RNA targeting compositions and methods of use

Abstract

An artificial exon junction complex comprising a fusion protein comprising an RNA-binding domain and an exon junction complex (EJC) mimicking domain, a guide RNA and a target mRNA, wherein the RNA-binding domain comprises an RNA-binding region of a CAS nuclease, the (EJC) mimicking domain comprises one or more regions derived from EJC components, the guide RNA comprises a sequence complementary to a target sequence in the target mRNA, the target mRNA comprises a premature stop codon (PTC) that is insensitive to nonsense-mediated mRNA decay (NMD), and the 5' end of the target sequence is located at least 10 nucleotides downstream of the PTC in the target mRNA. A method for inducing mRNA decay of a target mRNA that comprises a premature stop codon (PTC) that is insensitive to nonsense-mediated mRNA decay (NMD) processes.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 364,237, filed May 5, 2022.

[0002] This invention was made with Government support under Awards GM147719 and GM059614 from the National Institutes of Health. The Government has certain rights in this invention.

[0003] The present invention relates generally to the medical field, and in particular to compositions that specifically target mRNA. [Background technology]

[0004] All intracellular processes are not error-free. Thus, cells have developed complex quality control mechanisms aimed at dealing with their own errors. One particularly widespread and harmful error is the introduction of a premature stop codon (PTC) within a protein-coding exon. The resulting PTC-containing transcripts are best recognized and eliminated before the truncated proteins they encode accumulate in the cell. This failure to eliminate in humans can have pathological consequences, as evidenced by the fact that there are many dominantly inherited diseases caused by PTCs that fail to trigger NMD. Notably, about 33% of inherited and acquired diseases are the result of PTC acquisition.

[0005] Nonsense-mediated mRNA decay (NMD) is an intracellular mechanism that selectively degrades mRNAs with PTCs. However, the NMD process is only effective in eliminating mRNAs with PTCs located more than approximately 55 nt upstream of the last exon-exon junction (Figure 1). mRNAs with PTCs located in the last exon or less than 55 nt upstream of the last exon-exon junction are resistant to the NMD process and are the primary cause of dominantly inherited diseases caused by PTCs.

[0006] Nonsense-mediated mRNA decay (NMD) is an essential intracellular process that ensures proper regulation of gene expression, allows cells to respond to environmental changes, maintains mRNA quality, and prevents disease development. After pre-mRNA splicing, the newly synthesized mRNA is rapidly exported to the cytoplasm for translation. When translation terminates at a premature termination codon (PTC) located more than 55 nucleotides upstream of an exon-exon junction, the downstream exon junction complex (3'UTR EJC) is not removed by the ribosome (Figure 2). In such cases, the 3'UTR EJC interacts with the central NMD factor UPF1, which is phosphorylated by the serine / threonine kinase complex SMG1-SMG8-SMG9 (Figure 2). UPF1 phosphorylation triggers mRNA degradation by recruiting nucleases either directly, as in the case of endonucleolytic degradation by SMG6, or indirectly, as in the case of exonucleolytic degradation through the SMG5-SMG7 heterodimer, followed by the recruitment of the CCR4-NOT deadenylation complex to complete the subsequent round of mRNA degradation (Figure 2). Because 3'UTR EJC is the canonical activator of NMD, the majority of mRNAs containing 3'UTR EJC are rapidly degraded within 1 min of export into the cytoplasm. Summary of the Invention [Problem to be solved by the invention]

[0007] Thus, there is a need for compositions and methods that can effectively treat conditions caused by NMD-insensitive PTC. [Means for solving the problem]

[0008] One aspect of the invention relates to a fusion protein comprising an RNA-binding domain and an exon junction complex (EJC)-mimic domain, wherein the RNA-binding domain comprises a Cas nuclease or an RNA-binding region thereof, and the EJC-mimic domain comprises one or more regions derived from one or more components of the EJC.

[0009] In some embodiments, the fusion protein further comprises at least one nuclear export signal. In some embodiments, the nuclear export signal comprises SEQ ID NO: 18 or 19.

[0010] In some embodiments, the RNA-binding domain comprises a catalytically inactive Cas nuclease or an RNA-binding region thereof. In some embodiments, the RNA-binding domain comprises a catalytically inactive Cas13d nuclease or an RNA-binding region thereof.

[0011] In some embodiments, the component of the EJC is selected from the group consisting of eIF4A3, Y14 / RBM8A, MAGOH, RNPS1, SRSF1, UPF1, UPF2 and UPF3B / 3X.

[0012] In some embodiments, the fusion protein further comprises a linker between the RNA-binding domain and the EJC-mimic domain.

[0013] Another aspect of the present invention relates to an expression vector comprising a first nucleotide sequence encoding an RNA-binding domain of a CAS nuclease, a second nucleotide sequence encoding an exon junction complex (EJC) mimic domain derived from a component of the EJC, and a regulatory sequence operably linked to at least one of the first and second nucleotide sequences.

[0014] In some embodiments, the expression vector further comprises a nucleotide sequence encoding a guide RNA. In some embodiments, the guide RNA comprises a sequence complementary to a target sequence in an mRNA containing an NMD-insensitive PTC, and the 5' end of the target sequence is located at least 10 nucleotides downstream of the NMD-insensitive PTC in the mRNA. In some embodiments, the expression vector is a non-viral vector. In some embodiments, the expression vector is a viral vector. In some embodiments, the expression vector is an AAV vector.

[0015] Another aspect of the present invention relates to a pharmaceutical composition comprising the expression vector of the present application and a pharma- ceutically acceptable carrier.

[0016] Another aspect of the present invention relates to an artificial exon junction complex comprising a fusion protein comprising an RNA-binding domain and an exon junction complex (EJC) mimicking domain, a guide RNA, and a target mRNA, wherein the RNA-binding domain comprises the RNA-binding region of a CAS nuclease, the (EJC) mimicking domain comprises one or more regions derived from components of the EJC, the guide RNA comprises a sequence complementary to a target sequence in the target mRNA, the target mRNA comprises a premature termination codon (PTC) that is insensitive to nonsense-mediated mRNA decay (NMD), and the 5' end of the target sequence is located at least 10 nucleotides downstream of the PTC in the target mRNA. In some embodiments, the fusion protein further comprises a nuclear export signal.

[0017] Another aspect of the present invention relates to a method for treating a disease or condition caused by the translation of a target mRNA that contains a premature termination codon (PTC) that is insensitive to nonsense-mediated mRNA decay (NMD) process in a subject. The method includes the steps of introducing into a cell of the subject expressing the target mRNA (1) a fusion protein that includes an RNA-binding domain and an exon junction complex (EJC) mimicking domain, and (2) a guide RNA that specifically binds to a target region in the target mRNA, wherein the fusion protein and the guide RNA form a complex that binds to the target mRNA and causes the degradation of the target mRNA by NMD. In some embodiments, the fusion protein is introduced into the cell in the form of one or more expression vectors that express the fusion protein. In some embodiments, the expression vector further expresses a guide RNA.

[0018] Another aspect of the invention relates to a method for inducing mRNA degradation of a target mRNA that contains a premature termination codon (PTC) that is insensitive to the nonsense-mediated mRNA decay (NMD) process, comprising the steps of introducing into a cell expressing the target mRNA: (a) an expression vector comprising a first nucleotide sequence encoding an RNA-binding domain, a second nucleotide sequence encoding an EJC-mimic domain derived from one or more components of an exon junction complex (EJC), and a regulatory sequence operably linked to at least one of the first nucleotide sequence and the second nucleotide sequence; and (b) a guide RNA that specifically binds to a target region within the target mRNA, wherein the expression vector expresses in the cell a fusion protein comprising the RNA-binding domain and the EJC-mimic domain. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows the location of NMD-susceptible (blue) and NMD-insensitive (red) PTC regions within mRNA. [Diagram 2]FIG. 1 shows the molecular mechanism of NMD, depicting one of several possible EJC compositions and not showing SMG6-mediated endonucleolytic cleavage of the mRNA. [Diagram 3] FIG. 1 shows an embodiment of RNA-programmed NMD activation (RP-NMDA). The top panel shows dCas13d-mediated EJC anchored to a normal mRNA. This mRNA functions normally as the anchored protein is removed by the translating ribosome. The bottom panel shows dCas13d-mediated EJC anchored to an NMD-insensitive PTC-containing counterpart of the normal mRNA, and subsequent degradation of the NMD-insensitive PTC-containing counterpart of the normal mRNA. [Figure 4] FIG. 1 shows human beta-globin (HBB) reporter mRNA, EGFP-HBB-121Ter and mRFP-HBB-Norm, with the NMD-insensitive PTC at HBB codon 121 (121Ter) and the normal stop codon (Norm Ter). Exon-exon junctions are indicated by black vertical lines. The guide RNA target region on EGFP-HBB-121Ter mRNA is indicated by a light blue horizontal line. [Diagram 5] Figure 1. APC reporter transcripts. FLAG-tagged normal APC (FLAG-APC Norm), APC with a PTC at codon 1309 (FLAG-APC 1309Ter), and APC 1309 Ter with a 3'-intron (FLAG-APC 1309Ter+intron) are shown. Nucleotides encoding the mutation cluster region (MCR) are indicated by blue lines below the FLAG-APC Norm transcript. The NMD-insensitive PTC region is highlighted in cyan. [Figure 6]Figure 1. NMD-sensitive or insensitive nonsense mutations on HBB reporter constructs. (Panel A) Diagram of human beta-globin (HBB) reporter constructs containing normal stop codons (NormTer), NMD-sensitive (39Ter), or NMD-insensitive PTCs (121Ter and 127Ter). EGFP mRNA serves as a transfection control. (Panel B) Western blot analysis showing protein expression of specific HBB constructs. (Panel C) RT-qPCR analysis showing relative mRNA abundance of specific HBB constructs. Data represent mean with s.d. (n=3). ***p<0.001 (Student's t-test). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Reference will now be made in detail to certain aspects and exemplary embodiments of the present invention, illustrating the accompanying structures and figures. Aspects of the present invention will be described in conjunction with exemplary embodiments, including methods, materials, and examples, and such description is non-limiting, and the scope of the present invention is intended to include all equivalents, alternatives, and modifications that are generally known or incorporated herein. Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Those of ordinary skill in the art will recognize many techniques and materials similar or equivalent to those described herein that can be used to implement aspects and embodiments of the present invention. The aspects and embodiments described in the present invention are not limited to the methods and materials described.

[0021] Surprisingly, reconstitution of a 3'UTR EJC mimic downstream of the normal stop codon can induce NMD. By tethering EJC construct Y14 or EJC-associated factors such as UPF3B / 3X, but not cytoplasmic polyA-binding protein (PABPC1) sufficiently (>approximately 20-24 nt) downstream of the stop codon, non-NMD substrates are converted into targets for NMD by using the bacteriophage MS2 coat protein and lambda bacteriophage anti-terminator protein N-BoxB system. Thus, ultimately, a molecular tethering method utilizing the CRISPR-Cas system instead of the bacteriophage MS2 coat protein and lambda bacteriophage anti-terminator protein N-BoxB system is feasible to activate NMD for any NMD-insensitive transcript.

[0022] I. Definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

[0023] Ranges are expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will further be understood that each endpoint of a range is meaningful both in relation to the other endpoint, and independently of the other endpoint. It is also understood that several values ​​are disclosed herein, and that each value is also disclosed herein as "about" that particular value in addition to the value itself. For example, when a value "less than or equal to" a value is disclosed, it is also understood that "greater than or equal to" that value is disclosed, as would be well understood by one of ordinary skill in the art. For example, when a value "10" is disclosed, "less than or equal to 10" and "greater than or equal to 10" are also disclosed. When more than one value is disclosed, all possible ranges between any two values ​​are disclosed.

[0024] As used herein, the term "fusion protein" refers to a protein created by the joining of two or more protein domains that originally formed components of separate proteins.

[0025] As used herein, the term "exon junction complex (EJC)" refers to a protein complex formed upstream of the junction of two exons on the pre-messenger RNA strand that are joined during pre-mRNA splicing. The EJC is usually located approximately 20-24 nt upstream of the junction and has a major impact on the translation, surveillance and localization of the spliced ​​mRNA. The EJC is first located on the pre-mRNA during splicing and then transported on the spliced ​​mRNA into the cytoplasm. In the cytoplasm, the EJC plays a major role in the post-transcriptional regulation of mRNA. It is believed that the exon junction complex provides a position-specific memory of the splicing event as well as a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay mechanisms. The EJC consists of a stable heterotrimeric core that serves as a binding platform for other factors required for the mRNA pathway. The core of the EJC contains the protein eukaryotic initiation factor 4A3 (eIF4A3; a DEAD-box RNA helicase), bound to an adenosine triphosphate (ATP) analog, as well as additional proteins MAGOH and Y14 / RBM8A. The exon junction complex plays a major role in mRNA surveillance. More specifically, the exon junction complex functions in the nonsense-mediated decay pathway (NMD), in which mRNA transcripts with premature stop codons are degraded. In the translation of the majority of normal mRNAs, i.e., mRNAs that are not targeted by NMD, ribosomes bind to the transcript and begin elongating the amino acid chain. The ribosome continues to proceed until it reaches the location of the exon junction complex, which is then removed. This process is repeated until all exon junction complexes are removed. Translation is then completed when the ribosome reaches a stop codon. The EJC and its location act as a kind of regulator, determining whether or not a transcript should be targeted for NMD.

[0026] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by regulatory sequences such as promoters and / or enhancers.

[0027] As used herein, the term "expression vector" refers to a composition that contains a nucleotide sequence encoding a protein and / or RNA and can be used to deliver the nucleic acid sequence to the interior of a cell and express the encoded protein and / or RNA inside the cell. Expression vectors generally contain regulatory sequences for expressing the protein or RNA encoded by the nucleotide sequence.

[0028] As used herein, the term "encode" refers to the inherent property of a particular sequence of nucleotides within a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and other polymers and macromolecules in biological processes that have biological properties resulting therefrom. Thus, a gene encodes a protein when the protein is produced by transcription and translation of the mRNA corresponding to that gene in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually presented in a sequence listing, and the non-coding strand, which is used as a template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0029] The term "nucleotide sequence" includes all nucleotide sequences that are degenerate versions of each other and that code for the same amino acid sequence. Nucleotide sequences that code for proteins and RNA can contain introns.

[0030] As used herein, the term "regulatory sequence" refers to a nucleic acid sequence required for the expression of a coding sequence (either protein or RNA) operably linked to a promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also include enhancer sequences and other regulatory elements required for expression of a gene product. A promoter / regulatory sequence may, for example, be one that causes tissue-specific expression of a gene product. The term "promoter" as used herein is defined as a DNA sequence required to initiate the specific transcription of a polynucleotide sequence recognized by the synthetic machinery of a cell, or by an introduced synthetic machinery. A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the production of a gene product in a cell under most or all physiological conditions of the cell. An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the production of a gene product in a cell substantially only when an inducer corresponding to the promoter is present in the cell. A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoded or specified by a gene, causes production of a gene product in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0031] The term "operably linked" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, are in the same reading frame.

[0032] When referring to nucleobases, the terms "nucleobase complementarity" and "complementarity" refer to a nucleobase that can base pair with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). Complementarity can be partial or total. Partial complementarity occurs when one or more nucleobases do not match according to base pairing rules. Total or complete complementarity between nucleic acids occurs when every single nucleobase matches another base under base pairing rules. In certain embodiments, complementary nucleobase refers to a nucleobase of an antisense oligonucleotide that can base pair with the nucleobase of its target nucleic acid. For example, if the nucleobase present at a certain position of an antisense oligonucleotide can hydrogen bond with the nucleobase present at a certain position of a target nucleic acid, the hydrogen bond position of the oligonucleotide and the target nucleic acid is considered to be complementary with respect to the nucleobase pair. Nucleobases containing certain modifications may maintain the ability to pair with a corresponding nucleobase and thus still have nucleobase complementation capability.

[0033] As used herein, "treating" a disease or condition means reducing or eliminating the signs or symptoms of the disease or condition, stabilizing the disease or condition, and / or reducing or slowing the further progression of the disease or condition. In some embodiments, "treat," "treatment," or "treating" is intended to include prevention, amelioration, prevention, or cure of the disease or condition.

[0034] As used herein, the term "effective amount" refers to an amount necessary or sufficient to realize a desired biological effect or selected result, and such an amount can be determined by one of ordinary skill in the art based on routine experimentation.

[0035] As used herein, the term "pharmaceutical acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity and absorption delaying agents, and the like, that are physiologically compatible. Pharmaceutical compositions may include suitable solid or gel phase carriers or excipients. Exemplary carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycol. Exemplary pharmaceutical acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it is preferable to include an isotonicity agent, such as a sugar, a polyhydric alcohol, such as mannitol, sorbitol, or sodium chloride, in the composition. Pharmaceutically acceptable carriers may further include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the therapeutic agent.

[0036] II. Compositions of the Invention The present invention describes a novel approach to induce nonsense-mediated mRNA decay (NMD) of PTC-containing mRNAs that are resistant to NMD (also referred to as "NMD-insensitive PTC-containing mRNAs" or "target RNAs") by forming an artificial exon junction complex (EJC) on the target RNA. The formation of the artificial EJC then leads to the degradation of the target RNA by NMD. This approach can be used to treat diseases and conditions caused by NMD-insensitive PTC-containing mRNAs.

[0037] A. Fusion Proteins One aspect of the present invention relates to a fusion protein comprising (1) an RNA binding domain and (2) an exon junction complex (EJC) mimicking domain. The fusion protein of the present invention can bind to an NMD-insensitive PTC-containing mRNA by forming an artificial exon junction complex (EJC) on the NMD-insensitive PTC-containing mRNA in the presence of a guide RNA. In some embodiments, the fusion protein further comprises at least one nuclear export signal. In some embodiments, the fusion protein further comprises a linker.

[0038] RNA-binding domain In some embodiments, the RNA binding domain comprises a complete Cas nuclease or a Cas nuclease RNA binding region / sequence. As used herein, the term "Cas nuclease" refers to a CRISPR-associated (Cas) nuclease that is capable of cleaving the phosphodiester bond between nucleic acid nucleotides in an active state. As used herein, the term "Cas nuclease" also includes variants of Cas nuclease, such as catalytically inactive Cas nuclease or a Cas nuclease RNA binding region / sequence.

[0039] Examples of Cas nucleases include, but are not limited to, Cas9, Cas12 and Cas13. In some embodiments, the RNA-binding domain comprises the complete protein region or the RNA-binding region / sequence of a Cas13 family nuclease.

[0040] In some embodiments, the RNA-binding domain comprises the complete protein region or RNA-binding region / sequence of a Cas nuclease that does not require a protospacer adjacent sequence (PFS) for targeting by Cas.

[0041] In some embodiments, the RNA-binding domain comprises the complete protein region or RNA-binding region / sequence of Cas13d nuclease or its variants. Examples of Cas13d include, but are not limited to, Ruminoccocus flavefaciens XPD3002 (RfxCas13d) (SEQ ID NO: 1), Uncultured Ruminoccocus sp. (Ur) Cas13d (SEQ ID NO: 3), Ruminoccocus flavefaciens FD1 (Rff) Cas13d (SEQ ID NO: 4), Ruminoccocus albus (Ra) Cas13d (SEQ ID NO: 5), Anaerobic digester metagenome 15706 (Adm) Cas13d (SEQ ID NO: 6), Gut metagenome assembly P1E0-k21 (P1E0) Cas13d (SEQ ID NO: 7), Eubacterium siraeum DSM15702 (Es) Cas13d (SEQ ID NO: 8).

[0042] In some embodiments, the RNA-binding domain comprises the complete protein region or RNA-binding region / sequence of Cas13b nuclease or its variants. Examples of Cas13b nuclease include, but are not limited to, Prevotella sp. P5-125 (Psp) Cas13b (SEQ ID NO: 9), Porphyromonas gulae (Pgu) Cas13b (SEQ ID NO: 10), and Riemerella anatipestifer (Ran) Cas13b (SEQ ID NO: 11).

[0043] In some embodiments, the RNA-binding domain comprises a catalytically inactive Cas nuclease that does not cleave RNA binding but retains certain single-stranded RNA binding properties. In some embodiments, the catalytically inactive Cas nuclease is catalytically inactive Cas13d (dCas13d). In some embodiments, the catalytically inactive Cas nuclease is dRfxCas13d (SEQ ID NO: 2), which contains four positively charged amino acid substitutions (R295A, H300A, R849A and H854A).

[0044] It will be appreciated by those skilled in the art that the present invention encompasses all suitable Cas nuclease RNA-binding domains capable of binding to a guide RNA to form a fusion protein / guide RNA complex that is capable of initiating NMD of a target mRNA upon binding to the target mRNA.

[0045] EJC mimic domain. The EJC-mimicking domain can be any protein domain capable of forming an EJC or having the functional properties of an EJC that initiates NMD of a target RNA when the fusion protein of the invention binds to the target RNA. In some embodiments, the EJC-mimicking domain comprises a complete or partial region / sequence of one or more EJC proteins.

[0046] As used herein, the term "EJC protein" includes, but is not limited to, at least one of eIF4a3, Y14 / RBM8A, MAGOH, RNPS1, SRSF1, and NMD factors that associate with the EJC, such as UPF1, UPF2, and UPF3X / 3B.

[0047] In some embodiments, the EJC-mimicking domain comprises a complete or partial region / sequence of one or more of eIF4a3, Y14 / RBM8A and MAGOH. In some embodiments, the EJC-mimicking domain comprises a complete or partial region of one or more EJC proteins selected from the group consisting of eIF4A3 (SEQ ID NO: 12), Y14 / RBM8A (SEQ ID NO: 13), MAGOH (SEQ ID NO: 14), RNPS1 (SEQ ID NO: 27), SRSF1 (SEQ ID NO: 28), UPF1 (SEQ ID NO: 15), UPF2 (SEQ ID NO: 16) and UPF3X / 3B (SEQ ID NO: 17).

[0048] In some embodiments, the EJC mimetic domain comprises a complete or partial region of one or more of eIF4a3 (SEQ ID NO: 12), Y14 / RBM8A (SEQ ID NO: 13) and MAGOH (SEQ ID NO: 14).

[0049] Nuclear export signal As used herein, the term "nuclear export signal" refers to a short targeting peptide containing at least four hydrophobic residues within a protein that targets the protein for export from the cell nucleus to the cytoplasm using nuclear transport through the nuclear pore complex.

[0050] In some embodiments, the fusion protein comprises at least one nuclear export signal. In some embodiments, the nuclear export signal is attached to the N-terminus or C-terminus of the fusion protein of the present invention. The nuclear export signal localizes the protein to the cytoplasm for targeting cytoplasmic RNA. In some embodiments, the nuclear export signal comprises an amino acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15 or 16. In some embodiments, the nuclear export signal comprises the amino acid sequence of SEQ ID NO:18 or SEQ ID NO:19.

[0051] Linker In some embodiments, the RNA-binding domain and the EJC-mimicking domain are directly joined. In other embodiments, the RNA-binding domain and the EJC-mimicking domain are joined by a peptide linker of 2-40 amino acid residues. The linker forms a physical connection between the two protein domains without interfering with the function of either protein domain. In some embodiments, the linker comprises hydrophilic residues. In some embodiments, the linker is a remnant from the restriction cloning used to generate the fusion. Examples of linker sequences include, but are not limited to, GS (SEQ ID NO: 22), GSGGGGS (SEQ ID NO: 23), GGGGSGGGGSGGGGS (SEQ ID NO: 24), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 25), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 26).

[0052] B. Expression Vectors Another aspect of the present invention relates to an expression vector capable of expressing the fusion protein of the present invention in a cell. In some embodiments, the expression vector comprises a first nucleotide sequence encoding an RNA-binding domain, a second nucleotide sequence encoding an EJC-mimicking domain, and a regulatory sequence operably linked to at least one of the first nucleotide sequence and the second nucleotide sequence.

[0053] In some embodiments, the first nucleotide sequence encodes the entire Cas13b nuclease or its RNA-binding region or a variant thereof.

[0054] In some embodiments, the first nucleotide sequence encodes the complete protein region or the RNA binding region / sequence of a Cas13d nuclease or a variant thereof.

[0055] In some embodiments, the first nucleotide sequence encodes the complete protein region or RNA-binding domain / sequence of catalytically inactive Cas13d (dCas13d).

[0056] In some embodiments, the catalytically inactive Cas nuclease is dRfxCas13d (SEQ ID NO: 2) with four positively charged amino acid substitutions (R295A, H300A, R849A and H854A).

[0057] In some embodiments, the second nucleotide sequence encodes an EJC-mimic domain that includes a complete or partial region / sequence of one or more EJC proteins.

[0058] In some embodiments, the second nucleotide sequence encodes an EJC-mimic domain that includes a complete or partial region / sequence of one or more of eIF4a3, Y14 / RBM8A, and MAGOH. In some embodiments, the EJC-mimic domain includes a complete protein region or a partial region / sequence of one or more EJC proteins selected from the group consisting of eIF4A3 (SEQ ID NO: 12), Y14 / RBM8A (SEQ ID NO: 13), MAGOH (SEQ ID NO: 14), RNPS1 (SEQ ID NO: 27), SRSF1 (SEQ ID NO: 28), UPF1 (SEQ ID NO: 15), UPF2 (SEQ ID NO: 16), and UPF3X / 3B (SEQ ID NO: 17).

[0059] In some embodiments, the second nucleotide sequence encodes an EJC mimetic domain comprising a complete protein region or a partial region of one or more EJC proteins selected from the group consisting of eIF4A3 (SEQ ID NO: 12), Y14 / RBM8A (SEQ ID NO: 13), MAGOH (SEQ ID NO: 14), RNPS1 (SEQ ID NO: 27), SRSF1 (SEQ ID NO: 28), UPF1 (SEQ ID NO: 15), UPF2 (SEQ ID NO: 16) and UPF3X / 3B (SEQ ID NO: 17).

[0060] In some embodiments, the second nucleotide sequence encodes an EJC-mimicking domain comprising a complete or partial protein region of one or more of eIF4a3 (SEQ ID NO: 12), Y14 / RBM8A (SEQ ID NO: 13), and MAGOH (SEQ ID NO: 14).

[0061] In some embodiments, the expression vector further comprises a nucleotide sequence encoding a nuclear export signal. In some embodiments, the nuclear export signal comprises the amino acid sequence of SEQ ID NO: 18 or 19.

[0062] In some embodiments, the expression vector further comprises a nucleotide sequence encoding a guide RNA. As used herein, the term "guide RNA" refers to an RNA that functions as a guide for the RNA-binding domain of the present invention, so that the fusion protein of the present invention can bind to the NMD-insensitive PTC-containing mRNA and form an artificial exon junction complex (EJC) on the NMD-insensitive PTC-containing mRNA. The formation of the artificial EJC leads to the initiation of NMD and the degradation of the NMD-insensitive PTC-containing mRNA.

[0063] In some embodiments, the guide RNA comprises a nucleotide sequence complementary to a target sequence in an NMD-insensitive PTC-containing mRNA. In some embodiments, the 5' end of the target sequence is (1) at least 10, 15, 20, 25 or 30 nucleotides downstream of the NMD-insensitive PTC, and (2) within a normal protein-coding sequence of a normal mRNA translational reading frame. As used herein, the term "normal mRNA" refers to an mRNA that does not contain an NMD-insensitive PTC, but is otherwise identical to an NMD-insensitive PTC-containing mRNA. The term "normal protein" refers to a protein product translated from a "normal mRNA". In some embodiments, the 5' end of the target sequence is at least 20-24 nucleotides downstream of the NMD-insensitive PTC.

[0064] In some embodiments, the target sequence comprises at least 15, 18 or 21 nucleotides to maintain the binding specificity of the guide RNA. In some embodiments, the guide RNA comprises a hairpin structure. In some embodiments, the hairpin structure is 30-36 nt in length and contains a stem of 8-10 nucleotides with an A / U rich loop. In some embodiments, the hairpin structure comprises the sequence 5'-CACUAGUGCGAAUUUGCACUAGUCUAAAAC-3' (SEQ ID NO: 20).

[0065] The expression vector can be any vector suitable for expressing the fusion protein and / or guide RNA of the present invention in eukaryotes.Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters that are useful for regulating the expression of desired nucleic acid sequences.In some embodiments, one or more expression vectors are engineered to direct the expression of polynucleotide antagonist ubiquitously (constitutively). Exemplary promoters for ubiquitous expression include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the RSV LTR, the MoMLV LTR, the phosphoglycerate kinase-1 (PGK) promoter, the simian virus 40 (SV40) promoter, the CK6 promoter, the transthyretin promoter (TTR), the TK promoter, the tetracycline responsive promoter (TRE), the U6 promoter, the E2F promoter, the telomerase (hTERT) promoter, the H1 promoter, the cytomegalovirus enhancer / chicken beta actin / rabbit beta-globin promoter (CAG) promoter, the elongation factor 1-alpha promoter (EF1-α) promoter, the human beta-glucuronidase promoter, the chicken beta-actin (CBA) promoter, the retroviral Rous sarcoma virus (RSV) LTR promoter, the dihydrofolate reductase promoter, the beta-actin promoter, and the like.

[0066] In certain embodiments, one or more expression vectors are engineered to provide tissue-specific expression of the polynucleotides in targeted cells, including various B cell promoters known to be active in plasma cells, plasmablasts, lymphoplasmacytoid cells, memory B cells, B-2 cells, follicular (FO) B cells, marginal zone (MZ) B cells, B-1 cells, and regulatory B (Breg) cells.

[0067] In some embodiments, the expression vector is a non-viral vector, such as a plasmid, a phagemid, or a cosmid. In some embodiments, the expression vector is a plasmid vector. Examples of plasmid vectors include, but are not limited to, pXR002:EF1a-dCasRx-2A-EGFP, pXR003:CasRx gRNA, pXR004:CasRx pre-gRNA, and derivative vectors.

[0068] In some embodiments, the expression vector is in the form of a recombinant virus (viral vector). The term "recombinant virus" or "viral vector" is used herein to refer to a virus that has been genetically altered, for example, by the addition or insertion of a heterologous nucleic acid construct into the viral particle.

[0069] Viral vectors for expressing polynucleotide antagonists can be derived, for example, from adenoviruses, adeno-associated viruses (AAV), retroviruses (including lentiviruses such as HIV-1 and HIV-2), vaccinia viruses and other poxviruses, herpesviruses (e.g., herpes simplex viruses types 1 and 2), polioviruses, Sindbis and other RNA viruses, alphaviruses, astroviruses, coronaviruses, orthomyxoviruses, papovaviruses, paramyxoviruses, parvoviruses, picornaviruses, togaviruses, and others. Viral vectors may or may not contain sufficient viral genetic information and / or structural components for the production of infectious virus upon introduction into a host cell, i.e., viral vectors may be replication-competent or replication-deficient. In some embodiments, for example, when structural components for the production of infectious virus are missing, the necessary functional components can be supplied in trans from the host cell or another vector introduced into the cell if production of recombinant virus is desired. In a preferred embodiment, a replication-defective recombinant virus is administered for therapy. The nucleic acid to be delivered may be incorporated into a naturally occurring or modified viral genome (or part thereof) or may be present as a separate nucleic acid molecule within the viral capsid.

[0070] In some embodiments, viral vectors can be engineered to target specific cells, such as plasma cells or multiple myeloma cells, by using targeting features inherent to the viral vector or engineered into the viral vector.Specific cells can be "targeted" for polynucleotide delivery and expression.Thus, "targeting" in this case refers to the use of endogenous or heterologous binding agents in the form of capsids, envelope proteins, antibodies for delivery to specific cells, the use of tissue-specific regulatory elements to restrict expression to a specific subset(s) of cells, or both.

[0071] In some embodiments, the viral vector is an AAV vector.AAV vector can provide the preferred delivery system for the nucleic acid therapeutic agent of the present invention, since it can enable the long-lasting continuous expression of the fusion protein and / or guide RNA of the present invention.AAV vector can contain several different regulatory elements, including various promoters and / or enhancer elements for constitutive or cell type specific expression, or can be modified so that the expression of fusion protein and / or guide RNA is controlled under several different regulatory elements, including various promoters and / or enhancer elements for constitutive or cell type specific expression.

[0072] Any suitable AAV serotype or AAV pseudotype can be used to express the fusion protein and / or guide RNA of the present invention in vivo in cells. The type of vector for in vivo delivery is preferably selected based on the absence of immunity to the selected AAV subtype in the host and stable expression in vivo. In general, AAV vectors are derived from single-stranded (ss) DNA parvoviruses that are non-pathogenic to mammals. Among the serotypes of AAV isolated from humans or non-human primates, human serotype 2 is the first and best characterized AAV developed as a gene transfer vector. Other useful AAV serotypes include AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ8 and AAV-DJ.

[0073] In some embodiments, the AAV vector may be a pseudotyped AAV vector that contains sequences and / or components originating from at least two different AAV serotypes. Thus, the pseudotyped (or chimeric) AAV vector may include, for example, an AAV2-derived genome in an AAV1-derived or AAV6-derived capsid; or an AAV2-derived genome in an AAV4-derived capsid; or an AAV2-derived genome in an AAV9-derived capsid. Alternatively, the pseudotyped AAV vector may include a fusion of a portion of a capsid from one AAV serotype with a second portion of a different AAV serotype capsid, resulting in a vector encoding a pseudotyped AAV2 / AAV5 capsid. In other embodiments, the pseudotyped AAV vector may include a capsid from one serotype and an inverted terminal repeat (ITR) from another AAV serotype. Exemplary AAV vectors include recombinant pseudotyped AAV2 / 1, AAV2 / 2, AAV2 / 5, AAV2 / 7, AAV2 / 8, and AAV2 / 9 serotype vectors.

[0074] In some embodiments, an expression vector of the invention is an AAV vector or a derivative vector made from pAAV-hU6-DR30-BsaI_EFS-dCasRx-bghpA.

[0075] C. Pharmaceutical Compositions Another aspect of the present invention relates to a pharmaceutical composition comprising the expression vector of the present application and a pharma- ceutically acceptable carrier.

[0076] Exemplary carriers for delivery include buffers, nanoparticles, lipids, liposomes, micelles, polymers, polymeric micelles, emulsions, polyelectrolyte complexes, hydrogels, microcapsules, exosomes, combinations thereof, and pegylated derivatives thereof.

[0077] In some embodiments, the pharmaceutical composition comprises a viral expression vector and an aqueous buffer, including but not limited to physiological saline buffer, Hank's solution, Ringer's solution, sodium succinate buffer, sodium citrate buffer, sodium phosphate buffer, or potassium phosphate buffer.

[0078] In the case of utilizing a non-viral delivery system, an exemplary delivery vehicle is a liposome. The use of lipid formulations is intended for the introduction of nucleic acid into host cells (in vitro, ex vivo or in vivo). In another embodiment, the nucleic acid can be associated with lipid. The nucleic acid associated with lipid can be encapsulated in the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, attached to the liposome via a linking molecule that associates with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipid, mixed with lipid, combined with lipid, contained in a suspension in lipid, contained in or complexed with micelles, or otherwise associated with lipid. The composition associated with lipid, lipid / DNA or lipid / expression vector is not limited to any particular structure in solution.

[0079] In some embodiments, the pharmaceutical composition comprises a non-viral expression vector and a nanoparticle carrier. Exemplary nanoparticles include paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, polymeric nanoparticles, nanoworms, nanoemulsions, nanogels, fullerene-like materials, inorganic nanotubes, dendrimers (e.g., with covalently attached metal chelates), nanocapsules, nanospheres, nanofibers, nanohorns, nanoonions, nanorods, nanoropes, and quantum dots. Nanoparticles can produce detectable signals, for example, through absorption and / or emission of photons (including radio frequency and visible photons) and plasmon resonance. Nanoparticles can be biodegradable or non-biodegradable. In some embodiments, the nanoparticle carrier is coupled to a tissue-specific targeting peptide or antibody to facilitate carrier-mediated delivery of the expression vector.

[0080] In some embodiments, the pharmaceutical composition is in a lyophilized form and includes a cryoprotectant. Examples of cryoprotectants include, but are not limited to, sucrose (optimally 0.5-1.0%), trehalose, and lactose. In some embodiments, the pharmaceutical composition further includes a bulking agent. Examples of bulking agents include, but are not limited to, mannitol, glycine, and arginine.

[0081] In some embodiments, the pharmaceutical compositions of the invention are formulated for parenteral administration by injection, e.g., intravenous, intramyocardial, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

[0082] Pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions, and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions. The form should be sterile and fluid to the extent that easy injectability exists. The form should be stable under the conditions of manufacture and storage, and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The pharmaceutical carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants.

[0083] Parenteral compositions can be formulated into unit dosage form for ease of administration and uniformity of dosage.As used herein, unit dosage form refers to a physically separate unit suitable as a unit dosage for a subject to be treated, each unit containing a predetermined amount of active material calculated to produce a desired therapeutic effect, together with necessary pharmaceutical carrier.The specification of the unit dosage form of the present invention can be selected based on (a) the unique characteristics of the active material and the specific therapeutic effect to be achieved, and (b) the inherent limitations in the technical field of compounding such active material to treat a condition in a living subject having a condition that impairs physical health as described herein.

[0084] D. Artificial exon junction complex Another aspect of the present invention relates to an artificial exon junction complex comprising the fusion protein of the present application and a guide RNA. In some embodiments, the fusion protein comprises an RNA binding domain and an exon junction complex (EJC) mimicking domain, and the guide RNA comprises a nucleotide sequence that is complementary to a target sequence in a target mRNA that contains an NMD-insensitive PTC.

[0085] III. Treatment method Another aspect of the present invention relates to a method for preventing, treating, or ameliorating symptoms of a disease or condition caused by translation of an mRNA (hereinafter "target mRNA") having an NMD-insensitive PTC using the fusion protein and guide RNA of the present invention. The fusion protein and guide RNA can be expressed in situ in a target cell using one or more expression vectors.

[0086] In some embodiments, the method comprises introducing into a cell expressing a target mRNA (1) a fusion protein comprising an RNA-binding domain and an EJC-mimicking domain, and (2) a guide RNA that specifically binds to a target region in the target mRNA, wherein the fusion protein, the guide RNA, and the target mRNA form a complex that results in degradation of the target mRNA by NMD. In some embodiments, the fusion protein and the guide RNA are introduced into the cell by one or more expression vectors that express the fusion protein and the guide RNA in situ in the cell.

[0087] In some embodiments, the method comprises administering to a subject in need of such treatment an effective amount of one or more expression vectors of the invention described herein. In some embodiments, the method comprises administering to a subject in need of such treatment an effective amount of an expression vector comprising a first nucleotide sequence encoding an RNA-binding domain, a second nucleotide sequence encoding an EJC-mimicking domain derived from one or more components of an exon junction complex (EJC), and a regulatory sequence operably linked to at least one of the first nucleotide sequence and the second nucleotide sequence.

[0088] In some embodiments of the method, the first nucleotide sequence encodes the entire Cas13b nuclease or its RNA-binding region or a variant thereof.

[0089] In some embodiments of the method, the first nucleotide sequence encodes the complete protein region or the RNA binding region / sequence of the Cas13d nuclease or a variant thereof.

[0090] In some embodiments of the method, the first nucleotide sequence encodes the complete protein region or RNA-binding domain / sequence of catalytically inactive Cas13d (dCas13d).

[0091] In some embodiments of the methods, the catalytically inactive Cas nuclease is dRfxCas13d (SEQ ID NO: 2) with four positively charged amino acid substitutions (R295A, H300A, R849A and H854A).

[0092] In some embodiments of the method, the second nucleotide sequence encodes an EJC-mimic domain comprising a complete protein region or a partial region / sequence of one or more EJC proteins.

[0093] In some embodiments of the method, the second nucleotide sequence encodes an EJC-mimic domain comprising a complete protein region or a partial region / sequence of one or more of eIF4a3, Y14 / RBM8A, and MAGOH. In some embodiments, the EJC-mimic domain comprises a complete protein region or a partial region / sequence of one or more EJC proteins selected from the group consisting of eIF4A3 (SEQ ID NO: 12), Y14 / RBM8A (SEQ ID NO: 13), MAGOH (SEQ ID NO: 14), RNPS1 (SEQ ID NO: 27), SRSF1 (SEQ ID NO: 28), UPF1 (SEQ ID NO: 15), UPF2 (SEQ ID NO: 16), and UPF3X / 3B (SEQ ID NO: 17).

[0094] In some embodiments of the method, the second nucleotide sequence encodes an EJC mimetic domain comprising a complete protein region or a partial region of one or more EJC proteins selected from the group consisting of eIF4A3 (SEQ ID NO: 12), Y14 / RBM8A (SEQ ID NO: 13), MAGOH (SEQ ID NO: 14), RNPS1 (SEQ ID NO: 27), SRSF1 (SEQ ID NO: 28), UPF1 (SEQ ID NO: 15), UPF2 (SEQ ID NO: 16) and UPF3X / 3B (SEQ ID NO: 17).

[0095] In some embodiments of the method, the second nucleotide sequence encodes an EJC-mimicking domain comprising a complete or partial protein region of one or more of eIF4a3 (SEQ ID NO: 12), Y14 / RBM8A (SEQ ID NO: 13) and MAGOH (SEQ ID NO: 14).

[0096] In some embodiments of the method, the expression vector further comprises a nucleotide sequence encoding a nuclear export signal. In some embodiments, the nuclear export signal comprises the amino acid sequence of SEQ ID NO: 18 or 19.

[0097] In some embodiments of the method, the method further comprises administering to the subject a guide RNA as described herein. In some embodiments, the guide RNA comprises a sequence complementary to a target sequence in an mRNA containing an NMD-insensitive PTC, and the 5' end of the target sequence is located at least 10 nucleotides downstream of the NMD-insensitive PTC in the mRNA.

[0098] In some embodiments of the method, the guide RNA can be administered in combination with the expression vector that expresses the RNA binding domain and the EJC mimic domain.In some embodiments of the method, the guide RNA is expressed under the same expression vector that expresses the RNA binding domain and the EJC mimic domain.

[0099] In some embodiments of the method, the guide RNA is expressed in an expression vector separate from the expression vector expressing the RNA-binding domain and the EJC-mimic domain.

[0100] In some embodiments of the methods, the methods comprise administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising one or more expression vectors of the invention described herein.

[0101] In yet another aspect, the present invention provides a method for inducing mRNA degradation of a target mRNA that comprises a premature termination codon (PTC) that is insensitive to nonsense-mediated mRNA decay (NMD) process. In some embodiments, the method includes introducing into a cell expressing the target mRNA an expression vector comprising: (a) (1) a first nucleotide sequence encoding an RNA-binding domain; (2) a second nucleotide sequence encoding an EJC-mimicking domain derived from one or more components of an exon junction complex (EJC); and (3) a regulatory sequence operably linked to at least one of the first nucleotide sequence and the second nucleotide sequence; and (b) a guide RNA that specifically binds to a target region in the target mRNA, wherein the expression vector expresses in the cell a fusion protein comprising the RNA-binding domain and the EJC-mimicking domain.

[0102] In some embodiments of the method, the first nucleotide sequence encodes the entire Cas13b nuclease or its RNA-binding region or a variant thereof.

[0103] In some embodiments of the method, the first nucleotide sequence encodes the complete protein region or the RNA binding region / sequence of the Cas13d nuclease or a variant thereof.

[0104] In some embodiments of the method, the first nucleotide sequence encodes the complete protein region or RNA-binding domain / sequence of catalytically inactive Cas13d (dCas13d).

[0105] In some embodiments of the methods, the catalytically inactive Cas nuclease is dRfxCas13d (SEQ ID NO: 2) with four positively charged amino acid substitutions (R295A, H300A, R849A and H854A).

[0106] In some embodiments of the method, the second nucleotide sequence encodes an EJC-mimic domain that includes a complete or partial region / sequence of one or more EJC proteins.

[0107] In some embodiments of the method, the second nucleotide sequence encodes an EJC-mimic domain comprising a complete or partial protein region / sequence of one or more of eIF4a3, Y14 / RBM8A, and MAGOH. In some embodiments, the EJC-mimic domain comprises a complete or partial protein region / sequence of one or more EJC proteins selected from the group consisting of eIF4A3 (SEQ ID NO: 12), Y14 / RBM8A (SEQ ID NO: 13), MAGOH (SEQ ID NO: 14), RNPS1 (SEQ ID NO: 27), SRSF1 (SEQ ID NO: 28), UPF1 (SEQ ID NO: 15), UPF2 (SEQ ID NO: 16), and UPF3X / 3B (SEQ ID NO: 17).

[0108] In some embodiments of the method, the second nucleotide sequence encodes an EJC mimetic domain comprising a complete protein region or a partial region of one or more EJC proteins selected from the group consisting of eIF4A3 (SEQ ID NO: 12), Y14 / RBM8A (SEQ ID NO: 13), MAGOH (SEQ ID NO: 14), RNPS1 (SEQ ID NO: 27), SRSF1 (SEQ ID NO: 28), UPF1 (SEQ ID NO: 15), UPF2 (SEQ ID NO: 16) and UPF3X / 3B (SEQ ID NO: 17).

[0109] In some embodiments of the method, the second nucleotide sequence encodes an EJC-mimicking domain comprising a complete or partial protein region of one or more of eIF4a3 (SEQ ID NO: 12), Y14 / RBM8A (SEQ ID NO: 13) and MAGOH (SEQ ID NO: 14).

[0110] In some embodiments of the method, the expression vector further comprises a nucleotide sequence encoding a nuclear export signal. In some embodiments, the nuclear export signal comprises the amino acid sequence of SEQ ID NO: 18 or 19.

[0111] In some embodiments of the method, the method further comprises administering a guide RNA to the subject. In some embodiments, the guide RNA comprises a sequence complementary to a target sequence in the NMD-insensitive PTC-containing mRNA, and the 5' end of the target sequence is located at least 10 nucleotides downstream of the NMD-insensitive PTC in the mRNA.

[0112] In some embodiments of the method, the guide RNA can be administered in combination with the expression vector that expresses the RNA binding domain and the EJC mimic domain.In some embodiments, the guide RNA is expressed under the same expression vector that expresses the RNA binding domain and the EJC mimic domain.

[0113] In some embodiments of the method, the guide RNA is expressed in an expression vector separate from the expression vector expressing the RNA-binding domain and the EJC-mimic domain.

[0114] A. Target Diseases and Conditions Examples of diseases or conditions caused by translation of an mRNA having an NMD-insensitive PTC include, but are not limited to, the diseases and conditions listed in Table 1.

[0115] [Table 1] TIFF2025514983000003.tif255159TIFF2025514983000004.tif255156TIFF2025514983000005.tif46170

[0116] B. Guide RNA The guide RNA is designed to target the NMD-insensitive PTC-containing mRNA that causes the disease or condition. Specifically, the guide RNA comprises a nucleotide sequence that is complementary to a target sequence in the NMD-insensitive PTC-containing mRNA. In some embodiments, the 5' end of the target sequence is at least 10, 15, 20, 25 or 30 nucleotides downstream of the NMD-insensitive PTC, and the target sequence is within a normal protein-coding sequence of a normal mRNA translation reading frame. In some embodiments, the 5' end of the target sequence is at least 20-24 nucleotides downstream of the NMD-insensitive PTC.

[0117] In some embodiments, the target sequence comprises at least 21 nucleotides to maintain the binding specificity of the guide RNA. In some embodiments, the guide RNA comprises a hairpin structure. In some embodiments, the hairpin structure is 30-36 nt in length and contains a stem of 8-10 nucleotides with an A / U rich loop. An exemplary guide RNA sequence is shown in SEQ ID NO: 21. The guide RNA is specifically designed to treat colorectal cancer using the methods of the present application.

[0118] C. Route of Administration The fusion protein and guide RNA of the present invention can be introduced into target cells or administered to subjects using methods well known in the art.In some embodiments, the fusion protein and guide RNA of the present invention are introduced into target cells in the form of one or more expression vectors.The one or more expression vectors express the fusion protein and guide RNA in target cells, and then guide the degradation of NMD-insensitive PTC-containing mRNA in target cells.

[0119] In some embodiments, one or more expression vectors are administered to a subject by injection or infusion, for example, intravenous, intramyocardial, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection or infusion. EXAMPLES

[0120] Example 1 RNA-programmed NMD activation (RP-NMDA) system In the bacteriophage protein-tethered system, exogenous RNA-binding sequences must be implanted to target mRNA, which is a significant drawback for the application of this system in disease therapy. In contrast, the class 2 type VI CRISPR and Cas13 (CRISPR-Cas13) system is a robust and programmable molecular tool for RNA. We establish a new methodology for selective RNA degradation using CRISPR-Cas13 technology to target NMD-insensitive PTC-containing transcripts. The Cas13 family is the only family of class 2 Cas enzymes that cleaves only single-stranded RNA using two higher eukaryotic and prokaryotic nucleotide (HEPN)-binding domains.

[0121] Ruminococcus flavefaciens Cas13d (also known as CasRx) was chosen because it (1) has better on-target RNA specificity compared to other Cas13s and shRNAs, (2) is one of the smallest (~930 amino acids) class 2 Cas enzymes reported to date, and (3) does not require a protospacer adjacent sequence (PFS) at the 3' end of the spacer sequence, overcoming limitations on guide RNA sequence design.

[0122] To selectively digest PTC-containing mRNA, an RNA-programmable RP-NMDA system is established by mimicking the 3'UTR EJC-dependent NMD mechanism (Figure 2). In this example, catalytically inactive Cas13d (dCas13d) is used. dCas13d has four positively charged amino acid substitutions (R295A, H300A, R849A, and H854A) to inactivate Cas13d HEPN nuclease activity. By inactivating the nuclease activity, digestion of off-target transcripts derived from normal alleles can be avoided.

[0123] Additionally, we generate fusions of dCas13d with EJC constructs (eIF4a3, Y14 / RBM8A, or MAGOH) to activate the NMD pathway. We design guide RNAs that hybridize to a position between the NMD-insensitive PTC and the normal stop codon, following the "approximately 55-nt rule" of NMD. This position is preferably located at least 20-24 nt downstream of any PTC, since NMD sensitivity requires the PTC to be >20-24 nt from the downstream EJC at its 3' end, to efficiently activate NMD. Since ribosomes remove any proteins placed on the mRNA coding region, we expect that the EJC-dCas13d on the coding region of the normal mRNA will be efficiently removed by the migrating ribosome during the first round of translation (top panel of Figure 3). On the other hand, translating ribosomes do not proceed past or remove the PTC, leaving EJC-dCas13d positioned downstream of the PTC on NMD-insensitive mRNAs behind to induce NMD (Figure 3, bottom panel).

[0124] Because Cas13d does not require PFS, this RP-NMDA system is applicable to any location on a disease-causing NMD-insensitive transcript that meets the rules for NMD involvement. Fusion proteins are expressed using a mammalian expression vector encoding dCas13d (pXR002 / EF1a-dCasRx-2A-EGFP; Addgene), a mammalian expression vector encoding a guide RNA (pXR003 / CasRx gRNA cloning backbone; Addgene), and a mammalian expression vector encoding an EJC construct (pcDNA3-HA-eIF4a3; pcDNA-FLAG-Y14 / RBM8A and pcDNA-FLAG-MAGOH).

[0125] Example 2 Effects of the RP-NMDA system To evaluate the effect of RP-NMDA, we generate a reporter construct (pEGFP-HBB-121Ter) by using pEGFP-N1 (Clontech) and pFLAG-CMV2-HBB, which has an NMD-insensitive beta-thalassemia gene mutation (121 Ter) at position 121 of the amino acid sequence derived from the beta-globin (HBB) start codon (Figure 4). Due to the lack of 3'UTR EJC, mRNA with 121 Ter in the last exon of the HBB gene is insensitive to NMD, resulting in a dominantly inherited severe form of beta-thalassemia in which a truncated protein accumulates and insoluble inclusions are formed.

[0126] As a negative control, use pmRFP-N1 (Clontech) and pFLAG-CMV2-HBB to generate a reporter construct with normal HBB mRNA (pmRFP-HBB-Norm) (Figure 4). Use a human cervical cancer cell line (HeLa CCL-2; ATCC) and introduce these reporter plasmids into HeLa cells using Lipofectamine 2000 (Thermo Fisher Scientific) to establish stable cell lines expressing both reporter constructs (pEGFP-HBB-121Ter and pmRFP-HBB-Norm), selected by protein fluorescence and geneticin (G418) treatment.

[0127] By introducing EJC-dCas13d and gRNA into HeLa cells stably expressing the HBB mRNA reporter, mRNA abundance is measured by reverse transcription combined with quantitative PCR (RT-qPCR) using the QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific), and protein abundance is measured by Western blotting and fluorescence assay using a microplate reader (SpectraMax M4, Molecular Devices). Inclusion body formation is monitored using a fluorescence microscope (EVOS FL Cell Imaging System, Thermo Fisher Scientific) and an FV-1000 Confocal Laser Microscope (OLYMPUS).

[0128] Once the most effective EJC-dCas13d variant is found, deletion variants of the EJC construct will be generated to reduce the size of the effective protein, which may be important for future RP-NMDA applications, since a smaller size is required for packaging into low-capacity viral vectors that minimize immunogenicity in gene therapy treatments.

[0129] Example 3 Physiological effects of RNA-programmed NMD induction on human colon cancer cell lines Colorectal cancer is the third leading cause of cancer-related deaths in the United States, with a lifetime risk of approximately 1 in 23 (4.3%) for men and 1 in 25 (4%) for women. Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are one of the most frequently observed genetic alterations in colorectal cancer cells. Somatic mutations in the APC gene are associated with sporadic colorectal cancer (up to 80%), and germline APC mutations are associated with autosomal dominant familial adenomatous polyposis. Notably, more than 85% of APC mutations generate truncated APC proteins in colorectal cancer cell lines, and the majority of APC mutations (approximately 60%) reside within regions known as mutation cluster regions (MCRs). The truncated APC proteins promote the Wnt / β-catenin signaling pathway, thereby affecting cancer progression and severity. However, the underlying molecular mechanisms of how the truncated proteins disrupt normal cellular functions remain largely unknown.

[0130] Since the majority of somatic mutations in the APC gene are observed only in the last exon, which accounts for approximately 80% of the APC gene, the production of truncated APC protein in colorectal cancer is most likely due to insensitivity to NMD (Figure 1). To prove the NMD insensitivity of PTC in APC mRNA, a reporter construct expressing normal APC (pCMV-Neo-Bam APC; Addgene) and a reporter construct expressing mutant APC containing 1309 PTC (pCMV-Neo-Bam APC 1-1309; Addgene) are obtained. Then, for protein analysis, a FLAG tag sequence is inserted to generate pCMV-Neo-FLAG-APC and pCMV-Neo-FLAG-APC-1309Ter. To use pCMV-Neo-FLAG-APC-1309Ter and test NMD induction for this construct, the HBB intron 1 (130 bp) is further inserted downstream of APC 1309Ter to generate pCMV-Neo-FLAG-APC-1309Ter+intron (Figure 5).

[0131] The generated mammalian expression plasmid and the loading control plasmid pcDNA-EGFP are transfected into HeLa cells using Lipofectamine 2000. In parallel, either a negative control siRNA (silencer negative control #1 siRNA; Thermo Fisher Scientific) or UPF1 siRNA is introduced into HeLa cells expressing pCMV-Neo-FLAG-APC-1309Ter+intron using Lipofectamine RNAiMAX (Thermo Fisher Scientific) to test for NMD-dependence of any observed reporter mRNA degradation.

[0132] After 2 days, cells are harvested and mRNA abundance is measured by RT-qPCR and protein abundance is measured by semi-quantitative Western blotting using anti-FLAG HRP conjugated antibody (Sigma Aldrich).If mRNA abundance is reduced when APC 1309Ter+intron mRNA is used but not APC 1309Ter mRNA, and the mRNA reduction of APC 1309Ter+intron mRNA is restored by UPF1 downregulation, this is the proof of concept of RP-NMDA application.Then, the most effective EJC-dCas13d and guide RNA described herein are applied to HeLa cells expressing APC Norm or APC 1309Ter mRNA to evaluate the efficacy and target specificity of NMD induction for PTC-containing mRNA.

[0133] Next, the RP-NMDA approach is applied to colon cancer cell lines (SW480, CaCo2, DLD-1, Lovo, SW948, and HT-29; ATCC) by selectively designing guide RNAs to target transcripts encoding APC truncation in these cell types. CCD 841 CoN cells (ATCC) are used as a normal control cell line. The RP-NMDA constructs (pXR002 and pXR003) are transfected into colon cancer cell lines or normal control cell lines using the NEON Transfection System (Thermo Fisher Scientific). Protein abundance of factors involved in APC and Wnt / β-catenin signaling is measured by Western blotting using specific antibodies: anti-APC (Ali 12-28; Abcam), anti-β-catenin (E-5; Santa Cruz Biotechnologies), anti-Axin2 (76G6; Cell Signaling), and anti-Lgr5 (OTI2A2; Thermo Fisher Scientific). APC and other Wnt / β-catenin signaling pathway-related mRNA abundance is measured by RT-PCR, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays are performed using the Vybrant MTT Cell Proliferation Assay Kit (Thermo Fisher Scientific) to assess cell viability and proliferation. Cell migration and invasion are measured using the QCM Tumor Cell Trans-Endothelial Migration Assay Kit (Millipore). Wnt / β-catenin signaling activity will be assessed using the TCF / LEF Reporter Kit (BPS Bioscience).Furthermore, the effects of RP-NMDA will be thoroughly evaluated in colon cancer cell lines compared with normal control cell lines by transcriptome-wide RNA sequencing.

[0134] To avoid potential artifacts and off-target effects, three independent primer sets or siRNA sequences were used for each target. For statistics, a power analysis (power = 0.80 and α = 0.05, two-sided statistical test) determines the sample size required for reliable statistical judgment. Statistical significance is calculated using R version 4.0.5 using two-sided unpaired t-test, two-sided Wilcoxon rank sum test, one-way ANOVA Dunnett's multiple comparison test, Pearson correlation coefficient test, or Fisher's exact test. All R scripts used for data processing and statistical analysis are described in publications or are available upon request.

[0135] Example 4 NMD reporter systems with NMD-sensitive or NMD-insensitive PTCs In this study, we will determine whether NMD can be induced against disease-causing NMD-insensitive PTCs. In this study, we will first focus on the red blood cell disorder beta thalassemia as a disease model. The primary objectives are (i) to determine whether NMD can be induced against NMD-insensitive PTCs containing mRNA, and (ii) to define NMD factors that effectively induce NMD against NMD-insensitive PTCs. In this study, the following experiments will be performed.

[0136] First, this study utilizes the bacteriophage MS2 coat protein system as a proof of concept. As an NMD reporter, this study uses the human beta-globin (HBB) gene harboring NMD-insensitive nonsense mutations at codon 121 (E121X) and codon 127 (Q127X). These mutations cause dominantly inherited inclusion body beta-thalassemia, characterized by moderate anemia, jaundice, and splenomegaly.

[0137] In this study, NMD reporter plasmids expressing HBB mRNA containing a normal stop codon (NormTer), an NMD-sensitive PTC (39Ter), or an NMD-insensitive PTC (E121X:121Ter or Q127X:127Ter) fused to a FLAG tag (FLAG-HBB) were generated along with an NMD reporter plasmid expressing a transfection control EGFP mRNA (Figure 6, Panel A). In this study, these NMD reporter plasmids were transfected into human embryonic kidney (HEK) 293T cells. Western blotting analysis using HEK293T cell lysates shows that FLAG-HBB 121Ter or FLAG-HBB 127Ter expressed 34% or 47% of the wild-type levels of the truncated HBB protein, respectively (Figure 6, Panel B). Quantitative reverse transcription PCR (RT-qPCR) analysis indicates that FLAG-HBB 121Ter mRNA and FLAG-HBB 127Ter mRNA are insensitive to NMD (FIG. 6, panel C).

[0138] In this study, NMD reporter plasmids with multiple MS2 coat protein binding sequences (generating pFLAG-CMV2-HBB-6MS2bs NormTer, pFLAG-CMV2-HBB-6MS2bs 39Ter, pFLAG-CMV2-HBB-6MS2bs 121Ter, and pFLAG-CMV2-HBB-6MS2bs 127Ter) are introduced into HEK293T cells. To select the most effective NMD effector, this study tested several strong NMD activators, such as UPF1, eIF4a3, RBM8A / Y14, MAGOH, or SRSF1. In this study, one of the strong NMD activators downstream of the NMD-insensitive PTC on HBB mRNA was tethered using the bacteriophage MS2 coat protein and MS2 RNA binding system. In this study, we had already generated plasmids expressing fusions of MS2 coat protein with NMD effectors (pcNMS2-UPF1, pcNMS2-eIF4a3, pcNMS2-RBM8A / Y14, pcNMS2-MAGOH, and pcNMS2-SRSF1).

[0139] In this study, these plasmid constructs were used to introduce NMD reporters (pFLAG-CMV2-HBB-6MS2bs NormTer, pFLAG-CMV2-HBB-6MS2bs 121Ter, or pFLAG-CMV2-HBB-6MS2bs 127Ter), NMD effector plasmids (pcNMS2, pcNMS2-UPF1, pcNMS2-eIF4a3, pcNMS2-RBM8A / Y14, pcNMS2-MAGOH, pcNMS2-SRSF1), and a transfection control plasmid (pcDNA-EGFP) into HEK293T cells using the TransIT-X2 Dynamic Delivery system (Mirus Bio). After 2 days of incubation (48 hours), cells are harvested to measure HBB protein abundance by Western blotting using monoclonal anti-FLAG M2 antibody (Sigma-Aldrich) and HBB mRNA abundance by RT-qPCR using HBB-specific primers. As a transfection control, EGFP protein and mRNA are analyzed. HBB protein aggregates, HBB inclusions, are measured by immunofluorescence and confocal laser scanning microscopy (OLYMPUS FV-1000). Tethering of NMD activators downregulates HBB protein and mRNA, and reduces the number and size of HBB inclusions; this study concludes that NMD may be inducible for disease-causing NMD-insensitive PTC. In this study, by comparing several NMD activators, the most effective NMD activator is selected for application to downstream analysis.

[0140] Example 5 Methods for inducing NMD of dominant-negative transcripts Bacteriophage MS2 coat protein and MS2 RNA binding system require exogenous RNA binding sequences to be implanted to target mRNA, which is a significant drawback for the application of this system in disease therapy. In contrast, class 2 type VI CRISPR and Cas13 (CRISPR-Cas13) system is a robust and programmable molecular tool for RNA editing. To target NMD-insensitive PTC-containing transcripts, we first attempt to establish a new methodology for selective RNA degradation using CRISPR-Cas13 technology. The Cas13 family is the only family of class 2 Cas enzymes that uses two higher eukaryotic and prokaryotic nucleotide (HEPN) binding domains to cleave only single-stranded RNA. In this study, Ruminococcus flavefaciens Cas13d (also known as CasRx) was used because Cas13d (1) has better on-target RNA specificity compared to other Cas13s and shRNAs, (2) is one of the smallest (~930 amino acids) class 2 Cas enzymes reported to date, and (3) does not require a protospacer adjacent sequence (PFS) at the 3' end of the spacer sequence, overcoming limitations on guide RNA sequence design.

[0141] To selectively digest PTC-containing mRNAs, an RNA-programmed NMD-activated (RP-NMDA) system is established by mimicking the 3'UTR EJC-dependent NMD mechanism (Figure 2). A catalytically inactive Cas13d (dCas13d) with four positively charged amino acid substitutions (R295A, H300A, R849A, and H854A) is used to inactivate Cas13d HEPN nuclease activity. The use of dCas13d avoids digestion of transcripts derived from normal alleles. Furthermore, dCas13d is fused to EJC constructs (eIF4a3, Y14 / RBM8A, or MAGOH) or NMD activators (UPF1, RNPS1, or SRSF1) to activate the NMD pathway (Figure 3). Guide RNAs are designed to hybridize to a position between the NMD-insensitive PTC and the normal stop codon, following the "~55 nt rule" of NMD. This position is located at least 20-24 nt downstream of any PTC, since NMD sensitivity requires that the PTC be >20-24 nt from the downstream EJC at the 3' end, to efficiently activate NMD. Since ribosomes remove any proteins placed on mRNA coding regions, in this study, EJC-dCas13d on the coding region of normal mRNAs is predicted to be efficiently removed by the moving ribosome during the first round of translation (top panel of Figure 3). On the other hand, EJC-dCas13d located downstream of the PTC on NMD-insensitive mRNAs is left behind, and NMD is induced, since translating ribosomes do not proceed past and remove the PTC (bottom panel of Figure 3). Because Cas13d does not require PFS, this RP-NMDA system is theoretically applicable to any location on a disease-causing NMD-insensitive transcript that meets the rules for NMD engagement.

[0142] To evaluate the effect of RP-NMDA, this study utilizes reporter constructs, pFLAG-CMV2-HBB NormTer, pFLAG-CMV2-HBB 39Ter, pFLAG-CMV2-HBB 121Ter, or pFLAG-CMV2-HBB 127Ter (Figure 6, Panel A). This study uses pmRFP reference plasmid as a negative control. This study uses HEK293T cells, and these reporter plasmids are introduced into HEK293T cells using the TransIT-X2 Dynamic Delivery system (Mirus Bio).

[0143] In this study, dCas13d plasmids (pXR002-UPF1, eIF4a3, RBM8A / Y14, MAGOH, RNPS1, or SRSF1) and gRNAs are introduced into HEK293T cells expressing HBB mRNA reporter, and mRNA abundance is measured by RT-qPCR using the QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific), and protein abundance is measured by Western blotting and fluorescence assay using a microplate reader (SpectraMax M4, Molecular Devices). Inclusion body formation is monitored using a fluorescent microscope (EVOS FL Cell Imaging System, Thermo Fisher Scientific) and an FV-1000 Confocal Laser Microscope (OLYMPUS). In this study, we find the most effective dCas13d plasmid, and in this study, we generate deletion variants of the NMD effector to reduce the size of the effective protein. Size reduction may be important for future RP-NMDA applications, as smaller size is necessary for packaging into low-capacity viral vectors that minimize immunogenicity in gene therapy treatments.

[0144] [Table 2] TIFF2025514983000007.tif255155TIFF2025514983000008.tif255153TIFF2025514983 000009.tif255153TIFF2025514983000010.tif255157TIFF2025514983000011.tif86170

[0145] While various embodiments have been described above, it should be understood that such disclosure is presented by way of example only, and not limitation. Thus, the breadth and scope of the subject compositions and methods should not be limited by any of the above exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0146] The above description is intended to teach those skilled in the art how to carry out the invention, and does not detail all of the obvious modifications and variations that will become apparent to those skilled in the art upon reading the description. However, all such obvious modifications and variations are intended to be included within the scope of the invention as defined by the following claims. The claims are intended to cover any order of elements and steps that is effective for achieving the intended purpose, unless the context specifically indicates otherwise.

Claims

1. an RNA-binding domain; Exon junction complex (EJC) mimic domain A fusion protein comprising: The RNA-binding domain comprises a Cas nuclease or an RNA-binding region thereof, and the EJC-mimic domain comprises one or more regions derived from one or more components of the EJC; Fusion proteins.

2. The fusion protein of claim 1 , further comprising at least one nuclear export signal.

3. The fusion protein of claim 2 , wherein the nuclear export signal comprises SEQ ID NO: 18 or 19.

4. The fusion protein of any one of claims 1 to 3, wherein the RNA-binding domain comprises a catalytically inactive Cas nuclease or an RNA-binding region thereof.

5. The fusion protein of any one of claims 1 to 4, wherein the RNA-binding domain comprises a catalytically inactive Cas13d nuclease or an RNA-binding region thereof.

6. The fusion protein of claim 1 , wherein the RNA binding domain comprises SEQ ID NO:

2.

7. 7. The fusion protein of claim 1, wherein the EJC component is selected from the group consisting of eIF4A3, Y14 / RBM8A, MAGOH, RNPS1, SRSF1, UPF1, UPF2 and UPF3B / 3X.

8. The fusion protein of claim 7, wherein (1) eIF4A3 comprises SEQ ID NO: 12, (2) Y14 / RBM8A comprises SEQ ID NO: 13, (3) MAGOH comprises SEQ ID NO: 14, (4) UPF1 comprises SEQ ID NO: 15, (5) UPF2 comprises SEQ ID NO: 16, (6) UPF3B / 3X comprises SEQ ID NO: 17, (7) RNPS1 comprises SEQ ID NO: 27, and (8) SRSF1 comprises SEQ ID NO:

28.

9. The fusion protein of claim 1 , further comprising a linker between the RNA binding domain and the EJC mimetic domain.

10. a first nucleotide sequence encoding the RNA binding domain of a CAS nuclease; a second nucleotide sequence encoding an exon junction complex (EJC) mimic domain derived from a component of the EJC; a regulatory sequence operably linked to at least one of the first nucleotide sequence and the second nucleotide sequence; 1. An expression vector comprising:

11. The expression vector of claim 10, further comprising a nucleotide sequence encoding a nuclear export signal.

12. 12. The expression vector of claim 10 or 11, wherein the first nucleotide sequence encodes dCas13d.

13. 13. An expression vector according to any one of claims 10 to 12, wherein the second nucleotide sequence encodes one or more proteins or peptides or fragments thereof derived from eIF4A3, Y14 / RBM8A, MAGOH, RNPS1, SRSF1, UPF1, UPF2 or UPF3B / 3X, and the amino acid sequence is capable of activating NMD.

14. 14. The expression vector of claim 10, further comprising a nucleotide sequence encoding a guide RNA.

15. The expression vector of claim 14, wherein the guide RNA comprises a sequence complementary to a target sequence in an mRNA containing an NMD-insensitive PTC, and the 5' end of the target sequence is located at least 10 nucleotides downstream of the NMD-insensitive PTC in the mRNA.

16. 16. An expression vector according to any one of claims 10 to 15, which is a non-viral vector.

17. 17. An expression vector according to any one of claims 10 to 16, which is a viral vector.

18. The expression vector of claim 17 which is an AAV vector.

19. An expression vector according to any one of claims 10 to 18, A pharma- ceutically acceptable carrier; 23. A pharmaceutical composition comprising:

20. a fusion protein comprising an RNA-binding domain and an exon junction complex (EJC) mimic domain; A guide RNA; Target mRNA and An artificial exon junction complex comprising: The RNA-binding domain comprises an RNA-binding region of a CAS nuclease, and the (EJC) mimicking domain comprises one or more regions derived from a component of the EJC; The guide RNA comprises a sequence complementary to a target sequence in the target mRNA; the target mRNA contains a premature termination codon (PTC) that is insensitive to nonsense-mediated mRNA decay (NMD); The 5' end of the target sequence is located at least 10 nucleotides downstream of the PTC in the target mRNA; Artificial exon junction complex.

21. 21. The artificial exon junction complex of claim 20, wherein the fusion protein further comprises a nuclear export signal.

22. 1. A method for treating a disease or condition in a subject caused by the translation of a target mRNA that contains a premature termination codon (PTC) that is insusceptible to nonsense-mediated mRNA decay (NMD) processing, comprising: Into cells of a subject expressing a target mRNA, (1) a fusion protein comprising an RNA-binding domain and an exon junction complex (EJC) mimicking domain; and (2) A guide RNA that specifically binds to a target region in a target mRNA introducing The fusion protein and the guide RNA form a complex that binds to the target mRNA and leads to degradation of the target mRNA by NMD. A method comprising:

23. 23. The method of claim 22, wherein the RNA-binding domain comprises the RNA-binding region of a CAS nuclease.

24. 24. The method of claim 22 or 23, wherein the EJC-mimicking domain comprises a region derived from one or more components of the EJC and is capable of activating NMD.

25. 25. The method of any one of claims 22 to 24, wherein the 5' end of the target region is at least 10 nucleotides downstream of the PTC in the target mRNA.

26. 26. The method of any one of claims 22 to 25, wherein the disease is selected from the group consisting of the diseases listed in Table 1.

27. 27. The method of any one of claims 22 to 26, wherein the fusion protein is introduced into the cell in the form of one or more expression vectors that express the fusion protein.

28. 28. The method of claim 27, wherein the expression vector further expresses a guide RNA.

29. 1. A method for inducing mRNA degradation of a target mRNA that contains a premature termination codon (PTC) that is insensitive to nonsense-mediated mRNA decay (NMD) processes, comprising: Into cells expressing the target mRNA, (a) a first nucleotide sequence encoding an RNA-binding domain; a second nucleotide sequence encoding an EJC-mimicking domain derived from one or more components of the exon junction complex (EJC); a regulatory sequence operably linked to at least one of the first nucleotide sequence and the second nucleotide sequence; an expression vector comprising (b) a guide RNA that specifically binds to a target region within a target mRNA; introducing The expression vector expresses in the cell a fusion protein comprising the RNA-binding domain and the EJC mimetic domain. A method comprising:

30. 30. The method of claim 29, wherein the expression vector further comprises a nucleotide sequence encoding a nuclear export signal.

31. 31. The method of claim 29 or 30, wherein the first nucleotide sequence encodes dCas13d.

32. 32. The method of any one of claims 29 to 31, wherein the second nucleotide sequence encodes one or more proteins or peptides or fragments thereof derived from eIF4A3, Y14 / RBM8A, MAGOH, RNPS1, SRSF1, UPF1, UPF2 or UPF3B / 3X, and the amino acid sequence is capable of activating NMD.

33. 33. The method of any one of claims 29 to 32, wherein the guide RNA comprises a sequence complementary to a target sequence in an NMD-insensitive PTC-containing mRNA, and the 5' end of the target sequence is located at least 10 nucleotides downstream of the NMD-insensitive PTC in the mRNA.

34. 34. The method of claim 33, wherein the guide RNA is introduced into the cell in the form of a second expression vector comprising a nucleotide sequence encoding the guide RNA.

35. 34. The method of claim 33, wherein the guide RNA is included in the same expression vector that expresses the fusion protein.

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