Agents and methods for generating closed-ended DNA thread molecules

Abstract

The present disclosure relates to agents and methods for making closed-ended DNA thread (CEDT) molecules, as well as compositions and uses of CEDT molecules.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 486,491, filed February 23, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Statement Regarding Federally Sponsored Research This invention was made with government support under HL153988 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence Listing Reference This application has been filed with a Sequence Listing in electronic format. The Sequence Listing is provided in a file entitled SeqList-161118-04501.xml, created on February 20, 2023, and is 27,224 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0004] The present invention relates generally to agents and methods for generating closed-ended DNA thread (CEDT) molecules. [Background technology]

[0005] Nonsense mutations account for over 10% of all genetic diseases and approximately 1,000 genetic human disorders, including cancer, which affects approximately 300 million people worldwide. For example, approximately 22% of all cystic fibrosis patients have "class 1" premature termination codon (PTC) mutations (e.g., p.G542X, p.R553X, and p.W1282X), which result in near-complete loss of cystic fibrosis transmembrane conductance regulator (CFTR) function and severe clinical symptoms. Nonsense mutations typically change an amino acid codon to a PTC through a single base substitution, resulting in a defective, truncated protein and severe disease.

[0006] Due to the extremely high incidence and unified mechanism of nonsense-associated disease, concerted efforts have been made to develop PTC therapeutics. Aminoglycosides have been the primary focus of these efforts. However, ototoxicity and nephrotoxicity with long-term use limit their clinical use. Synthetic aminoglycoside derivatives are currently being investigated to reduce off-target effects, but they often have low read-through efficiency. Non-aminoglycoside small molecules (i.e., tylosin, ataluren) have also been identified as promising PTC read-through compounds with reduced toxicity. However, these approaches face many challenges, including the insertion of near-cognate tRNAs, which often leads to the generation of missense mutations at the original PTC site. Furthermore, some of the compounds have low efficiency of PTC inhibition in human primary cells, which led to ataluren's failure in phase 3 clinical trials.

[0007] Thus, there is a need for agents and methods for treating diseases or disorders associated with or caused by nonsense mutations. Summary of the Invention

[0008] The present disclosure addresses the aforementioned needs in several aspects. In one aspect, the present disclosure provides an oligonucleotide set comprising: (a) a first hairpin oligonucleotide comprising, from its 5' to 3' end, a first antisense strand, a first loop, and a first sense strand that is complementary to the first antisense strand; and (b) a second hairpin oligonucleotide comprising, from its 5' to 3' end, a second sense strand, a second loop, and a second antisense strand that is complementary to the second sense strand, wherein the first sense strand and the second sense strand are adapted to join together to form a nucleic acid sequence that encodes an RNA molecule.

[0009] In some embodiments, when the first sense strand and the second sense strand are joined, the first hairpin oligonucleotide and the second hairpin oligonucleotide form a closed-ended DNA thread (CEDT) molecule, which is sometimes also referred to as a picovector.

[0010] In some embodiments, the first hairpin oligonucleotide comprises a nucleic acid sequence encoding a tRNA leader, hi some embodiments, the second hairpin oligonucleotide comprises a nucleic acid sequence encoding an RNA polymerase III termination signal.

[0011] In some embodiments, the oligonucleotide set further comprises a third sense strand and a third antisense strand having a sequence complementary to the third sense strand, hi some embodiments, the first sense strand, the third sense strand, and the second sense strand are adapted to be joined together in order to form a second nucleic acid sequence encoding the RNA molecule.

[0012] In another aspect, the present disclosure provides an oligonucleotide set comprising: (i) a first hairpin oligonucleotide comprising, from its 5' to 3' end, a first antisense strand, a first loop, and a first sense strand that is complementary to the first antisense strand; (ii) a second hairpin oligonucleotide comprising, from its 5' to 3' end, a second sense strand, a second loop, and a second antisense strand that is complementary to the second sense strand; and (iii) a third sense strand and a third antisense strand having a sequence complementary to the third sense strand, wherein the first sense strand, the third sense strand, and the second sense strand are adapted to be joined together in an order to form a second nucleic acid sequence encoding an RNA molecule.

[0013] In some embodiments, when the first sense strand, the third sense strand, and the second sense strand are joined, the first hairpin oligonucleotide, the third sense and antisense strands, and the second hairpin oligonucleotide form a CEDT molecule.

[0014] In some embodiments, the third sense strand comprises a nucleic acid sequence encoding a tRNA leader. In some embodiments, the third sense strand comprises a nucleic acid sequence encoding an RNA polymerase III termination signal.

[0015] In some embodiments, the RNA molecule comprises a tRNA. In some embodiments, the tRNA comprises an anti-codon editing tRNA (ACE-tRNA). In some embodiments, the ACE-tRNA causes the ribosome to read through one or more stop codons during translation. In some embodiments, the one or more stop codons comprise a premature termination codon (PTC). Examples of PTCs include disease-causing PTCs or PTCs that cause nonsense-associated diseases. In some embodiments, the PTC is present in a nucleic acid sequence encoding the cystic fibrosis transmembrane conductance regulator (CFTR).

[0016] In some embodiments, the tRNA is selected from the group consisting of Arg-tRNA-UGA, Gln-tRNA-UAA, Glnt-RNA-UAG, Trp-tRNA-UGA, Trp-tRNA-UAG, Glu-tRNA-UAA, Glu-tRNA-UAG, Cys-tRNA-UGA, Tyr-tRNA-UAG, Tyr-tRNA-UAA, Leu-tRNA-UGA, Leu-tRNA-UAG, Leu-tRNA-UAA, Lys-tRNA-UAG, Lys-tRNA-UGA, Ser-tRNA-UGA, Ser-tRNA-UAG, and Ser-tRNA-UAA.

[0017] In some embodiments, the nucleic acid sequence comprises the polynucleotide sequence of SEQ ID NOs: 4, 8, 13, 15, and 17, or a polynucleotide sequence having at least 85% sequence identity to the polynucleotide sequence of SEQ ID NOs: 4, 8, 13, 15, and 17.

[0018] In some embodiments, the first hairpin oligonucleotide comprises the polynucleotide sequence of SEQ ID NOs: 1, 3, 7, and 11, or a polynucleotide sequence having at least 85% sequence identity to the polynucleotide sequence of SEQ ID NOs: 1, 3, 7, and 11, and / or the second hairpin oligonucleotide comprises the polynucleotide sequence of SEQ ID NOs: 2, 6, 10, and 12, or a polynucleotide sequence having at least 85% sequence identity to the polynucleotide sequence of SEQ ID NOs: 2, 6, 10, and 12.

[0019] In some embodiments, the nucleic acid sequence has a size of between 200 nucleotides and 1,000 nucleotides.

[0020] In some embodiments, the first loop or the second loop or another portion of the oligonucleotide is linked to an agent. In some embodiments, the agent comprises a labeling agent, a peptide, a bioactive agent, or a combination thereof. In some embodiments, the labeling agent comprises any one of N-hydroxysuccinimide (NHS), thiol-maleimide, and azido-dibenzocyclooctyne (DBCO). Such agents can be linked to the oligonucleotide via any suitable method known in the art, such as bioorthogonal chemistry and click chemistry. Exemplary reactions may include native chemical ligation and Staudinger ligation, copper-catalyzed azide-alkyne cycloaddition, strain-promoted [3 + 2] reactions, tetrazine ligation, metal-catalyzed coupling reactions, oxime and hydrazone ligation, and photoinduced bioorthogonal reactions.

[0021] In some embodiments, the first hairpin oligonucleotide or the second hairpin oligonucleotide comprises one or more chemically modified nucleotides. In some embodiments, the one or more chemically modified nucleotides comprise a 2'-O-methyl modified sugar moiety. In some embodiments, the one or more chemically modified nucleotides comprise a modified internucleoside linkage.

[0022] Also within the scope of this disclosure are compositions comprising the oligonucleotide sets described herein.

[0023] In another aspect, the present disclosure also provides kits comprising an oligonucleotide set described herein and, optionally, a ligase. In some embodiments, the ligase is T4 DNA ligase.

[0024] In yet another aspect, the present disclosure further provides a method for producing a CEDT molecule. In some embodiments, the method includes providing an oligonucleotide set described herein and ligating the components of the oligonucleotide set to thereby obtain a CEDT molecule.

[0025] In some embodiments, the oligonucleotide set is chemically synthesized. In some embodiments, the oligonucleotide set is synthesized with chemically modified nucleotides.

[0026] In some embodiments, the CEDT is further linked to a labeling agent, a peptide, a bioactive agent, or a combination thereof.

[0027] In another aspect, the present disclosure provides a CEDT molecule produced according to the methods described herein.

[0028] In yet another aspect, the present disclosure further provides a method for treating a PTC-related disease in a subject in need thereof, in some embodiments, the method comprises administering to the subject a CEDT molecule or a pharmaceutical composition thereof as described herein.

[0029] In some embodiments, the disease is cystic fibrosis, Duchenne and Becker muscular dystrophy, retinoblastoma, neurofibromatosis, ataxia-telangiectasia, Tay-Sachs disease, Wilms' tumor, hemophilia A, hemophilia B, Menkes disease, Ullrich's disease, b-thalassemia, von Willebrand's disease types 2A and 3, Robinow syndrome, brachydactyly type B (shortened fingers and metacarpals), genetic susceptibility to mycobacterial infections, inherited retinal diseases, inherited bleeding tendencies, hereditary blindness, congenital sensorineural hearing loss and enteric ganglion cell deficiencies, and inherited neurodevelopmental disorders including sensorineural hearing loss, enteric ganglion cell deficiencies, peripheral neuropathies, and central hypomyelination, Liddle's syndrome, The disease is selected from the group consisting of xeroderma pigmentosum, Fanconi anemia, anemia, hypothyroidism, p53-related cancer, esophageal cancer, bone cancer, ovarian cancer, hepatocellular carcinoma, breast cancer, hepatocellular carcinoma, fibrous histiocytoma, ovarian cancer, SRY sex reversal, triosephosphate isomerase anemia, diabetes mellitus, rickets, Hurler syndrome, Dravet syndrome, spinal muscular dystrophy, Usher syndrome, aniridia, congenital choroideremia, ophthalmic coloboma, retinitis pigmentosa, dystrophic epidermolysis bullosa, pseudoxanthoma elasticum, Alagille syndrome, Waardenburg-Schaa syndrome, childhood neuronal ceroid lipofuscinosis, cystinosis, X-linked nephrogenic diabetes insipidus, McArdle disease, and polycystic kidney disease.

[0030] The foregoing summary is not intended to define all aspects of the present disclosure; additional aspects are described in other sections, such as the following detailed description. It should be understood that the entire specification is intended to be related as a unified disclosure, and that all combinations of features described herein are contemplated, even if the combinations of features are not found together in the same sentence, paragraph, or section of the specification. Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0031] [Figure 1] An exemplary scheme for the generation of ArgTGA CEDT using a synthetic hairpin (synthHP) is shown, also referred to as the "1+1 scheme." [Figure 2] 1 shows the incorporation of bi-orthogonal reactive groups via ArgTGA synthHP generation. [Figure 3] Electrophoretic analysis after ligation of a synthetic hairpin (synthHP) to generate ArgTGA CEDT. [Figure 4] We show that ArgTGA CEDT synthHP rescues PTC in vivo. [Figure 5] Quantification of the 200 bp ArgTGA CEDT generated by ligation of a synthetic hairpin (synthHP) is shown. [Figure 6] An exemplary scheme for the generation of ArgTGA CEDT by ligation of synthetic hairpins (synthHP) is shown, also referred to as the "2+2 scheme." [Figure 7] An exemplary scheme for the generation of ArgTGA CEDT by ligation of a synthetic hairpin (synthHP) is shown, also referred to as the "4P scheme." [Figure 8] Figures 8A, 8B, and 8C show the generation of labeled ACE-tRNA DNA picovectors and the effect of labeling on ACE-tRNA function. Figure 8A shows the assembly scheme for labeled ACE-tRNA picovectors. Figure 8B shows the electrophoresis results of the labeled ACE-tRNA DNA picovector products. Figure 8C shows that labeled ACE-tRNAArg TGA picovectors were effective in nonsense suppression. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present disclosure relates to novel agents and methods for generating closed-end DNA thread (CEDT) molecules. CEDT molecules can be used for the delivery and expression of RNA molecules, such as anti-codon editing tRNA (ACE-tRNA). ACE-tRNA can convert nonsense mutations (e.g., premature termination codons (PTCs)) back to amino acids during mRNA translation. Therefore, ACE-tRNA can be used to treat genetic diseases associated with nonsense mutations.

[0033] Oligonucleotides and methods for generating CEDT molecules Oligonucleotide sets for generating CEDT molecules Accordingly, the present disclosure provides an oligonucleotide set comprising: (a) a first hairpin oligonucleotide comprising, from its 5' to 3' end, a first antisense strand, a first loop, and a first sense strand that is complementary to the first antisense strand; and (b) a second hairpin oligonucleotide comprising, from its 5' to 3' end, a second sense strand, a second loop, and a second antisense strand that is complementary to the second sense strand, wherein the first sense strand and the second sense strand are adapted to join together to form a nucleic acid sequence that encodes an RNA molecule.

[0034] Examples of oligonucleotide sets disclosed herein are illustrated in FIGS.

[0035] As used herein, "oligonucleotide" refers to a compound comprising multiple linked nucleosides. In some embodiments, an oligonucleotide may comprise one or more unmodified RNA and / or unmodified DNA and / or one or more modified nucleosides.

[0036] As used herein, "hairpin," "hairpin loop," or "terminal hairpin" refers to a structure formed when two regions of the same strand, usually complementary in nucleotide sequence, when read in opposite directions, advance base-pairing to form a double helix that ends in an unpaired loop.

[0037] As used herein, terms such as "first," "second," and "third" are used to modify nouns; such use is intended merely to distinguish one item from another and is not intended to require sequential ordering unless specifically stated.

[0038] As used herein, "antisense strand" refers to a nucleic acid strand that is complementary to the "sense" strand. The notation (-) (i.e., "negative") is sometimes used in reference to the antisense strand, and the notation (+) is sometimes used in reference to the sense (i.e., "positive") strand.

[0039] As used herein, the terms "complementary" or "complementarity" refer to "polynucleotides" and "oligonucleotides" (which are interchangeable terms referring to sequences of nucleotides) related by the base-pairing rules. For example, the sequence "CAGT" is complementary to the sequence "GTCA." Complementarity can be "partial" or "full." "Partial" complementarity is when one or more nucleic acid bases do not match according to the base-pairing rules. "Full" or "complete" complementarity between nucleic acids is when each and every nucleic acid base matches another base under the base-pairing rules. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions and detection methods that rely on binding between nucleic acids.

[0040] As used herein, a "self-complementary sequence" refers to a first nucleic acid sequence on a first oligonucleotide, and a second oligonucleotide may contain a second nucleic acid sequence in reverse order to the first nucleic acid. In this manner, the first and second nucleic acid sequences are complementary and can hybridize, thereby annealing the first and second oligonucleotides.

[0041] In some embodiments, the first hairpin may comprise a first segment of a nucleic acid sequence encoding an RNA molecule (e.g., an ACE-tRNA), and the second hairpin may comprise a second segment of a nucleic acid sequence encoding an RNA molecule, such that when the first hairpin and the second hairpin are joined together, directly or indirectly, the first and second segments of the nucleic acid sequence form a complete nucleic acid sequence encoding the RNA molecule.

[0042] In some embodiments, when the first and second sense strands are joined, the first hairpin oligonucleotide and the second hairpin oligonucleotide form a CEDT molecule. In some embodiments, the first and second sense strands can be joined directly or indirectly, for example, by ligation.

[0043] As used herein, the terms "ligate," "ligating," or "ligation" refer to any method or composition in which two different double-stranded nucleotides are joined into a single oligonucleotide strand by a chemical reaction. Generally, a ligase enzyme (e.g., T4 DNA ligase, T3 DNA ligase) can be used to facilitate the joining process.

[0044] In some embodiments, the first sense strand and the second sense strand can be directly joined. For example, the first sense strand and the second sense strand can be directly joined via ligatable ends, including blunt ends or sticky ends. In some embodiments, the first hairpin oligonucleotide and the second hairpin oligonucleotide can include blunt ends or sticky ends. For this purpose, the first antisense strand and the first sense strand can have the same length or different lengths. Similarly, the second antisense strand and the second sense strand can have the same length or different lengths.

[0045] As used herein, the term "blunt end" or "blunt-ended oligonucleotide" refers to an oligonucleotide that does not have an overhang. As used herein, the term "sticky end" refers to a double-stranded polynucleotide molecule end that may include a sequence overhang. In some embodiments, a sticky end can be a nucleic acid molecule end having a 5' or 3' sequence overhang. In some embodiments, the sticky ends of the present disclosure can hybridize with a compatible sticky end of the same or another molecule. Thus, in some embodiments, a sticky end at the 3' end of a first DNA fragment can hybridize with a compatible sticky end on a second DNA fragment. In some embodiments, these hybridized sticky ends can be joined together by a ligase. In other embodiments, the sticky ends may require overhang extension to complete the dsDNA molecule prior to ligation. In some embodiments, the first and second sense strands can be joined indirectly via a spacer, such as a spacer nucleic acid sequence (e.g., a double-stranded DNA segment) that bridges the first and second sense strands.

[0046] In some embodiments, the first hairpin oligonucleotide can include a nucleic acid sequence encoding a tRNA leader. For example, the tRNA leader can be encoded by double-stranded DNA, and the sense and antisense strands of the double-stranded DNA can be disposed on the first sense and first antisense strands of the first hairpin oligonucleotide, respectively.

[0047] In some embodiments, the second hairpin oligonucleotide can include a nucleic acid sequence encoding a transcription termination signal, hi some embodiments, the transcription termination signal is an RNA polymerase III termination signal.

[0048] As shown in Figure 5, the first hairpin oligonucleotide and the second hairpin oligonucleotide can also be joined together via one or more double-stranded nucleic acid segments. Thus, in some embodiments, the oligonucleotide set can include a third sense strand and a third antisense strand having a sequence complementary to the third sense strand. In some embodiments, the first sense strand, the third sense strand, and the second sense strand are adapted to be joined together in order to form a second nucleic acid sequence encoding an RNA molecule.

[0049] In another aspect, the present disclosure provides an oligonucleotide set comprising: (i) a first hairpin oligonucleotide comprising, from its 5' to 3' end, a first antisense strand, a first loop, and a first sense strand that is complementary to the first antisense strand; (ii) a second hairpin oligonucleotide comprising, from its 5' to 3' end, a second sense strand, a second loop, and a second antisense strand that is complementary to the second sense strand; and (iii) a third sense strand and a third antisense strand having a sequence complementary to the third sense strand, wherein the first sense strand, the third sense strand, and the second sense strand are adapted to be joined together in an order to form a second nucleic acid sequence encoding an RNA molecule.

[0050] Exemplary oligonucleotide sets described herein are illustrated in FIGS.

[0051] In some embodiments, when the first sense strand, the third sense strand, and the second sense strand are joined, the first hairpin oligonucleotide, the third sense and antisense strands, and the second hairpin oligonucleotide form a CEDT molecule.

[0052] CEDT molecule As used herein, "closed-end DNA thread," "CEDT," "CEDT molecule," or "CEDT minivector" or "picovector" refers to a closed linear DNA molecule. Such a closed-end linear DNA molecule may be considered a single-stranded circular molecule. CEDT molecules may contain covalently closed ends, also described as hairpin loops, in which there is no base pairing between complementary DNA strands. Hairpin loops join the ends of complementary DNA strands. This type of structure typically forms at the telomeric ends of chromosomes and protects chromosomal DNA from loss or damage by sequestering the terminal nucleotides in a closed structure. In some examples of CEDT molecules described herein, the hairpin loops are adjacent to complementary base-paired DNA strands, forming a "doggy-bone"-shaped structure, as shown, for example, in Figures 1-2 and 5-7.

[0053] A CEDT molecule typically comprises a linear double-stranded segment of DNA with covalently closed ends, i.e., hairpin ends. The hairpin joins the ends of the linear double DNA strand so that, when the molecule is fully denatured, a single-stranded circular DNA molecule is generated. As described herein, CEDT can be essentially perfectly complementary in sequence, although some slight variations or "wobbles" can be tolerated by the structure. Thus, a closed linear DNA or CEDT can be at least 75%, 80%, 85%, 90%, or 95% complementary in sequence, or at least 96, 97, 98, 99, or 100% complementary. When denatured, it is a substantially circular molecule containing both forward (sense or plus) and reverse (antisense or minus) strands adjacent to each other. This is in contrast to plasmid DNA or minicircle (MC) DNA, in which the complementary sequences (minus and plus) are on separate circular strands.

[0054] Bases within the apex (end or turn) of the hairpin may not be able to form base pairs due to conformational stresses on the DNA strand at this point. For example, at least two base pairs at the apex of the apex may not form base pairs, but the exact conformation is likely subject to variation depending on the conditions under which the DNA is maintained and the exact sequence around the hairpin. Thus, despite their complementary nature, two or more bases may not be able to pair if there is structural distortion involved. Some "wobble" of non-complementary bases within the length of the hairpin may not affect the structure. The wobble may interrupt the palindrome, but the sequence may remain complementary. In some embodiments, the sequence of the hairpin is fully self-complementary. Complementarity describes how each polynucleotide base in a sequence (5' to 3') is in a hydrogen-bonded pair with a complementary base, A for T (or U) and C for G, on the antiparallel (3' to 5') strand, which may be on the same strand (internal complementary sequence) or a different strand. This definition applies to any aspect or embodiment of the invention. In some embodiments, the sequences in the hairpin are 90% complementary, such as 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99%, or 100% complementary.

[0055] A CEDT can contain any sequence within a double-stranded sequence, either naturally occurring or artificial. It can contain at least one processing enzyme target sequence, such as one, two, three, or more processing enzyme target sites. Such target sequences allow the DNA to be optionally further processed after synthesis. A processing enzyme is an enzyme that recognizes its target site and processes the DNA. A processing enzyme target sequence can be a target sequence for a restriction enzyme. Restriction enzymes, i.e., restriction endonucleases, bind to and cleave a target sequence at a specific point. A processing enzyme target sequence can be a target for a recombinase. Recombinases catalyze the directionality of DNA exchange reactions between short (30-40 nucleotide) target site sequences specific to each recombinase. Examples of recombinases include Cre recombinase (which has loxP as its target sequence) and FLP recombinase (which has a short flippase recognition target (FRT) site). The processing enzyme target sequence can be a target for a site-specific integrase, such as phiC31 integrase.

[0056] The processing enzyme target sequence can be a target sequence for an RNA polymerase so that the CEDT serves as a template for RNA synthesis. In this case, the processing enzyme target site is a promoter, such as a eukaryotic promoter. To that end, the CEDT can include an expression cassette comprising, or consisting essentially of, a eukaryotic promoter operably linked to a sequence surrounding an RNA (e.g., tRNA) or protein of interest, and, optionally, a eukaryotic transcription termination sequence. A "promoter" is a nucleotide sequence that initiates and regulates transcription of a polynucleotide. "Operably linked" refers to the arrangement of elements such that the components so described are configured to perform their normal functions. Thus, a given promoter operably linked to a nucleic acid sequence can affect the expression of that sequence when the appropriate enzymes are present. The term "operably linked" is intended to encompass any spacing or orientation of the promoter element and DNA sequence of interest that allows initiation of transcription of the DNA sequence of interest upon recognition of the promoter element by a transcription complex.

[0057] CEDTs can be of any suitable length. For example, CEDTs can have a size of up to 4 kb, such as 100 bp to 2 kb, 200 bp to 1 kb, or 200 bp to 800 bp. In some embodiments, CEDTs of 200 bp or more can accommodate multiple ACE-tRNA cassettes / copies, allowing for higher ACE-tRNA expression from each CEDT unit. Having multiple copies of ACE-tRNA from each CEDT allows for one unit to contain more than one sequence. For example, a leucine ACE-tRNA and a tryptophan ACE-tRNA can be included in a single CEDT molecule. Both of these ACE-tRNAs can be effective in cystic fibrosis to rescue or suppress mutant W1282X-CFTR, and because they utilize different tRNA aminoacyl synthetases, they can significantly enhance suppression activity.

[0058] ACE-tRNA In some embodiments, the tRNA may include an ACE-tRNA. ACE-tRNA is an engineered tRNA molecule that can convert PTCs back to the original missing amino acid or a different amino acid. Such engineered tRNAs allow for the "reediting" of disease-causing nonsense codons into specific amino acids. The small size of these tRNA molecules, with the tRNA and promoter combined, is only approximately 300 bp, allowing for their rapid expression. To this end, oligonucleotides can be synthesized to contain the structural components of tRNA genes that are functional in human cells. The sequence of this oligonucleotide can be designed based on known sequences with substitutions made within the anticodon region of the tRNA, allowing a specific tRNA to recognize nonsense or other specific mutations. Examples of ACE-tRNAs include those described in WO2019 / 090154, WO2019 / 090169, WO2021 / 252354A1, and Lueck, J.D. et al. Nature Communications 10, 822 (2019), the contents of which are incorporated herein by reference.

[0059] Generally, ACE-tRNA has a four-arm structure including a T-arm, a D-arm, an anticodon arm, and an acceptor arm (see, for example, Figure 2 of WO2019 / 090169). The T-arm is composed of a "T-stem" and a "TYE loop." In some embodiments, the T-stem is modified to increase the stability of the tRNA. In some embodiments, the ACE-tRNA has a modified T-stem that suppresses the termination site compared to the endogenous T-stem sequence, thereby increasing biological activity.

[0060] ACE-tRNAs can be used to suppress PTCs. This ACE-tRNA approach offers several key advantages over other readthrough strategies, including (1) codon specificity, (2) ACE-tRNA suppression of PTCs resulting in seamless rescue, thus abolishing spurious effects on protein stability, folding, trafficking, and function, and (3) in vitro delivery of these ACE-tRNAs resulting in significant functional rescue of affected proteins, such as CFTR channels carrying the p.G542X or p.W1282X CF mutations. ACE-tRNAs have been shown to be efficient at suppressing PTCs in several cDNA genes with various PTC locations in multiple cell types. Because ACE-tRNAs exhibit high efficiency in suppressing PTCs without known adverse effects, they can be used as therapeutic agents.

[0061] ACE-tRNA can be produced according to the methods described in WO2019 / 090154, WO2019 / 090169, WO2021 / 252354A1, and Lueck, J.D. et al., Nature Communications 10, 822 (2019). Using the described methods, a wide library of ACE-tRNAs can be generated for the effective rescue of PTC in cell culture. Other engineered human tRNA sequences for suppressing disease-causing PTC include those described in WO2019 / 090154, WO2019 / 090169, WO2021 / 252354A1, and Lueck, J.D. et al., Nature Communications 10, 822 (2019), the contents of which are incorporated herein by reference.

[0062] In some embodiments, the ACE-tRNA can be any one of Arg-tRNA-UGA, Gln-tRNA-UAA, Glnt-RNA-UAG, Trp-tRNA-UGA, Trp-tRNA-UAG, Glu-tRNA-UAA, Glu-tRNA-UAG, Cys-tRNA-UGA, Tyr-tRNA-UAG, Tyr-tRNA-UAA, Leu-tRNA-UGA, Leu-tRNA-UAG, Leu-tRNA-UAA, Lys-tRNA-UAG, Lys-tRNA-UGA, Ser-tRNA-UGA, Ser-tRNA-UAG, and Ser-tRNA-UAA. [Table 1-1] [Table 1-2] [Table 1-3]

[0063] In some embodiments, the third sense strand may comprise the polynucleotide sequences of SEQ ID NOs: 4, 8, 13, 15, and 17, or may comprise a polynucleotide sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the polynucleotide sequences of SEQ ID NOs: 4, 8, 13, 15, and 17.

[0064] In some embodiments, the third antisense strand may comprise the polynucleotide sequence of SEQ ID NOs: 5, 9, 14, 16, and 18, or may comprise a polynucleotide sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the polynucleotide sequence of SEQ ID NOs: 5, 9, 14, 16, and 18.

[0065] In some embodiments, the nucleic acid sequence may comprise the polynucleotide sequence of SEQ ID NOs: 4, 8, 13, 15, and 17, or may comprise a polynucleotide sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the polynucleotide sequence of SEQ ID NOs: 4, 8, 13, 15, and 17.

[0066] In some embodiments, the second nucleic acid sequence may comprise the polynucleotide sequence of SEQ ID NOs: 4, 8, 13, 15, and 17, or may comprise a polynucleotide sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the polynucleotide sequence of SEQ ID NOs: 4, 8, 13, 15, and 17.

[0067] In some embodiments, the first hairpin oligonucleotide may comprise the polynucleotide sequence of SEQ ID NOs: 1, 3, 7, and 11, or may comprise a polynucleotide sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the polynucleotide sequence of SEQ ID NOs: 1, 3, 7, and 11.

[0068] In some embodiments, the second hairpin oligonucleotide may comprise the polynucleotide sequence of SEQ ID NOs: 2, 6, 10, and 12, or may comprise a polynucleotide sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the polynucleotide sequence of SEQ ID NOs: 2, 6, 10, and 12.

[0069] In some embodiments, CEDT molecules formed from the disclosed oligonucleotide sets can have a size of 200 nucleotides (nt) to 1,000 nt (e.g., 200, 250, 300, 350, 400, 45, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000 nt). In some embodiments, the ACE-tRNA-encoding duplex segment has a size of, for example, less than 200 bp, less than 250 bp, less than 300 bp, less than 350 bp, less than 400 bp, less than 450 bp, less than 500 bp, less than 550 bp, less than 600 bp, less than 650 bp, less than 700 bp, less than 750 bp, less than 800 bp, less than 850 bp, less than 900 bp, or less than 950 bp.

[0070] In some embodiments, one or more of the first loop or / and the second loop or / and another portion of the oligonucleotide are linked to a drug or different drugs, hi some embodiments, the drug or drugs may comprise a labeling agent, a peptide, a bioactive agent, or a combination thereof.

[0071] As used herein, the terms "labeling agent," "label," or "detectable label" refer to any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Such labels include biotin for staining with labeled streptavidin conjugates, magnetic beads (e.g., Dynabeads®), fluorescent dyes (e.g., fluorescein, Texas Red, rhodamine, green fluorescent protein, etc.), radioactive labels (e.g., 3 H, 125 I, 35 S, 14 C, or 32P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and others commonly used in ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. The use of such labels is described, for example, in U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, the relevant contents of which are incorporated herein by reference. Labels can be detected by a number of methods. For example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent markers can be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, whereas calorimetric labels are detected by simply visualizing the colored label. As shown in Example 3 and Figures 8A-8C, the addition of labels or modifications to hairpins or CEDT or picovectors does not negatively affect PTC inhibition. Therefore, such labeled hairpins or labeled CEDT / picovectors can be used in a variety of suitable applications, such as identifying nuclear localization signal sequences and / or cell-penetrating peptides to improve picovector delivery and subcellular localization.

[0072] As used herein, the term "bioactive agent" refers to a substance that can be used in connection with applications that are therapeutic or diagnostic in nature, such as, for example, a method for diagnosing the presence or absence of a disease in a patient or a method for treating a disease in a patient.

[0073] In some embodiments, the labeling agent may include N-hydroxysuccinimide (NHS), thiol-maleimide, azido-dibenzocyclooctyne (DBCO), or a combination thereof.

[0074] In some embodiments, nucleic acid molecules such as the first hairpin oligonucleotide, second hairpin oligonucleotide, or CEDT molecule described herein may contain one or more chemically modified nucleotides, such as 2'-O-methyl modified sugar moieties. For example, chemically modified nucleotides may contain modified internucleoside linkages. As used herein, "modified oligonucleotide" refers to an oligonucleotide containing at least one modified nucleoside and / or at least one modified internucleoside linkage. Examples of modified oligonucleotides include single-stranded and double-stranded compounds, such as antisense compounds, siRNA, shRNA, ssRNA, and proprietary-based compounds.

[0075] As used herein, "nucleoside" refers to a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (found in DNA and RNA) and modified nucleosides. Nucleosides can be linked to a phosphate moiety.

[0076] As used herein, "chemical modification" refers to the chemical difference in a compound compared with its naturally occurring counterpart.Chemical modification of oligonucleotides can include nucleoside modification (such as sugar moiety modification and nucleobase modification) and internucleoside linkage modification.As used herein, "internucleoside linkage" refers to the covalent bond between adjacent nucleosides in an oligonucleotide.With respect to oligonucleotides, chemical modification does not only include differences in nucleobase sequence.

[0077] As used herein, "sugar moiety" refers to the naturally occurring sugar moiety or a modified sugar moiety of a nucleoside. As used herein, "modified sugar moiety" refers to a substituted sugar moiety or sugar surrogate. As used herein, "substituted sugar moiety" refers to a furanosyl that is not a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to, furanosyl containing a substituent at the 2'-, 3'-, 5'-, and / or 4'-position. Certain substituted sugar moieties are bicyclic sugar moieties. As used herein, a "2'-substituted sugar moiety" refers to a furanosyl containing a substituent at the 2'-position other than H or OH. Unless otherwise specified, a 2'-substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2'-substituent of the 2'-substituted sugar moiety does not form a bridge with another atom of the furanosyl ring).

[0078] In some embodiments, chemical modifications may provide certain desirable properties, such as enhanced nuclease stability or increased binding affinity to a target nucleic acid, compared to molecules having only nucleosides containing naturally occurring sugar moieties. In some embodiments, the modified sugar moiety is a substituted sugar moiety. In some embodiments, the modified sugar moiety is a bicyclic or tricyclic sugar moiety. In some embodiments, the modified sugar moiety is a sugar surrogate. Such sugar surrogates may contain one or more substitutions corresponding to those of the substituted sugar moiety.

[0079] In some embodiments, the modified sugar moiety is a substituted sugar moiety containing one or more substituents, including, but not limited to, substituents at the 2' and / or 5' positions. Examples of suitable sugar substituents at the 2' position include, but are not limited to, 2'-F,2'-OCH3 ("OMe" or "O-methyl"), and 2'-O(CH2)2OCH3 ("MOE"). In some embodiments, the sugar substituent at the 2' position is allyl, amino, azido, thio, O-allyl, O-C1-C 10 Alkyl, O-C1-C 10 Substituted alkyl, O-C1-C 10 Alkoxy, O-C1-C 10substituted alkoxy, OCF, O(CH)SCH, O(CH)-ON(R)(R), and O-CH-C(=O)-N(R)(R), wherein each R and R is independently H or a substituted or unsubstituted C-C 10 and alkyl. Examples of sugar substituents at the 5'-position include, but are not limited to, 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy. In some embodiments, the substituted sugar can contain two or more non-bridging sugar substituents, such as a 2'-F-5'-methyl sugar moiety (see PCT International Application No. WO2008 / 101157 for additional 5',2'-bis-substituted sugar moieties and nucleosides).

[0080] Nucleosides that include a 2'-substituted sugar moiety are referred to herein as 2'-substituted nucleosides. In some embodiments, 2'-substituted nucleosides include halo, allyl, amino, azido, O-C-C 10 Alkoxy;O-C1-C 10 Substituted alkoxy, SH, CN, OCN, CF3, OCF3, O-alkyl, S-alkyl, N(R m )-alkyl; O-alkenyl, S-alkenyl, or N(R m )-alkenyl; O-alkynyl, S-alkynyl, N(R m )-alkynyl; O-alkynyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O-(CH2)2-ON(R m )(R n ) or O-CH2-C(=O)-N(R m )(R n ), wherein each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C-C 10These 2'-substituents can be further substituted with one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl.

[0081] In some embodiments, the 2'-substituted nucleoside is selected from the group consisting of F, NH, N, OCF, O-CH, O(CH)NH, CH-CH=CH, O-CH-CH=CH, OCHCHOCH, O(CH)SCH, O-(CH)-ON(R m )(R n ), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamides (O-CH2-C(=O)-N(R m )(R n ), wherein each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C-C 10 and alkyl. In some embodiments, 2'-substituted nucleosides can comprise a sugar moiety that includes a 2'-substituent selected from F, OCF, O-CH, OCHCHOCH, O(CH)SCH, O-(CH)-ON(CH), -O(CH)O(CH)N(CH), and O-CH-C(=O)-N(H)CH. In some embodiments, 2'-substituted nucleosides can comprise a sugar moiety that includes a 2'-substituent selected from F, O-CH, and OCHCHOCH.

[0082] In some embodiments, the modified sugar moiety may include a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In some embodiments, the bicyclic sugar moiety may include a bridge between the 4' and 2' furanose ring atoms. Examples of such 4' to 2' sugar substituents include -[C(R a )(R b )] n -, -[C(R a )(R b )] n -O-, -C(Ra R b )-N(R)-O-, or -C(R a R b )-ON(R)-; 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (cEt), and 4'-CH(CHOCH3)-O-2', and analogs thereof (see, e.g., U.S. Patent No. 7,399,845 issued July 15, 2008); 4'-C(CH3)(CH3)-O-2' and analogs thereof (see, e.g., WO 2009 / 006478 published January 8, 2009); 4'-CH2-N(OCH3)-2' and analogs thereof (see, e.g., WO 2008 / 150729 published December 11, 2008); 4'-CH2-ON(CH3)-2' (see, e.g., US 2004 / 0171570 published September 2, 2004); 4'-CH2-ON(R)-2', and 4'-CH2-N(R)-O-2'- (wherein each R is independently H, a protecting group, or C1-C 12 alkyl); 4'-CH2-N(R)-O-2' (wherein R is H, C1-C 12 alkyl, or a protecting group) (see U.S. Patent No. 7,427,672 issued September 23, 2008); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' and analogs thereof (see published PTC International Application No. WO2008 / 154401 published December 8, 2008).

[0083] Methods for making CEDT In yet another aspect, the present disclosure further provides a method for producing a CEDT molecule. In some embodiments, the method can include providing an oligonucleotide set described herein and ligating components of the oligonucleotide set, thereby obtaining a closed-end DNA thread molecule. In some embodiments, ligation of components of the oligonucleotide set can be assisted by an enzyme such as a ligase. The ligase can be a eukaryotic ligase. The ligase can be a prokaryotic ligase. The ligase can be a single-stranded ligase. The ligase can be a double-stranded ligase. The ligase can be a DNA ligase. The DNA ligase can be T4 DNA ligase, Taq DNA ligase, T7 DNA ligase, T3 DNA ligase, 9°N™ DNA ligase, and E. coli DNA ligase.

[0084] In some embodiments, the nucleic acid sequence may comprise the polynucleotide sequence of SEQ ID NOs: 4, 8, 13, 15, and 17, or may comprise a polynucleotide sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the polynucleotide sequence of SEQ ID NOs: 4, 8, 13, 15, and 17.

[0085] In some embodiments, the second nucleic acid sequence may comprise the polynucleotide sequence of SEQ ID NOs: 4, 8, 13, 15, and 17, or may comprise a polynucleotide sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the polynucleotide sequence of SEQ ID NOs: 4, 8, 13, 15, and 17.

[0086] In some embodiments, the first hairpin oligonucleotide may comprise the polynucleotide sequence of SEQ ID NOs: 1, 3, 7, and 11, or may comprise a polynucleotide sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the polynucleotide sequence of SEQ ID NOs: 1, 3, 7, and 11.

[0087] In some embodiments, the second hairpin oligonucleotide may comprise the polynucleotide sequence of SEQ ID NOs: 2, 6, 10, and 12, or may comprise a polynucleotide sequence having at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the polynucleotide sequence of SEQ ID NOs: 2, 6, 10, and 12.

[0088] In some embodiments, the oligonucleotide set can be chemically synthesized. In some embodiments, the oligonucleotide set can be synthesized with chemically modified nucleotides. In some embodiments, the first hairpin oligonucleotide, the second hairpin oligonucleotide, or the CEDT molecule can include one or more chemically modified nucleotides, such as 2'-O-methyl modified sugar moieties. For example, the chemically modified nucleotide can include a modified internucleoside linkage.

[0089] In some embodiments, the closed-ended DNA thread molecule is further linked to a labeling agent, a peptide, a bioactive agent, or a combination thereof.

[0090] Compositions and Kits Nucleic acid molecules such as oligonucleotide sets, hairpin oligonucleotides, or CEDT molecules generated from the disclosed oligonucleotide sets can be provided in compositions (e.g., pharmaceutical compositions) or kits. In some embodiments, the composition can include an oligonucleotide set described herein. In some embodiments, the composition can include a first hairpin oligonucleotide and / or a second hairpin oligonucleotide as described herein. In some embodiments, the composition can include a CEDT molecule prepared from an oligonucleotide set described herein.

[0091] Formulation of nucleic acids (e.g., DNA) as conventional pharmaceutical preparations can be carried out using standard pharmaceutical formulation chemistry and methodology available to those skilled in the art. Any pharmaceutically acceptable carrier or excipient may be used. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in the excipient or vehicle. These excipients, vehicles, and auxiliary substances are generally pharmaceuticals that can be administered without undue toxicity and, in the case of vaccine compositions, do not induce an immune response in the individual receiving the composition. A suitable carrier may be liposomes. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol. Pharmaceutically acceptable salts may also be included, for example, inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, and the like, and organic acid salts such as acetate, propionate, malonate, benzoate, and the like. The preparation may also include pharmaceutically acceptable excipients that function as stabilizers, particularly when the composition includes peptides, proteins, or other similar molecules. Examples of suitable carriers that also function as stabilizers for the peptides include, but are not limited to, pharmaceutical grade dextrose, sucrose, lactose, trehalose, mannitol, sorbitol, inositol, dextran, etc. Other suitable carriers include, but are not limited to, starch, cellulose, sodium or calcium phosphate, citric acid, tartaric acid, glycine, high molecular weight polyethylene glycol (PEG), or combinations thereof. A thorough discussion of pharmaceutically acceptable excipients, vehicles, and auxiliary substances is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991), which is incorporated herein by reference.

[0092] Compositions containing active ingredients, such as oligonucleotides or CEDT molecules, can be prepared by procedures known in the art using well-known and readily available ingredients.For CEDT molecules, the compositions can be formulated as solutions suitable for parenteral administration, for example, intramuscular, subcutaneous, or intravenous routes.The compositions can be in the form of aqueous or anhydrous solutions or dispersions, or in the form of emulsions or suspensions.Alternatively, the compositions can be in powder form, obtained by aseptic isolation of sterile solids or by lyophilization from solution, to be constituted with a suitable vehicle, for example, sterile, pyrogen-free water, before use.

[0093] In some embodiments, the compositions disclosed herein can be formulated as lipid nanoparticles (LNPs), such as those described in WO2020 / 263883, WO2013 / 123523, WO2012 / 170930, WO2011 / 127255, WO2008 / 103276, and US2013 / 0171646, each of which is incorporated herein by reference in its entirety. Thus, the present disclosure provides nanoparticle compositions comprising a lipid composition comprising at least one nucleic acid, such as a CEDT molecule, and a delivery agent. In such nanoparticle compositions, the lipid composition disclosed herein can encapsulate nucleic acids.

[0094] Nanoparticle compositions are typically submicrometer in size and can contain lipid bilayers. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, nanoparticle compositions can be liposomes with lipid bilayers having a diameter of 500 nm or less.

[0095] Nanoparticle compositions include, for example, lipid nanoparticles, liposomes, and lipoplexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In some embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by aqueous compartments. The lipid bilayers can be functionalized and / or crosslinked to each other. The lipid bilayer can comprise one or more ligands, proteins, or channels.

[0096] In one embodiment, the lipid nanoparticles may comprise an ionizable lipid, a structured lipid, a phospholipid, and a nucleic acid of interest. In some embodiments, the lipid nanoparticles may comprise an ionizable lipid, a PEG-modified lipid, a sterol, and a structured lipid. In some embodiments, the lipid nanoparticles have a molar ratio of about 20-60% ionizable lipid, about 5-25% structured lipid, about 25-55% sterol, and about 0.5-15% PEG-modified lipid. In some embodiments, the lipid nanoparticles have a polydispersity value of less than 0.4. In some embodiments, the lipid nanoparticles have a net neutral charge at neutral pH. In some embodiments, the lipid nanoparticles have an average diameter of 50-150 nm. In some embodiments, the lipid nanoparticles have an average diameter of 80-100 nm.

[0097] As generally defined herein, the term "lipid" refers to a small molecule having hydrophobic or amphipathic properties. Lipids can be naturally occurring or synthetic. Examples of lipid classes include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides, and prenol lipids. In some cases, the amphipathic properties of some lipids allow them to form liposomes, vesicles, or membranes in aqueous media.

[0098] In some embodiments, the nucleic acid can be formulated in lipid nanoparticles having a diameter of about 10 to about 100 nm. In some embodiments, the nanoparticles have a diameter of about 10 to 500 nm. In some embodiments, the nanoparticles have a diameter greater than 100 nm. In some embodiments, the largest dimension of the nanoparticle composition is 1 pm or less (e.g., 1 pm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or less). As used herein, "size" or "average size" in the context of a nanoparticle composition refers to the average diameter of the nanoparticles.

[0099] The nanoparticle composition can be relatively homogeneous. The polydispersity index can be used to indicate the uniformity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0 to about 0.25, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the nanoparticle compositions disclosed herein can be about 0.10 to about 0.20.

[0100] In some embodiments, the nucleic acids described herein can be formulated for controlled release and / or targeted delivery. As used herein, "controlled release" refers to a pharmaceutical composition or compound release profile that conforms to a specific release pattern to produce a therapeutic outcome. In one embodiment, the nucleic acid can be encapsulated in a delivery agent described herein and / or known in the art for controlled release and / or targeted delivery. As used herein, the term "encapsulation" means to surround, enclose, or encase. When referring to a formulation of a nucleic acid of the present disclosure, encapsulation can be substantial, complete, or partial. The term "substantially encapsulated" means that at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, or more than 99% of the pharmaceutical composition or nucleic acid of the present disclosure can be enclosed, surrounded, or encased within the delivery agent. By "partially encapsulated" is meant that fewer than 10, 10, 20, 30, 40, 50 or fewer pharmaceutical compositions or nucleic acids of the disclosure may be enclosed, surrounded, or encased within the delivery agent.

[0101] In some embodiments, the compositions can be formulated for sustained release. As used herein, "sustained release" refers to a pharmaceutical composition or compound that is adapted to a release rate over a specific period of time. The period can include, but is not limited to, hours, days, weeks, months, and years. As non-limiting examples, the sustained release nanoparticle compositions described herein can be formulated as disclosed in WO2010 / 075072, US2010 / 0216804, US2011 / 0217377, US2012 / 0201859, and US2013 / 0150295, each of which is incorporated herein by reference in its entirety. In some embodiments, nanoparticle compositions can be formulated to be target specific, such as those described in WO2008 / 121949, WO2010 / 005726, WO2010 / 005725, WO2011 / 084521, WO2011 / 084518, US2010 / 0069426, US2012 / 0004293, and US2010 / 0104655, each of which is incorporated herein by reference in its entirety.

[0102] Nucleic acid molecules (e.g., oligonucleotide sets, hairpin oligonucleotides, CEDT molecules) or compositions thereof, as described herein, can be provided in kits. In some embodiments, the kits include a container containing at least one nucleic acid molecule or composition thereof, and optionally, informational material. The informational material can be descriptive, instructional, marketing, or other material related to the methods described herein and / or the use of the agent for therapeutic benefit. For example, the kits can include instructions regarding manufacture, the treatment regimen to be used, and the duration of administration. In some embodiments, the kits can also include an additional therapeutic agent. The kits can include one or more containers, each with a different reagent. For example, the kits can include a first container containing the composition and a second container for an additional agent, such as a therapeutic agent.

[0103] The container may contain a unit dosage of the pharmaceutical composition. In addition to the composition, the kit may include other ingredients, such as a solvent or buffer, an adjuvant, a stabilizer, a preservative, or a combination thereof. The kit may optionally include a device suitable for administering the composition, such as a syringe or other suitable delivery device. The device may be provided pre-loaded with one or both agents, or may be empty but suitable for loading.

[0104] Treatment method In yet another aspect, the present disclosure additionally provides methods for treating a disease or disorder associated with PTC in a mammal (such as a human). In some embodiments, the method may include administering to the mammal a CEDT molecule prepared from the disclosed oligonucleotide set.

[0105] The disclosed method is advantageous because it provides improved stop codon suppression specificity. The therapeutic ACE-tRNA of the present disclosure can target specific stop codons, such as TGA, thus reducing off-target effects at stop codons unrelated to the disease. It is also advantageous because it provides amino acid specificity. The expressed tRNA is engineered to specifically replace the amino acid lost through the insertion of a disease-associated stop codon, thus negating any spurious effects on protein stability, folding, and transport. Furthermore, the method can be "personalized" to correct all possible disease PTCs. For example, the human genome contains nine individual tryptophan tRNAs recognized by Trp synthase, all of which suppress the mRNA UGG codon. Therefore, each of these nine Trp tRNAs offers an opportunity for codon re-editing resistance (e.g., to UGG, UGA). Additionally, given their proximity to stop codons in the genetic code, mutation of arginine codons to PTC nonsense codons is common in PTC-related diseases. There are over 30 Arg tRNAs that can be used, and the ACE-tRNA encoding arginine is a viable treatment for all Arg->PTC mutations, regardless of gene. In fact, 35% of Leber congenital amaurosis (LCA) cases are caused by nonsense mutations, the majority of which are nonsense mutations to the arginine stop codon. A further advantage of the disclosed method is that the entire system (tRNA + promoter sequence) is compact, providing easy expression and cell-specific delivery.

[0106] Diseases or disorders caused by or associated with PTCs include, but are not limited to, Duchenne muscular dystrophy and Becker muscular dystrophy variants caused by PTCs of dystrophin, retinoblastoma caused by PTCs of RBI, neurofibromatosis caused by PTCs of NF1 or NF2, ataxia-telangiectasia caused by PTCs of ATM, Tay-Sachs disease caused by PTCs of HEXA, cystic fibrosis caused by PTCs of CFTR, Wilms tumor caused by PTCs of WT1, hemophilia A caused by PTCs of factor VIII, hemophilia B caused by PTCs of factor IX, p53-related cancers caused by PTCs of p53, Menkes disease, Ullrich disease, b-thalassemia caused by PTCs of beta-globin, von Willebrand disease types 2A and 3 caused by PTCs of Willebrand factor, Robinow syndrome, brachydactyly type B ( hereditary neurodevelopmental disorders including congenital sensorineural hearing loss and intestinal dysganglionosis due to PTC of SOX10, and sensorineural hearing loss, intestinal dysganglionosis, peripheral neuropathy, and central hypomyelination due to PTC of SOX10; Liddle syndrome; xeroderma pigmentosum; Fanconi anemia; anemia; hypothyroidism; p53-related cancers (e.g., p53 squamous cell carcinoma, p53 hepatocellular carcinoma, p53 ovarian cancer); esophageal cancer; bone cancer; ovarian cancer; hepatocellular carcinoma; breast cancer; hepatocellular carcinoma; fibrous histiocytoma; ovarian cancer; SRY sex reversal; triosephosphate isomerase anemia; diabetes mellitus; and rickets.

[0107] In some embodiments, methods may include treating diseases or disorders such as cystic fibrosis by reversing the effects of existing mutations associated with nonsense mutations via the CEDT molecules of the present disclosure. Other diseases or disorders may include Hurler syndrome, Dravet syndrome, spinal muscular dystrophy, Usher syndrome, aniridia, congenital choroideremia, ophthalmic coloboma, retinitis pigmentosa, dystrophic epidermolysis bullosa, pseudoxanthoma elasticum, Alagille syndrome, Waardenburg-Schar syndrome, childhood neuronal ceroid lipofuscinosis, cystinosis, X-linked nephrogenic diabetes insipidus, and polycystic kidney disease.

[0108] Additional diseases or disorders associated with PTC that can be treated by the disclosed methods can include ocular diseases. Examples of ocular diseases can include those associated with one or more mutations in genes, such as cone dystrophies (Stargardt disease (STGD1), cone-rod dystrophy, retinitis pigmentosa (RP), and increased susceptibility to age-related macular degeneration): KCNV2 Glut43X; KCNV2 Glu306X; KCNV2 Gln76X; KCNV2 Glul48X;CACNA2D4, Tyr802X;CACNA2D4, Arg628X;RP2, Argl20X;Rho, Ser334X, Rpe65, Arg44X;PDE6A, Lys455X; Congenital stop night blindness 2 (CSNB2): CACNA1F, Arg958X; CACNA1F, Arg830X; Congenital stop night blindness 1 (CSNB1): TRPMl, GlnllX, TRPMl, Lys294X ;TRPMl, Arg977X;TRPMl, Ser882X;NYX, W350X;Best disease or BVMD, BEST1, Tyr29X;BEST1, Arg200X;BEST1, Ser517X;Leber congenital amaurosis (LCA):KCNJ13, Trp53X;KCNJ13, Argl66X;CEP290, Argl51X;CEP290, Glyl890X;CEP290, Lysl575X;CEP29 0, Argl271X;CEP290, Argl782X;CRB1, Cysl332X;GUCY2D, Ser448X;GUCY2D, Arg41091X;LCA5, Gln279X;RDH12, Tyrl94X;RDH12, Glu275X;SPATA7, Argl08X;TULP1, Gln301X;Usher syndrome 1:USH1C, Arg31X;PCDH15, Arg3X;PCDH15, A rg245X; PCDH15, Arg643X; PCDH15, Arg929X; IQCB1, Arg461X; IQCB1, Arg489X; PDE6A, Gln69X; ALMS1, Ser999X; ALMS1, Arg3804X; aniridia: Pax6, Glyl94X; ocular coloboma: Pax2, Argl39X; Lambl, Arg524X; and congenital choroideremia: REP1, Gln32X.

[0109] The composition can be administered in one or more doses by techniques well known to those skilled in the medical arts, taking into account factors such as the age, sex, weight, and condition of the particular subject, as well as the route of administration. The dosage of the composition can be from 1 pg to 10 mg of active ingredient per kg of body weight per dose, and can be from 20 pg to 10 mg of ingredient per kg of body weight per dose. The composition can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of doses of the composition for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0110] The agent or composition can be administered prophylactically or therapeutically. In therapeutic applications, the agent or composition is administered to a subject in need thereof in an amount sufficient to induce a therapeutic effect. An amount sufficient to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use will depend, for example, on the particular composition of the administered composition regimen, the mode of administration, the stage and severity of the disease, the subject's general health, and the judgment of the prescribing physician.

[0111] The agent or composition can be administered by methods well known in the art, such as those described in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)), U.S. Pat. No. 5,580,859, U.S. Pat. No. 5,703,055, and U.S. Pat. No. 5,679,647, the contents of which are incorporated herein by reference. The DNA of the composition can be complexed to particles or beads that can be administered to an individual using, for example, a vaccine gun. Those skilled in the art will know that the selection of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the expression vector. The composition can be delivered via various routes. Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular, or subcutaneous delivery. Other routes include oral, intranasal, and intravaginal administration. The composition can be delivered to the interstitial space of an individual's tissue (U.S. Pat. Nos. 5,580,859 and 5,703,055, the contents of all of which are incorporated herein by reference in their entirety). The composition can also be administered intramuscularly, or transdermally, such as via intradermal or subcutaneous injection, or by iontophoresis. Epidermal administration of the composition can also be used. Epidermal administration can include mechanically or chemically irritating the outermost layer of the epidermis to stimulate an immune response to the irritant (U.S. Pat. No. 5,679,647).

[0112] In some embodiments, the composition can be formulated for administration via the nasal cavity. Formulations suitable for intranasal administration, in which the carrier is a solid, can include, for example, a coarse powder having a particle size ranging from about 10 to about 500 microns, which is administered in the manner of snuffing, i.e., by rapid inhalation through the nasal passages from a container of powder held close to the nose. Formulations can be nasal sprays, nasal drops, or aerosolized by nebulizer. Formulations can include aqueous or oily solutions of the composition.

[0113] The composition can be a liquid preparation such as a suspension, syrup, or elixir. The composition can also be a preparation for parenteral, subcutaneous, intradermal, intramuscular, or intravenous administration (e.g., injectable administration), such as a sterile suspension or emulsion.

[0114] The compositions can be incorporated into liposomes, microspheres, or other polymer matrices (U.S. Pat. No. 5,703,055; Gregoriadis, Liposome Technology, Vols. Ito III (2nd ed. 1993), the contents of which are incorporated herein by reference in their entirety). Liposomes can be composed of phospholipids or other lipids and can be non-toxic, physiologically acceptable, metabolizable carriers that are relatively simple to prepare and administer.

[0115] The composition can be administered by various routes, including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, by inhalation, buccal administration, intrapleurally, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intrathecally, intraarticularly, or a combination thereof. For veterinary use, the composition can be administered in a suitably acceptable formulation in accordance with standard veterinary practice. A veterinarian can readily determine the dosage regimen and route of administration that is most appropriate for a particular animal. The composition can be administered by a conventional syringe, a needleless injection device, a "microparticle gene gun," or other physical methods such as electroporation ("EP"), "hydrodynamic methods," or ultrasound.

[0116] In some embodiments, the composition can be delivered to a mammal by several well-known techniques, including DNA injection with or without in vivo electroporation, liposome-mediated delivery, nanoparticle facilitation, and recombinant vectors such as recombinant adenovirus, recombinant adenovirus-associated virus, and recombinant vaccinia. ACE-tRNA or a nucleic acid molecule encoding ACE-tRNA can be delivered via DNA injection in conjunction with in vivo electroporation.

[0117] Additional Definitions To aid in understanding the detailed description of the compositions and methods according to the present disclosure, some explicit definitions are provided to facilitate a clear disclosure of the various aspects of the present disclosure. 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 disclosure belongs.

[0118] Nucleic acid or polynucleotide refers to a DNA molecule (e.g., cDNA or genomic DNA), an RNA molecule (e.g., mRNA), or a DNA or RNA analog. DNA or RNA analogs can be synthesized from nucleotide analogs. A nucleic acid molecule can be single-stranded or double-stranded. An "isolated nucleic acid" refers to a nucleic acid whose structure is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid. Thus, the term encompasses, for example, (a) a DNA having the sequence of a portion of a naturally occurring genomic DNA molecule, but which is not flanked by both coding sequences that flank that portion of the molecule in the genome of the naturally occurring organism; (b) a nucleic acid incorporated into a vector or into genomic DNA of a prokaryote or eukaryote in such a manner that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. The nucleic acids described above can be used to express the tRNA of the present invention. To this end, the nucleic acid can be operably linked to suitable regulatory sequences to generate an expression vector.

[0119] As used herein, "translation" refers to the process by which a polypeptide (e.g., a protein) is translated from an mRNA. In some embodiments, an increase in translation refers to an increase in the number of polypeptide molecules (e.g., proteins) made per copy of the mRNA encoding that polypeptide.

[0120] As used herein, "non-complementary" with respect to nucleobases means a pair of nucleobases that do not form hydrogen bonds with one another.

[0121] As used herein, "mismatch" means a nucleobase of a first oligomeric compound that is unable to pair with a nucleobase at a corresponding position in a second oligomeric compound when the first and second oligomeric compounds are aligned. Either or both of the first and second oligomeric compounds can be oligonucleotides.

[0122] As used herein, a vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. A vector may or may not be capable of autonomous replication or integration into host DNA. Examples of vectors include plasmids, cosmids, or viral vectors. A vector contains a nucleic acid in a form suitable for expression of a nucleic acid of interest in a host cell. Preferably, a vector contains one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed.

[0123] As used herein, "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include sequences that direct constitutive expression of nucleotide sequences, as well as tissue-specific regulatory sequences and / or inducible sequences. The design of an expression vector may depend on factors such as the choice of host cell to be transformed and the level of expression of the desired protein or RNA. The expression vector can be introduced into host cells to produce the desired RNA or polypeptide. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one that initiates RNA at a high frequency.

[0124] As used herein, a "promoter" is a nucleotide sequence that initiates and regulates transcription of a polynucleotide. Promoters can include inducible promoters (expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. The terms "promoter" or "control element" are intended to include full-length promoter regions and functional (e.g., transcription or translation controlling) segments of these regions.

[0125] As used herein, "operably linked" refers to an arrangement of elements such that the components so described are configured to perform their normal functions. Thus, a given promoter operably linked to a nucleic acid sequence can affect the expression of that sequence when the appropriate enzymes are present. The promoter need not be contiguous with the sequence, so long as it functions to direct its expression. Thus, for example, intervening untranslated but transcribed sequences can be present between the promoter sequence and the nucleic acid sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence. Thus, the term "operably linked" is intended to encompass any spacing or orientation of the promoter element and DNA sequence of interest that allows initiation of transcription of the DNA sequence of interest upon recognition of the promoter element by the transcription complex.

[0126] As used herein, "expression cassette" refers to a nucleic acid sequence capable of directing the expression of a particular nucleotide sequence in an appropriate host cell, which may include a promoter operably linked to the nucleotide sequence of interest, which may be operably linked to a termination signal. It may also include sequences necessary for proper translation of the nucleotide sequence. The coding region typically encodes an RNA or protein of interest. An expression cassette containing a nucleotide sequence of interest may be chimeric. An expression cassette may also be naturally occurring but obtained in a recombinant form useful for heterologous expression. Expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or a regulatable promoter that initiates transcription only when the host cell is exposed to some specific stimulus. In the case of multicellular organisms, the promoter may also be specific to a particular tissue or organ, or a developmental stage. In some embodiments, the promoter is a PGK, CMV, RSV, HI, or U6 promoter (Pol II and Pol III promoters). A "nucleic acid fragment" is a portion of a given nucleic acid molecule. The term "substantial identity" of a polynucleotide sequence means that the polynucleotide comprises a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity compared to a reference sequence using one of the alignment programs described using standard parameters.

[0127] As used herein, "minivector" refers to a miniature circular DNA vector system, such as a double-stranded circular DNA (e.g., a minicircle) or a closed linear DNA molecule (e.g., a CEDT), lacking a bacterial replication origin and an antibiotic selection gene and ranging in size from about 100 bp to about 5 kbp. It can be obtained, for example, by site-specific recombination of a parental plasmid to remove plasmid sequences outside the recombination site. It contains, for example, a nucleic acid molecule containing only a promoter and a transgene expression cassette containing a nucleic acid sequence of interest, such as an ACE-tRNA for inhibiting PTC. Importantly, the nucleic acid sequence is not of bacterial origin.

[0128] The term "disease" as used herein is intended to be generally synonymous with, and used interchangeably with, the terms "disorder" and "condition" (in medical conditions), in that both reflect an abnormal condition of the human or animal body or parts thereof that impairs normal function, is typically manifested by clear signs and symptoms, and reduces the duration or quality of human or animal life.

[0129] As used herein, "subject" or "subject in need thereof" refers to humans and non-human animals. Examples of non-human animals include all vertebrates, e.g., mammals, such as non-human mammals, non-human primates (particularly higher primates), dogs, rodents (e.g., mice or rats), guinea pigs, cats, and rabbits, as well as non-mammals such as birds, amphibians, and reptiles. In some embodiments, the subject is a human. In other embodiments, the subject is an animal suitable as a laboratory animal or a disease model.

[0130] A disease or disorder associated with a PTC or nonsense mutation, a PTC-related disease, or a PTC-related disease refers to any condition caused by or characterized by one or more nonsense mutations that change an amino acid codon to a PTC by a single base substitution, resulting in a defective, truncated protein.

[0131] As used herein, "treat" or "treatment" refers to the administration of a compound or agent to a subject having or at risk of developing a disorder with the intent to cure, alleviate, relieve, relieve, delay the onset of, prevent, or ameliorate the disorder, symptoms of the disorder, conditions secondary to the disorder, or predisposition to the disorder. The terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the probability of developing a disorder or condition in a subject who does not have the disorder or condition but who is at risk of or susceptible to developing the disorder or condition. "Ameliorating" generally refers to a reduction in the number or severity of signs or symptoms of a disease or disorder.

[0132] As used herein, the terms "prevent," "preventing," and "prevention" generally refer to a reduction in the occurrence of a disease or disorder in a subject. Prevention may be complete, e.g., the complete absence of a disease or disorder in a subject. Prevention may also be partial, e.g., the occurrence of a disease or disorder in a subject is less than that which would have occurred without an embodiment of the invention. "Preventing" a disease, as used herein, generally refers to inhibiting the completeness of the disease.

[0133] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier, making the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic use. A "pharmaceutically acceptable carrier" does not cause undesirable physiological effects after administration to or on a subject. A carrier in a pharmaceutical composition must also be "acceptable" in the sense that it must be compatible with and able to stabilize the active ingredient. One or more solubilizing agents may be used as pharmaceutical carriers for delivering the active compound. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a usable composition in dosage form. Other examples of carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate.

[0134] As used herein, the term "agent" refers to a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, antibody, protein, or portion thereof, e.g., a peptide), or an extract made from biological material such as a bacterial, plant, fungal, or animal (e.g., mammalian) cell or tissue. The activity of such an agent may make it suitable as a "therapeutic agent," which is a biologically, physiologically, or pharmacologically active substance or substances that act locally or systemically in a subject.

[0135] As used herein, the terms "therapeutic agent," "therapeutic agent," or "therapeutic agent" are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject. Beneficial effects include enabling a diagnostic determination; alleviating a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally combating a disease, symptom, disorder, or pathological condition.

[0136] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that is relatively non-toxic without abolishing the biological activity or properties of the composition, i.e., the material may be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0137] As used herein, the term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable salts, pharmaceutically acceptable materials, compositions, or carriers, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, that are involved in carrying or transporting a compound of the invention within or to a subject so that it can perform its intended function. Typically, such compounds are carried or transported from one organ or body part to another. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, almond oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; glycerin, sorbitol, mannitol, and polysaccharides. Examples of suitable non-toxic, compatible substances include polyols such as ethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solution; diluents; granulating agents; lubricants; binders; disintegrating agents; wetting agents; emulsifying agents; coloring agents; release agents; coating agents; sweeteners; flavoring agents; fragrances; preservatives; antioxidants; plasticizers; gelling agents; thickening agents; hardening agents; fixatives; suspending agents; surfactants; humectants; carriers; stabilizers; and other non-toxic, compatible substances used in pharmaceutical formulations, or any combination thereof. As used herein, "pharmaceutically acceptable carrier" also includes any and all coating agents, antibacterial and antifungal agents, and absorption delaying agents that are compatible with the activity of the compound and physiologically acceptable to the subject. Supplementary active compounds can also be incorporated into the composition.

[0138] As used herein, the terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, there is nucleotide sequence identity in at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known algorithm for sequence identity, such as FASTA, BLAST, or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0139] As applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using predetermined gap weighting, share at least 90% sequence identity, and even more preferably 95%, 98%, or 99% sequence identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions will not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, 2) aliphatic-hydroxyl side chains: serine and threonine, 3) amide-containing side chains: asparagine and glutamine, 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, 5) basic side chains: lysine, arginine, and histidine, 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. Conservative amino acid substitution groups include valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443 45, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0140] Sequence similarity in polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters. The GCG Version 6.1 program. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of best overlap between the query and search sequences (Pearson (2000) supra). When comparing the sequences of the present invention to a database containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

[0141] Doses are often expressed relative to body weight. Thus, a dose expressed as [g, mg, or other unit] / kg (or g, mg, etc.) usually refers to [g, mg, or other unit] "per kg (or g, mg, etc.) body weight," even if the term "body weight" is not explicitly mentioned.

[0142] As used herein, the term "in vitro" refers to events that take place in an artificial environment, such as in a test tube or reaction vessel, cell culture, etc., rather than within a multicellular organism.

[0143] As used herein, the term "in vivo" refers to events that take place within a multicellular organism, such as a non-human animal.

[0144] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0145] The terms "including," "comprising," "containing," or "having," and their conjugations, unless otherwise stated, are intended to encompass the subsequently listed items and equivalents thereof, as well as additional subject matter.

[0146] The phrases "in some embodiments," "in various embodiments," "in some embodiments," and the like are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but may refer to the same embodiment unless the context clearly indicates otherwise.

[0147] The term "and / or" or " / " means any one of the items, any combination of the items, or all of the items with which this term is associated.

[0148] The word "substantially" does not exclude "completely", for example, a composition that is "substantially free" of Y may be completely free of Y. If desired, the word "substantially" may be omitted from the definition of the invention.

[0149] As used herein, the term "approximately" or "about," when applied to one or more subject values, refers to a value similar to the referenced value. In some embodiments, the term "approximately" or "about" refers to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referenced value in either direction (greater or less), unless otherwise stated or clear from the context (except when such number exceeds 100% of the possible values). Unless otherwise indicated herein, the term "about" is intended to include values, e.g., weight percent, that are close to the referenced range and are equivalent in terms of the functionality of the individual component, composition, or embodiment.

[0150] Where values ​​and ranges are provided herein, it should be understood that all values ​​and ranges subsumed within those values ​​and ranges are intended to be encompassed within the scope of the present invention. Furthermore, all values ​​falling within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.

[0151] As used herein, the term "each," when used in reference to a collection of items, is intended to identify each individual item in the collection, but does not necessarily refer to every item in the collection. Exceptions may occur where express disclosure or context clearly dictates otherwise.

[0152] Any and all examples provided herein, or the use of exemplary language (e.g., "such as"), are intended merely to better illustrate the invention and do not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. As used herein, the term "exemplary" is intended to mean "by way of example" and is not intended to indicate that any particular exemplary item is preferred or required.

[0153] All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. For any of the methods provided, the method steps may occur simultaneously or sequentially. When method steps occur sequentially, the steps may occur in either order unless otherwise stated.

[0154] In cases where the method includes a combination of steps, unless otherwise stated herein, each and every combination or subcombination of the steps is encompassed within the scope of the present disclosure.

[0155] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety to the extent not inconsistent with this disclosure. The publications disclosed herein are provided solely for their disclosure prior to the filing date of the present invention. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates of the publications provided may be different from the actual publication dates, which may need to be independently confirmed.

[0156] It will be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the scope of the appended claims. [Example]

[0157] Example 1 This example describes the materials and methods used in the subsequent examples.

[0158] ArgTGA synthHP generation conditions:

[0159] a. SynthHP Oligo Annealing Conditions SynthHP Oligo 1 and Oligo 2 were delivered as desalted oligos (Integrated DNA Technologies (IDT)), dissolved in TE buffer at 2 μg / μL, and annealed in a 30 μL reaction containing 12 μL of molecular biology-grade HO, 15 μL of dissolved oligo (2 μg / μL), and 3 μL of 10× annealing buffer (1 M potassium acetate and 300 mM HEPES, pH 7.5). The annealed sample was heated to 95°C for 10 minutes in a thermocycler and then cooled to 4°C over 30 minutes.

[0160] b. SynthHP ligation reaction conditions Each annealed oligo was added to a ligation reaction (10 μL of annealed oligo, final concentration 100 ng / μL). The reaction also contained 10 μL of 10× T4 DNA ligase buffer (New England Biolabs (NEB)), 5 μL of T4 DNA ligase (NEB) or 5 μL of HO for a no-ligase control, and 65 μL of HO. The reaction proceeded overnight at room temperature.

[0161] c. T5 exonuclease reaction conditions: Five microliters of T5 exonuclease (NEB) or 5 microliters of HO for the no-T5 control was added to the completed ligation reaction and incubated at 37°C for 1 hour, followed by 10 minutes at 80°C. After the T5 exonuclease reaction, the products were resolved on a 1% agarose gel (left panel). A T5-resistant product appeared in the ligase-added reaction, indicating that intact ArgTGA synthHP was formed in the ligation reaction, whereas no product was evident in the no-ligase control reaction after T5 exonuclease treatment. The yield after T5 exonuclease treatment and anion exchange purification was approximately 4 μg of synthHP for 10 μg of oligo input.

[0162] SynthHP oligonucleotides can be chemically synthesized and can include any of several chemical modifications. SynthHP allows for the attachment of defined chemical moieties, such as bioorthogonal or other specific reactive groups, to the synthHP and assembled ArgTGA synthHP CEDT. Molecules of interest can be ligated using reactive pairs, denoted herein as R-R' pairs, and the retrosynthetic approach also allows for the attachment of two different chemical moieties with the same R-R' pair. For example, in this case, a cell-targeting peptide and a fluorescent tag are covalently attached to the completed synthHP ArgTGA CEDT. The R-R' pair can be either amine-NHS (N-hydroxysuccinimide), thiol-maleimide, or azide-DBCO (dibenzocyclooctyne), among others. This approach can employ many commercially available labeling reagents.

[0163] Example 2 This example demonstrates that ArgTGA synthHP functioned in vivo.

[0164] ArgTGA synthHP or pUC GG negative control plasmid was co-transfected into HEK293T cells with reporter plasmids (UbC-Fluc WT, CMV Nluc-TGA-PTC) in black 96-well cell culture plates using lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. After transfection, cells were maintained at 37°C in a CO2 incubator. After 24 hours of incubation, the medium was removed by aspiration, and 15 μL of PBS was added to each well. Expression levels of Fluc and Nluc were determined using the Dual-Glo Assay kit (Promega) and measured using a Synergy2 multimode microplate reader (BioTek Instruments). Data are shown as the Nluc signal from each well normalized to the Fluc signal from the same well. Data are shown as the mean ± standard error of the mean of triplicate wells. ArgTGA synthHP exhibits 386-fold rescue over background.

[0165] Example 3 This example shows that adding modifications to CEDT or picovectors does not affect their PTC suppression.

[0166] More specifically, an ACE-tRNA DNA picovector was generated and labeled in the manner shown in Figure 8A. Synthetic DNA hairpins corresponding to the 5' and 3' ends of the picovector containing chemical functional groups for site-specific labeling were prelabeled and assembled in a T4 DNA ligase-dependent reaction.

[0167] In one picovector product, the 5' hairpin was labeled with Alexa Fluor 488, while the 3' hairpin was unlabeled. The labeled ACE-tRNA DNA picovector products were resolved by electrophoresis using 5'-Alexa Fluor 488 (AF488), including products that exhibited AF488 fluorescence. The results are shown in Figure 8B. As shown in the figure, a band representing the picovector DNA was evident under the conditions for imaging AF488.

[0168] In another picovector product, the 3' hairpin was labeled with a scrambled peptide sequence: {azidoLys}GGPSEVANKEPQQTAEGKEGKTRAKRDEDQ (SEQ ID NO: 25), while the 5' hairpin was unlabeled. Electrophoresis results are similarly shown in Figure 8B. As shown in the figure, the 3'-peptide-labeled product exhibited an electrophoretic mobility shift when imaged with EtBr, with the band representing the picovector DNA shifting upward.

[0169] Labeled ACE-tRNA in nonsense suppression Arg TGA To examine the efficiency of the picovector, the following labeled ACE-tRNA was used: Arg TGA HEK293T cells were transfected with the picovectors, and the corresponding nonsense suppression was examined in the manner described herein, using a dual-luciferase reporter system (firefly luciferase for transfection normalization, NanoLook-PTC) to assay nonsense suppression. [Table 2]

[0170] As shown in Figure 8C, each of the labeled picovectors exhibited similar nonsense suppression efficiencies when assayed. These results suggest that the addition of the modifications does not negatively affect PTC suppression.

[0171] The foregoing examples and description of preferred embodiments should be construed as illustrating, but not limiting, the present disclosure, which is defined by the claims. As will be readily understood, numerous variations and combinations of the features described above can be utilized without departing from the present disclosure as set forth in the claims. Such variations will not be considered a departure from the scope of the present disclosure, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entirety.

Claims

1. A set of oligonucleotides, From the 5' end to the 3' end, a first antisense strand, A first loop, and a first hairpin oligonucleotide comprising a first sense strand that is complementary to the first antisense strand; From the 5' end to the 3' end, a second sense strand, A second loop, and a second hairpin oligonucleotide comprising a second antisense strand that is complementary to the second sense strand; The set of oligonucleotides is adapted such that the first sense strand and the second sense strand join together to form a nucleic acid sequence that encodes an RNA molecule.

2. A set of oligonucleotides, From the 5' end to the 3' end, a first antisense strand, A first loop, and a first hairpin oligonucleotide comprising a first sense strand that is complementary to the first antisense strand; From the 5' end to the 3' end, a second sense strand, A second loop, and a second hairpin oligonucleotide comprising a second antisense strand that is complementary to the second sense strand; a third sense strand and a third antisense strand having a sequence complementary to the third sense strand; a set of oligonucleotides adapted to join together in an order such that the first sense strand, the third sense strand, and the second sense strand form a second nucleic acid sequence that encodes an RNA molecule.

3. 2. The oligonucleotide set of claim 1, wherein when the first sense strand and the second sense strand join, the first hairpin oligonucleotide and the second hairpin oligonucleotide form a closed-ended DNA thread (CEDT) molecule.

4. The oligonucleotide set of claim 1 or 3, wherein the first hairpin oligonucleotide comprises a nucleic acid sequence encoding a tRNA leader.

5. The oligonucleotide set according to any one of claims 1 and 3 to 4, wherein the second hairpin oligonucleotide comprises a nucleic acid sequence encoding an RNA polymerase III termination signal.

6. The oligonucleotide set according to any one of claims 1 and 3 to 5, further comprising a third sense strand and a third antisense strand having a sequence complementary to the third sense strand.

7. 7. The oligonucleotide set of claim 6, wherein the first sense strand, the third sense strand, and the second sense strand are adapted to be joined together in an order to form a second nucleic acid sequence that encodes an RNA molecule.

8. 3. The oligonucleotide set of claim 2, wherein when the first sense strand, the third sense strand, and the second sense strand are joined, the first hairpin oligonucleotide, the third sense and antisense strands, and the second hairpin oligonucleotide form a closed-ended DNA thread (CEDT) molecule.

9. The oligonucleotide set of claim 2 or 8, wherein the third sense strand comprises a nucleic acid sequence encoding a tRNA leader.

10. The oligonucleotide set according to any one of claims 2 and 8 to 9, wherein the third sense strand comprises a nucleic acid sequence encoding an RNA polymerase III termination signal.

11. 10. The oligonucleotide set of any one of the preceding claims, wherein the RNA molecule comprises a tRNA.

12. 10. The oligonucleotide set of any one of the preceding claims, wherein the tRNA comprises an anticodon-edited tRNA (ACE-tRNA).

13. The oligonucleotide set according to claim 11 or 12, wherein the tRNA is selected from the group consisting of Arg-tRNA-UGA, Gln-tRNA-UAA, Glnt-RNA-UAG, Trp-tRNA-UGA, Trp-tRNA-UAG, Glu-tRNA-UAA, Glu-tRNA-UAG, Cys-tRNA-UGA, Tyr-tRNA-UAG, Tyr-tRNA-UAA, Leu-tRNA-UGA, Leu-tRNA-UAG, Leu-tRNA-UAA, Lys-tRNA-UAG, Lys-tRNA-UGA, Ser-tRNA-UGA, Ser-tRNA-UAG, and Ser-tRNA-UAA.

14. The oligonucleotide set according to claim 12 or 13, wherein the ACE-tRNA causes a ribosome to read through one or more stop codons during translation.

15. 15. The oligonucleotide set of claim 14, wherein the one or more stop codons comprise a premature termination codon (PTC).

16. 16. The oligonucleotide set of claim 15, wherein the premature termination codon (PTC) is present in a nucleic acid sequence encoding a cystic fibrosis transmembrane conductance regulator (CFTR).

17. 10. The oligonucleotide set of any one of the preceding claims, wherein the nucleic acid sequence or the second nucleic acid sequence comprises the polynucleotide sequences of SEQ ID NOs: 4, 8, 13, 15, and 17, or comprises a polynucleotide sequence having at least 85% sequence identity to the polynucleotide sequences of SEQ ID NOs: 4, 8, 13, 15, and 17.

18. the first hairpin oligonucleotide comprises the polynucleotide sequence of SEQ ID NOs: 1, 3, 7, and 11, or comprises a polynucleotide sequence having at least 85% sequence identity to the polynucleotide sequence of SEQ ID NOs: 1, 3, 7, and 11; and / or 2. The oligonucleotide set of any one of the preceding claims, wherein the second hairpin oligonucleotide comprises a polynucleotide sequence of SEQ ID NO: 2, 6, 10, and 12, or a polynucleotide sequence having at least 85% sequence identity to a polynucleotide sequence of SEQ ID NO: 2, 6, 10, and 12.

19. 10. The set of oligonucleotides according to any one of the preceding claims, wherein the nucleic acid sequences have a size of between 200 and 1,000 nucleotides.

20. 10. The oligonucleotide set of any one of the preceding claims, wherein the first loop or the second loop or another portion of the oligonucleotide is linked to an agent.

21. 21. The oligonucleotide set of claim 20, wherein the agent comprises a labeling agent, a peptide, a bioactive agent, or a combination thereof.

22. 22. The oligonucleotide set of claim 21, wherein the labeling agent comprises any one of N-hydroxysuccinimide, thiol-maleimide, and azido-dibenzocyclooctyne.

23. 10. The oligonucleotide set of claim 1, wherein the first hairpin oligonucleotide or the second hairpin oligonucleotide comprises one or more chemically modified nucleotides.

24. 24. The oligonucleotide set of claim 23, wherein the one or more chemically modified nucleotides comprise a 2'-O-methyl modified sugar moiety.

25. 25. The oligonucleotide set of claim 23 or 24, wherein the one or more chemically modified nucleotides comprise a modified internucleoside linkage.

26. A composition comprising the oligonucleotide set of any one of the preceding claims.

27. A kit comprising the oligonucleotide set according to any one of claims 1 to 26 and optionally a ligase.

28. 28. The kit of claim 27, wherein the ligase is T4 DNA ligase.

29. 1. A method for making closed-ended DNA thread (CEDT) molecules, comprising: Providing an oligonucleotide set according to any one of claims 1 to 25; ligating members of said oligonucleotide set, thereby obtaining said CEDT.

30. 30. The method of claim 29, wherein the oligonucleotide set is chemically synthesized.

31. 31. The method of claim 29 or 30, wherein the oligonucleotide set is synthesized with chemically modified nucleotides.

32. 32. The method of any one of claims 29 to 31, wherein the closed-ended DNA thread molecule is further linked to a labeling agent, a bioactive agent, or a combination thereof.

33. A closed-ended DNA thread (CEDT) molecule produced according to the method of any one of claims 29 to 32.

34. 34. A method for treating a disease associated with a premature termination codon (PTC) in a subject in need thereof, comprising administering to the subject a closed-ended DNA thread (CEDT) molecule of claim 33 or a pharmaceutical composition thereof.

35. The disease may be cystic fibrosis, Duchenne and Becker muscular dystrophy, retinoblastoma, neurofibromatosis, ataxia-telangiectasia, Tay-Sachs disease, Wilms' tumor, hemophilia A, hemophilia B, Menkes disease, Ullrich's disease, b-thalassemia, von Willebrand's disease types 2A and 3, Robinow syndrome, brachydactyly type B (shortened fingers and metacarpals), genetic susceptibility to mycobacterial infections, inherited retinal diseases, inherited bleeding tendencies, hereditary blindness, congenital sensorineural deafness and intestinal ganglion cell deficiencies, and inherited neurodevelopmental disorders including sensorineural deafness, intestinal ganglion cell deficiencies, peripheral neuropathy, and central hypomyelination, Liddle's syndrome, xeroderma pigmentosum, Funko syndrome, 35. The method of claim 34, wherein the disease is selected from the group consisting of: Ni anemia, anemia, hypothyroidism, p53-related cancer, esophageal cancer, bone cancer, ovarian cancer, hepatocellular carcinoma, breast cancer, hepatocellular carcinoma, fibrous histiocytoma, ovarian cancer, SRY sex reversal, triosephosphate isomerase anemia, diabetes mellitus, rickets, Hurler syndrome, Dravet syndrome, spinal muscular dystrophy, Usher syndrome, aniridia, congenital choroideremia, ophthalmic coloboma, retinitis pigmentosa, dystrophic epidermolysis bullosa, pseudoxanthoma elasticum, Alagille syndrome, Waardenburg-Scher syndrome, childhood neuronal ceroid lipofuscinosis, cystinosis, X-linked nephrogenic diabetes insipidus, McArdle disease, and polycystic kidney disease.

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