WO2024255817A1

WO2024255817A1 - Systems and method of inhibiting nonsense-mediated decay with circular rnas

Info

Publication number: WO2024255817A1
Application number: PCT/CN2024/099113
Authority: WO
Inventors: Wensheng Wei; Huixian Tang; Zongyi YI
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2023-06-16
Filing date: 2024-06-14
Publication date: 2024-12-19
Anticipated expiration: 2025-12-16
Also published as: TW202504646A

Abstract

Provided are methods and systems for inhibiting nonsense-mediated mRNA decay in a gene-specific manner, for example in the treatment of diseases or disorders related to premature termination codons (PTCs).

Description

SYSTEMS AND METHOD OF INHIBITING NONSENSE-MEDIATED DECAY WITH CIRCULAR RNAS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of International Patent Application No. PCT/CN2023/100757, filed June 16, 2023, the content of which is incorporated herein by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The content of the electronic sequence listing (165392001641seqlist. xml; Size: 69,820 bytes; and Date of Creation: May 28, 2024) is herein incorporated by reference in its entirety.

FIELD

The present disclosure relates generally to methods and compositions for inhibiting nonsense mediated decay (NMD) using an inhibitory RNA (ihRNA) .

BACKGROUND

Genetic diseases are caused by a variety of mutations and changes to the genome. One such mutation is the introduction of a stop codon before the end of a gene, known as a premature termination codon (PTC) . Premature termination codons (PTCs) may arise from single nucleotide mutations that convert a canonical triplet nucleotide codon into one of three stop codons, e.g., TAG, TGA, or TAA. Aberrant transcripts harboring a premature termination codon (PTC) can also be generated by abnormal or inefficient biogenesis of mRNAs. Since the termination codon stops translation before a full-length protein is produced, the protein may not be as effective, or it will be degraded following translation termination. This can result in a disease phenotype. According to the Human Gene Mutation Database (HGMD; www. hgmd. org) , more than 20%of the disease-associated single base-pair substitutions are nonsense mutations, which account for approximately 11%of all described gene lesions causing human inherited diseases.

PTC activates the nonsense-mediated mRNA decay (NMD) pathway to degrade most PTC-containing mRNAs. The NMD pathway is a quality-control step during gene expression that is relevant to genetic diseases because it degrades PTC-containing transcripts to prevent the synthesis of truncated, potentially deleterious proteins. Generally, transcripts containing PTCs located more than 50-55 nucleotides upstream of the last exon-exon junction elicit NMD.

In recent years, efforts have been made to develop translational readthrough therapies for treatment of diseases caused by nonsense mutations. Such therapies include small molecule based readthrough (such as aminoglycoside antibiotics) (Kadunc et al., FEBS, 2020) , tRNA based readthrough (Porter et al., Wiley Interdiscip Rev RNA, 2021) , and targeted pseudouridylation (Adachi et al., Molecular Cell, 2023; Song et al., Molecular Cell, 2023) .

The efficiency of readthrough of PTCs by aminoglycoside antibiotics (such as ataluren) and other agents is relatively low, but it is beneficial in diseases in which even low levels of full-length protein are sufficient to improve cell function (completely or partially) . The outcome of these kinds of treatment is highly variable among patients. At the gene level, the variability can be due in part to the identity and sequence context of the PTC (e.g., UGA>UAG>UAA) . However, this cannot explain the variability observed in response to readthrough agents among patients participating in the same study who carry the same mutation. These observations led to the suspicion that variability NMD could influence the outcome of the treatment.

Pre-mRNA splicing is a prerequisite for NMD. This is the case because, concomitantly or immediately after two exons are spliced together, a complex of proteins termed the exon junction complex (EJC) is deposited typically upstream (e.g., from about 20 to about 24 nucleotides) of each exon-exon junction. EJC does not only bind to the canonical deposition site, 20–24 nt upstream of an exon-exon junction, but also at other positions in exons. The EJC consists of four core components, the anchor EIF4A3, Y14, Magoh and MLN51 proteins, and associated proteins that are involved in mRNA export. The core tetramer facilitates the recruitment of the first two NMD factors, UPF3B and UPF2. Subsequently, during a pioneer round of translation, when a ribosome encounters a stop codon upstream of an EJC, the third and key NMD factor, UPF1, is recruited to the transcript through interaction with UPF2, marking the transcript for degradation. Thus, it is presumed that the presence of a downstream EJC distinguishes a PTC from a normal stop codon, thereby signaling (marking it) for NMD.

The efficiency with which NMD degrades PTC-containing transcripts is variable. This variability has been observed not only for different mutations, but also for the same mutation in different cell types, or among patients. The efficiency of NMD can have a direct effect on disease severity and/or the response to readthrough therapies, at least in part because the availability of PTC-containing transcripts is key to the success of readthrough agents. In some cases, NMD contributes to more severe phenotypes because the truncated protein is still functional, but NMD can lead to the complete non-expression of the truncated protein. For example, variations in the efficiency of NMD between individuals might result in variable degrees of sensitivity to C-terminally truncating mutations (e.g., in Duchenne/Becker muscular dystrophy (DMD/BMD) and other diseases) . Several methods have been developed to inhibit NMD targeting SMG1 (Serine/Threonine-protein kinase) , one of the NMD-factor that activates RNase H1 mediated mRNA degradation. However, this approach relies on general inhibition of the NMD machinery as mentioned above, which may interfere with global RNA surveillance. Therefore, there exists a need to provide more efficient methods and systems for transcript-specific inhibition of NMD for the target gene, thereby increasing the levels of mRNA expression. The present invention addresses this need and related needs in the field.

Summary

In one aspect, provided herein is a method of specifically inhibiting nonsense-mediated decay (NMD) of a premature termination codon (PTC) -containing mRNA in a host cell, the method comprising introducing into the host cell an inhibitory RNA (ihRNA) or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.

In one aspect, provided herein is a method of inhibiting deposition of EJC on a PTC-containing mRNA in a host cell, the method comprising introducing into the host cell an ihRNA or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.

In one aspect, provided herein is a method of increasing the efficacy of a PTC correction agent in a host cell, the method comprising introducing into the host cell an ihRNA or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.

In one aspect, provided herein is a method of increasing expression of a PTC-containing mRNA in a host cell, the method comprising introducing into the host cell an ihRNA or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.

In any of the embodiments herein, the method can comprise introducing a construct encoding the ihRNA into the host cell.

In any of the embodiments herein, the ihRNA can be a circular RNA. In any of the embodiments herein, the ihRNA can be a linear RNA. In some embodiments, the linear ihRNA forms a circular RNA in the host cell.

In any of the embodiments herein, the targeting sequence can be at least about 15 nucleotides in length.

In any of the embodiments herein, the target sequence can comprises an EJC deposition site immediately downstream of the PTC or a portion thereof. In any of the embodiments herein, the target sequence can comprises two or more EJC deposition sites or portions thereof. In any of the embodiments herein, the EJC deposition site can be identified by mapping EIF4A3 binding site on the PTC-containing mRNA sequence. In some embodiments, the mapping comprises CLIP-seq. In any of the embodiments herein, the target sequence can be located about 1 to about 50 nucleotides upstream of an exon-exon junction. In any of the embodiments herein, the PTC can result from a mutation. In any of the embodiments herein, the PTC-containing mRNA can be a IDUA mRNA comprising a PTC.

In any of the embodiments herein, the host cell can be a eukaryotic cell. In any of the embodiments herein, the eukaryotic cell can be in an individual. In some embodiments, the individual is a human individual.

In any of the embodiments herein, the method can further comprise introducing into the host cell an effective amount of a PTC correction agent.

In one aspect, provided herein is a method of treating an individual having a disease associated with a mutation that introduces a PTC in an mRNA to produce an PTC-containing mRNA, comprising administering to the individual an effective amount of a PTC correction agent and an ihRNA or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting RNA sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.

In any of the embodiments herein, the PTC correction agent can be a readthrough drug. In some embodiments, the readthrough drug is an aminoglycoside. In some embodiments, the aminoglycoside is amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, apramycin, G418 (geneticin) , lividomycin, or an aminoglycoside analog chosen from NB30, NB54, or NB84.

In any of the embodiments herein, the PTC correction agent can be a suppressor tRNA.

In any of the embodiments herein, the PTC correction agent can be a targeted pseudouridylation agent.

In any of the embodiments herein, the PTC correction agent can be an RNA editing agent. In some embodiments, the RNA editing agent is deaminase-recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding the dRNA, wherein the dRNA comprises a correction RNA sequence that is at least partially complementary to a PTC region of the PTC-containing mRNA, wherein the dRNA recruits an adenosine deaminase acting on RNA (ADAR) to the PTC, wherein the recruitment of the ADAR converts a target adenosine in the PTC to a sense codon. In some embodiments, the dRNA does not comprises an ADAR recruiting domain. In some embodiments, the dRNA comprises an ADAR recruiting domain.

In any of the embodiments herein, the correction sequence can be about 70 to about 150 nucleotides in length. In any of the embodiments herein, the dRNA can be a circular dRNA. In any of the embodiments herein, the dRNA can be a linear RNA. In some embodiments, the dRNA is a linear RNA that forms a circRNA within a host cell.

In any of the embodiments herein, the dRNA may but does not need to be chemically modified. In any of the embodiments herein, the dRNA can be chemically modified.

In any of the embodiments herein, the disease can be selected from the group consisting of Hurler syndrome, β-thalassemia, and Rett syndrome.

In one aspect, provided herein is a system for specifically inhibiting nonsense-mediated decay (NMD) of a PTC-containing mRNA in a host cell, comprising an ihRNA or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.

In one aspect, provided herein is a system of inhibiting deposition of EJC on a PTC-containing mRNA in a host cell, comprising an ihRNA or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.

In any of the embodiments herein, the system can comprise a construct encoding the ihRNA into the host cell.

In any of the embodiments herein, the ihRNA can be a circular RNA.

In any of the embodiments herein, the targeting RNA sequence can be at least about 15 nucleotides in length. In any of the embodiments herein, the target sequence can comprise an EJC deposition site immediately downstream of the PTC or a portion thereof. In any of the embodiments herein, the target sequence can comprise two or more EJC deposition sites or portions thereof. In any of the embodiments herein, the EJC deposition site can be identified by mapping EIF4A3 binding site on the PTC-containing mRNA sequence. In some embodiments, the mapping comprises CLIP-seq. In any of the embodiments herein, the target sequence can be located about 1 to about 50 nucleotides upstream of an exon-exon junction.

In any of the embodiments herein, the PTC can result from a mutation. In any of the embodiments herein, the PTC-containing mRNA can be a IDUA mRNA comprising a PTC.

In any of the embodiments herein, the host cell can be a eukaryotic cell. In some embodiments, the eukaryotic cell is in an individual. In some embodiments, the individual is a human individual.

In any of the embodiments herein, the system can further comprise a PTC correction agent.

In some embodiments, the PTC correction agent is a readthrough drug. In some embodiments, the readthrough drug is an aminoglycoside. In some embodiments, the aminoglycoside is amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, apramycin, G418 (geneticin) , lividomycin, or an aminoglycoside analog chosen from NB30, NB54, or NB84. In some embodiments, readthrough drug is a suppressor tRNA.

In some embodiments, the PTC correction agent is an RNA editing agent. In some embodiments, the RNA editing agent is deaminase-recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding the dRNA, wherein the dRNA comprises a correction RNA sequence that is at least partially complementary to a PTC region of the PTC-containing mRNA, wherein the dRNA recruits an adenosine deaminase acting on RNA (ADAR) to the PTC, wherein the recruitment of the ADAR converts a target adenosine in the PTC to a sense codon.

In some embodiments, the dRNA does not comprises an ADAR recruiting domain. In some embodiments, the dRNA comprises an ADAR recruiting domain.

In any of the embodiments herein, the correction sequence can be about 70 to about 150 nucleotides in length.

In any of the embodiments herein, the dRNA can be a circular dRNA. In any of the embodiments herein, the dRNA can be a linear RNA. In some embodiments, the dRNA is a linear RNA that forms a circRNA within a host cell.

Brief Description of the Drawings

FIGs. 1A-1C show circular antisense RNAs targeting the IDUA^W402X mRNA and the expression level of IDUA in a primary cell line GM06214. FIG. 1A shows the design of circular antisense RNAs targeting an EIF4A3 binding site. FIG. 1B shows the fold change in the expression level of IDUA in GM06214 cells treated with the designed circular antisense RNAs that target an EIF4A3 binding site. FIG. 1C shows the expression level of IDUA in GM06214 cells treated with the designed circular antisense RNAs that target canonical EJC deposition sites in various exons downstream of the PTC.

FIGs. 2A-2E show circular antisense RNAs targeting an idua mini gene in idua^W392X reporter cells. FIG. 2A shows that the reporter cells responded to G418, which acts as a translational readthrough drug, indicating that the cells are suitable for testing the effect of circular antisense RNAs on inhibiting NMD. FIG. 2B shows the tiling design of circ-antisense₅₀ targeting exons downstream PTC in idua^W392X reporter cells. FIG. 2C shows the expression levels (fold change) of the idua^W392X minigene in cells treated with the circ-antisense₅₀ RNAs. FIG. 2D shows inhibition efficiency of NMD within exons by the circ-antisense₅₀ RNAs. FIG. 2E shows expression levels (fold change) of the idua^W392X minigene RNA and restored protein by the combination of circ-arRNA₁₅₁-idua^W392X and circ-antisense₅₀ RNA targeting exon 9.

FIGs. 3A-3E show circ-antisense RNA rescues IDUA enzyme activity in Hurler syndrome mice (W392X) by elevating idua expression, and increasing RNA editing efficiency by inhibition of NMD factor EIF4A3. FIG. 3A illustrates the experimental design for in vivo assessment of circ-antisense RNA. FIG. 3B shows the abundance of idua mRNA in mice treated with circ-arRNA alone or circ-arRNA combined with circ-antisense RNA compared to a negative control and wildtype. FIG. 3C shows RNA editing efficiency in mice treated with circ-arRNA alone or circ-arRNA combined with circ-antisense RNA compared to a negative control. FIG. 3D shows IDUA enzyme activity in mice treated with circ-arRNA alone or circ-arRNA combined with circ-antisense RNA compared to a negative control and wildtype. FIG. 3E shows relative enrichment of EIF4A3 in mice treated with circ-arRNA alone or circ-arRNA combined with circ-antisense RNA.

FIGs. 4A-4C show combinations of antisense RNA targeting CFTR R1162X mutation in 16HBEge cell line. FIG. 4A shows CFTR expression levels in cells treated with different combinations of antisense RNA. FIG. 4B shows CFTR protein expression in cells treated with different combinations of antisense RNA in combination with a suppressor tRNA. FIG. 4C shows CFTR protein expression in cells treated with different combinations of antisense RNA in combination with a suppressor tRNA.

Detailed Description

The invention described herein relates, in part, to methods of inhibiting NMD in a gene-specific manner using circular antisense RNA, thus obviating concerns relating to global NMD inhibition. Inhibition of NMD increases the availability of PTC-containing transcripts, which increases the efficacy of therapies such as readthrough drugs because there are more PTC-containing transcript molecules on which the drugs can act. This results in the production of more full-length protein than would occur in the absence of inhibition of NMD. In addition, inhibition of NMD in a gene-specific manner is beneficial to patients with nonsense mutations that result in production of a truncated protein that retains normal or partial function. Promoting stabilization of the mRNA and therefore translation of a truncated protein by NMD inhibition (without co- treatment with other therapies such as readthrough drugs) could also significantly improve the outcome of a disease.

In some embodiments, the methods or systems disclosed herein inhibits NMD by utilizing an inhibitory RNA (ihRNA) , which is capable of hybridizing to a target sequence that is located downstream of the PTC in the PTC-containing mRNA. In some instances, the target sequence comprises a sequence on which an EJC is deposited onto (e.g. an EJC deposition site or a portion thereof) . By hybridizing to the target sequence, the ihRNA inhibits EJC deposition. Without the EJC deposited downstream of the PTC, the mRNA is not marked for NMD, and therefore the amount or expression of the mRNA in the cell is increased.

One aspect of the invention relates to a method or a system of specifically inhibiting nonsense-mediated decay (NMD) of a premature termination codon (PTC) -containing mRNA in a host cell, the method comprising introducing into the host cell an inhibitory RNA (ihRNA) or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.

Another aspect of the invention relates to a method or a system of inhibiting deposition of EJC on a PTC-containing mRNA in a host cell, the method comprising introducing into the host cell an ihRNA or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.

Another aspect of the invention relates to a method or a system of increasing the efficacy of a PTC correction agent, the method comprising introducing into the host cell an ihRNA or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.

Yet another aspect of the invention relates to a method or a system of increasing the expression of a PTC-containing mRNA, the method comprising introducing into the host cell an ihRNA or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC- containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.

All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

I. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entireties. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in a patent, application, or other publication that is herein incorporated by reference, the definition set forth in this section prevails over the definition incorporated herein by reference.

It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to particular method steps, reagents, or conditions are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed.

As used herein and in the appended claims, the singular forms “a” , “an” , and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely, ” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

The terms “deaminase-recruiting RNA” , “dRNA” , “ADAR-recruiting RNA” and “arRNA” are used herein interchangeably to refer to an engineered RNA capable of recruiting an ADAR to a target exon and/or a flanking 5’ intron.

“Transfer ribonucleic acid” or “tRNA” is a nucleic acid molecule that helps translate mRNA to protein. tRNA have a distinctive folded structure, comprising three hairpin loops; one of these loops comprises a “stem” portion that encodes an anticodon. The anticodon recognizes the corresponding codon on the mRNA. Each tRNA is “charged with” an amino acid corresponding to the mRNA codon; this “charging” is accomplished by the enzyme tRNA synthetase. Upon tRNA recognition of the codon corresponding to its anticodon, the tRNA transfers the amino acid with which it is charged to the growing amino acid chain to form a polypeptide or protein. Endogenous tRNA can be charged by endogenous tRNA synthetase. Accordingly, endogenous tRNA are typically charged with canonical amino acids. Orthogonal tRNA, derived from an external source, require a corresponding orthogonal tRNA synthetase. Such orthogonal tRNAs may be charged with both canonical and non-canonical amino acids. In some embodiments, the amino acid with which the tRNA is charged may be detectably labeled to enable detection in vivo. Techniques for labeling are known in the art and include, but are not limited to, click chemistry wherein an azide/alkyne containing unnatural amino acid is added by the orthogonal tRNA/synthetase pair and, thus, can be detected using alkyne/azide comprising fluorophore or other such molecule.

The terms “polynucleotide” , “nucleotide sequence” and “nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.

The terms “adenine” , “guanine” , “cytosine” , “thymine” , “uracil” and “hypoxanthine” as used herein refer to the nucleobases as such. The terms “adenosine” , “guanosine” , “cytidine” , “thymidine” , “uridine” and “inosine” refer to the nucleobases linked to the ribose or deoxyribose sugar moiety. The term “nucleoside” refers to the nucleobase linked to the ribose or deoxyribose. The term "nucleotide" refers to the respective nucleobase-ribosyl-phosphate or nucleobase-deoxyribosyl-phosphate. Sometimes the terms adenosine and adenine (with the abbreviation, “A”) , guanosine and guanine (with the abbreviation, “G” ) , cytosine and cytidine (with the abbreviation, “C” ) , uracil and uridine (with the abbreviation, “U” ) , thymine and thymidine (with the abbreviation, “T” ) , inosine and hypo-xanthine (with the abbreviation, “I” ) , are used interchangeably to refer to the corresponding nucleobase, nucleoside or nucleotide. Sometimes the terms nucleobase, nucleoside and nucleotide are used interchangeably, unless the context clearly requires differently.

The term “introducing” or “introduction” used herein means delivering one or more polynucleotides, such as ihRNAs or one or more constructs including vectors as described herein, one or more transcripts thereof, to a host cell. The methods of the present application can employ many delivery systems, including but not limited to, viral, liposome, electroporation, microinjection and conjugation, to achieve the introduction of the ihRNA or construct as described herein into a host cell. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding ihRNA of the present application to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a construct described herein) , naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either epitomal or integrated genomes for delivery to the host cell.

“Pre-mRNA” used in the present application refers to the primary transcript of a gene that can contain intron and exons and requires further splicing to produce mature mRNA molecules containing only exons.

In the context of the present application, the term "target sequence" refers to a sequence in an RNA to which an ihRNA sequence is designed to have perfect complementarity or substantial complementarity and is downstream of a premature termination codon (PTC) in a premature termination codon (PTC) -containing mRNA. In the context of the present application, a target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.

The term “EJC deposition site” refers to a region on an mRNA molecule on which the EJC is deposited to the mRNA molecule and assembles. In some instances, the EJC is deposited approximately 20-24 nucleotides upstream of the splice junction (where two exons are joined) . Other than the canonical deposition site, EJC may also be deposited at non-canonical sites on mRNAs. The number of EJC deposition sites and the intensity by which the EJC is deposited are different among different exons and genes.

The term “stop codon” intends a three nucleotide contiguous sequence within messenger RNA that signals a termination of translation. Non-limiting examples include in RNA, UAG, UAA, UGA and in DNA, TAG, TAA or TGA. Unless otherwise noted, the term also includes nonsense mutations within DNA or RNA that introduce a premature stop codon, causing any resulting protein to be abnormally shortened. tRNA that correspond to the various stop codons are known by specific names: amber (UAG) , ochre (UAA) , and opal (UGA) .

“Premature termination codon” (PTC) used in the present application refers to a stop (termination) codon upstream of the normal stop codon. When the PTC-containing mRNA is translated into a protein, the resulting protein is incomplete and shorter than normal. PTCs can arise from nonsense mutations, or can be a result of abnormal or inefficient biogenesis of mRNAs.

By “mutation” , it is meant that a particular nucleic acid (e.g., a gene, transcript expressed from such gene) differs by one or more nucleotides from a wild type nucleotide sequence and encodes one or more amino acid substitutions, additions, or in some cases, deletions or truncations, in the protein expressed therefrom. Mutations include, but are not limited to, point mutations (affecting a single nucleotide) , nucleotide substitutions, insertions, deletions (including truncations) , or combinations thereof. “Nonsense mutations” include any mutation that results in (premature) introduction of a stop (termination) codon upstream of the normal stop codon. Nonsense mutations, in some aspects, cause and are thus interchangeably referred to as “premature termination codons” (PTCs) .

The term “PTC correction agent” refers to an agent that can promote readthrough of a PTC or correct a PTC introduced by a nonsense mutation. The PTC correction agent can be a readthrough drug or an RNA editing agent.

Polynucleotides (e.g., ihRNA, oligonucleotides, mRNA, etc. ) are “complementary” to one another when hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides. A double-stranded polynucleotide can be “complementary” to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second. Complementarity (the degree to which one polynucleotide is complementary with another) is quantifiable in terms of the proportion (e.g., the percentage) of bases in opposing strands that are expected to form hydrogen bonds with each other, according to generally accepted base-pairing rules.

As used herein, "complementarity" refers to the ability of a nucleic acid to form hydrogen bond (s) with another nucleic acid by traditional Watson-Crick base-pairing. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (i.e., Watson-Crick base pairing) with a second nucleic acid (e.g., about 5, 6, 7, 8, 9, 10 out of 10, being about 50%, 60%, 70%, 80%, 90%, and 100%complementary respectively) . "Perfectly complementary" means that all the contiguous residues of a nucleic acid sequence form hydrogen bonds with the same number of contiguous residues in a second nucleic acid sequence. "Substantially complementary" as used herein refers to a degree of complementarity that is at least about any one of 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%over a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

“Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.

The term “effective amount” refers to a quantity sufficient to achieve a desired effect. In the context of therapeutic or prophylactic applications, the effective amount will depend on the type and severity of the condition at issue and the characteristics of the individual subject, such as general health, age, sex, body weight, and tolerance to pharmaceutical compositions. In the context of an immunogenic composition, in some embodiments the effective amount is the amount sufficient to result in a protective response against a pathogen. In other embodiments, the effective amount of an immunogenic composition is the amount sufficient to result in antibody generation against the antigen. In some embodiments, the effective amount is the amount required to confer passive immunity on a subject in need thereof. With respect to immunogenic compositions, in some embodiments the effective amount will depend on the intended use, the degree of immunogenicity of a particular antigenic compound, and the health/responsiveness of the subject’s immune system, in addition to the factors described above. The skilled artisan will be able to determine appropriate amounts depending on these and other factors. In the case of an in vitro application, in some embodiments the effective amount will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the in vitro target and the methods in use. The skilled artisan will be able to determine the effective amount based on these and other considerations. The effective amount may comprise one or more administrations of a composition depending on the embodiment.

As used herein, the terms “including” , “containing” , and “comprising” are used in their open, non-limiting sense. It is also understood that aspects and embodiments of the invention described herein may include “consisting” and/or “consisting essentially of” aspects and embodiments.

It is understood that, whether the term “about” is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and/or measurement conditions for such given value.

II. METHODS OF INHIBITING NONSENSE-MEDIATED DECAY

The present application in one aspect provides a method of specifically inhibiting nonsense-mediated decay (NMD) of a premature termination codon (PTC) -containing mRNA in a host cell, the method comprising introducing into the host cell an inhibitory RNA (ihRNA) or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof. In some embodiments, there is provided a method of inhibiting deposition of EJC on a PTC-containing mRNA in a host cell, the method comprising introducing into the host cell an inhibitory RNA (ihRNA) or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof. In some embodiments, there is provided a method of increasing the efficacy of a PTC correction agent in correcting a PTC-containing mRNA, the method comprising introducing into the host cell an inhibitory RNA (ihRNA) or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof, wherein the PTC correction agent is administered prior to, simultaneously, or after the administration of the ihRNA or construct encoding the ihRNA. In some embodiments, there is provided a method of increasing expression of a PTC-containing mRNA, the method comprising introducing into the host cell an inhibitory RNA (ihRNA) or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof. In some embodiments, the target sequence comprises an EJC deposition site (or a portion thereof) immediately downstream of the PTC. In some embodiments, the target sequence comprises two or more EJC deposition sites or portions thereof. In some embodiments, the target sequence is located about 1 to about 50 nucleotides upstream from an exon-exon junction. In some embodiments, the EJC deposition site is identified by mapping EIF4A3 binding site on the PTC-containing mRNA sequence. In some embodiments, the host cell is in an individual. In some embodiments, the individual is a human.

In some embodiments, there is provided a method of specifically inhibiting nonsense-mediated decay (NMD) of a premature termination codon (PTC) -containing mRNA in a host cell, the method comprising introducing into the host cell a construct encoding an ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof. In some embodiments, there is provided a method of inhibiting deposition of EJC on a PTC-containing mRNA in a host cell, the method comprising introducing into the host cell a construct encoding an ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof. In some embodiments, there is provided a method of increasing the efficacy of a PTC correction agent in correcting a PTC-containing mRNA, the method comprising introducing into the host cell a construct encoding an ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof, wherein the PTC correction agent is administered prior to, simultaneously, or after the administration of the ihRNA or construct encoding the ihRNA. In some embodiments, there is provided a method of increasing expression of a PTC-containing mRNA, the method comprising introducing into the host cell a construct encoding an ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof. In some embodiments, the target sequence comprises an EJC deposition site (or a portion thereof) immediately downstream of the PTC. In some embodiments, the target sequence comprises two or more EJC deposition sites or portions thereof. In some embodiments, the target sequence is located about 1 to about 50 nucleotides upstream from an exon-exon junction. In some embodiments, the EJC deposition site is identified by mapping EIF4A3 binding site on the PTC-containing mRNA sequence. In some embodiments, the host cell is in an individual. In some embodiments, the individual is a human.

In some embodiments, there is provided a method of specifically inhibiting nonsense-mediated decay (NMD) of a premature termination codon (PTC) -containing mRNA in a host cell, the method comprising introducing into the host cell a construct encoding an ihRNA, wherein in ihRNA is capable of circularizing with the host cell, wherein the circularized ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof. In some embodiments, there is provided a method of inhibiting deposition of EJC on a PTC-containing mRNA in a host cell, the method comprising introducing into the host cell a construct encoding the ihRNA, wherein in ihRNA is capable of circularizing with the host cell, wherein the circularized ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof. In some embodiments, there is provided a method of increasing the efficacy of a PTC correction agent in correcting a PTC-containing mRNA, the method comprising introducing into the host cell a construct encoding the ihRNA, wherein in ihRNA is capable of circularizing with the host cell, wherein the circularized ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof, wherein the PTC correction agent is administered prior to, simultaneously, or after the administration of the ihRNA or construct encoding the ihRNA. In some embodiments, there is provided a method of increasing expression of a PTC-containing mRNA, the method comprising introducing into the host cell a construct encoding the ihRNA, wherein in ihRNA is capable of circularizing with the host cell, wherein the circularized ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof. In some embodiments, the target sequence comprises an EJC deposition site (or a portion thereof) immediately downstream of the PTC. In some embodiments, the target sequence comprises two or more EJC deposition sites or portions thereof. In some embodiments, the target sequence is located about 1 to about 50 nucleotides upstream from an exon-exon junction. In some embodiments, the EJC deposition site is identified by mapping EIF4A3 binding site on the PTC-containing mRNA sequence. In some embodiments, the host cell is in an individual. In some embodiments, the individual is a human.

In some embodiments, there is provided a method of specifically inhibiting nonsense-mediated decay (NMD) of a premature termination codon (PTC) -containing mRNA encoding IDUA in a host cell, the method comprising introducing into the host cell a construct encoding an ihRNA, wherein in ihRNA is capable of circularizing with the host cell, wherein the circularized ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof. In some embodiments, there is provided a method of inhibiting deposition of EJC on a PTC-containing mRNA encoding IDUA in a host cell, the method comprising introducing into the host cell a construct encoding the ihRNA, wherein in ihRNA is capable of circularizing with the host cell, wherein the circularized ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof. In some embodiments, there is provided a method of increasing the efficacy of a PTC correction agent in correcting a PTC-containing mRNA encoding IDUA, the method comprising introducing into the host cell a construct encoding the ihRNA, wherein in ihRNA is capable of circularizing with the host cell, wherein the circularized ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof, wherein the PTC correction agent is administered prior to, simultaneously, or after the administration of the ihRNA or construct encoding the ihRNA. In some embodiments, there is provided a method of increasing expression of a PTC-containing mRNA encoding IDUA, the method comprising introducing into the host cell a construct encoding the ihRNA, wherein in ihRNA is capable of circularizing with the host cell, wherein the circularized ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof. In some embodiments, the target sequence comprises an EJC deposition site (or a portion thereof) immediately downstream of the PTC. In some embodiments, the target sequence comprises two or more EJC deposition sites or portions thereof. In some embodiments, the target sequence is located about 1 to about 50 nucleotides upstream from an exon-exon junction. In some embodiments, the target sequence is located about 1 to about 10 nucleotides upstream from an exon-exon junction. In some embodiments, the target sequence is located about 11 to about 20 nucleotides upstream from an exon-exon junction. In some embodiments, the target sequence is located about 21 to about 30 nucleotides upstream from an exon-exon junction. In some embodiments, the target sequence is located about 31 to about 40 nucleotides upstream from an exon-exon junction. In some embodiments, the target sequence is located about 41 to about 50 nucleotides upstream from an exon-exon junction. In some embodiments, the target sequence is located about 22 nucleotides upstream from an exon-exon junction to about 28 nucleotides downstream from the exon-exon junction. In some embodiments, the target sequence is located about 12 nucleotides upstream from an exon-exon junction to about 38 nucleotides downstream from the exon-exon junction. In some embodiments, the target sequence is located about 2 nucleotides upstream from an exon-exon junction to about 48 nucleotides downstream from the exon-exon junction. In some embodiments, the target sequence is located about 1 to about 50 nucleotides downstream from an exon-exon junction. In some embodiments, the target sequence is located about 22 nucleotides upstream from an exon-exon junction to about 50 nucleotides downstream from the exon-exon junction. In some embodiments, the EJC deposition site is identified by mapping EIF4A3 binding site on the PTC-containing mRNA sequence. In some embodiments, the host cell is in an individual. In some embodiments, the individual is a human.

A. Inhibitory RNA (ihRNA) characteristics

One embodiment of the present disclosure is a method of specifically inhibiting nonsense-mediated decay (NMD) of a premature termination codon (PTC) -containing mRNA in a host cell, the method comprising introducing into the host cell an inhibitory RNA (ihRNA) . One embodiment of the present disclosure is a method of inhibiting deposition of EJC on a PTC-containing mRNA in a host cell, the method comprising introducing into the host cell an ihRNA.

The ihRNA can bind to a specific region of an mRNA transcript and interfere with the binding of one or more components of the EJC. The ihRNA can block or inhibit the binding or deposition of one or more EJC components to an mRNA and do so in a gene-specific manner; ihRNAs are designed so that they bind (hybridize) to a target nucleic acid (e.g., a, mRNA transcript) and remain hybridized under physiological conditions. Design of an ihRNA can take into consideration the occurrence of the target sequence or a sufficiently similar nucleic acid sequence in other locations in the genome or cellular mRNA/transcriptome, such that the likelihood the ihRNA will bind other sites and cause “off-target” effects is limited.

In some embodiments, the ihRNA binds a target sequence located downstream of (3′to) a premature termination codon (PTC) , such as a PTC resulting from a nonsense mutation in a particular gene or mRNA. In some embodiments, the ihRNA binds a target sequence located downstream of (3′to) a PTC, such as a PTC resulting from abnormal or inefficient biogenesis of mRNAs. In some embodiments, the ihRNA binds a target sequence upstream of an exon-exon junction that is located downstream of (3′to) a premature termination codon (PTC) , such as a PTC resulting from a nonsense mutation in a particular gene or mRNA. In some examples, the target sequence is upstream of the first exon-exon junction that is downstream of the PTC. In some embodiments, the target sequence immediately flanks an exon-exon junction that is downstream of the PTC. In some embodiments, the target sequence is about 30 nucleotides upstream to about 50 nucleotides downstream of an exon-exon junction that is downstream of the PTC. In some embodiments, the target sequence is about 22 nucleotides upstream to about 28 nucleotides downstream of an exon-exon junction that is downstream of the PTC. In some embodiments, the target sequence is about 12 nucleotides upstream to about 38 nucleotides downstream of an exon-exon junction that is downstream of the PTC. In some embodiments, the target sequence is about 2 nucleotides upstream to about 48 nucleotides downstream of an exon-exon junction that is downstream of the PTC. In some embodiments, the target sequence is about 1 to about 50 nucleotides downstream of an exon-exon junction that is downstream of the PTC. In some embodiments, the target sequence is about 22 nucleotides upstream to about 50 nucleotides downstream of an exon-exon junction that is downstream of the PTC. In other examples, the target sequence is upstream of one or more exon-exon junctions other than the first exon-exon junction that is downstream of the PTC (e.g., as long as they are downstream of the PTC) . In one embodiment, the target sequence is a region that, if bound by the ihRNA, inhibition or blocking of one or more components of the EJC occurs, and the region that, when otherwise bound by the EJC, is one that marks or signal the mRNA to be degraded by NMD. In another embodiment, the target sequence that is targeted by the ihRNA to block deposition of the EJC (by blocking the binding or formation of the EJC) is adjacent to the region that would otherwise be bound by the EJC. Thus, for example, and without being bound by any particular mechanism, binding or hybridizing of an ihRNA to an mRNA displaces, blocks, or otherwise prevents the EJC from binding to or forming on the mRNA in a functional manner (e.g., at a region that is located downstream of a PTC and upstream of or flanks an exon-exon junction) , resulting in inhibition of NMD of the mRNA.

In some embodiments, the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA. The targeting sequence may be of any length suitable for specific binding and effective inhibition of EJC binding or deposition. In some embodiments, the targeting sequence is at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 nucleotides in length. In some embodiments, the targeting sequence is about 15 to about 80, about 15 to about 70, about 15 to about 60, about 15 to about 50, about 15 to about 40, about 15 to about 30, or about 15 to 20 nucleotides in length. In some embodiments, the targeting sequence is at least about 15 nucleotides in length. In some embodiments, the targeting sequence is about 50 nucleotides in length. In some embodiments, the targeting sequence is about 72 nucleotides in length. Generally, the ihRNA is designed to bind to a region downstream of the PTC and be of a certain length so as to specifically block EJC binding or deposition but not interfere with mRNA splicing.

In some embodiments, the targeting sequence and the target sequence are at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%or at least about 99%complementary to each other. In some embodiments, the targeting sequence and the target sequence are fully complementary to each other. In some embodiments, the targeting sequence has sufficient complementarity to bind the target sequence and inhibit binding of one or more EJC components. Percent complementarity between a targeting sequence and a target sequence can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) .

An ihRNA need not hybridize to all nucleobases in a target sequence and the nucleobases to which it does hybridize may be contiguous or noncontiguous. ihRNAs may hybridize over one or more segments of a target sequence, such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure may be formed) . In certain embodiments, an ihRNA hybridizes to noncontiguous nucleobases in a target sequence. For example, an ihRNA can hybridize to nucleobases in a target sequence that are separated by one or more nucleobase (s) to which the ihRNA does not hybridize.

In some embodiments, the ihRNA comprises more than one targeting sequences that is each at least partially complementary to a corresponding target sequence downstream of the PTC in the PTC-containing mRNA.

In some embodiments, the ihRNA is a circular RNA. In some embodiments, the ihRNA is a linear RNA. In some embodiments, the linear ihRNA forms a circular RNA in the host cell. The processes by which the linear ihRNA forms a circular RNA in the host cell are further detailed in Section III.

In certain embodiments, the mRNA expression level of the PTC-containing RNA is increased as NMD is inhibited by the ihRNA. In some embodiments, the expression level is increased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or higher. Methods for measuring or quantifying mRNA levels are well known in the art, and include, for example, RT-PCR, RT-qPCR, microarray analysis, northern blot analysis, RNase-protection analysis, or any other suitable method, for example as described in Rio, D. C., RNA: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2011, which is incorporated herein in its entirety.

In certain embodiments, the expression level of the protein encoded by the PTC-containing RNA is increased as NMD is inhibited by the ihRNA. In some embodiments, the protein expression level is increased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or higher. Methods of measuring or quantifying protein levels are well known in the art, and include, for example, western blot analysis, immunocytochemistry, flow cytometry, mass spectrometry, or any other suitable method, for example as described in Link, A. J., Proteomics: A Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 2009, which is incorporated herein in its entirety.

In some embodiments, the ihRNA does not affect global RNA surveillance while specifically inhibiting NMD of the target PTC-containing mRNA, thus regulation and quality control of non-target genes/mRNAs is not affected.

In some embodiments, the ihRNA is not chemically modified. In some embodiments, the ihRNA is chemically modified. In some embodiments, the ihRNA comprises one or more modifications, such as 2′-O-methylation and/or phosphorothioation.

B. Methods of delivery and construct encoding the ihRNA

In some embodiments, the ihRNA is encoded by a nucleic acid that can be delivered to a host cell via viral or non-viral based methods. Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, electroporation, nanoparticles, exosomes, microvesicles, or gene-gun, naked DNA and artificial virions. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) .

The use of RNA or DNA viral based systems for the delivery of nucleic acids has high efficiency in targeting a virus to specific cells and trafficking the viral payload to the cellular nuclei.

In certain embodiments according to any one of the methods described herein, the method comprises introducing a viral vector (such as an AAV or a lentiviral vector) encoding the ihRNA to the host cell. In some embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the construct is flanked by one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the construct is flanked by two AAV ITRs. In some embodiments, the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs. In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, the vector further comprises a stuffer nucleic acid. In some embodiments, the stuffer nucleic acid is located upstream or downstream of the nucleic acid encoding the dRNA. In some embodiments, the vector is a self-complementary rAAV vector. In some embodiments, the vector comprises first nucleic acid sequence encoding the ihRNA and a second nucleic acid sequence encoding a complement of the ihRNA, wherein the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence along most or all of its length. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutated AAV ITR, wherein the mutated AAV ITR comprises a deletion of the D region and comprises a mutation of the terminal resolution sequence. In some embodiments, the vector is encapsidated in a rAAV particle. In some embodiments, the AAV viral particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV2 V708K, AAV2-HBKO, AAVDJ8, AAV-PHP. B, AAV-PHP. eB, AAV-BR1, AAVHSC15, AAVHSC17, goat AAV, AAV1/AAV2 chimeric, bovine AAV, mouse AAV, or rAAV2/HboV1 serotype capsid.

In some embodiments, the method comprises introducing a plasmid encoding the ihRNA to the host cell. In some embodiments, the method comprises electroporation of the ihRNA (e.g., synthetic ihRNA) into the host cell. In some embodiments, the method comprises transfection of the ihRNA into the host cell.

C. mRNA and target sequence

In some embodiments, the PTC-containing mRNA is an mRNA transcript expressed in a cell, such as a eukaryotic cell. In some embodiments, the PTC-containing mRNA contains a nonsense mutation, which results in a PTC. Typically, mRNAs with PTCs are targets of NMD. In some embodiments, the nonsense mutation is a disease-causing mutation (such as a disease-causing mutation described herein) . The disease can be caused by the rapid turnover (e.g., by NMD) of the mRNA, a lack or reduced production of functional protein due to a truncated protein product, insufficient levels of a truncated protein product having normal or partial function, or combinations thereof. In some embodiments, the PTC is a PTC resulting from abnormal or inefficient biogenesis of mRNAs, e.g., the PTC is not the result of a mutation.

In some aspects, PTCs comprise a triplet nucleotide sequence, for example UGA (e.g., TGA in DNA) , UAG (e.g., TAG in DNA) , or UAA (e.g., TAA in DNA) . For example, mutations (e.g., in DNA, mRNA (RNA) , or both) that result in a PTC include, but are not limited to: (1) single base pair substitutions that change a sense codon to an in-frame PTC (e.g., nonsense mutations) ; (2) insertion or deletion mutations that alter the ribosomal reading frame, causing translating ribosomes to encounter a PTC; (3) an insertion mutation that maintains the proper distal reading frame but introduces an in-frame PTC; and (4) mutations that lead to mRNA splicing defects that cause retention of an intron (or part of an intron) that alters the reading frame, leading translating ribosomes to encounter a PTC. In some aspects, mutations resulting in a PTC have important consequences on gene expression, such as in the context of disease. For example, a PTC will terminate mRNA translation prior to completion of a full-length polypeptide, leading to production of truncated proteins that are often nonfunctional and/or unstable and/or have detrimental function. In addition, PTC-containing mRNAs are also frequently unstable because the mRNAs are degraded by NMD, resulting in a severe reduction in steady-state mRNA levels. In some examples, the combination of these PTC-induced events reduce the level of functional protein produced to such an extent that a severe disease state results.

The PTC-containing mRNA described herein can be transcribed from any gene of interest. In some embodiments, the PTC-containing RNA is transcribed from a gene selected from the group consisting of IDUA, CFTR, DMD, HBB, and MECP2. In some embodiments, the PTC-containing RNA is transcribed from IDUA. Additional nonsense mutations can be found in the Human Gene Mutation Database (HGMD) .

In some embodiments, the target sequence comprises an EJC deposition site immediately downstream of the PTC or a portion thereof. In some embodiments, the EJC deposition site is about 10 nt, 20 nt, 30 nt, 40 nt, 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, or 100 nt downstream of the PTC. In some embodiments, the EJC deposition site is about 1-10 nt, 11-20 nt, 21-30 nt, 31-40 nt, 41-50 nt, 51-60 nt, 61-70 nt, 71-80 nt, 81-90 nt, or 91-100 nt downstream of the PTC. In some embodiments, the target sequence comprises a portion of an EJC deposition site immediately downstream of the PTC. In some embodiments, the target sequence comprises 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt of an EJC deposition site immediately downstream of the PTC. In some embodiments, the target sequence comprises two or more EJC deposition sites or portions thereof. In some embodiments, the target sequence comprises two EJC deposition sites or portions thereof. In some embodiments, the target sequence comprises three EJC deposition sites or portions thereof. In some embodiments, the target sequence comprises four EJC deposition sites or portions thereof. In some embodiments, the target sequence comprises five EJC deposition sites or portions thereof.

In some embodiments, the EJC deposition site is identified by mapping EIF4A3 binding site on the PTC-containing mRNA sequence. The DEAD-box protein EIF4A3 is the main RNA-binding component in the EJC, and has been shown to be essential for NMD (Shibuya et al., 2006) . Methods for mapping the site where proteins or protein complexes bind to mRNA are known in the art. In some embodiments, the mapping comprises RNA immune-precipitation (RIP) or crosslinking-immunoprecipitation (CLIP) .

In some embodiments, the mapping comprises crosslinking and immunoprecipitation of RNA–protein complexes sequencing (CLIP-seq) . CLIP-seq combines UV crosslinking with immunoprecipitation in order to identify RNA binding sites of proteins on a transcriptome-wide scale. CLIP begins with the in-vivo cross-linking of RNA-protein complexes using ultraviolet light (UV) . Upon UV exposure, covalent bonds are formed between proteins and nucleic acids (e.g., mRNAs) that are in close proximity (on the order of Angstroms apart) . The cross-linked cells are then lysed, RNA is fragmented, and the protein of interest is isolated via immunoprecipitation. In order to allow for priming of reverse transcription, RNA adapters are ligated to the 3'ends, and RNA fragments are labelled to enable the analysis of the RNA-protein complexes after they have been separated from free RNA using gel electrophoresis and membrane transfer. Proteinase K digestion is then performed in order to remove protein from the crosslinked RNA, which leaves a few amino acids at the crosslink site. cDNA is then synthesized via RT-PCR followed by high-throughput sequencing followed by mapping the reads back to the transcriptome and other computational analyses to study the interaction sites.

In some embodiments, the target sequence is located about 1 to about 50 nucleotides upstream of an exon-exon junction. In some embodiments, the target sequence is located about 5 to about 45 nucleotides upstream of an exon-exon junction. In some embodiments, the target sequence is located about 10 to about 40 nucleotides upstream of an exon-exon junction. In some embodiments, the target sequence is located about 15 to about 35 nucleotides upstream of an exon-exon junction. In some embodiments, the target sequence is located about 20 to about 30 nucleotides upstream of an exon-exon junction. In some embodiments, the target sequence is located about 20 to about 25 nucleotides upstream of an exon-exon junction. In some embodiments, the target sequence is located about 20 to about 24 nucleotides upstream of an exon-exon junction. In some embodiments, the target sequence immediately flanks an exon-exon junction that is downstream of the PTC. In some embodiments, the target sequence is located about 1 to about 10 nucleotides upstream from an exon-exon junction that is downstream of the PTC. In some embodiments, the target sequence is located about 11 to about 20 nucleotides upstream from an exon-exon junction that is downstream of the PTC. In some embodiments, the target sequence is located about 21 to about 30 nucleotides upstream from an exon-exon junction that is downstream of the PTC. In some embodiments, the target sequence is located about 31 to about 40 nucleotides upstream from an exon-exon junction that is downstream of the PTC. In some embodiments, the target sequence is located about 41 to about 50 nucleotides upstream from an exon-exon junction that is downstream of the PTC. In some embodiments, the target sequence is about 22 nucleotides upstream to about 28 nucleotides downstream of an exon-exon junction that is downstream of the PTC. In some embodiments, the target sequence is about 12 nucleotides upstream to about 38 nucleotides downstream of an exon-exon junction that is downstream of the PTC. In some embodiments, the target sequence is about 2 nucleotides upstream to about 48 nucleotides downstream of an exon-exon junction that is downstream of the PTC. In some embodiments, the target sequence is about 1 to about 50 nucleotides downstream of an exon-exon junction that is downstream of the PTC. In some embodiments, the target sequence is about 22 nucleotides upstream to about 50 nucleotides downstream of an exon-exon junction that is downstream of the PTC.

D. Host cell

In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is a eukaryotic cell. Preferably, the host cell is a mammalian cell. Most preferably, the host cell is a human cell. In some embodiments, the host cell is a murine cell. In some embodiments, the host cell is a plant cell or a fungal cell. In some embodiments, the host cell is a diseased cell. In some embodiments, the host cell comprises one or more mutations, such as a nonsense mutation.

In some embodiments, the host cell is a cell line, such as Neuro-2a, HEK293T, HT29, A549, HepG2, RD, SF268, SW13 and HeLa cell. In some embodiments, the host cell is a primary cell, such as fibroblast, epithelial, or immune cell. In some embodiments, the host cell is a T cell. In some embodiments, the host cell is a post-mitosis cell. In some embodiments, the host cell is a cell of the central nervous system (CNS) , such as a brain cell, e.g., a cerebellum cell.

In some embodiments, the host cell is in an individual, such as a human individual. In some embodiments, the host cell is an ex vivo cell population.

III. SYSTEMS COMPRISING IHRNA OR CONSTRUCT ENCODING THE IHRNA

Also provided herein are systems comprising ihRNAs or constructs useful for any one of the methods described herein. NMD is inhibited using specifically designed RNAs, namely, ihRNAs which are either circular (circRNA) or linear ihRNA, optionally an ihRNA that can form circRNA within a cell. The ihRNA contains a targeting sequence that is at least partially complementary to a target sequence of a PTC-containing mRNA transcript and interferes with the binding of one or more components of the EJC. In some embodiments, interference with the binding of one or more components of the EJC specifically inhibits NMD of the PTC-containing mRNA transcript. In some embodiments, interference with the binding of one or more components of the EJC inhibits deposition of EJC on the PTC-containing mRNA transcript. In some embodiments, interference with the binding of one or more components of the EJC increases the efficacy of a PTC correction agent. In some embodiments, interference with the binding of one or more components of the EJC increases expression of the PTC-containing mRNA.

The present application thus in some embodiments provides a system for specifically inhibiting nonsense-mediated decay (NMD) of a PTC-containing mRNA in a host cell, comprising an ihRNA or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.

In some embodiments, there is provided a system for inhibiting deposition of EJC on a PTC-containing mRNA in a host cell, comprising an ihRNA or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.

Any one of the ihRNAs or constructs described in this section may be used in the systems and methods of specifically inhibiting NMD and treatment described herein. It is intended that any of the features and parameters described herein for ihRNAs or constructs can be combined with each other, as if each and every combination is individually described. In some embodiments, the ihRNAs described herein do not comprise a tracrRNA, crRNA or gRNA used in a CRISPR/Cas system.

In some embodiments, there is provided a construct comprising any one of the ihRNAs described herein. In certain embodiments, the construct is a viral vector (preferably a lentivirus vector) or a plasmid. In some embodiments, the construct encodes a single ihRNA. In some embodiments, the construct encodes a plurality (e.g., about any one of 1, 2, 3, 4, 5, 10, 20 or more) ihRNAs.

In some embodiments, there is provided a library comprising a plurality of the ihRNAs or a plurality of the constructs described herein.

In one aspect, the present application provides a system or a host cell comprising the dRNA or the construct described herein. In certain embodiments, the host cell is a prokaryotic cell or a eukaryotic cell. Preferably, the host cell is a mammalian cell. Most preferably, the host cell is a human cell.

A. Circular ihRNAs and constructs

In some embodiments, the ihRNA is a linear RNA that is capable of forming a circRNA. In some embodiments, the ihRNA is a circRNA. In some embodiments, the ihRNA is circulated by the Tornado method. In some embodiments, the ihRNA transcript is also flanked a 5’ and/or 3’ litigation sequences which are then optionally flanked by the 5’ -Twister ribozyme and/or 3’ -Twister ribozymes, respectively. In some embodiments, the 3’ ligation sequence and the 5’ ligation sequence are at least partially complementary to each other. In some embodiments, the 3’ ligation sequence and the 5’ ligation sequence are at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%or at least about 99%complementary to each other. In some embodiments, the 3’ ligation sequence and the 5’ ligation sequence are fully complementary to each other.

In some embodiments, the 3’ ligation sequence and the 5’ ligation sequence are independently at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides or at least about 100 nucleotides in length. In some embodiments, the 3’ ligation sequence and the 5’ ligation sequence are independently about 20-30 nucleotides, about 30-40 nucleotides, about 40-50 nucleotides, about 50-60 nucleotides, about 60-70 nucleotides, about 70-80 nucleotides, about 80-90 nucleotides, about 90-100 nucleotides, about 100-125 nucleotides, about 125-150 nucleotides, about 20-50 nucleotides, about 50-100 nucleotides or about 100-150 nucleotides in length.

In some embodiments, the ihRNA is circularized by an RNA ligase. In some embodiments, the RNA ligase is expressly endogenously in the host cell. In some embodiments, the RNA ligase is RNA ligase RtcB. In some embodiments, the RNA ligase RtcB is expressly endogenously in the host cell. In some embodiments, the ihRNA is circularized through in vitro enzymatic ligation (e.g., using RNA or DNA ligase) or chemical ligation (e.g., using cyanogen bromide or a similar condensing agent) .

Also provided herein is a construct comprising a nucleic acid encoding the ihRNA. The term “construct” as used herein refers to DNA or RNA molecules that comprise a coding nucleotide sequence that can be transcribed into RNAs or expressed into proteins. In some embodiments, the construct contains one or more regulatory elements operably linked to the nucleotide sequence encoding the RNA or protein. When the construct is introduced into a host cell, under suitable conditions, the coding nucleotide sequence in the construct can be transcribed or expressed.

In some embodiments, the ihRNA is introduced by a construct comprising a nucleic acid encoding the ihRNA. In some embodiments, the construct further comprises a 3’ twister ribozyme sequence linked to the 3’ end of the nucleic acid encoding the ihRNA and a 5’ twister ribozyme sequence linked to the 5’ end of the nucleic acid encoding the ihRNA. In some embodiments, the 3’ twister sequence is twister P3 U2A and the 5’ twister sequence is twister P1. In some embodiments, wherein the 5’ twister sequence is twister P3 U2A and the 3’ twister sequence is twister P1. In some embodiments, the ihRNA undergoes autocatalytic cleavage. In some embodiments, the catalyzed ihRNA product comprises a 5′-hydroxyl group and a 2′, 3′-cyclic phosphate at the 3′terminus. In some embodiments, the catalyzed ihRNA product is ligated by ubiquitous endogenous RNA ligase (e.g., RNA ligase RtcB) . In some embodiments, the construct is a plasmid or a viral vector.

In some embodiments, the construct comprises a promoter that is operably linked to the coding nucleotide sequence, such that the promoter controls the transcription or expression of the coding nucleotide sequence. A promoter may be positioned 5' (upstream) of a coding nucleotide sequence under its control. The distance between the promoter and the coding sequence may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function. In some embodiments, the construct comprises a 5’ UTR and/or a 3’ UTR that regulates the transcription or expression of the coding nucleotide sequence. In some embodiments, the promoter is a Pol III promoter (such as U6 promoter) . In some embodiments, the promoter is a Pol II promoter (such as a CMV promoter or a U7 promoter) .

In some embodiments, the construct is a vector encoding any one of the ihRNAs disclosed in the present application. The term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular) ; nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a "plasmid, " which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) . Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the transcription or expression of coding nucleotide sequences to which they are operatively linked. Such vectors are referred to herein as “expression vectors” .

Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for transcription or expression of the nucleic acid in a host cell. In some embodiments, the recombinant expression vector includes one or more regulatory elements, which may be selected on the basis of the host cells to be used for transcription or expression, which is operatively linked to the nucleic acid sequence to be transcribed or expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element (s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell) .

In some embodiments, the vector is a rAAV vector. In some embodiments, the rAAV vector is a vector derived from an AAV serotype, including without limitation, AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV capsid serotype or the like. In some embodiments, the nucleic acid in the AAV comprises an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV capsid serotype or the like. In some embodiments, the nucleic acid in the AAV further encodes a dRNA as described herein. Use of any AAV serotype is considered within the scope of the present disclosure. In some embodiments, the vector is encapsidated in a rAAV particle. In some embodiments, the AAV viral particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV2 V708K, AAV2-HBKO, AAVDJ8, AAV-PHP. B, AAV-PHP. eB, AAV-BR1, AAVHSC15, AAVHSC17, goat AAV, AAV1/AAV2 chimeric, bovine AAV, mouse AAV, or rAAV2/HboV1 serotype capsid.

IV. METHODS OF TREATMENT

The NMD inhibition methods and systems described herein may be used to treat or prevent a disease or condition in an individual. In one aspect, provided herein is a method of treating an individual having a disease associated with a mutation that introduces a PTC in an mRNA to produce an PTC-containing mRNA, comprising administering to the individual an effective amount of a PTC correction agent and an ihRNA or a construct encoding the ihRNA, wherein the PTC correction agents can promote readthrough of the PTC or correct the PTC introduced by a nonsense mutation, the ihRNA comprises a targeting RNA sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.

In one aspect of the present disclosure, a method for increasing the efficacy of a PTC correction agent is provided. The present methods increase the efficacy of PTC correction agents by, e.g., inhibiting NMD in a gene-specific manner, thereby providing increased levels of transcripts available for readthrough or correction of PTCs contained therein.

“Individual, ” as used herein, refers to an individual organism. In some aspects, “individual” may be used interchangeably with “subject, ” or “patient. ” In some embodiments, an individual is a mammal, for example, a human, a nonhuman primate, a mouse, a rat, a cat, a dog, a cattle, a goat, a pig, a sheep, or a plant. In some embodiments, the individual is a human having or at increased risk of having a disease or disorder caused by a nonsense mutation. If an individual is “at an increased risk” of having a disease or disorder caused by a nonsense mutation, the method involves preventative or prophylactic treatment. For example, an individual may be at an increased risk of having such a disease or disorder because of family history of the disease (e.g., the individual has a genetic predisposition) . Many of the diseases and disorders described herein are primarily, if not entirely, genetic based diseases, e.g., mucopolysaccharidosis type I (MPS I) (mutations in IDUA) , β-thalassemia (nonsense mutations in HBB) , Rett syndrome (nonsense mutations in MECP2) , cystic fibrosis (nonsense mutations in CFTR) , and Duchenne/Becker muscular dystrophy (nonsense mutations in DMD) . Thus, in some aspects, an individual having one or more nonsense mutations in these genes (or others associated with diseases or disorder caused by nonsense mutations) but not yet diagnosed with such a disease or disorder, is an individual at increased risk of having a disease or disorder caused by a nonsense mutation. Typically, individuals at an increased risk of having such a disease or disorder benefit from prophylactic treatment (e.g., by preventing or delaying the onset or progression of the disease or disorder) .

A. PTC correction agents

In some embodiments, the method further involves contacting, introducing, or delivering (e.g., as described herein) an effective amount of a PTC correction agent.

In some embodiments, the PTC correction agent can promote readthrough of the PTC-containing mRNA. By “promoting readthrough, ” it is meant that the PTC correction agent affects translation of a PTC-containing mRNA, resulting in the incorporation of an amino acid at the PTC in the nascent growing polypeptide chain, rather than termination of translation and generation of a truncated protein, which would otherwise occur) . For example, PTC correction agents, in some cases, enhance the ability of near-cognate aminoacyl tRNAs to compete with the release factor complex for binding PTCs in the ribosomal A site. By increasing the frequency at which PTCs are recoded into sense codons, enough full-length, functional protein may be produced to provide a therapeutic benefit to individuals who carry disease-causing nonsense mutations, as described herein. Methods for determining whether a compound or composition is promoting readthrough are known in the art, and include, for example, western blot analysis, immunohistochemistry, flow cytometry, as well as cell-based reporter assays such as those described in the Examples section. In some aspects, improvement in one or more clinical parameters allows for determining whether a PTC correction agent is effectively increasing levels of full-length protein. In some aspects, even modest or slight increases in the amount of full-length protein are beneficial in alleviating some disease states. For example, the lysosomal storage disease mucopolysaccharidosis type I-Hurler (MPS I-H, caused by nonsense mutation resulting in decreased levels of iduronidase encoded by the IDUA gene) , has a low threshold for correction, since <1%of wild-type iduronidase function can significantly moderate the clinical phenotype (Ashton et al., Am. J. Hum. Genet. 1992, 50: 787-794, Bunge et al., Biochim. Biophys. Acta. 1998, 1407: 249-256) . Thus, increasing the amount of full-length protein, in some aspects, to reach 1%of wild-type levels, is beneficial in treating some diseases caused by nonsense mutations. In some examples, the method results in an increase of at least 5%, such as 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or a 100%increase, in the amount of full length protein produced, for example as compared to wild type levels (e.g., levels of expression of the wild-type protein wherein the gene/mRNA does not contain a nonsense mutation) . In other examples, such as the disease β-thalassemia, increased levels of the full-length beta chains of hemoglobin leads to improved or ameliorated disease states, such as decreased or no anemia, decreased tiredness, decreased breathlessness, and increased exercise tolerance. Methods for monitoring improvement in the β-thalassemia disease state are known, and include, for example, pulse oximetry, hemoglobin electrophoresis; serum transferrin, ferritin, Fe binding capacity analysis; urine urobilin &urobilinogen assays; peripheral blood smear test; hematocrit analysis; and serum bilirubin analysis.

In some embodiments, the PTC correction agent is a readthrough drug. In some embodiments, the readthrough drug is ataluren (PTC124, PTC Therapeutics, South Plainfield, N.J. ) , or an aminoglycoside (e.g., drugs that generally consist of two to three aminosugars joined to a 2-deoxystreptamine ring by glycosidic linkages) , such as amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, apramycin, G418 (geneticin) , and lividomycin, or salts and derivatives thereof. However, at certain doses, these drugs have been shown to be toxic. Thus in some aspects, the readthrough drug is an aminoglycoside analog having lower or no toxicity, for example NB30, a derivative of paromomycin; NB54, which combines components of paromomycin and amikacin; and NB84, which is composed of structural elements from paromomycin, amikacin, and G418. In some embodiments, these compounds show more than a 10-fold reduction in cellular toxicity compared to the classical aminoglycosides, and each of the compounds was found to restore a significant amount of functional protein in mammalian cells carrying PTCs related to Usher syndrome, Rett syndrome, cystic fibrosis, and mucopolysaccharidosis I-Hurler (MPS I-H) . Other drugs or compounds that promote readthrough include the dipeptide antibiotic negamycin, as well as compounds identified in a screen performed by Du et al., (J. Exp. Med. 2009; 206 (10) : 2285-97) , e.g., N- (sec-butyl) -N′-phenylthiourea; 1, 2-di-2-furyl-2-hydroxyethanone; 1-methyl-9-oxo-9H-indeno [2, 1-b] pyridinium iodide; 2, 2′- [1, 4-phenylenebis (methylylidenenitrilo) ] bis (5-methylphenol) ; 3-methyl-5- { [5- (2-nitrophenyl) -2-furyl] methylene} -2-thioxo-1, 3-thiazolidin-4-one; 5-benzyl-2-methyl-2- (4-nitrophenyl) -2, 3-dihydro-1, 3, 4-thiadiazole; 5-hydroxy-5-methyl-2-phenyl-3-isoxazolidinone; 2- (3-pyridinylmethylene) -1-benzothiophen-3 (2H) -one; 2-imino-5- { [5- (2-nitrophenyl) -2-furyl] methylene} -1, 3-thiazolidin-4-one; 4-tert-butyl-2- [ (3-nitrobenzylidene) amino] phenol; [4- (difluoromethoxy) benzylidene] (phenyl) azane oxide; and 1- [ (4-nitrophenyl) sulfonyl] -1H-pyrrole.

In some embodiments, the readthrough drug is not an aminoglycoside. Examples of readthrough drugs that promote readthrough but are not aminoglycosides are known in the art and include, for example, negamycin, clitocine, acetylaminobenzoic acids such as 3- [2- (4-tertbutyl-phenoxy) -acetylamino] -benzoic acid and 3- {2- [4- (1, 1-dimethylpropyl) -phenoxy] acetylamino} -benzoic acid, readthrough compount (RTC) #13, RTC #14, erythromycin, oleandomycin, tylosin, spiramycin, and josamycin. Salts, analogs, or derivatives of any of the aforementioned drugs may be used to practice the methods described herein.

In some embodiments, the PTC correction agent is a suppressor tRNA. Transfer RNAs (tRNAs) translate mRNA into a protein on a ribosome. Each tRNA contains an "anti-codon" region that hybridizes with a complementary codon on the mRNA. A tRNA that carries its designated amino acid is called a "charged" tRNA. If the tRNA is one of the 61 amino-acid-associated (i.e., not a stop-signal-associated) tRNAs, it will normally attach its amino acid to the growing peptide. The structural gene of tRNA is about 72-90 nucleotides long and folds into a cloverleaf structure. tRNAs are transcribed by RNA polymerase III and contain their own intragenic split promoters that become a part of the mature tRNA coding sequence (Sharp S. J., Schaack J., Coolen L., Burke D. J. and Soil D., "Structure and transcription of eukaryotic tRNA genes" , Crit. Rev. Biochem, 19: 107-144 (1985) ; Geiduschek E. O., and Tocchini-Valentini, "Transcription by RNA polymerase III, Annu. Rev. Biochem. 57: 873-914 (1988) ) .

A "suppressor tRNA" is one whose anti-codon is complementary with a codon that would otherwise terminate translation, so that detectable read-through occurs under the conditions of the experiment. Standard termination codons are amber (UAG) , ochre (UAA) , and opal (UGA) codons. However, non-standard termination codons (e.g., 4-nucleotide codons) have also been employed in the literature (see, for example, Moore et al., J. Mol. Biol. 298: 195, 2000; Hohsaka et al., J. Am. Chem. Soc. 121: 12194, 1999) .

In a few species, the detrimental effects of pervasive nonsense mutations are kept low by suppressor tRNAs, which commonly arise by mutation in a tRNA’s anticodon to decode the newly arising stop codon. However, through the exchange of the anticodon triplet only few natural tRNAs can be repurposed into suppressor tRNAs generating tRNAs with fairly modest effectivity in decoding stop codons and correcting nonsense mutations (see, for example, Kiselevet al., Mol. Biol. (Mosk. ) 2002; Buvoli et al., JMol. Cell. Biol., 2000; Bordeira-et al, , Eur. J. Hum. Genet. 2014; Panchal et al., Hum. Gene Ther. 1999; Temple et al., Nature, 1982; Lueck et al., Nat. Commun. 2019) . Multiple rounds of random nucleotide mutagenesis in the tRNA body and combinatorial changes of different tRNA segments have been shown to improve suppression efficiency, implying that other tRNA elements, in addition to the anticodon, can modulate this efficiency (see, for example, Ogawa et al., J. Nucleic Acids 2012; Ogawa et al., Org. Biomol. Chem. 2011; Albers et al., Nat. Commun. 2021) . In some embodiments, the tRNA is a modified endogenous tRNA charged with a canonical amino acid. In some embodiments, the canonical amino acid is serine. In some embodiments, the tRNA is an orthogonal tRNA charged with a non-canonical amino acid. In some embodiments, the tRNA targets an amber codon. In some embodiments, the tRNA targets an ochre codon. In some embodiments, the tRNA targets an opal codon.

In some embodiments, the PTC correction agent is a targeted pseudouridylation agent. As the most abundant modification in RNA, pseudouridine (Ψ) possesses a similar base-pairing property as uridine (U) . Replacing uridine with pseudouridine in stop codons suppresses translation termination, which could be harnessed to mediate readthrough of premature termination codons (PTCs) . Site-specific pseudouridylation of rRNA and snRNA is catalyzed primarily by H/ACA box snoRNP (ribonucleoprotein) . The H/ACA box snoRNP machinery can be leveraged to achieve precise and targeted pseudouridylation at PTCs (see, for example, Song et al., Molecular Cell. 2023; Adachi et al., Molecular Cell. 2023) .

In some embodiments, the PTC correction agent is an RNA editing agent. In some embodiments, the RNA editing agent can edit the PTC to a non-stop codon. In some embodiments, the RNA editing agent is deaminase-recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding the dRNA, wherein the dRNA comprises a correction RNA sequence that is at least partially complementary to a PTC region of the PTC-containing mRNA, wherein the dRNA recruits an adenosine deaminase acting on RNA (ADAR) to the PTC, wherein the recruitment of the ADAR converts a target adenosine in the PTC to a sense codon. The PTC region of the PTC-containing mRNA is a sequence encompassing a PTC.

The term “ADAR” as used herein refers to an adenosine deaminase that can convert adenosines (A) to inosines (I) in an RNA sequence. ADAR1 and ADAR2 are two exemplary species of ADAR that are involved in mRNA editing in vivo. Non-limiting exemplary sequences for ADAR1 may be found under the following reference numbers: HGNC: 225; Entrez Gene: 103; Ensembl: ENSG 00000160710; OMIM: 146920; UniProtKB: P55265; and GeneCards: GC01M154554, as well as biological equivalents thereof. Non-limiting exemplary sequences for ADAR2 may be found under the following reference numbers: HGNC: 226; Entrez Gene: 104; Ensembl: ENSG00000197381; OMIM: 601218; UniProtKB: P78563; and GeneCards: GC21P045073, as well as biological equivalents thereof.

In some embodiments, the correction sequence is about 70 to about 150 nucleotides in length. In some embodiments, the correction sequence is about 70 nucleotides in length. In some embodiments, the correction sequence is about 80 nucleotides in length. In some embodiments, the correction sequence is about 90 nucleotides in length. In some embodiments, the correction sequence is about 100 nucleotides in length. In some embodiments, the correction sequence is about 110 nucleotides in length. In some embodiments, the correction sequence is about 120 nucleotides in length. In some embodiments, the correction sequence is about 130 nucleotides in length. In some embodiments, the correction sequence is about 140 nucleotides in length. In some embodiments, the correction sequence is about 150 nucleotides in length.

In some embodiments according to any one of the methods or use described herein, the dRNA does not comprise an ADAR-recruiting domain. In some embodiments, the dRNA comprises an ADAR recruiting domain. “ADAR-recruiting domain” can be a nucleotide sequence or structure that binds at high affinity to ADAR, or a nucleotide sequence that binds to a binding partner fused to ADAR in an engineered ADAR construct. Exemplary ADAR-recruiting domains include, but are not limited to, GluR-2, GluR-B (R/G) , GluR-B (Q/R) , GluR-6 (R/G) , 5HT2C, and FlnA (Q/R) domain; see, for example, Wahlstedt, Helene, and Marie, "Site-selective versus promiscuous A-to-I editing. " Wiley Interdisciplinary Reviews: RNA 2.6 (2011) : 761-771, which is incorporated herein by reference in its entirety. In some embodiments, the recruitment of the ADAR results in editing of the PTC and/or correcting the nonsense mutation. In some embodiments, the recruitment of the ADAR converts a target adenosine in the PTC to a sense codon In some embodiments, the ADAR is naturally present in a host cell, such as a eukaryotic cell. In some embodiments, the ADAR is introduced into the host cell.

In some embodiments, the dRNA is a circRNA. In some embodiments, the dRNA is a linear dRNA. In some embodiments the dRNA is a linear dRNA that forms a circRNA within the host cell.

In some embodiments, the dRNA is circularized before being introduced to the host cell. In some embodiments, the dRNA is a circRNA formed using a linear RNA in vitro by autocatalysis of a Group I intron comprising a 5’ catalytic Group I intron fragment and a 3’ catalytic Group I intron fragment. In some embodiments, the linear RNA comprises the 3’ catalytic Group I intron fragment flanking the 5’ end of a 3’ exon sequence recognizable by the 3’ catalytic Group I intron fragment, and the 5’ catalytic Group I intron fragment flanking the 3’ end of a 5’ exon sequence recognizable by the 5’ catalytic Group I intron fragment. In some embodiments, the linear RNA further comprises a 5’ homology sequence flanking the 5’ end of the 3’ catalytic Group I intron fragment, and a 3’ homology sequence flanking the 3’ end of the 5’ catalytic Group I intron fragment. In some embodiments, said forming a circRNA comprises: (a) subjecting the linear RNA to a condition that activates autocatalysis of the 5’ catalytic Group I intron fragment and the 3’ catalytic Group I intron fragment to provide a circularized RNA product; and (b) isolating the circularized RNA product, thereby providing the circRNA. Methods of forming circRNA in vitro (including forming circRNA by chemical ligation) are further described in International Patent Applications WO2022150974 and WO2022037692, which are specifically incorporated herein by reference.

In some embodiments, the dRNA is circularized by an RNA ligase. Non-limiting examples of RNA ligase include: RtcB, T4 RNA Ligase 1, T4 RNA Ligase 2, Rnl3 and Trl1. In some embodiments, the RNA ligase is expressed endogenously in the host cell. In some embodiments, the RNA ligase is RNA ligase RtcB. In some embodiments, the method further comprises introducing an RNA ligase (e.g., RtcB) into the host cell. In some embodiments, the dRNA is circularized through in vitro enzymatic ligation (e.g., using RNA or DNA ligase) or chemical ligation (e.g., using cyanogen bromide or a similar condensing agent) .

The circRNA can be formed using a linear RNA in vitro by a ligase. In some embodiments, the ligase is selected from the group consisting of a T4 DNA ligase (T4 Dnl) , a T4 RNA ligase 1 (T4 Rnl1) and a T4 RNA ligase 2 (T4 Rnl2) . In some embodiments, the linear RNA comprises a 5’ ligation sequence at the 5’ end of a nucleic acid sequence encoding the circRNA, and a 3’ ligation sequence at the 3’ end of the nucleic acid sequence encoding the circRNA, wherein the 5’ ligation sequence and the 3’ ligation sequence can be ligated to each other via the ligase. In some embodiments, said forming a circRNA comprises: (a) contacting the linear RNA with a single-stranded adaptor nucleic acid comprising from the 5’ end to the 3’ end: a first sequence complementary to the 3’ ligation sequence and a second sequence complementary to the 5’ ligation sequence, and wherein the 5’ ligation sequence and the 3’ ligation sequence hybridize to the single-stranded adaptor nucleic acid to provide a duplex nucleic acid intermediate comprising a single strand break between the 3’ end of the 5’ ligation sequence and the 5’ end of the 3’ ligation sequence; (b) contacting the intermediate with an RNA ligase under a condition that allows ligation of the 5’ ligation sequence to the 3’ ligation sequence to provide a circularized RNA product; and (c) isolating the circularized RNA product, thereby providing the circRNA. In some embodiments, said forming a circRNA comprises: (a) contacting the linear RNA with an RNA ligase under a condition that allows ligation of the 5’ ligation sequence to the 3’ ligation sequence to provide a circularized RNA product; and (b) isolating the circularized RNA product, thereby providing the circRNA. In some embodiments, the method further comprises obtaining the linear RNA by in vitro transcription of a nucleic acid construct comprising a nucleic acid sequence encoding the linear RNA. In some embodiments, the method further comprises purifying the circRNA. Methods for forming circRNAs in vitro are described in WO2021008447A1 and WO2022037692A1, each of which is incorporated herein in its entirety by reference.

In one aspect, the dRNA is a linear RNA that is capable of forming a circRNA. Linear RNA capable of forming circRNA are described in WO2021008447, which is specifically incorporated herein by reference. In some embodiments, the circulation is performed using the Tornado expression system ( “Twister-optimized RNA for durable overexpression” ) as described in Litke, J. L. &Jaffrey, S. R. Highly efficient expression of circRNA aptamers in cells using autocatalytic transcripts. Nat Biotechnol 37, 667-675 (2019) , which is hereby incorporated herein by reference in its entirety. Briefly, Tornado-expressed transcripts contain an RNA of interest flanked by Twister ribozymes. A twister ribozyme is any catalytic RNA sequences that are capable of self-cleavage. The ribozymes rapidly undergo autocatalytic cleavage, leaving termini that are ligated by an RNA ligase.

In some embodiments, the dRNA is introduced by a construct comprising a nucleic acid encoding the dRNA. In some embodiments, the construct further comprises a 3’ twister ribozyme sequence linked to the 3’ end of the nucleic acid encoding the dRNA and a 5’ twister ribozyme sequence linked to the 5’ end of the nucleic acid encoding the dRNA. In some embodiments, the 3’ twister sequence is twister P3 U2A and the 5’ twister sequence is twister P1. In some embodiments, wherein the 5’ twister sequence is twister P3 U2A and the 3’ twister sequence is twister P1. In some embodiments, the dRNA undergoes autocatalytic cleavage. In some embodiments, the catalyzed dRNA product comprises a 5′-hydroxyl group and a 2′, 3′-cyclic phosphate at the 3′terminus. In some embodiments, the catalyzed dRNA product is ligated by ubiquitous endogenous RNA ligase (e.g., RNA ligase RtcB) . In some embodiments, the construct is a plasmid or a viral vector.

In some embodiments, the dRNA transcript is also flanked a 5’ and/or 3’ ligation sequences which are then flanked by the 5’ -Twister ribozyme and/or 3’ -Twister ribozymes, respectively. In some embodiments, the dRNA further comprises a 3’ ligation sequence and a 5’ ligation sequence. In some embodiments, the 3’ ligation sequence and the 5’ ligation sequence are at least partially complementary to each other. In some embodiments, the 3’ ligation sequence and the 5’ ligation sequence are at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%or at least about 99%complementary to each other. In some embodiments, the 3’ ligation sequence and the 5’ ligation sequence are fully complementary to each other.

In some embodiments, the dRNA is not chemically modified. In some embodiments, the dRNA is chemically modified. In some embodiments, the dRNA comprises one or more modifications, such as 2′-O-methylation and/or phosphorothioation. Chemically modified dRNAs are provided in WO2022/150974, which is specifically incorporated herein by reference.

The PTC correction agent may be introduced in sufficient quantity, or in an effective amount. Increasing readthrough or correction of the PTC results in an increase of full-length protein as compared, for example, to levels of full-length protein in the absence of the composition (e.g., as described herein) . In some aspects, the method is ameliorative or preventative of disease or condition, such as those provided herein, for example when the method is performed on cells in an individual, e.g., a human.

The PTC correction agent may be administered simultaneously or sequentially with the ihRNA (s) . In some embodiments, the PTC correction agent is administered simultaneously with the ihRNA (s) . In some embodiments, the PTC correction agent is administered prior to the ihRNA (s) . In some embodiments, the PTC correction agent is administered after the ihRNA (s) .

B. Diseases and conditions

Any nonsense mutation occurring downstream of the first exon and at least 50-55 nucleotides upstream of the last exon-exon junction can be treated by the methodology and compositions provided herein. Mutations that follow this rule are referred to as “treatable” mutations. EJC deposition is blocked at at least one exon-exon junction and, in some embodiments, at more than one exon-exon junction downstream of a PTC, simultaneously, to provide for enhanced inhibition of NMD. The vast majority of genes in the human genome have at least two exons (the average number of exons per gene in the human genome is approximately eight) , which means that nonsense mutations in many disease-associated genes are treatable mutations that can be targeted by the disclosed methodology.

In some embodiments, the compositions and methodology are useful for inhibiting NMD of transcripts derived from a disease-associated gene, such as CFTR, DMD, HBB, MECP2 and IDUA. Mutations in these genes cause, respectively, cystic fibrosis (1/2500 live births) , Duchenne/Becker muscular dystrophy (1/4000 male live births) , β-thalassemia (1/158-1/25, 000 live births, Rett syndrome (1/10, 000-15, 000 female live births) , and mucopolysaccharidosis type 1-Hurler (1/100, 000) .

Any disease associated with a nonsense allele may be treated using the compositions and methods provided herein. Other diseases or disorders that are also treatable using the compositions and methods provided herein include, but are not limited to, Shwachman-Diamond syndrome, Usher syndrome, ataxia telangiectasia, hemophilia A and B, Hailey-Hailey disease, Ullrich disease, methylmalonic acidemia, carnitine palmitoyltransferase 1A deficiency, peroxisome biogenesis disorders, limb girdle muscular dystrophy, Schmid metaphyseal chondrodysplasia, Sandhoff disease, Marfan syndrome, anemia, epidermolysis bullosa simplex, Tay-Sachs disease, triose phosphate isomerase deficiency, Alzheimer's disease, long-QT syndrome, insulin resistance, maple syrup urine disease, hereditary fructose intolerance, X-linked severe combined immunodeficiency, infantile neuronal ceroid lipofuscinosis, cystinosis, X-linked nephrogenic diabetes insipidus, polycystic kidney disease, Liddle's syndrome, xeroderma pigmentosum, Fanconi's anemia, p53-associated cancers (e.g., p53 squamal cell carcinoma, p53 hepatocellular carcinoma, p53 ovarian carcinoma) , esophageal carcinoma, osteocarcinoma, ovarian carcinoma, hepatocellular carcinoma, breast cancer, hepatocellular carcinoma, fibrous histiocytoma, ovarian carcinoma, SRY sex reversal, triosephosphate isomerase-anemia, inherited cancers such as those due to BRCA1 nonsense mutations, carbohydrate metabolism disorders, amino acid metabolism disorders, lipoprotein metabolism disorders, lipid metabolism disorders, lysomal enzymes metabolism disorders, steroid metabolism disorders, purine metabolism disorders, pyrimidine metabolism disorders, metal metabolism disorders, porphyrin metabolism disorders, and heme metabolism disorders.

V. COMPOSITIONS, KITS, AND ARTICLES OF MANUFACTURE

Also provided herein are compositions (such as pharmaceutical compositions) comprising any one of the ihRNAs, constructs, libraries, or host cells described herein.

In some embodiments, there is provided a pharmaceutical composition comprising any one of the ihRNAs or constructs encoding the ihRNA described herein, and a pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) ) . Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol) ; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as olyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes) ; and/or non-ionic surfactants such as TWEEN^TM, PLURONICS^TM or polyethylene glycol (PEG) . In some embodiments, lyophilized formulations are provided. Pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished by, e.g., filtration through sterile filtration membranes.

Further provided are kits useful for any one of the methods of inhibiting NMD or methods of treatment described herein, comprising any one of the ihRNAs, constructs, compositions, libraries, or host cells as described herein.

The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags) , and the like. Kits may optionally provide additional components such as transfection or transduction reagents, cell culturing medium, buffers, and interpretative information.

The present application thus also provides articles of manufacture. The article of manufacture can comprise a container and a label or package insert on or associated with the container. Suitable containers include vials (such as sealed vials) , bottles, jars, flexible packaging, and the like. In some embodiments, the container holds a pharmaceutical composition, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) . The container holding the pharmaceutical composition may be a multi-use vial, which allows for repeat administrations (e.g. from 2-6 administrations) of the reconstituted formulation. Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such products. Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI) , phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

The kits or article of manufacture may include multiple unit doses of the pharmaceutical compositions and instructions for use, packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.

VI. EXEMPLARY EMBODIMENTS

Among the provided embodiments are:

Embodiment 1. A method of specifically inhibiting nonsense-mediated decay (NMD) of a premature termination codon (PTC) -containing mRNA in a host cell, the method comprising introducing into the host cell an inhibitory RNA (ihRNA) or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.

Embodiment 2. A method of inhibiting deposition of EJC on a PTC-containing mRNA in a host cell, the method comprising introducing into the host cell an ihRNA or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.

Embodiment 3. The method of Embodiment 1 or 2, wherein the method comprises introducing a construct encoding the ihRNA into the host cell.

Embodiment 4. The method of any one of Embodiments 1-3, wherein the ihRNA is a circular RNA.

Embodiment 5. The method of any one of Embodiments 1-3, wherein the ihRNA is a linear RNA.

Embodiment 6. The method of Embodiment 5, wherein the linear ihRNA forms a circular RNA in the host cell.

Embodiment 7. The method of any one of Embodiments 1-6, wherein the targeting sequence is at least about 15 nucleotides in length.

Embodiment 8. The method of any one of Embodiments 1-7, wherein the target sequence comprises an EJC deposition site immediately downstream of the PTC or a portion thereof.

Embodiment 9. The method of any one of Embodiments 1-8, wherein the target sequence comprises two or more EJC deposition sites or portions thereof.

Embodiment 10. The method of any one of Embodiments 1-9, wherein the EJC deposition site is identified by mapping EIF4A3 binding site on the PTC-containing mRNA sequence.

Embodiment 11. The method of Embodiment 10, wherein the mapping comprises CLIP-seq.

Embodiment 12. The method of any one of Embodiments 1-11, wherein the target sequence is located about 1 to about 50 nucleotides upstream of an exon-exon junction.

Embodiment 13. The method of any one of Embodiments 1-12 wherein the PTC results from a mutation.

Embodiment 14. The method of any one of Embodiments 1-13, wherein the PTC-containing mRNA is an IDUA mRNA comprising a PTC.

Embodiment 15. The method of any one of Embodiments 1-14, wherein the host cell is a eukaryotic cell.

Embodiment 16. The method of any one of Embodiments 15, wherein the eukaryotic cell is in an individual.

Embodiment 17. The method of Embodiment 16, wherein the individual is a human individual.

Embodiment 18. The method of any one of Embodiments 1-17, further comprising introducing into the host cell an effective amount of a PTC correction agent.

Embodiment 19. A method of treating an individual having a disease associated with a mutation that introduces a PTC in an mRNA to produce an PTC-containing mRNA, comprising administering to the individual an effective amount of a PTC correction agent and an ihRNA or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting RNA sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.

Embodiment 20. The method of Embodiment 18 or 19, wherein the PTC correction agent is a readthrough drug.

Embodiment 21. The method of Embodiment 20, wherein the readthrough drug is an aminoglycoside.

Embodiment 22. The method of Embodiment 21, wherein the aminoglycoside is amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, apramycin, G418 (geneticin) , lividomycin, or an aminoglycoside analog chosen from NB30, NB54, or NB84.

Embodiment 23. The method of Embodiment 18 or 19, wherein the PTC correction agent is a suppressor tRNA.

Embodiment 24. The method of Embodiment 18 or 19, wherein the PTC correction agent is a targeted pseudouridylation agent.

Embodiment 25. The method of Embodiment 18 or 19, wherein the PTC correction agent is an RNA editing agent.

Embodiment 26. The method of Embodiment 25, wherein the RNA editing agent is deaminase-recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding the dRNA, wherein the dRNA comprises a correction RNA sequence that is at least partially complementary to a PTC region of the PTC-containing mRNA, wherein the dRNA recruits an adenosine deaminase acting on RNA (ADAR) to the PTC, wherein the recruitment of the ADAR converts a target adenosine in the PTC to a sense codon.

Embodiment 27. The method of Embodiment 26, wherein the dRNA does not comprises an ADAR recruiting domain.

Embodiment 28. The method of Embodiment 26, wherein the dRNA comprises an ADAR recruiting domain.

Embodiment 29. The method of any one of Embodiments 26-28, wherein the correction sequence is about 70 to about 150 nucleotides in length.

Embodiment 30. The method of any one of Embodiments 26-29, wherein the dRNA is a circular dRNA.

Embodiment 31. The method of any one of Embodiments 26-29, wherein the dRNA is a linear RNA.

Embodiment 32. The method of Embodiment 31, wherein the dRNA is a linear RNA that forms a circRNA within a host cell.

Embodiment 33. The method of any one of Embodiments 26-32, wherein the dRNA is not chemically modified.

Embodiment 34. The method of any one of Embodiments 26-32, wherein the dRNA is chemically modified.

Embodiment 35. The method of any one of Embodiments 19-34, wherein the disease is selected from the group consisting of Hurler syndrome, β-thalassemia, and Rett syndrome.

Embodiment 36. A system for specifically inhibiting nonsense-mediated decay (NMD) of a PTC-containing mRNA in a host cell, comprising an ihRNA or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.

Embodiment 37. A system of inhibiting deposition of EJC on a PTC-containing mRNA in a host cell, comprising an ihRNA or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.

Embodiment 38. The system of Embodiment 36 or 37, wherein the system comprises a construct encoding the ihRNA.

Embodiment 39. The system of any one of Embodiments 36-38, wherein the ihRNA is a circular RNA.

Embodiment 40. The system of any one of Embodiments 36-39, wherein the targeting RNA sequence is at least about 15 nucleotides in length.

Embodiment 41. The system of any one of Embodiments 36-40, wherein the target sequence comprises an EJC deposition site immediately downstream of the PTC or a portion thereof.

Embodiment 42. The system of any one of Embodiments 36-41, wherein the target sequence comprises two or more EJC deposition sites or portions thereof.

Embodiment 43. The system of any one of Embodiments 36-42, wherein the EJC deposition site is identified by mapping EIF4A3 binding site on the PTC-containing mRNA sequence.

Embodiment 44. The system of Embodiment 43, wherein the mapping comprises CLIP-seq.

Embodiment 45. The system of any one of Embodiments 36-44, wherein the target sequence is located about 1 to about 50 nucleotides upstream of an exon-exon junction.

Embodiment 46. The system of any one of Embodiments 36-45 wherein the PTC results from a mutation.

Embodiment 47. The system of any one of Embodiments 36-46, wherein the PTC-containing mRNA is an IDUA mRNA comprising a PTC.

Embodiment 48. The system of any one of Embodiments 36-47, wherein the host cell is a eukaryotic cell.

Embodiment 49. The system of any one of Embodiments 48, wherein the eukaryotic cell is in an individual.

Embodiment 50. The method of Embodiment 49, wherein the individual is a human individual.

Embodiment 51. The system of any one of Embodiments 36-50, further comprising a PTC correction agent.

Embodiment 52. The system of Embodiment 51, wherein the PTC correction agent is a readthrough drug.

Embodiment 53. The system of Embodiment 52, wherein the readthrough drug is an aminoglycoside.

Embodiment 54. The system of Embodiment 53, wherein the aminoglycoside is amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, apramycin, G418 (geneticin) , lividomycin, or an aminoglycoside analog chosen from NB30, NB54, or NB84.

Embodiment 55. The system of Embodiment 52, wherein readthrough drug is a suppressor tRNA.

Embodiment 56. The system of Embodiment 51, wherein the PTC correction agent is an RNA editing agent.

Embodiment 57. The system of Embodiment 56, wherein the RNA editing agent is deaminase-recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding the dRNA, wherein the dRNA comprises a correction RNA sequence that is at least partially complementary to a PTC region of the PTC-containing mRNA, wherein the dRNA recruits an adenosine deaminase acting on RNA (ADAR) to the PTC, wherein the recruitment of the ADAR converts a target adenosine in the PTC to a sense codon.

Embodiment 58. The system of Embodiment 57, wherein the dRNA does not comprises an ADAR recruiting domain.

Embodiment 59. The system of Embodiment 57, wherein the dRNA comprises an ADAR recruiting domain.

Embodiment 60. The system of any one of Embodiments 56-59, wherein the correction sequence is about 70 to about 150 nucleotides in length.

Embodiment 61. The system of any one of Embodiments 56-60, wherein the dRNA is a circular dRNA.

Embodiment 62. The system of any one of Embodiments 56-61, wherein the dRNA is a linear RNA.

Embodiment 63. The system of Embodiment 62, wherein the dRNA is a linear RNA that forms a circRNA within a host cell.

Embodiment 64. The system of any one of Embodiments 57-63, wherein the dRNA is not chemically modified.

Embodiment 65. The system of any one of Embodiments 57-63, wherein the dRNA is chemically modified.

VII. EXAMPLES

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1 Inhibition of NMD in GM06214 Primary Cells

This example demonstrates that circular antisense RNAs can inhibit NMD of mutated IDUA mRNA.

GM06214 primary cells derived from Hurler syndrome patients harbor IDUA^W402X in exon 9. Circular antisense RNAs with a length of 50 nucleotides (Circ-antisense₅₀) at 10 bp intervals and 1 circular antisense RNA with a length of 72 nucleotides circ-antisense₇₂ RNA were designed to target an EIF4A3 binding site (SEQ ID NO: 1) spanning across exon 12 and exon 13 since no binding peak of EIF4A3 was found on exon 9, 10, or 11 (FIG. 1A) . Circ-antisense₅₀-1 (SEQ ID NO: 4) targets 22 nt upstream to 28 nt downstream of exon 12-13 junction. Circ-antisense₅₀-2 (SEQ ID NO: 5) targets 12 nt upstream to 38 nt downstream of exon 12-13 junction. Circ-antisense₅₀-3 (SEQ ID NO: 6) targets 2 nt upstream to 48 nt downstream of exon 12-13 junction. Circ-antisense₅₀-4 (SEQ ID NO: 7) targets 1-50 nt downstream of exon 12-13 junction. Circ-antisense₇₂ (SEQ ID NO: 3) targets 22 nt upstream to 50 nt downstream of exon 12-13 junction. A circular RNA with a length of 151 nucleotides (Ctrl RNA₁₅₁) (SEQ ID NO: 2) was used as the control. As shown in FIG. 1B, the expression level of IDUA was elevated by circ-antisense RNAs targeting the EJC binding site. In particular, Circ-antisense₇₂ RNA that covering all binding region of EIF4A3 and circ-antisense₅₀-3 achieved about 3 fold of increase in expression. Circular antisense RNA₅₀ (SEQ ID NOs: 8-13) targeting exon 9, 10, or 11, or canonical EJC deposition sites 24 nt upstream of the exon junctions (which don’ t contain EIF4A3 binding peaks) did not elevate the expression level of IDUA (FIG. 1C) . These results indicate that circ-antisense RNAs can increase the expression level of the target transcript by covering strongest EJC deposition sites.

Example 2 Inhibition of NMD in idua mouse reporter cell line

This example demonstrates that circular antisense RNAs can inhibit NMD of mutated IDUA mRNA in a mouse reporter cell line.

To make a reporter cell line, an idua minigene was first constructed, which comprised IDUA^W392X exons 8-14 and a FLAG tag. The minigene was then introduced into Neuro-2a cells. As shown in FIG. 2A, the expression level of the idua minigene in the transfected Neuro-2a cells was increased by aminoglycoside geneticin G418, which acts as a translational readthrough drug, thereby confirming that the idua^W392X cell line is suitable for modeling NMD and be used to test inhibition of NMD.

To test if circular antisense RNAs can inhibit NMD of the idua minigene in the idua^W392X cell line, different circ-antisense RNAs (SEQ ID NOs: 14-67) targeting exons downstream the PTC in idua^W392X were designed in a tiling manner due to the lack of CLIP data of EIF4A3 in mice (FIG. 2B) . A circular RNA with a length of 151 nucleotides (Ctrl RNA₁₅₁) was used as the control.

As shown in FIG. 2C, the designed circ-antisense₅₀ RNAs elevated the expression level of idua by up to 6-fold compared to the control RNA. The results also showed that targeting the flanking regions in exon are more likely to increase the expression level of the target transcript whether in IDUA or idua (FIG. 2D) . Together with CLIP data of EJC complex, circular antisense RNA can be engineered to elevate expression of PTC-containing target transcripts by inhibiting nonsense-mediated decay.

A circular arRNA (SEQ ID NO: 68) with a length of 151 nucleotides (circ-arRNA₁₅₁) was designed to target W392X in idua. When used in combination with circ-arRNA₁₅₁, the circ-antisense₅₀ RNAs can increase the protein level by up to 6-fold, and the restored protein level was correlated with RNA level in exon 9 (FIGs. 2E-2F) .

Example 3 Inhibition of NMD in vivo

This example shows that circular antisense RNAs can inhibit NMD in vivo.

In vivo experiments were carried out in Hurler syndrome mice as illustrated in FIG. 3A. Circ-antisense₅₀ (SEQ ID NO: 69) was delivered together with the circ-arRNA₁₅₁ targeting W392X in idua (SEQ ID NO: 68) (FIG. 3A) . The circ-antisense₅₀ was shown to increase the RNA editing efficiency compared to the circ-arRNA₁₅₁ alone (FIG. 3C) . Protein level indicated by enzyme activity was shown to increase by 1.4-fold, and the restored protein level is correlated with RNA level (FIGs. 3B and 3D) . Additionally, RIP-qPCR confirms that the circ-antisense RNA affected the loading of the NMD machinery (EIF4A3) (FIG. 3E) .

Example 4 Inhibition of NMD of CFTR

This example demonstrates that circular antisense RNAs can inhibit NMD of mutated CFTR mRNA in 16HBEge cell line.

Different combinations of antisense RNAs (SEQ ID NOs: 70-73) were designed targeting mRNA downstream of CFTR R1162X mutation in 16HBEge cells. Combination of antisense RNAs intro one circ-antisense RNA elevated the CFTR expression level (FIG. 4A) . When used together with a suppressor tRNA (SEQ ID NO: 74) , CFTR protein was restored with a 3.3-fold change compared to suppressor tRNA treatment alone, and the restoration of CFTR reached 50%of WT (FIGs. 4B and 4C) . These results show that engineered circ-antisense RNA has the potential to elevate expression by inhibition of NMD of PTC-containing transcripts.

The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.

SEQUENCES

Claims

A method of specifically inhibiting nonsense-mediated decay (NMD) of a premature termination codon (PTC) -containing mRNA in a host cell, the method comprising introducing into the host cell an inhibitory RNA (ihRNA) or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.
A method of inhibiting deposition of EJC on a premature termination codon (PTC) -containing mRNA in a host cell, the method comprising introducing into the host cell an inhibitory RNA (ihRNA) or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.
The method of claim 1 or 2, wherein the method comprises introducing a construct encoding the ihRNA into the host cell.
The method of any one of claims 1-3, wherein the ihRNA is a circular RNA.
The method of any one of claims 1-3, wherein the ihRNA is a linear RNA.
The method of claim 5, wherein the linear ihRNA forms a circular RNA in the host cell.
The method of any one of claims 1-6, wherein the targeting sequence is at least about 15 nucleotides in length.
The method of any one of claims 1-7, wherein the target sequence comprises an EJC deposition site immediately downstream of the PTC or a portion thereof.
The method of any one of claims 1-8, wherein the EJC deposition site is identified by mapping EIF4A3 binding site on the PTC-containing mRNA sequence.
The method of any one of claims 1-9, wherein the target sequence is located about 1 to about 50 nucleotides upstream of an exon-exon junction.
The method of any one of claims 1-10, wherein the PTC results from a mutation.
The method of any one of claims 1-11, wherein the PTC-containing mRNA is an IDUA mRNA comprising a PTC or a CFTR mRNA comprising a PTC.
The method of any one of claims 1-12, wherein the host cell is a eukaryotic cell.
The method of any one of claims 1-13, further comprising introducing into the host cell an effective amount of a PTC correction agent.
A method of treating an individual having a disease associated with a mutation that introduces a premature termination codon (PTC) in an mRNA to produce an PTC-containing mRNA, comprising administering to the individual an effective amount of a PTC correction agent and an ihRNA or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting RNA sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.
The method of claim 14 or 15, wherein the PTC correction agent is a readthrough drug.
The method of claim 14 or 15, wherein the PTC correction agent is a suppressor tRNA or a targeted pseudouridylation agent or an RNA editing agent.
The method of claim 17, wherein the RNA editing agent is deaminase-recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding the dRNA, wherein the dRNA comprises a correction RNA sequence that is at least partially complementary to a PTC region of the PTC-containing mRNA, wherein the dRNA recruits an adenosine deaminase acting on RNA (ADAR) to the PTC, wherein the recruitment of the ADAR converts a target adenosine in the PTC to a sense codon.
The method of claim 18, wherein the correction sequence is about 70 to about 150 nucleotides in length.
The method of any one of claims 15-19, wherein the disease is selected from the group consisting of Hurler syndrome, β-thalassemia, cystic fibrosis, and Rett syndrome.
A system for specifically inhibiting nonsense-mediated decay (NMD) of a premature termination codon (PTC) -containing mRNA in a host cell, comprising an ihRNA or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.
A system of inhibiting deposition of exon junction complexes (EJC) on a premature termination codon (PTC) -containing mRNA in a host cell, comprising an ihRNA or a construct encoding the ihRNA, wherein the ihRNA comprises a targeting sequence that is at least partially complementary to a target sequence downstream of the PTC in the PTC-containing mRNA, wherein the target sequence comprises an exon junction complexes (EJC) deposition site or a portion thereof.
The system of claim 21 or 22, wherein the system comprises a construct encoding the ihRNA.
The system of any one of claims 21-23, wherein the ihRNA is a circular RNA.
The system of any one of claims 21-24, wherein the targeting RNA sequence is at least about 15 nucleotides in length.
The system of any one of claims 21-25, wherein the target sequence comprises an EJC deposition site immediately downstream of the PTC or a portion thereof, and/or wherein the target sequence comprises two or more EJC deposition sites or portions thereof.
The system of any one of claims 21-26, wherein the EJC deposition site is identified by mapping EIF4A3 binding site on the PTC-containing mRNA sequence.
The system of any one of claims 21-27, wherein the target sequence is located about 1 to about 50 nucleotides upstream of an exon-exon junction.
The system of any one of claims 21-28, wherein the PTC results from a mutation.
The system of any one of claims 21-29, wherein the PTC-containing mRNA is an IDUA mRNA comprising a PTC or a CFTR mRNA comprising a PTC.
The system of any one of claims 21-30, wherein the host cell is a eukaryotic cell.
The system of any one of claims 21-31, further comprising a PTC correction agent.
The system of claim 32, wherein the PTC correction agent is a readthrough drug.
The system of claim 33, wherein readthrough drug is a suppressor tRNA.
The system of claim 32, wherein the PTC correction agent is an RNA editing agent.
The system of claim 35, wherein the RNA editing agent is deaminase-recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding the dRNA, wherein the dRNA comprises a correction RNA sequence that is at least partially complementary to a PTC region of the PTC-containing mRNA, wherein the dRNA recruits an adenosine deaminase acting on RNA (ADAR) to the PTC, wherein the recruitment of the ADAR converts a target adenosine in the PTC to a sense codon.
The system of claim 35 or 36, wherein the correction sequence is about 70 to about 150 nucleotides in length.