some aspects, the complementary oligonucleotide hybridizes 3' to the premature stop codon of the target gene. In some aspects, the complementary oligonucleotide hybridizes to a 3' untranslated region of the target gene.

[0106] In some aspects, the complementary oligonucleotide hybridizes 5' to the premature stop codon of the target gene. In some aspects, the complementary oligonucleotide hybridizes to a 5' untranslated region of the target gene.

C. Linker

[0107] In some aspects, the polynucleotide compositions described herein do not include a linker.

[0108] In some aspects, the polynucleotide compositions described herein further comprises a linker. In some aspects, the linker is an oligonucleotide linker or a chemical linker. i. Oligonucleotide Linkers

[0109] In some aspects, the linker of the polynucleotide compositions described herein is an oligonucleotide. In some aspects, the linker comprises 1-50 linked nucleotides. In some aspects, the linker comprises 1-40 linked nucleotides, 1-30 linked nucleotides, 1-20 linked nucleotides, or 1-10 linked nucleotide.

[0110] In some aspects, the linker of the polynucleotide compositions described herein is single-stranded. In some aspects, the linker of the polynucleotide compositions described herein comprises at least one duplex region. In some aspects, the linker of the polynucleotide compositions described herein comprises at least one hairpin.

[OHl] In some aspects, the linker of the polynucleotide compositions described herein comprise a linker comprising a nucleotide sequence selected from the group consisting of UAU, UGUUAA, GCUGAUAGA, TAT, TGTTAA, SEQ ID NOs: 171-173, and SEQ ID NOs: 184-186. In some aspects, the linker of the polynucleotide compositions described herein comprise a linker consisting of a nucleotide sequence selected from the group consisting of UAU, UGUUAA, GCUGAUAGA, TAT, TGTTAA, SEQ ID NOs: 171-173, and SEQ ID NOs: 184-186. ii. Chemical Linkers

[0112] In some aspects, the linker of the polynucleotide compositions described herein is a chemical linker.

[0113] Linkers typically include a direct bond or an atom such as oxygen or sulfur, a unit such as NR. ⁸ , C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroaryl alkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R ⁸ ), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; wherein R ⁸ is hydrogen, acyl, aliphatic or substituted aliphatic. In one aspect, the chemical linker of the polynucleotide compositions described herein is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.

[0114] In some aspects, the polynucleotide compositions described herein comprises a chemical linker that is cleavable. A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together.

[0115] Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential, or the presence of degradative molecules. Examples of such degradative agents include: redox agents which are selective for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

[0116] A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

[0117] A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

[0118] Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes. a. Redox Cleavable Linking Groups

[0119] In one aspect, the cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group ( — S — S — ). b. Phosphate-Based Cleavable Linking Groups

[0120] In another aspect, the cleavable linker includes a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. c. Acid Cleavable Linking Groups

[0121] In another aspect, the cleavable linker includes an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In certain aspects, an acid cleavable linking group is cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula — C=NN — , C(O)O, or — OC(O). One aspect is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. d. Ester-Based Linking Groups

[0122] In another aspect, the cleavable linker can be an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula — C(O)O — , or — OC(O) — . e. Peptide-Based Cleaving Groups

[0123] In yet another aspect, the cleavable linker includes a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group ( — C(O)NH — ). The amide group can be formed between any alkylene, alkenylene, or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide-based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula — NHCHR ^A C(O)NHCHR ^B C(O) — , where R ^A and R ^B are the R groups of the two adjacent amino acids.

[0124] In some aspects, the chemical linker comprises glycerol or a glycerol homolog of the formula HO — (CH2)o — CH(OH) — (CH2)p — OH, wherein o and p independently are integers from 1 to 6, from 1 to 4 or from 1 to 3.

[0125] In some aspects, the chemical linker is a polyethylene glycol (PEG). There are two classes of PEG, monodispersed and poly-dispersed. A monodispersed PEG linker has an exact number of PEG units with a specific chemical structure and a precise molecular weight, whereas a poly-dispersed PEG is a polymer with an averaged molecular weight. In some aspects, the PEG linker is selected from a group consisting of alkyne PEG, amino PEG, aminooxy PEG, APN PEG, benzyl-PEG, biotin PEG, Bis-PEG-acid, Bis-PEG-NHS, Boc- PEG, Bromo PEG, Fmoc PEG, DNP-PEG, hydroxyl PEG, iodo PEG, lipid PEG, PEG aldehyde, PEG azide, PEG hydrazide, tetraethylene glycol, sugar PEG, and combinations thereof.

[0126] In some aspects, the chemical linker is a non-cleavable linker. In certain aspects, the non-cleavable linker is succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-lcarboxylate (SMCC).

[0127] In some aspects, the chemical linker can be crosslinkers that are used to connect two functional motifs of a PROTAC, a target protein binder and an E3 ligase recruiter. Linkers that are commonly used for PROTAC include Azido-PEG12-NHS ester, 4-hydroxy- thalidomide, thalidomide-acid, thalidomide-O-PEG-acid, thalidomide-O-PEG-Amine, thalidomide-O-PEG-azide, thalidomide-O-PEG-NHS ester, thalidomide-O-PEG-propargyl, dBETl, thalidomide-O-PEG-t-butyl ester, thalimide-O-PEG-tosyl, 4-fluoro-thalidomide, pomalidomide, thalidomide-O-acetamido-C4-amine, thalidomide-O-amido-PEG4-azide, thalidomide-O-amido-PEG4-propargyl, pomalideomide-PEG-NH-Boc, pomalidomide-PEG- Ph-NH2, P131, ARV-825, pomalidomide-PEG-azide, pomalidomide 4'-PEG-azide, D-biotin- PEG-thalidomide, D-amino-PEG-thalidomide, VHL ligand 1, and EGFR PROTAC. D. Oligonucleotide Comprising a SECIS Element

[0128] The polynucleotide compositions described herein comprise a SECIS element. In some aspects, the oligonucleotide comprising a SECIS element comprises at least one ribonucleotide. In some aspects, the oligonucleotide comprising a SECIS element comprises at least two ribonucleotides. In some aspects, the oligonucleotide comprising a SECIS element comprises at least 10% ribonucleotides. In some aspects, the oligonucleotide comprising a SECIS element comprises at least 20% ribonucleotides. In some aspects, the oligonucleotide comprising a SECIS element comprises at least 30% ribonucleotides. In some aspects, the oligonucleotide comprising a SECIS element comprises at least 40% ribonucleotides. In some aspects, the oligonucleotide comprising a SECIS element comprises at least 50% ribonucleotides. In some aspects, the oligonucleotide comprising a SECIS element comprises at least 60% ribonucleotides. In some aspects, the oligonucleotide comprising a SECIS element comprises at least 70% ribonucleotides. In some aspects, the oligonucleotide comprising a SECIS element comprises at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% ribonucleotides. In some aspects, the oligonucleotide comprising a SECIS element comprises substantially all ribonucleotides. In some aspects, the oligonucleotide comprising a SECIS element comprises 100% ribonucleotides.

[0129] In some aspects, the oligonucleotide comprising a SECIS element comprises at least one deoxyribonucleotide. In some aspects, the oligonucleotide comprising a SECIS element comprises at least two deoxyribonucleotides. In some aspects, the oligonucleotide comprising a SECIS element comprises at least 10% deoxyribonucleotides. In some aspects, the oligonucleotide comprising a SECIS element comprises at least 20% deoxyribonucleotides. In some aspects, the oligonucleotide comprising a SECIS element comprises at least 30% deoxyribonucleotides. In some aspects, the oligonucleotide comprising a SECIS element comprises at least 40% deoxyribonucleotides. In some aspects, the oligonucleotide comprising a SECIS element comprises at least 50% deoxyribonucleotides. In some aspects, the oligonucleotide comprising a SECIS element comprises at least 60% deoxyribonucleotides. In some aspects, the oligonucleotide comprising a SECIS element comprises at least 70% deoxyribonucleotides. In some aspects, the oligonucleotide comprising a SECIS element comprises at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% deoxyribonucleotides. In some aspects, the oligonucleotide comprising a SECIS element comprises substantially all deoxyribonucleotides. In some aspects, the oligonucleotide comprising a SECIS element comprises 100% deoxyribonucleotides.

[0130] In some aspects, the oligonucleotide comprising a SECIS element comprises at least one hairpin. In some aspects, the oligonucleotide comprising a SECIS element comprises at least one duplex region.

[0131] In some aspects, the oligonucleotide comprising a SECIS element has a Kd for SBP2 of less than 100 nM when measured by an RNA electromobility shift assay. In some aspects, the oligonucleotide comprising a SECIS element has a Kd for SBP2 of less than 95 nM, less than 90 nM, less than 85 nM, less than 80 nM, less than 75 nM, less than 70 nM, less than 65 nM, less than 60 nM, less than 55 nM, less than 50 nM, less than 45 nM, less than 40 nM, less than 35 nM, less than 30 nM, less than 25 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM when measured by an RNA electromobility shift assay.

[0132] The RNA electromobility shift assay to determine the Kd for SBP2 is to be performed as set forth in Latreche, L., et al., "Novel structural determinants in human SECIS elements modulate the translational recoding of UGA as selenocysteine," Nucleic Acids Research, 37:5868-80 (2009).

[0133] In some aspects, the polynucleotide composition comprises an oligonucleotide comprising the SECIS element comprises the sequence of

T nn ATGAT GnCnnnnnCnn AAA, wherein: T is thymine; A is adenine; G is guanine; C is cytosine; and n is independently selected from thymine, adenine, guanine, cytosine, and uracil.

[0134] In some aspects, the polynucleotide composition comprises an oligonucleotide comprising the SECIS element comprises the sequence of:

ATGAnGnCnnnnnCCnAAAnCCTC, wherein: T is thymine; A is adenine; G is guanine; C is cytosine; and n is independently selected from thymine, adenine, guanine, cytosine, and uracil.

[0135] In some aspects, the polynucleotide composition comprises an oligonucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementarity to SEQ ID NOs: 5-31. In some aspects, the polynucleotide composition comprises an oligonucleotide comprising the SECIS element comprises one or more sequences selected from the group consisting of SEQ ID NOs: 5-31. In some aspects, the polynucleotide composition comprises an oligonucleotide comprising the SECIS element consisting one or more sequences selected from the group consisting of SEQ ID NOs: 5-31.

[0136] In some aspects, the polynucleotide composition comprises an oligonucleotide comprising the SECIS element comprises the sequence of:

Unn AU GAU GnCnnnnnCnn A A A, wherein: U is uracil; A is adenine; G is guanine; C is cytosine; and n is independent selected from thymine, adenine, guanine, cytosine, and uracil.

[0137] In some aspects, the polynucleotide composition comprises an oligonucleotide comprising the SECIS element comprises the sequence of:

AUGAnGnCnnnnnCCnAAAnCCUC, wherein: U is uracil; A is adenine; G is guanine; C is cytosine; and n is independent selected from thymine, adenine, guanine, cytosine, and uracil.

[0138] In some aspects, the polynucleotide composition comprises an oligonucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementarity to SEQ ID NOs: 32-58. In some aspects, the polynucleotide composition comprises an oligonucleotide comprising the SECIS element comprises one or more sequences selected from the group consisting of SEQ ID NOs: 32-58. In some aspects, the polynucleotide composition comprises an oligonucleotide comprising the SECIS element consisting one or more sequences selected from the group consisting of SEQ ID NOs: 32-58.

[0139] In some aspects, the polynucleotide composition comprises an oligonucleotide comprising the SECIS element comprises 20-200, 30-190, 40-180, 50-170, 60-160, 70-150, 80-140, 90-130, 100-120, or 110 nucleotides.

E. Configurations

[0140] As previously described, the polynucleotide composition comprises, among other possible components, a complementary oligonucleotide, an optional linker, and an oligonucleotide comprising a SECIS element. In some aspects, a single oligonucleotide comprises the complementary oligonucleotide, optional linker and the oligonucleotide comprising a SECIS element, for example, as depicted in FIG. 2A. In one aspect, the polynucleotide composition has the following configuration (from 5’ to 3’):

Complementary oligonucleotide - Linker (optional) - oligonucleotide comprising SECIS element. [0141] In another aspect, the polynucleotide composition has the following configuration (from 5’ to 3’):

Oligonucleotide comprising SECIS element - Linker (optional) - Complementary oligonucleotide.

[0142] In another aspect the polynucleotide composition comprises two separate oligonucleotides, one comprising the complementary oligonucleotide, and the other comprising a portion or all of a SECIS element, for example, as depicted in FIG. 2B, FIG. 2C, FIG. 3B or FIG. 3C. In this aspect, one or both of the oligonucleotides can further comprise a linker. The oligonucleotide comprising a SECIS element and complementary oligonucleotide can share a region of complementarity either in the linker (FIG. 2B and FIG. 3B) or in the oligonucleotide comprising a SECIS element (FIG. 2C and FIG. 3C). In one aspect, the polynucleotide composition comprising the complementary oligonucleotide and the oligonucleotide comprising part or all of a SECIS element have a region of at least 10 consecutive nucleobases having at least 80% complementarity to one another. In another aspect, the polynucleotide composition comprising the complementary oligonucleotide and the oligonucleotide comprising part or all of a SECIS element have a region of at least 12 consecutive nucleobases having at least 90% complementarity to one another. In still another aspect, the polynucleotide comprising the complementary oligonucleotide and the oligonucleotide comprising part or all of the SECIS element have a region of at least 12 consecutive nucleobases having 100% complementarity to one another.

[0143] In yet another aspect, the composition comprises a complementary olionucleotide (complementarity region) and a SECIS element that are joined via a chemical linker (FIG. 4A).

[0144] In still another aspect, the polynucleotide composition comprises a circular configuration, for example, as depicted in FIG. 4C.

[0145] In still another aspect, the polynucleotide composition comprises a complementary oligonucleotide comprising at least one alternative intemucleoside linkage selected from a phosphorothioate linkage and 2’-alkoxy linkage, and the oligonucleotide comprising a SECIS element comprises at least one ribonucleotide. In one aspect, the polynucleotide composition comprises a complementary oligonucleotide wherein at least 50% of the internucleotide linkages are phosphorothioate linkages, and at least 50% of the nucleotides within the SECIS element are ribonucleotides. F. Targeting Moiety

[0146] In some aspects, the polynucleotide composition further comprises a targeting moiety. In some aspects, the targeting moiety is selected from a group consisting of an antibody, cholesterol, peptide, aptamer, and combinations thereof. In some aspects, the targeting moiety is attached to the 5' end of the polynucleotide composition. In some aspects, the targeting moiety is attached to the 3' end of the polynucleotide composition. In some aspects, the targeting moiety is attached to the polynucleotide composition using a linker that is an oligonucleotide linker or a chemical linker. In some aspects, the linker is the same or different as the linker attaching the complementary oligonucleotide to the oligonucleotide comprising a SECIS element.

G. Alternative Oligonucleosides

[0147] In some aspects, one or more of the complementary oligonucleotide, linker that is an oligonucleotide, and oligonucleotide comprising a SECIS element of the polynucleotide composition comprises at least one alternative nucleobase. In some aspects, one or more of the complementary oligonucleotide, linker that is an oligonucleotide, and oligonucleotide comprising a SECIS element of the polynucleotide composition comprises at least one alternative sugar moiety and/or internucleoside linkage. In some aspects, one or more of the complementary oligonucleotide, linker that is an oligonucleotide, and oligonucleotide comprising a SECIS element of the polynucleotide composition comprises at least one alternative nucleobase and at least one alternative sugar moiety and/or internucleoside linkage.

1. Alternative Nucleobases

[0148] One or more of the complementary oligonucleotide, linker that is an oligonucleotide, and oligonucleotide comprising a SECIS element of the polynucleotide composition comprises at least one alternative nucleobase. Alternative nucleobases include synthetic and natural nucleobases such as 5-methylcytidine, 5-hydroxymethylcytidine, 5- formylcytidine, 5-carboxycytidine, pyrrolocytidine, dideoxycytidine, uridine, 5- methoxyuridine, 5-hydroxy deoxyuridine, dihydrouridine, 4-thiourdine, pseudouridine, 1- methyl-pseudouridine, deoxyuridine, 5-hydroxybutynl -2’ -deoxyuridine, xanthine, hypoxanthine, 7-deaza-xanthine, thienoguanine, 8-aza-7-deazaguanosine, 7-methylguanosine, 7-deazaguanosine, 6-aminomethyl-7-deazaguanosine, 8-aminoguanine, 2,2,7- trimethylguanosine, 8-methyladenine, 8-azidoadenine, 7-methyladenine, 7-deazaadenine, 3- deazaadenine, 2,6-diaminopurine, 2-aminopurine, 7-deaza-8-aza-adenine, 8-amino-adenine, thymine, dideoxythymine, 5-nitroindole, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouridine, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5- propynyl uridine and cytidine, 6-azo uridine, cytidine and thymine, 4-thiouridine, 8-halo, 8- amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5- halo, particularly 5-bromo, 5 -trifluoromethyl and other 5-substituted uridines and cytidines, 8-azaguanine and 8-azaadenine, 3 -deazaguanine, and combinations thereof.

2. Alternative Sugar Moieties and Internucleoside Linkages

[0149] One or more of the complementary oligonucleotide, linker that is an oligonucleotide, and oligonucleotide comprising a SECIS element of the polynucleotide composition comprises at least one alternative sugar moiety and/or internucleoside linkages. Alternative sugar moieties and internucleoside linkages include morpholino, phosphorothioates, 2' O-methyl (2'-0-Me), phosphorodiamidate morpholino, peptide nucleic acid (PNA), locked nucleic acid (LNA), 2' O-methoxyethyl, 2'-fluoro, 2'0,4'C-ethylene- bridged nucleic acids (ENAs), tricyclo-DNAs, tricyclo-DNA phosphorothioate, 2'-O-[2-(N- methylcarbamoyl)ethyl], and combinations thereof.

II. Vectors

[0150] Vectors comprising a polynucleotide composition described herein are also provided. In some aspects, the vector is a viral vector. In some aspects, the vector is a retrovirus vector. In some aspects, the vector is an AAV vector.

Ill Pharmaceutical Compositions

[0151] The polynucleotide composition, or pharmaceutically acceptable salt thereof, described herein can be formulated into pharmaceutical compositions for administration to a subject (e.g., a human) in a biologically compatible form suitable for administration in vivo.

[0152] The polynucleotide compositions described herein can be used in free form or in the form of salts, solvates, and as prodrugs. All forms are within the methods described herein. In accordance with the methods described herein, the described polynucleotide compositions or salts, solvates, or prodrugs thereof can be administered to a patient in a variety of forms depending on the selected route of administration such as by oral, parenteral, intrathecal, intracerebroventricular, intraparenchymal, buccal, sublingual, intraocular (subretinal, intravitreal), intra cisterna magna (ICM), intranasal, rectal, patch, pump, or transdermal administration. Parenteral administration includes intravenous, intraperitoneal, intra-articular, subcutaneous, intramuscular, transepithelial, intranasal, intrapulmonary, intrathecal, intraocular, intracerebroventricular, intraparenchymal, rectal, and topical modes of administration.

IV. Pharmaceutical Uses

[0153] Methods of increasing expression of a protein encoded by a gene containing a premature stop codon in a subject in need thereof, comprising administering to the subject one or more of the polynucleotide compositions, vectors, or pharmaceutical compositions described herein are also provided. In some aspects, there is an increase of at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, of functional expression of the protein when compared with a control.

[0154] Methods of treating, preventing, or delaying the progression of a condition caused by one or more premature stop mutations in a subject in need thereof, comprising administering to the subject one or more of the polynucleotide compositions, vectors, or pharmaceutical compositions described herein are also provided. In some aspects, the condition is a cancer. In some aspects, the condition is Rett syndrome, cystic fibrosis, autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, Duchenne muscular dystrophy, Becker's muscular dystrophy, cystinosis, tuberous sclerosis, colorectal cancer, choroideremia, laminopathies, retinitis pigmentosa, Stargardt disease, phenylketonuria, ataxia telangiectasia, fabry, niemann-Pick A/B, gangliosidosis type 1, MPS I-H, Hunter syndrome (MPS IIIB), Maroteaux-Lamy syndrome (MPS VI), neuronal ceroid lipofuscinoses (NCLs), hemophilia A, hemophilia B, spinal muscular atrophy, Leber congenital amaurosis (LCA) type 2, ocular coloboma, usher syndrome type 1, congenital aniridia, cancer, Greig cephalopolysyndactyly syndrome, achalasia, adrenocortical insufficiency, or alacrimia.

[0155] Methods of reducing the level of premature termination in a cell comprising contacting the cell with one or more of the polynucleotide compositions, vectors, or pharmaceutical compositions described herein are also provided. In some aspects, the cell is in a subject. In some aspects, the subject is a human. In some aspects, the subject is identified as having premature termination.

[0156] Polynucleotide compositions, vectors, or pharmaceutical compositions described herein for use in preventing or delaying the progression of a premature termination disease in a subject are also provided.

[0157] Use of one or more of the polynucleotide compositions, vectors, or pharmaceutical compositions provided herein in the preparation for a medicament for use in preventing or delaying the progression of a premature termination disease in a subject are also provided.

EXAMPLE

Example 1. Optimization of Bifunctional SECTS

[0158] Nonsense mutations change a codon into a premature termination codon (UAA, UAG, or UGA). These mutations can cause a subset of genetic diseases such as cystic fibrosis, Rett syndrome, Duchenne muscular dystrophy, and polycystic kidney disease. For those nonsense mutations resulting in a premature UGA stop codon, a strategy utilizing selenocysteine insertion can be applied. Selenocysteine is the 21 ^st amino acid and is incorporated into at least 25 selenoproteins. The 3’ untranslated region (3’ UTR) of selenoprotein transcripts contains a RNA element called a selenocysteine insertion sequence (SECIS), which recruits trans-acting factors including the SECIS binding protein 2 (SBP2), a selenocysteine tRNA ^Sec (which recognizes the UGA codon), an elongation factor (EFsec), and ribosomal protein L30. SBP2 is a RNA-binding protein that dictates the expression of selenoproteins. The resulting translated proteins will effectively read through a premature UGA stop codon, incorporating a selenocysteine at that position.

[0159] Testing and evaluating many sequences in an iterative manner both in a cell-free system and using a cellular testing system will lead to improved readthrough efficiency over the natural SECIS sequences. Also, identifying the minimally required sequence and structure is important for overall therapeutic characteristics.

[0160] To test the importance of various parameters driving efficacy of polynucleotide compositions in UGA readthrough, a number of permutations are tested with respect to position of the complementarity region relative to the UGA stop codon, the size of the linker in between the complementarity region and the SECIS element, the SECIS element itself, and modifications in the oligonucleotide.

[0161] To identify the importance of the location of the complementarity region, antisense oligos are designed to anneal to regions upstream of the UGA codon (i.e., in the 5’ untranslated region or within the coding region) and regions downstream of the UGA codon (i.e. within the coding sequence or in the 3’ untranslated region). Also, the nucleic acid-based spacer between the complementarity region and the SECIS element is varied from 0 to 30 nucleotides in length to identify the relationship between the spacer and readthrough efficiency.

Materials and Methods

A. Reporter Construct

[0162] The coding sequence of firefly luciferase from Luciola cruciata (SEQ ID NO: 1 and 2) is modified at Leu ²⁵⁸ to contain an in frame UGA codon (UUA UGA) (as previously described: Latreche et al (2009) Nucleic Acids Res, 37(17): 5868-5880 doi: 10.1093/nar/gkp635) or firefly luciferase from Photinus pyralis (SEQ ID NO: 375 and 376) is modified at Cys ²⁵⁸ to contain an in-frame UGA codon (UGU UGA), to allow the quantitative analysis of various polynucleotide compositions on UGA recoding efficiency. Briefly, incorporation of selenocysteine into UGA at codon 258 of Luciola cruciata firefly luciferase or at codon 258 of Photinus pyralis firefly luciferase restores luciferase activity that can be quantitatively measured.

[0163] Exemplary constructs that can be used for the transient transfection assays described below comprise the sequence of SEQ ID NO: 4 or 378, and comprise the Luciola cruciata UGA ²⁵⁸ luciferase (SEQ ID NO: 3) or the Photinus pyralis UGA ²⁵⁸ luciferase (SEQ ID NO: 377), respectively, and further comprise the 5’ UTR and intron of the P-globin gene, the BGH 3’ UTR and a polyadenylation sequence. The exemplary polynucleotides comprising SEQ ID NO: 4 or 378 are cloned into the pcDNA3.1 vector (Thermo Fisher Scientific) driven by the CMV promoter, and the resulting plasmid vectors are used for the transient transfection assays as described below. A companion control vector, in which the BGH 3’ UTR is replaced with the SECIS element (SEQ ID NO: 19) of the glutathione peroxidase 4 (GPX4) (NCBI Reference Sequence: NM_002085), which encodes a selenocysteine-containing protein and includes a SECIS element in its 3’ UTR, is used as a control to compare efficiency of selenocysteine incorporation of the polynucleotide compositions containing the SECIS element in cis compared with providing the SECIS element in trans.

[0164] The ability of the UGA ²⁵⁸ reporter and companion constructs to produce functional luciferase after in vitro transcription and translation, or after transfection of constructs in cells, are measured by luminescence detection as previously described (see, for example, Mehta et al (2004) J. Biol. Chem., 279: 37852-37859, which is incorporated by reference in its entirety).

[0165] An additional luciferase-based reporter construct was created to allow for signal normalization and a quantitative comparison across oligonucleotides. SEQ ID NO: 270 contains the Renilla reniformis luciferase followed by a sequence encoding a 2A selfcleaving peptide (T2A) followed by firefly luciferase containing UGA ²⁵⁸ followed by the BGH 3’UTR. This DNA fragment was cloned into the pcDNA3.1 vector driven by the CMV promoter via BamHl and Notl (FIG. 5 A).

[0166] A mCherry and GFP reporter construct was also created. SEQ ID NO: 271 contains two cassettes. Cassette 1 is mCherry driven by the PGK promoter and cassette 2 is eGFP driven by the CMV promoter. A UGA stop codon is incorporated into three potential sites Cys ⁴⁸ , Trp ⁵⁷ , and Cys70 (FIG. 5B). The purpose of this reporter construct is to demonstrate readthrough in a different reporter system with more sequence diversity.

B. Polynucleotide Compositions

[0167] As described above, the polynucleotide compositions of the present invention comprise at least two functional elements: a complementarity region that targets the gene-of- interest (GO I) with the premature UGA stop codon and a second arm that contains a SECIS element. These two elements are joined by an optional linker (e.g., UAU, UGUUAA, GCUGAUAGA, TAT, TGTTAA, or SEQ ID NOs: 171-173, 184-186). One key difference with incorporation of selenocysteines in nature, which occurs in a transcript containing the SECIS element in cis, is that the polynucleotide compositions described herein act in trans to provide selenocysteine incorporation and, in so doing, read through the premature stop codon (e.g., premature UGA stop codon). In the examples to follow, a number of parameters and variables are tested in order to determine the optimal placement and configuration of the polynucleotide composition for maximum selenocysteine incorporation. 1. Complementarity region

[0168] In this experiment, complementarity regions are selected corresponding to various regions of the transcript, 5’ untranslated region (UTR), the coding region, and 3’ UTR are tested to determine the optimum location of the complementarity region with respect to the UGA stop codon targeted for selenocysteine incorporation. As shown in Table 2, complementarity regions corresponding to various regions of the reporter transcript are designed. For purposes of this study, oligonucleotides that are complementary to a 17-21 nucleotide stretch are used. However, in practice, longer or shorter oligonucleotides can be used depending on the predicted melting temperature, specificity required, etc. Table 2 below shows representative complementarity sequences (SEQ ID Nos: 71-97, 175-180, 366-368) targeting different portions of the luciferase reporter constructs of SEQ ID NO: 4,270, or 378 described above.

Table 2

[0169] To identify the optimum locations of the complementarity sequence within the transcript, several complementarity sequences (C) listed in Table 2 above are synthesized and joined to a representative SECIS element (S), connected via a linker (L) comprising 5 nucleotides (adenosine), generating polynucleotides (SEQ ID NOs: 99-108) as shown in Table 3 below:

Table 3

[0170] Each of the complementarity sequence is chosen for its particular location within the transcript. For example, SEQ ID NO: 71 is from the intron within the 5’ UTR, whereas SEQ ID NOs: 85 and 88 are upstream and downstream of the UGA codon within the coding sequence, respectively.

2. SECIS element

[0171] In Table 4 below, SEQ ID NOs 5-58 correspond to the nucleobase sequence of the SECIS consensus sequences.

[0172] The human genome is believed to contain at least 26 SECIS element sequences from the following: Gpxl-Gpx6, Trxrl-Trxr2, Diol-Dio3, MsrBl, Sei 15, SelH, Sell, SelK, SelM, SelNl, SelN2, SelPl, SelP2, SelS, SelT, SelW, SelX, and Sps2. Each of these SECIS elements is tested by generating the SECIS chimeric constructs (SEQ ID NO: 109-135) using an overlapping oligonucleotide containing the designed mutations (e.g., SEC ID NOs: 94 or 95) shown in Table below:

Table 4

[0173] As with the previous experiment, oligonucleotide constructs in Table 4 above are either transfected into cells that are either stably expressing the luciferase construct, or cotransfected with the luciferase construct. Restoration of luciferase activity is measured and compared with control samples. SECIS elements providing the most robust luciferase activity when provided in trans are identified, and the relationship between the sequence and activity are analyzed. Further studies are conducted using either chimeric sequences (e.g., SEQ ID NOs: 59-70), consisting of components of different SECIS elements and/or by performing site-directed mutagenesis of the SECIS elements within constructs and tested for effects on luciferase activity. The resulting data are analyzed to result in a consensus sequence for SECIS elements yielding the most potent selenocysteine incorporation when provided in trans.

[0174] Further optimizations will be performed to identify the optimum distance between the UGA codon targeted for selenocysteine incorporation (read through) and the SECIS element. While the distance can be partly/largely set by selecting the complementarity region to a certain distance (e.g., 100, 200, 300, 400 or more, nucleotides upstream (5’ of) or downstream (3’ of) the targeted UGA codon, further optimizations can be achieved by using a linker of different lengths between the complementarity region and the SECIS element. Table 5 below provides some examples of testing the effect of linkers on the efficacy of luciferase restoration and, hence, UGA readthrough. SEQ ID NOs: 136-142 are the sequence of linkers used for the optimization.

Table 5

[0175] A circular RNA containing a SECIS element and complementarity region is another approach to carry out readthrough. The concept is depicted in FIG. 4B. Several constructs were designed to demonstrate whether this approach is successful. These circular RNAs included the Dio3 and Trxrl expanded SECIS elements (SEQ ID NO: 189 or 144, and 187 or 174, respectively), a 6-nucleotide linker (UGUUAA), a single complementarity region targeting the beta-globin 3’UTR (SEQ ID NO: 162), and a backbone fragment of 21, 50, or 100 nucleotides in length. Alternative single complementarity region targeting the betaglobin could be used (e.g., SEQ ID NOs: 163-167). Alternatively, any of the 26 different SECIS element could be included in these circular RNAs, for example the GPX4 or SelN SECIS elements (e.g., SEQ ID NO: 188 or 143, and 190 or 145, respectively). Short sequences may create sufficient strain to the overall structure of the RNA bifunctional oligonucleotide that eliminates the readthrough activity. The details of the constructs and their components are described below in Table 6. The circular RNAs (SEQ ID NO: 369-374) are to be ordered and synthesized by a manufacturer skilled in the art. Similar co-transfection and measurement of luminescence experiments to those described in Studies 1-5 are to be conducted.

Table 6

3. Construct designs to evaluate Complementarity Regions, SECIS elements, Linkers, and order

[0176] In study #1, 10 SECIS elements chosen based on Latreche et al (2009) Nucleic Acids Res, 37(17): 5868-5880 doi:10.1093/nar/gkp635 were cloned into the 3’UTR of the dual luciferase reporter construct to evaluate the relative recoding or readthrough efficiency across SECIS elements. These 10 SECIS elements include Diol (SEQ ID NO: 23), Dio3 (SEQ ID NO:22), Sell 5 (SEQ ID NO: 28), SelN (SEQ ID NO: 25), SelPlA (SEQ ID NO: 26), SelPlB (SEQ ID NO: 15), SelRl (SEQ ID NO: 31), Sps2 (SEQ ID NO: 27), Trxrl (SEQ ID NO: 11), and Trxr2 (SEQ ID NO: 21). These plasmid constructs are transfected using the standard transfection protocol and evaluated for relative luminescence.

[0177] In study #2, several RNA constructs using different complementarity regions and SECIS elements were synthesized and tested to demonstrate that recoding of the luciferase reporter can be enabled. These polynucleotides do not contain a linker and are described in Table 7 below. SEQ ID NO: 150-161 were ordered and synthesized at Integrated DNA Technologies (IDT DNA) as pure RNA without any modifications:

Table 7

[0179] In study #3, the composition of the specific order SECIS element-linker- complementarity region was investigated. These constructs were produced using a different approach than ordering and synthesizing at Integrated DNA Technologies. The doublestranded DNA fragments (SEQ ID NO: 193-210) were designed to contain a T7 promoter and filler DNA to meet the minimum length requirement of 300 nucleotides of the manufacturer (Twist Bioscience, Inc.). These polynucleotides and their components are described below in Table 8:

Table 8

[0180] In study #4, the composition of the specific order complementarity region-linker- SECIS element was investigated. Similar to the study associated with study #3, doublestranded DNA fragments (SEQ ID NO: 211-222) were ordered and synthesized at Twist Bioscience, Inc. These polynucleotides and their components are described below in Table 9.

Table 9 [0181] Another approach to delivering polynucleotides to enable UGA stop codon readthrough is to deliver double stranded DNA, linear or as a plasmid, that contains a promoter and a DNA sequence that is transcribed into the functional RNA oligonucleotide. In study #5, DNA sequences (SEQ ID NO: 254-261) were cloned into the plasmid SEQ ID NO: 253 via Sall and Xbal downstream of the U6 promoter. The resulting plasmids can be delivered through many modalities including transfection, electroporation, viral, ultrasound, liposomes, nanoparticles, etc. As a proof-of-concept study, transfection was used. Details on the plasmid constructs and its components are described below in Table 10:

Table 10

Optimization of the SECIS element

[0182] The 26 SECIS element sequences found in the human genome database are used to design primers for RT-PCR amplification and cloning. To have SECIS elements of similar size that would encompass the minimum active domain for all selenoprotein mRNAs, 103 -nt long sequences are selected starting 30 bp before the highly conserved ATGA motif. PCR primers are designed to contain restriction sites for subcloning into a luciferase reporter vector. Total RNA is extracted from Hek293 and HepG2 cells. Total RNAs (5 mg) are annealed with oligodT and used for reverse transcription. An aliquot of this reaction is used for the PCR amplification. PCR products and a luciferase reporter vector are digested with the appropriate restriction enzymes, purified and ligated with T4 DNA Ligase after dephosphorylation of the vector. Quick change site-directed mutagenesis is performed to generate SECIS mutants and luciferase (UAA ²⁵⁸ , UGU ²⁵⁸ , or UGA ²⁵⁸ ) mutants using overlapping oligonucleotides containing the designed. Diverse sequence variations of SECIS elements are created with synthesized oligonucleotides with the appropriate restriction sites to be incorporated into the luciferase reporter construct.

Cell Culture and Transfection

[0183] HEK293 cells were grown and maintained in 100 mm plates in Dulbecco’s Modified Eagle Medium (DMEM). Media was supplemented with 10% fetal bovine serum (FBS), and 2 mM L-glutamine. Cells were cultured in 5% CO2 at 37°C in a humidified atmosphere. Cells were cultured in 5% CO2 at 37 °C in a humidified atmosphere.

[0184] For all experiments involving transfecting readthrough oligonucleotides (Tables 2- 6, 8), HEK293 cells were seeded in 96-well plates. Cells are ready for transfection when at 70-90% confluent. Lipofectamine™ 3000 reagent (Thermo Fisher Scientific) is used for the co-transfection of the dual luciferase reporter construct (SEQ ID NO: 270) in the pCDNA3.1(+) vector and the oligonucleotide of interest. Mixture A was made with 5 microliters of Opti-MEM™ Medium (Thermo Fisher Scientific) is combined with 0.3 microliters of Lipofectamine™ 3000 reagent (Thermo Fisher Scientific) on a per well basis. Mixture B was composed of 5 microliters of Opti-MEM™ Medium, 0.8 micrograms of the dual luciferase reporter plasmid, and 1.6 microliters of Lipofectamine™ 3000 reagent. Mixtures A and B were combined, the oligonucleotide-of-interest was added such that the final, diluted concentration is the desired test concentration (e.g., 500, 1000, 1500 nM) and incubated for 15 minutes at room temperature prior to the addition to cells in 90 microliters of growth media.

[0185] For all experiments involving co-transfecting a plasmid encoding a readthrough RNA oligonucleotide, HEK293 cells were seeded in 96-well plates. Cells are ready for transfection when at 70-90% confluent. Lipofectamine™ 3000 reagent (Thermo Fisher Scientific) is used for the co-transfection of the dual luciferase reporter construct (SEQ ID NO: 270) in the pCDNA3.1(+) vector and the oligonucleotide of interest. Mixture A was made with 5 microliters of Opti-MEM™ Medium (Thermo Fisher Scientific) is combined with 0.3 microliters of Lipofectamine™ 3000 reagent (Thermo Fisher Scientific) on a per well basis. Mixture B was composed of 5 microliters of Opti-MEM™ Medium, 0.8 micrograms of the dual luciferase reporter plasmid, 0.8 micrograms of the plasmid encoding a readthrough RNA oligonucleotide (SEQ ID NO: 72-79 cloned into SEQ ID NO: 253) and 1.6 microliters of Lipofectamine™ 3000 reagent. Mixtures A and B were combined and incubated for 15 minutes at room temperature prior to the addition to cells in 90 microliters of growth media.

[0186] Co-transfections were incubated for 24 hours and the Dual Luciferase Reporter Assay was conducted using a GloMax® Plate Reader (Promega) per the manufacturer’s protocol. In short, growth media and/or co-transfection mixtures were removed. PBS was added and removed to rinse the cells. 20 microliters of the IX Passive Lysis Buffer was dispensed into each well. Lysis was carried out on a gently shaker for 15 minutes at room temperature. Lysate was transferred into white 96-well plates. 100 microliters of reconstituted Luciferase Assay II Reagent (LAR II) was added and the firefly luciferase signal was read over a 10-second integration period. 100 microliters of Stop & Gio® Reagent was added and the Renilla signal was measured over a 10-second integration period.

In vitro transcription to generate RNA bifunctional oligonucleotides

[0187] The 300 nucleotide double-stranded DNA fragments (SEQ ID NO: 193-222) were in vitro transcribed using the HiScribe® T7 High Yield RNA Synthesis Kit (New England Biolabs, Inc.) per the manufacturer’s protocol. In short, 30 ng of DNA, 2 microliters ATP, 2 microliters CTP, 2 microliters GTP, 2 microliters UTP, 2 microliters 10X T7 buffer, 2 microliters T7 polymerase mix, and water were mixed together for a final volume of 20 microliters. The reaction was incubated overnight at 37 degrees Celsius. Afterwards, 2 microliters of 10X DNAse incubation buffer and 1 microliter of DNAse 1 (Roche Catalog #04716728001) was added and incubated for 15 minutes at room temperature. The reaction was diluted with water to 60 microliters and ethanol precipitated using 0.1 volumes of 7.5 M ammonium acetate and 2.5 volumes of 100% ethanol. This was incubated overnight at -80 degrees Celsius. The mixture was centrifuged at 16,000 x g for 10 minutes at 4 degrees Celsius. The supernatant was removed and the pellet was washed by adding 500 microliters of 70% ethanol and centrifuged again at 16,000 x g for 10 minutes at 4 degrees Celsius. The supernatant was removed and the RNA pellet was left to dry for 1 hour at room temperature and resuspended in 10 mM Tris, 0.1 mM EDTA pH 8.0. Resulting RNA bifunctional oligonucleotide sequences are listed (SEQ ID NO: 223-252).

Modifications

[0188] RNA oligonucleotides are tested to develop optimum construct design.

Modifications of the nucleic acids and the impact on readthrough efficiency is explored to identify strategies to increase stability. 2’ ribose modifications include 2’0-methyl, 2’fluoro, 2’0-methyoxy ethyl, arabinose. 5’ phosphate modifications include 5’methyl phosphonate, 5’E-vinyl phosphonate, 5’ (S)-Methyl w/phosphate, and 5’phosphorothioate. Constrained ribose modifications include locked nucleic acids (LNA), (S)-constrained ethyl LNA, bridged nucleic acid (BNA ^C0C , BNA ^NC ), tricyclo DNA, and unlocked nucleic acid. Lastly, the phosphorodiamidate morpholino oligomer is also tested.

Results and discussion

[0189] The luminescence readout from the many bifunctional oligonucleotide constructs leads us to identify the most potent SECIS element, sequence and structural requirements of a minimum-sized SECIS element, and where the complementarity region needs to be located for optimal UGA readthrough.

[0190] Latreche et al (2009) Nucleic Acids Res, 37(17): 5868-5880 doi: 10.1093/nar/gkp635 reported a 250-fold difference in cloned-in SECIS elements using a system that was not particularly amenable to normalization. Having Renilla luciferase to normalize to in the dual luciferase reporter system allowed for more robust conclusions. Normalized luminescence results of Study 1 are shown in FIG. 6 as the ratio of firefly to Renilla luciferase luminescence. There are some differences in normalized luminescence suggesting SECIS elements like SelN, Diol, and Dio3 are more effective at recruiting SBP2 thereby enabling recoding.

[0191] Studies 2, 3, and 4 evaluated different permutations of SECIS elements, linkers, and complementarity regions. Also, the SECIS-linker-complementarity region and complementarity region-linker-SECIS order was investigated. Changing between SECIS elements (Dio3, SelN, Trxrl), varying linkers from 0 to 24 nucleotides in length, and targeting to different sites of the beta-globin 3’UTR yielded a range of translational readthrough. None completely failed. The normalized data from studies 2, 3, and 4 are shown in FIGs. 7, 8, and 9. In study 2 (FIG. 7), readthrough was not impacted significantly by the SECIS element, but the complementarity region had a mild effect. Under these conditions, 1000-1500 nM RNA bifunctional oligonucleotide resulted in the most normalized firefly luminescence. Studies 3 and 4 (FIGs. 8 and 9) shows that not all bifunctional oligonucleotides enable readthrough. SEQ ID Nos: 231, 232, 233, 234, 240, 241, and 246 had the most induction of readthrough. The majority of these RNA bifunctional oligonucleotides contained the SelN SECIS element. [0192] Study 5 utilized DNA plasmids to express the RNA bifunctional oligonucleotide within cells. After having co-transfected both plasmids (SEQ ID NO: 254-261 in SEQ ID NO: 253 and SEQ ID NO: 270 in pcDNA3.1(+), luminescence was measured for readthrough activity. The normalized data is shown in FIG. 10. Plasmids 254, 257, and 260 had the highest levels of luminescence. These three varied in SECIS element, linker length, and complementarity region.

Example 2: Chemical Linkers

[0193] Bifunctional oligonucleotide therapeutics are composed of two distinct functional molecule arms that are linked together by chemical matter. In the case of UGA stop-codon suppressor bifunctional oligonucleotide therapeutics, one arm targets the oligonucleotide therapeutic to the mRNA transcript of the gene of interest (GOI), and the other arm contains a selenocysteine insertion sequence (SECIS) element. While each arm has independent molecular interaction functions, when linked together the arms induce proximity of the GOI transcript and the cellular machinery that incorporates selenocysteine at UGA codons in order to suppress disease causing mutant stop codons.

[0194] Alternative designs include utilizing a chemical linker to connect the SECIS element to the complementarity region. Also, an approach using overlapping, complementing of separately synthesized SECIS and complementarity region elements resulting in a double stranded RNA linker is tested. A graphical description of these strategies is described above in FIG. 2 A and FIG. 2B.

Materials and methods

Linker design

[0195] To determine the optimal chemical linker composition, a set of bifunctional oligonucleotide therapeutics are synthesized with the general structure shown in FIG IB. These bifunctional oligonucleotides are then tested for therapeutic function in in vitro translation assays that measure selenocysteine incorporation at UGA codons which are described elsewhere.

[0196] To determine the appropriate linker length, alkyl groups are used to create linkers of different lengths. Alkyl chain linkers of lengths beginning at 6 carbon atoms increasing by 3 carbon atoms at a time up to 30 carbon atoms are tested. [0197] To determine if total polar surface area (TPSA) and lipophilicity of the linker impact bifunctional oligonucleotide function, PEG groups are used. Repeating PEG units beginning at 3 PEG motifs increasing by 3 PEG motifs up to 15 PEG units are tested.

Cell-type and tissue-type delivery testing

[0198] For biodistribution studies, animals (n = 2-3 per group) are euthanized at 48 h after injection and perfused with PBS. Brains are harvested and post-fixed overnight with 10% formalin and processed for paraffin embedding. For efficacy studies, animals (n = 6-8 per group) are euthanized at different time points.

Example 3: Disease Genes

[0199] Translating efficacy of the bifunctional oligonucleotides from a reporter transcript to a therapeutically-relevant disease gene is needed. Two strategies can be used. One is to introduce a UGA stop codon into the coding region using genome editing tools. The UGA stop codon can be the same mutation that is present in human disease. The second strategy is to use a reporter assay where GFP is fused to the target gene and transiently delivered into HEK293 cells. Further studies can use patient-derived cells that already contain nonsense mutations preventing full-length protein translation.

[0200] Another aspect of translation is understanding another potential source of variability. UGA stop codons can occur at different locations in the mRNA transcript and the target gene can be very long. As an example, dystrophin mRNA is over 13,000 nucleotides in length. Using a series of UGA nonsense mutations across the gene, the readthrough efficiency relationship between the complementarity region and the UGA location is characterized.

Isolation of patient-derived nonsense mutation carrying cells:

[0201] Patient-derived cells are accessed through collaboration and disease foundation centers. In some cases, disease-relevant cells are acquired from post-mortem tissue or via non-invasive means (e.g., airway epithelial cells from bronchoalveolar lavage fluid, myoblasts from muscle biopsy). In other cases, patient-derived fibroblasts are acquired, tested as fibroblasts, or are used to generate induced pluripotent stem cells that are differentiated into disease-relevant cells. Examples of disease-relevant cells would be renal cells in polycystic kidney disease, airway epithelial cells in cystic fibrosis, neurons in Rett syndrome, and myoblasts in Duchenne’s or Becker muscular dystrophy.

Induced pluripotent stem cell generation and differentiation into disease-relevant cells:

[0202] Patient-derived fibroblasts from patients with UGA nonsense mutations can be acquired and treated to generate induced pluripotent stem cells through standard methods. Oftentimes, these fibroblasts are pre-differentiated by research centers to facilitate research. If not, these cells are then treated to differentiate into disease-relevant cells. Examples of such include airway epithelial cells for cystic fibrosis, myoblasts for Duchenne’s and Becker muscular dystrophy, renal cells for polycystic kidney disease, and neurons for Rett syndrome.

Generation of cell line clones:

[0203] UGA stop codons mutations are introduced for the disease genes CFTR, MeCP2, PKD1, PKD2, dystrophin from representative diseases cystic fibrosis, Rett syndrome, polycystic kidney disease, and Duchenne muscular dystrophy, respectively. CRISPR/Cas9 is used to introduce point mutations.

[0204] To introduce mutations to disease genes, guide RNAs oligonucleotides are ligated to the Bbsl-linearized plasmid vector pX458 [plasmid pSpCas9(BB)-2A-GFP (PX458), (Addgene #48138), Cambridge MA] at 16°C for 12-15 h. The ligation reaction is used to transform Escherichia coli DH5alpha cells, plated on a Luria broth-ampicillin plate, and incubated overnight at 37°C. Multiple colonies from each bacterial plate are marked and selected for colony PCR to test for successful ligation of the annealed oligo- nucleotides into the plasmid vector. After the desired sequence of these plasmids are confirmed by Sanger sequencing, these plasmids are used for transfection of HEK-293 cells (American Type Culture Collection, Manassas, VA).

Reporter fusion constructs:

[0205] Fusion constructs between GFP and dystrophin, CFTR, PKD1, PKD2 are created in plasmid vectors. UGA stop codons introduced at different positions in the transcript to characterize the readthrough activity relationship with UGA location. For example, UGA stop codons are introduced at every 1000 nucleotides across the dystrophin cDNA resulting in 13 different constructs. Bifunctional oligonucleotide testing in cellular models:

[0206] Oligonucleotides with the best UGA stop codon readthrough from Example 1 are used here to assess translatability to genes of interest. RNA bifunctional oligonucleotides, circular RNAs, bifunctional RNAs connected by chemical linkers, and others are delivered using transfection reagents and/or carriers such as lipid nanoparticles, extracellular vesicles, or other methods. Double-stranded DNA plasmids encoding RNA bifunctional oligonucleotides can be delivered by transfection reagents as well, but can also include viral (e.g., adeno-associated virus (AAV), lentivirus, others), ultrasound, lipid nanoparticles, extracellular vesicles or other means. In patient-derived cellular models, cellular models with mutated endogenous genes, or models requiring transfection of mutated genes, UGA readthrough efficiency is measured by the level of full-length protein expression using ELISA or western blots relative to wildtype levels. In cellular models containing reporter fusion constructs (i.e. those containing GFP, luciferase, or others), UGA readthrough efficiency is assessed using fluorescence and/or luminescence.

[0207] RNA bifunctional oligonucleotide approaches were designed firstly to investigate the relative efficacy of different SECIS elements and 6 complementarity regions targeting the 3’UTR of MeCP2 because the 3’UTR of MeCP2 is over 10 kilobases in length. Similar to study #3, double-stranded DNA fragments (SEQ ID NOs: 278-295) were ordered and synthesized at Twist Bioscience, Inc. In vitro transcription yielded RNA sequences (SEQ ID NOs: 316-333). These polynucleotides and their components are described below in Table 11 :

Table 11

[0208] The subsequent set of RNA bifunctional oligonucleotides were designed and created to understand the benefit, detriment, or lack of effect on MeCP2 readthrough from incorporating a linker between the SECIS element and the complementarity region. These oligonucleotides have this order: SECIS-linker-complementarity region. Similar to study #3, double-stranded DNA fragments (SEQ ID NO: 296-303) were ordered and synthesized at Twist Bioscience, Inc. In vitro transcription yielded RNA sequences (SEQ ID NO: 334-341). These polynucleotides and their components are described below in Table 12:

Table 12

[0209] The subsequent set of RNA bifunctional oligonucleotides were designed and created to understand whether the order of complementarity region-linker-SECIS element has an impact on MeCP2 readthrough as compared to the order of SECIS-linker-complementarity region. Similar to study #3, double-stranded DNA fragments (SEQ ID NO: 304-315) were ordered and synthesized at Twist Bioscience, Inc. In vitro transcription yielded RNA sequences (SEQ ID NO: 342-353). These polynucleotides and their components are described below in Table 13:

Table 13

[0210] Another approach to delivering polynucleotides to enable UGA stop codon readthrough is to deliver double stranded DNA, linear or as a plasmid, that contains a promoter and a DNA sequence that is transcribed into the functional RNA oligonucleotide. DNA sequences (SEQ ID NO: 354-359) were cloned into the plasmid SEQ ID NO: 253 via Sall and Xbal downstream of the U6 promoter. The resulting plasmids can be delivered through many modalities including transfection, electroporation, viral, ultrasound, liposomes, nanoparticles, etc. Details on the plasmid constructs and its components are described below in Table 14:

Table 14

[0211] Testing of RNA bifunctional oligonucleotides and plasmids described in Tables 11-14 are to be carried out by transfection into immortalized Rett mouse ear tip fibroblast cells that are wildtype for MeCP2 (Applied Biological Materials, Catalog #T0869) or are mutated R294X or R255X (Applied Biological Materials, Catalog #T0871, #T0870). Characterization of readthrough efficiency is conducted via western blot analysis probing for MeCP2 protein (MeCP2 D4F3 XP Rabbit mAb, Cell Signaling #3456) normalizing to the TATA-binding protein (anti-TATA binding protein TBP antibody, Abeam #ab300656).

Animal models:

[0212] Mouse animal models of disease are acquired or created with appropriate UGA nonsense mutations in disease genes of interest (e.g., dystrophin, CFTR, MeCP2, PKD1, PKD2). Candidate bifunctional oligonucleotide therapeutics are administered by the appropriate route (e.g., intracerebroventricular, intrathecal, intravenous, intraperitoneal, intraarticular, intravitreal, inhalation, intramuscular). Carriers to enable delivery such as lipid nanoparticles, other nanoparticles, liposomes, extracellular vesicles, or bifunctional oligonucleotides with a targeting moiety is utilized. A variety of assessments will be used to evaluate the efficiency and efficacy of UGA stop codon readthrough in these animal models. These include ELISA, western blot, and functional measurements relevant to the given disease model.

Results and discussion:

[0213] Based on the results of testing the bifunctional oligonucleotides and UGA stop codon readthrough in disease-relevant cellular assays, oligos can progress to in vivo testing. Testing in animal models will demonstrate the degree of functional restoration of the mutated disease-causing gene. Also, we will have assessed the impact of efficacy on the distance between the disease-causing UGA stop codon and where the bifunctional oligonucleotide targets the transcript of interest and recruits the stop codon readthrough efficiency.

OTHER ASPECTS

[0214] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.

[0215] While the invention has been described in connection with specific aspects thereof, it will be understood that invention is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and can be applied to the essential features hereinbefore set forth, and follows in the scope of the claimed.

Sequences