Compositions, systems and methods for RNA editing using DKC1

Abstract

Provided are methods, compositions, and systems for targeted pseudouridylation of RNA. In some embodiments, provided are methods for editing target RNA (e.g., mRNA) in a host cell, comprising introducing an engineered guide small nucleolar RNA (gsnoRNA) into the host cell, wherein the gsnoRNA recruits DKC1 protein to modify targeted uridine residues in the target RNA to pseudouridine residues. In some embodiments, the DKC1 protein has a cytoplasmic localization in the host cell.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the priority benefit of International Patent Application No. PCT / CN2021 / 096122, filed May 26, 2021, the contents of which are incorporated by reference in their entirety herein.

[0002] INCORPORATION BY REFERENCE TO SEQUENCE LISTING The following submission in an ASCII text file: Computer Readable Form (CRF) of the Sequence Listing (Filename: 165392000441SEQLIST.TXT, Date Recorded: May 24, 2022, Size: 75,959 bytes) is incorporated herein by reference in its entirety.

[0003] The present application relates to compositions, systems, and methods for editing RNA via targeted pseudouridylation using DKC1. [Background technology]

[0004] Pseudouridine (Ψ) is the most abundant post-transcriptionally modified nucleotide in stable RNAs (including tRNA, rRNA, snRNA, and mRNA), constituting approximately 5% of all ribonucleotides. Conversion of uridine to Ψ (pseudouridation) requires two separate chemical reactions: the breaking of the C1'-N1 glycosidic bond and the formation of a new carbon-glycosidic (C1'-C5) bond that relinks the base to the sugar. Pseudouridation is a true isomerization reaction that generates an extra hydrogen bond donor and affects various functional aspects (such as protein synthesis and increased stop codon read-through) depending on the type of RNA that carries Ψ and its position within the RNA sequence (Yu and Meier, 2014, RNA Biology 11:1483-1494). Many of the Ψ mRNAs are located in coding regions, and the majority of them respond to environmental stress and exhibit functional significance (Carlile et al. 2014, Nature 515:143).

[0005] In eukaryotes and archaea, pseudouridylation can be introduced by box H / ACA ribonucleoproteins (RNPs), each of which contains a unique small RNA (box H / ACA It contains RNA (one of two major classes of small nucleolar RNAs or "snoRNAs") and four core proteins (dyskerin (DKC1), NHP2, NOP10, and GAR1). Dyskerin (DKC1; also known as NAP57 / CBF5) is a highly conserved multifunctional protein that acts as an RNA-guided pseudouridine synthetase, directing the enzymatic conversion of specific uridines to pseudouridines. It is concentrated in the nucleolus and Cajal bodies (CBs), where it associates with three other highly conserved proteins (Nop10, Nhp2, Gar1) to form a tetramer that can enter into the composition of different nuclear RNPs that perform important biological functions. Within the nucleolus, the tetramer associates with H / ACA small nucleolar RNAs (snoRNAs) to regulate rRNA processing and pseudouridylate RNA targets by snoRNA-guided base complementation, H / ACA tetramers. Within the CB, the tetramer associates with CB-specific small RNAs (scaRNAs) to form scaRNPs that direct the pseudouridylation of spliceosomal snRNAs. NAP57 / dyskerin (DKC1) / CBF5 catalyzes the chemical reaction to convert the target uridine to Ψ. The RNA component acts as a guide to specify the target uridine for pseudouridylation through base-pairing interactions with the substrate RNA (Ge and Yu, 2013, Trends Biochem Sci 38(4):210-218). Based on this guide-substrate base-pairing scheme, Karijolich and Yu (2011, Nature 474:395-398) designed an artificial box H / ACA RNA to introduce Ψ into mRNA at a premature termination codon (PTC) in S. cerevisiae. They showed that Ψ is a TRM4 RNA that binds to the PTC. We demonstrated that it was indeed incorporated into mRNA.Pseudouridylated PTCs promoted nonsense suppression by altering ribosomal decoding (Fernandez et al. 2013, Nature 500:107-110; Wu et al. 2015, Methods in Enzymology 560:187-217; US8,603,457). Using a similar strategy, others have shown that artificial H / ACA RNAs can site-specifically pseudouridylate pre-mRNAs after microinjection into Xenopus oocytes (Chen et al. 2010, Mol Cell Biol 30:4108-4119). In both instances, the artificial H / ACA RNAs were modified to alter the loops that act as guide sequences, but otherwise these snoRNAs were not altered. Site-specific pseudouridylation or targeting RNA is a potentially powerful technique, but the methods available so far have resulted in low editing efficiency of target RNA.Therefore, there is a need in the art for optimized gsnoRNA, gsnoRNA-based gene editing systems, and methods for editing target RNA by pseudouridylation. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Yu and Meier,2014,RNA Biology 11:1483-1494 [Non-Patent Document 2] Carlile et al.2014,Nature 515:143 [Non-Patent Document 3] Ge and Yu,2013,Trends Biochem Sci 38(4):2l0-218 Summary of the Invention [Means for solving the problem]

[0007] The present application provides methods for editing target RNA in a host cell using gsnoRNA and DKC1 protein. Method embodiments, also referred to herein as "RESTART" methods, can be used to enable read-through of RNA transcripts with premature termination codons (PTCs).

[0008] In some aspects, the present application provides a method for editing a target RNA in a host cell, comprising introducing into the host cell a nucleic acid molecule encoding a guide small nucleolar RNA (gsnoRNA) and a DKC1 protein, wherein the gsnoRNA comprises a guide sequence that hybridizes to a sequence comprising a target uridine residue in the target RNA, and the gsnoRNA recruits the DKC1 protein to modify the target uridine residue in the target RNA to a pseudouridine residue. In some embodiments, the gsnoRNA comprises a scaffold sequence derived from a wild-type H / ACA-snoRNA selected from the group consisting of ACA19, ACA2b, ACA36, ACA44, ACA27, E2, ACA3, and ACA17.

[0009] In some embodiments, a method for editing target RNA in a host cell is provided herein, comprising introducing an engineered gsnoRNA into the host cell, wherein the gsnoRNA comprises a guide sequence that hybridizes to a sequence that comprises a target uridine residue in the target RNA, the gsnoRNA comprises a scaffold sequence derived from wild-type ACA2b, ACA36, ACA44, ACA27, E2, ACA3 or ACA17, and the gsnoRNA recruits DKC1 protein in the host cell to modify the target uridine residue in the target RNA to a pseudouridine residue. In some embodiments, the DKC1 protein is an endogenous DKC1 protein of the host cell. In some embodiments, the method further comprises introducing a nucleic acid encoding a DKC1 protein into the host cell.

[0010] In some embodiments, provided herein is a method for editing a target RNA in a host cell, comprising introducing an engineered gsnoRNA into the host cell, wherein the gsnoRNA comprises a guide sequence that hybridizes to a sequence comprising a target uridine residue in the target RNA, wherein the gsnoRNA comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences provided in Table 2, Table 3, or Table 4, and wherein the gsnoRNA recruits DKC1 protein in the host cell to modify the target uridine residue in the target RNA to a pseudouridine residue.

[0011] In some embodiments, provided herein is a method for editing a target RNA in a host cell comprising introducing an engineered gsnoRNA into the host cell, wherein the gsnoRNA comprises a guide sequence that hybridizes to a sequence comprising a target uridine residue in the target RNA, wherein the gsnoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4-6, 9-12, 15-19, 22-36, and 177-179, and wherein the gsnoRNA recruits DKC1 protein in the host cell to modify the target uridine residue in the target RNA to a pseudouridine residue.

[0012] In some aspects, provided herein is a method for editing a target RNA in a host cell, comprising introducing an engineered gsnoRNA into the host cell, wherein the gsnoRNA comprises a guide sequence that hybridizes to a sequence comprising a target uridine residue in the target RNA, the gsnoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 20-21 and 145-150, and the gsnoRNA recruits a DKC1 protein in the host cell to modify the target uridine residue in the target RNA to a pseudouridine residue. In some embodiments, the DKC1 protein is an endogenous DKC1 protein of the host cell. In some embodiments, the method further comprises introducing a nucleic acid encoding the DKC1 protein into the host cell.

[0013] In some embodiments according to any of the methods described above, the DKC1 protein has cytoplasmic localization in the host cell.

[0014] In some embodiments according to any of the methods described above, the DKC1 protein comprises a DKC1 protein fragment corresponding to amino acid residues 41 to 420 of human DKC1 isoform 3 protein, wherein the amino acid numbering is according to SEQ ID NO:2.

[0015] In some embodiments according to any of the methods described above, the DKC1 protein comprises an amino acid sequence having at least 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity to SEQ ID NO: 88. In some embodiments, the DKC1 protein comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments according to any of the methods described above, the DKC1 protein comprises a naturally occurring DKC1 isoform that is localized in the cytoplasm in a host cell.

[0016] In some embodiments, provided herein is a method for editing a target RNA in a host cell, comprising introducing an engineered gsnoRNA into the host cell, wherein the gsnoRNA comprises a guide sequence that hybridizes to a sequence comprising a target uridine residue in the target RNA, wherein the host cell expresses a DKC1 isoform with cytoplasmic localization, and wherein the gsnoRNA recruits the DKC1 isoform to modify the target uridine residue in the target RNA to a pseudouridine residue.

[0017] In some aspects, provided herein is a method for editing a target RNA in a host cell, comprising: (a) introducing an engineered gsnoRNA and (b) a splice-switching antisense oligonucleotide (ASO) into the host cell, wherein the gsnoRNA comprises a guide sequence that hybridizes to a sequence that comprises a target uridine residue in the target RNA, and the ASO promotes the expression of DKC1 protein, an endogenous DKC1 isoform with cytoplasmic localization, in the host cell, and the gsnoRNA recruits the DKC1 protein to modify the target uridine residue in the target RNA to a pseudouridine residue. In some embodiments, the gsnoRNA comprises a scaffold sequence derived from a wild-type H / ACA-snoRNA selected from the group consisting of ACA19, ACA2b, ACA36, ACA44, ACA27, E2, ACA3, and ACA17.

[0018] In some embodiments according to any of the methods described above, the DKC1 isoform corresponds to isoform 3 of the human DKC1 protein.

[0019] In some embodiments according to any of the methods described above, the DKC1 protein comprises an amino acid sequence having at least 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity to SEQ ID NO: 2. In some embodiments, the DKC1 protein comprises the amino acid sequence of SEQ ID NO: 2.

[0020] In some embodiments according to any of the methods described above, the DKC1 protein comprises a DKC1 protein fragment corresponding to amino acid residues 1 to 419 of full-length human DKC1 isoform 1 protein, where the amino acid numbering is according to SEQ ID NO:1.

[0021] In some embodiments according to any of the methods described above, the gsnoRNA comprises a scaffold sequence derived from ACA2b. In some embodiments according to any of the methods described above, the gsnoRNA comprises a scaffold sequence derived from ACA36. In some embodiments, the gsnoRNA comprises a mutation in the 3' hairpin of the ACA36 scaffold.

[0022] In some embodiments according to any of the methods described above, the gsnoRNA comprises a scaffold sequence derived from ACA19.

[0023] In some embodiments according to any of the methods described above, the gsnoRNA comprises one or more guide sequences, each located in a region corresponding to a hairpin structure of the wild-type H / ACA-snoRNA. In some embodiments, at least one of the one or more guide sequences is located in a hairpin structure at the 3' end of the wild-type H / ACA-snoRNA (also referred to herein as a "3' hairpin structure"). In some embodiments, at least one of the one or more guide sequences is located in a hairpin structure at the 5' end of the wild-type H / ACA-snoRNA (also referred to herein as a "5' hairpin structure"). In some embodiments, the gsnoRNA comprises a single guide sequence. In some embodiments, the gsnoRNA comprises two or more (e.g., 2, 3, 4, 5, 6, or more) guide sequences.

[0024] In some embodiments according to any of the methods described above, the gsnoRNA comprises one or more mutations (e.g., substitutions, insertions, and / or deletions) in one or more hairpin structures (e.g., the 3' hairpin structure and / or the 5' hairpin structure) of wild-type ACA19.

[0025] In some embodiments according to any of the methods described above, the engineered gsnoRNA comprises one or more substitution mutations in the nucleotides of a polyU sequence in the wild type H / ACA-snoRNA, wherein the polyU sequence comprises at least four consecutive U residues.

[0026] In some embodiments according to any of the methods described above, the engineered gsnoRNA comprises one or more insertion or deletion mutations located between the nucleotide residue in the guide region that hybridizes to the target uridine and the H / ACA box of the wild type H / ACA snoRNA, whereby the engineered gsnoRNA comprises 14 or 15 nucleotides between the nucleotide residue in the guide region that hybridizes to the target uridine and the H / ACA box.

[0027] In some embodiments, the one or more mutations are selected from the group consisting of substitution of residues 26-29 with UUCU, substitution of residues 26-29 with UGUU, addition of a G to the 3' hairpin structure after residue 115, and addition of a dinucleotide sequence (XX, e.g., CU) to the 5' hairpin after residue 8, where X is a nucleotide selected from A, U and C and G, numbering according to SEQ ID NO: 37. In some embodiments, the dinucleotide sequence is part of a guide RNA designed to hybridize to a target RNA.

[0028] In some embodiments according to any of the methods described above, the gsnoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3-12, 15-19, 22-36, and 177-179. In some embodiments, the gsnoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15-19.

[0029] In some embodiments according to any of the methods described above, the gsnoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 20-21 and 145-150.

[0030] In some embodiments according to any of the methods described above, the method comprises introducing a nucleic acid molecule encoding a gsnoRNA into a host cell. In some embodiments, the nucleic acid molecule encoding a gsnoRNA is under a small RNA promoter. In some embodiments, the nucleic acid molecule encoding a gsnoRNA is under a promoter selected from the group consisting of a U6 promoter (e.g. transcribed by polymerase III) and a U1 promoter (e.g. transcribed by polymerase II). In some embodiments, the nucleic acid molecule encoding a gsnoRNA is embedded in an intron sequence located between a first exon sequence and a second exon sequence, the first exon sequence, the intron sequence, and the second exon sequence being derived from a naturally occurring gene. In some embodiments, the intron sequence is from an intron of an endogenous gene in the host cell, the gene being selected from the group consisting of EIF3A, SNHG12, RPL21, and RPSA. In some embodiments, the intron sequence is from an intron of an exogenous gene (such as HBB). In some embodiments, the nucleic acid encoding a gsnoRNA is not embedded in an intron sequence.

[0031] In some embodiments according to any of the methods described above, the nucleic acid molecule encoding the DKC1 protein is present in a viral vector. In some embodiments, the nucleic acid molecule encoding the gsnoRNA is present in a viral vector. In some embodiments according to any of the methods described above, the method comprises introducing into a host cell a vector comprising a first nucleic acid sequence encoding the DKC1 protein and a second nucleic acid sequence encoding the gsnoRNA. In some embodiments, the nucleic acid molecule encoding the DKC1 protein and the nucleic acid molecule encoding the gsnoRNA are present in separate vectors. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an adeno-associated virus (AAV) vector.

[0032] In some embodiments according to any of the methods described above, the gsnoRNA comprises one or more chemically modified nucleosides and / or internucleoside linkages. In some embodiments, the gsnoRNA comprises one or more nucleosides with a 2'-OMe or 2'-MOE modification. In some embodiments, the gsnoRNA comprises no more than 10, no more than 8, no more than 6, or no more than 4 chemically modified nucleosides. In some embodiments, the gsnoRNA comprises one or more phosphorothioate internucleoside linkages. In some embodiments, the gsnoRNA comprises no more than 10, no more than 9, no more than 8, or no more than 6 phosphorothioate internucleoside linkages. In some embodiments, the gsnoRNA comprises a 5' cap modification. In some embodiments, the 5' cap modification is 7-methylguanosine (m 7 G) Cap. In some embodiments, the gsnoRNA does not contain one or more chemically modified nucleosides or internucleoside linkages. Cap.

[0033]

[0034] In some embodiments according to any of the methods described above, the efficiency of editing the target RNA is at least 10% (e.g., at least about any one of 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, or more).

[0035] In some embodiments according to any of the methods described above, where the sequence containing the target uridine in the target RNA is a premature stop codon in a sequence encoding a protein, the method results in expression of a full-length protein in the host cell that is at least 4% (e.g., at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10%) of the expression level of the full-length protein without the premature stop codon.

[0036] In some embodiments according to any of the methods described above, where the sequence containing the target uridine in the target RNA is a premature stop codon in a protein-encoding sequence, the method results in expression of a full-length protein, and the expression of the protein is detectable without enrichment (e.g., without enrichment by immunoprecipitation). In some embodiments, the protein is detected via a tag (e.g., via a fluorescent tag). In some embodiments, the protein is detected by immunostaining according to methods known in the art.

[0037] In some embodiments according to any of the methods described above, where the sequence containing the target uridine in the target RNA is a premature stop codon in a protein-encoding sequence, the method results in expression of the full-length protein in at least 20% of the host cells (e.g., at least 25%, at least 30%, or at least 35%, at least 40%, at least 45%, or at least 50% of the host cells).

[0038] In some embodiments according to any of the methods described above, the target RNA is not a ribosomal RNA (rRNA) (such as endogenous rRNA of the host cell).

[0039] In some embodiments according to any of the methods described above, the target RNA is a messenger RNA (mRNA). In some embodiments, the sequence containing the target uridine in the target RNA is a stop codon, and the modification of the target uridine to pseudouridine causes the stop codon to be translated as a coding codon. In some embodiments, the stop codon is a premature stop codon (PTC). In some embodiments, the PTC is associated with an inherited disease or condition. In some embodiments, the sequence containing the target uridine in the target RNA is a stop codon, and the modification of the target uridine to pseudouridine reduces or prevents nonsense-mediated decay (NMD).

[0040] In some embodiments according to any of the methods described above, the DKC1 protein is part of a ribonucleoprotein (RNP) complex that associates with gsnoRNA. In some embodiments, the RNP complex comprises NOP10, GAR1, and NHP2.

[0041] In some embodiments according to any of the methods described above, the host cell is an archaeal cell. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a human cell.

[0042] In some embodiments according to any of the methods described above, the method is performed in vivo. In some embodiments, the method is performed ex vivo.

[0043] In some embodiments, provided herein is a method of treating a disease or condition associated with a PTC in a target RNA in a subject, comprising editing the target RNA in a cell of the subject using any of the methods described above, wherein the gsnoRNA comprises a guide sequence that hybridizes to the PTC in the target RNA, and modification of the uridine residue in the PTC to a pseudouridine residue causes translational read-through of the PTC in the target RNA, thereby treating the disease or condition in the subject.

[0044] In some embodiments, the disease or condition is cystic fibrosis, Hurler syndrome, alpha-1-antitrypsin (A1AT) deficiency, Parkinson's disease, Alzheimer's disease, albinism, amyotrophic lateral sclerosis, asthma, 8-thalassemia, Cadasil syndrome, Charcot-Marie-Tooth disease, chronic obstructive pulmonary disease (COPD), distal spinal muscular atrophy (DSMA), Duchenne / Becker muscular dystrophy, dystrophic epidermolysis bullosa, bullous epidermolysis, Fabry disease, factor V Leiden-related disorder, familial adenomatous polyposis, galactosemia, Gaucher disease, glucose-6-phosphate dehydrogenase, hemophilia, hereditary hemochromatosis, Hunter syndrome, Huntington's disease, inflammatory bowel disease (IBD), hereditary polyaggregation syndrome, syndrome, Leber's congenital amaurosis, Lesch-Nyhan syndrome, Lynch syndrome, Marfan syndrome, mucopolysaccharidoses, muscular dystrophies, myotonic dystrophy types I and II, neurofibromatosis, Niemann-Pick disease types A, B, and C, NY-esol-associated cancer, Peutz-Jeghers syndrome, phenylketonuria, Pompe disease, primary ciliary diseases, prothrombin mutation-associated disorders (such as prothrombin G20210A mutation), pulmonary hypertension (autosomal dominant), retinitis pigmentosa, Sandhoff disease, severe combined immunodeficiency syndrome (SCID), sickle cell anemia, spinal muscular atrophy, Stargardt disease, Tay-Sachs disease, Usher syndrome, X-linked immunodeficiency, Sturge-Weber syndrome, and cancer.

[0045] In some aspects, provided herein is an engineered gsnoRNA comprising a guide sequence that hybridizes to a sequence comprising a target uridine residue in a target RNA in a host cell. In some embodiments, the gsnoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4-6, 9-12, 15-19, 22-36, and 177-179, and the gsnoRNA is capable of recruiting DKC1 protein in a host cell to modify a target uridine residue in a target RNA to a pseudouridine residue. In some embodiments, the gsnoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 20-21 and 145-150, and the gsnoRNA is capable of recruiting DKC1 protein in a host cell to modify a target uridine residue in a target RNA to a pseudouridine residue.

[0046] In some embodiments, provided herein is an engineered gsnoRNA comprising a guide sequence that hybridizes to a sequence comprising a target uridine residue in a target RNA in a host cell, wherein the gsnoRNA comprises a scaffold sequence derived from a wild-type H / ACA-snoRNA selected from the group consisting of ACA2b, ACA36, ACA44, ACA27, E2, ACA3, and ACA17, and wherein the gsnoRNA is capable of recruiting DKC1 protein in the host cell to modify the target uridine residue in the target RNA to a pseudouridine residue.

[0047] In some embodiments according to any of the engineered gsnoRNAs described above, the gsnoRNA comprises a 5' cap modification. In some embodiments, the 5' cap modification is 7-methylguanosine (m 7G) a cap. In some embodiments according to any of the engineered gsnoRNAs described above, the gsnoRNA comprises one or more chemically modified nucleosides and / or internucleoside linkages. In some embodiments according to any of the engineered gsnoRNAs described above, the gsnoRNA comprises one or more nucleosides with 2'-OMe or 2'-MOE modifications. In some embodiments, the gsnoRNA comprises 10 or less, 8 or less, 6 or less, or 4 or less chemically modified nucleosides. In some embodiments, the gsnoRNA comprises one or more phosphorothioate internucleoside linkages. In some embodiments, the gsnoRNA comprises 10 or less, 9 or less, 8 or less, or 6 or less phosphorothioate internucleoside linkages.

[0048] In some embodiments according to any of the engineered gsnoRNAs described above, the gsnoRNA comprises a scaffold sequence derived from ACA2b. In some embodiments according to any of the engineered gsnoRNAs described above, the gsnoRNA comprises a scaffold sequence derived from ACA36. In some embodiments, the gsnoRNA comprises a mutation in the 3' hairpin of the ACA36 scaffold.

[0049] In some embodiments according to any of the engineered gsnoRNAs described above, the gsnoRNA comprises a scaffold sequence derived from ACA19.

[0050] In some embodiments according to any of the engineered gsnoRNAs described above, the gsnoRNA comprises one or more guide sequences, each located in a region corresponding to a hairpin structure of the wild-type H / ACA-snoRNA. In some embodiments, at least one of the one or more guide sequences is located in a hairpin structure at the 3'-end of the wild-type H / ACA-snoRNA. In some embodiments, at least one of the one or more guide sequences is located in a hairpin structure at the 5'-end of the wild-type H / ACA-snoRNA. In some embodiments, the gsnoRNA comprises a single guide sequence. In some embodiments, the gsnoRNA comprises two or more (e.g., 2, 3, 4, 5, 6, or more) guide sequences.

[0051] In some embodiments according to any of the engineered gsnoRNAs described above, the gsnoRNA comprises one or more mutations (e.g. substitutions, insertions, and / or deletions) in one or more hairpin structures (e.g. 3' hairpin structure and / or 5' hairpin structure) of wild type ACA19.

[0052] In some embodiments according to any of the engineered gsnoRNAs described above, the engineered gsnoRNA comprises one or more substitution mutations in the nucleotides of the polyU sequence in the wild type H / ACA-snoRNA, wherein the polyU sequence comprises at least four consecutive U residues.

[0053] In some embodiments according to any of the engineered gsnoRNAs described above, the engineered gsnoRNA comprises one or more insertion or deletion mutations located between the nucleotide residue in the guide region that hybridizes to the target uridine and the H / ACA box of the wild type H / ACA snoRNA, whereby the engineered gsnoRNA comprises 14 or 15 nucleotides between the nucleotide residue in the guide region that hybridizes to the target uridine and the H / ACA box.

[0054] In some embodiments, the one or more mutations are selected from the group consisting of substitution of residues 26-29 with UUCU, substitution of residues 26-29 with UGUU, addition of a G to the 3' hairpin structure after residue 115, and addition of a dinucleotide sequence (XX, e.g. CU) to the 5' hairpin after residue 8, where X is a nucleotide selected from A, U and C and G, numbering according to SEQ ID NO: 37. In some embodiments, the engineered gsnoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15-19.

[0055] In some embodiments, there is provided herein an engineered gsnoRNA comprising a guide sequence that hybridizes to a sequence comprising a target uridine residue in a target RNA in a host cell, wherein the gsnoRNA comprises a scaffold sequence derived from wild type ACA19, and the engineered gsnoRNA comprises one or more insertion or deletion mutations located between the nucleotide residue in the guide region that hybridizes to the target uridine and the H / ACA box of wild type ACA19, whereby the engineered gsnoRNA comprises 14 or 15 nucleotides between the nucleotide residue in the guide region that hybridizes to the target uridine and the H / ACA box, and the gsnoRNA is capable of recruiting DKC1 protein in the host cell to modify the target uridine residue in the target RNA to a pseudouridine residue. In some embodiments, the one or more mutations are selected from the group consisting of substitution of residues 26-29 with UUCU, substitution of residues 26-29 with UGUU, addition of a G to the 3' hairpin structure after residue 115, and addition of a dinucleotide sequence (XX, e.g. CU) to the 5' hairpin after residue 8, where X is a nucleotide selected from A, U and C and G, numbering according to SEQ ID NO: 37. In some embodiments, the gsnoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15-19.

[0056] In some aspects, provided herein is an isolated nucleic acid molecule comprising a sequence encoding the engineered gsnoRNA of any of the preceding embodiments. In some embodiments, provided herein is a vector (e.g., a viral vector) comprising the nucleic acid molecule.

[0057] In some embodiments, provided herein is an engineered RNA editing system comprising: (a) a gsnoRNA, or a nucleic acid molecule encoding a gsnoRNA, comprising a guide sequence that hybridizes to a sequence comprising a target uridine residue in a target RNA in a host cell; and (b) a DKC1 protein, or a nucleic acid molecule encoding a DKC1 protein, wherein the gsnoRNA is capable of recruiting the DKC1 protein to modify the target uridine residue in the target RNA to a pseudouridine residue.

[0058] In some embodiments, the DKC1 protein has a cytoplasmic localization in the host cell. In some embodiments according to any of the methods described above, the DKC1 protein comprises a DKC1 protein fragment corresponding to amino acid residues 41-420 of human DKC1 isoform 3 protein, the amino acid numbering being according to SEQ ID NO:2.

[0059] In some embodiments according to any of the methods described above, the DKC1 protein comprises an amino acid sequence having at least 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity to SEQ ID NO: 88. In some embodiments, the DKC1 protein comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments according to any of the methods described above, the DKC1 protein comprises a naturally occurring DKC1 isoform that is localized in the cytoplasm in a host cell.

[0060] In some embodiments according to any of the engineered RNA editing systems described above, the DKC1 isoform corresponds to isoform 3 of the human DKC1 protein.

[0061] In some embodiments according to any of the engineered RNA editing systems described above, the DKC1 protein comprises an amino acid sequence having at least 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity to SEQ ID NO: 2. In some embodiments, the DKC1 protein comprises the amino acid sequence of SEQ ID NO: 2.

[0062] In some embodiments according to any of the engineered RNA editing systems described above, the DKC1 protein comprises a DKC1 protein fragment corresponding to amino acid residues 1 to 419 of full-length human DKC1 isoform 1 protein, with amino acid numbering according to SEQ ID NO:1.

[0063] In some embodiments according to any of the engineered RNA editing systems described above, the gsnoRNA comprises a scaffold sequence derived from ACA2b. In some embodiments according to any of the engineered RNA editing systems described above, the gsnoRNA comprises a scaffold sequence derived from ACA36. In some embodiments, the gsnoRNA comprises a mutation in the 3' hairpin of the ACA36 scaffold.

[0064] In some embodiments according to any of the engineered RNA editing systems described above, the gsnoRNA comprises a scaffold sequence derived from ACA19.

[0065] In some embodiments according to any of the engineered RNA editing systems described above, the gsnoRNA comprises one or more guide sequences, each located in a region corresponding to a hairpin structure of the wild-type H / ACA-snoRNA. In some embodiments, at least one of the one or more guide sequences is located in a hairpin structure at the 3'-terminal end of the wild-type H / ACA-snoRNA. In some embodiments, at least one of the one or more guide sequences is located in a hairpin structure at the 5'-terminal end of the wild-type H / ACA-snoRNA. In some embodiments, the gsnoRNA comprises a single guide sequence. In some embodiments, the gsnoRNA comprises two or more (e.g., 2, 3, 4, 5, 6, or more) guide sequences.

[0066] In some embodiments according to any of the engineered RNA editing systems described above, the gsnoRNA comprises one or more mutations (e.g., substitutions, insertions, and / or deletions) in one or more hairpin structures (e.g., the 3' hairpin structure and / or the 5' hairpin structure) of wild-type ACA19.

[0067] In some embodiments according to any of the engineered RNA editing systems described above, the engineered gsnoRNA comprises one or more substitution mutations in the nucleotides of the polyU sequence in the wild type H / ACA-snoRNA, wherein the polyU sequence comprises at least four consecutive U residues.

[0068] In some embodiments according to any of the engineered RNA editing systems described above, the engineered gsnoRNA comprises one or more insertion or deletion mutations located between the nucleotide residue in the guide region that hybridizes to the target uridine and the H / ACA box of the wild type H / ACA snoRNA, whereby the engineered gsnoRNA comprises 14 or 15 nucleotides between the nucleotide residue in the guide region that hybridizes to the target uridine and the H / ACA box.

[0069] In some embodiments of the engineered RNA editing system, the DKC1 protein is part of a ribonucleoprotein (RNP) complex that associates with gsnoRNA. In some embodiments, the ribonucleoprotein complex includes NOP10, GAR1, and / or NHP2.

[0070] In some embodiments, the one or more mutations are selected from the group consisting of a substitution of residues 26-29 with UUCU, a substitution of residues 26-29 with UGUU, the addition of a G to the 3' hairpin structure after residue 115, and the addition of a dinucleotide sequence (XX, e.g., CU) to the 5' hairpin after residue 8, where X is a nucleotide selected from A, U and C and G, and the numbering is according to SEQ ID NO:37.

[0071] In some embodiments according to any of the engineered RNA editing systems described above, the gsnoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3-12, 15-19, 22-36, and 177-179. In some embodiments, the gsnoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15-19.

[0072] In some embodiments according to any of the engineered RNA editing systems described above, the gsnoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 20-21 and 145-150.

[0073] In some embodiments, provided herein is a pharmaceutical composition comprising any of the gsnoRNAs, nucleic acid molecules, or engineered RNA editing systems described above and a pharma- ceutically acceptable carrier.

[0074] In some embodiments, provided herein is a host cell comprising any of the gsnoRNAs, nucleic acid molecules, or engineered RNA editing systems described above.

[0075] In some embodiments, provided herein are kits for editing target RNA in a host cell comprising any of the gsnoRNAs, nucleic acid molecules, or engineered RNA editing systems described above.

[0076] Compositions, kits, and articles of manufacture for use in any one of the methods described above are also provided. [Brief description of the drawings]

[0077] [Figure 1] The read-through of a premature stop codon mediated by an engineered guide snoRNA is shown. A provides a schematic of the "RESTART" method design. The snoRNP complex is shown in the dashed box. B provides a schematic showing the structure of reporter-1 and guide snoRNA constructs. In reporter-1, 15 bases are inserted into the position between the codons of the 154th and 155th amino acids. The DNA sequence of the 15 bases is shown, and the premature stop codon (PTC) site (TAG) is indicated. A positive control (Venus-GGT) is included. In C-D, HEK293T cells were co-transfected with reporter-1 and guide snoRNA constructs. Venus expression was detected by a high content imaging system. C shows a representative fluorescent image of cells showing the expression level of Venus. Bar, 200 μm. D shows a dot plot showing the relative percentage of Venus positive cells. (E) Western blot analysis showing the expression levels of DKC1 protein upon stable knockdown of DKC1. (F) Bar plot showing the relative percentage of Venus positive cells in sh-control and stable knockdown of DKC1 cells co-transfected with reporter-1 and gsnoRNA constructs.

[0078] [Diagram 2]Figure 1 shows the PTC read-through effect mediated by gsnoRNAs of different constructs. Dot plots show the relative percentage of Venus positive cells co-transfected with reporter-1 and gsnoRNA in a host intron (A), gsnoRNA in the HBB intron (B), or gsnoRNA transcribed from a small RNA promoter (C). The structure of the gsnoRNA constructs is shown at the bottom of each panel.

[0079] [Diagram 3] The predicted secondary structures of the gsnoRNA scaffolds used in Figures 1A-1F are shown. Secondary structures and base pairing probabilities are predicted using the RNAfold server as described in Gruber et al. (The Vienna RNA websuite. Nucleic Acids Res 36, W70-4 (2008)), the contents of which are incorporated herein by reference in their entirety.

[0080] [Figure 4] Figure 2 shows that optimization of gsnoRNA scaffolds improves the efficiency of PTC read-through. A, Predicted secondary structures of gACA19, gACA2b, and gACA36 scaffolds. Secondary structures and base pairing probabilities are predicted using RNAfold server. Seven mutations are shown in the structure of gACA19 scaffold. B, Structure of gsnoRNA construct. C, Dot plot showing the relative percentage of Venus positive cells co-transfected with reporter-1 and gsnoRNA of construct (B). D, Representative fluorescence image of cells co-transfected with reporter-1 and gsnoRNA of construct (B). Bar, 200 μm. E, Dot plot showing the relative percentage of Venus positive cells co-transfected with reporter-1 and engineered gACA19 scaffolds with different mutations. Engineered positions of gACA19 are annotated in (A). F, Representative fluorescence image of (E). Bar, 200 μm.

[0081] [Diagram 5] The predicted secondary structures of the gsnoRNA scaffolds used in Figures 4A-4F are shown. Secondary structures and base pairing probabilities are predicted using the RNAfold server.

[0082] [Figure 6] Figure 2 shows the engineering of gACA36 scaffold. A is the predicted secondary structure of the engineered gACA36 scaffold. Secondary structure and base pairing probability are predicted using RNAfold server. B is a dot plot showing the relative percentage of Venus positive cells co-transfected with reporter-1 and different gsnoRNA constructs.

[0083] [Figure 7A] Figure 1 shows that exogenous DKC1 isoform 3 protein improves the efficiency of PTC-RT. Structures of the two isoforms of the human DKC1 transcript. Exons are numbered on top, coding regions are represented by solid boxes, and UTRs are represented by white boxes. NLS, nuclear localization signal. [Figure 7B] Figure 1 shows that exogenous DKC1 isoform 3 protein improves the efficiency of PTC-RT. Schematic diagram showing the structure of the reporter-3 construct, in which gsnoRNA is arranged in tandem with the reporter. The sequences surrounding the PTC site and the PTC site (TAA / TAG / TGA) are shown. [Figure 7C] Figure 1 shows that exogenous DKC1 isoform 3 protein improves the efficiency of PTC-RT. Western blot analysis showing the expression level of DKC1 protein in HEK293T DKC1 stable overexpressing cells. Santa Cruz and Abcam anti-DKC1 antibodies target the C-terminal and N-terminal regions of DKC1 protein, respectively. [Figure 7D]Figure 1 shows that exogenous DKC1 isoform 3 protein improves the efficiency of PTC-RT. The indicated reporter-3 constructs were transfected into control HEK293T, cells stably overexpressing DKC1 isoform 1, and cells stably overexpressing DKC1 isoform 3, respectively. Representative fluorescent images of cells. Bar, 200 μm. [Figure 7E] Figure 2 shows that exogenous DKC1 isoform 3 protein improves the efficiency of PTC-RT. The indicated reporter-3 constructs were transfected into control HEK293T, cells stably overexpressing DKC1 isoform 1, and cells stably overexpressing DKC1 isoform 3, respectively. Bar plot showing the relative percentage of EGFP-positive cells. [Figure 7F] Figure 2 shows that exogenous DKC1 isoform 3 protein improves the efficiency of PTC-RT. The indicated reporter-3 constructs were transfected into control HEK293T, cells stably overexpressing DKC1 isoform 1, and cells stably overexpressing DKC1 isoform 3, respectively. Bar plots showing the relative percentage of EGFP intensity. [Figure 7G] Figure 1 shows that exogenous DKC1 isoform 3 protein improves the efficiency of PTC-RT.Figure 2 is a bar plot showing the relative percentage of EGFP-positive cells in HEK293T cells transfected with different reporter-3 constructs and cotransfected with different reporter-3 and DKC1 isoform 3 (200 ng) constructs. [Figure 7H] Figure 1 shows that exogenous DKC1 isoform 3 protein improves the efficiency of PTC-RT.Figure 2 is a bar plot showing the relative percentage of EGFP intensity in HEK293T cells transfected with different reporter-3 constructs and cotransfected with different reporter-3 and DKC1 isoform 3 (200 ng) constructs. [Figure 7I]We show that exogenous DKC1 isoform 3 protein improves the efficiency of PTC-RT. Locus-specific Ψ modifications in reporter-3 transcripts were detected by a qPCR-based method without radioisotope labeling. Curves were obtained by high-resolution melting analysis. Ψ sites were specifically labeled by CMC chemicals, and after reverse transcription, Ψ-CMC adducts cause mutations / deletions at or around the Ψ sites in the cDNA, thus resulting in a shift in the melting temperature. HEK293T cells were co-transfected with different reporter-3 and DKC1 isoform 3 (200 ng) constructs.

[0084] [Figure 8] Figure 2 shows that exogenous DKC1 isoform 3 protein improves the efficiency of PTC-RT. A-C, Reporter-1 constructs were transfected into control HEK293T, cells stably overexpressing DKC1 isoform 1, and cells stably overexpressing DKC1 isoform 3, respectively. A, Representative fluorescent images of cells. Bar, 200 μm. B, Bar plot showing the relative percentage of Venus positive cells. C, Bar plot showing the relative percentage of Venus intensity. D, Dot plot showing the relative percentage of EGFP positive cells cotransfected with Reporter-3 together with empty vector (Vec), DKC1 isoform 1 (DKC1 iso1), or DKC1 isoform 3 (DKC1 iso3) constructs. Statistical analysis of bar plots is unpaired Student's t test.

[0085] [Figure 9] Figure 1 shows the read-through efficiency of different truncations of DKC1 isoform 3. Figure 1 shows dot plots showing the relative percentage of EGFP positive cells co-transfected with reporter-3 and different DKC1 isoform 3 truncation constructs.

[0086] [Figure 10]Comparison of read-through efficiency for different stop codons. A, Representative fluorescence images of cells transfected with different reporter-3 constructs. Bar, 200 μm. B, Representative fluorescence images of cells cotransfected with the indicated reporter-3 and DKC1 isoform 3 (200 ng) constructs. Bar, 200 μm. C-E, Bar plots showing the relative percentage of EGFP-positive cells cotransfected with reporter-3-TAA (C), reporter-3-TAG (D), or reporter-3-TGA (E) with decreasing amounts of DKC1 isoform 3 constructs.

[0087] [Figure 11] Detection of locus-specific Ψ modifications. HEK293T cells were co-transfected with different reporter-3 and DKC1 isoform 3 (200 ng) constructs. Locus-specific Ψ modifications within reporter-3 transcripts (A) and Ψ1045 site in 18S rRNA (B-C) were detected by a qPCR-based method without radioisotope labeling. Curves were obtained by high-resolution melting analysis.

[0088] [Figure 12] The guide snoRNA targets the genetic disorder caused by a nonsense mutation. Schematic of the PTC disease reporter and gsnoRNA constructs. Region of complementarity between the gsnoRNA (top) and the target site in the PTC disease gene (bottom).

[0089] [Figure 13] Region of complementarity between gsnoRNA (top) and the target site in the PTC disease gene (bottom).

[0090] [Figure 14A]Figure 1 shows that RESTART corrects nonsense mutations that can cause genetic disorders. Figure 2 shows dot plots showing the relative percentage of EGFP positive cells co-transfected with the indicated gsnoRNAs and the RESTART v1 PTC disease reporter construct. [Figure 14B] 1 shows that RESTART corrects nonsense mutations that can cause genetic disorders. FIG. 1 is a dot plot showing the relative percentage of EGFP positive cells co-transfected with the indicated gsnoRNA and RESTART v2 DKC1 isoform 3 constructs. [Figure 14C] 1 shows that RESTART corrects nonsense mutations that can cause genetic disorders.Bar plot showing the relative percentage of EGFP positive cells co-transfected with the indicated gsnoRNAs and PTC disease reporters with or without DKC1 isoform 3 constructs.

[0091] [Figure 15A] 1 shows delivery of RESTART by RNA oligonucleotides. Structure of gsnoRNA prepared by in vitro transcription. [Figure 15B] 1 shows delivery of RESTART by RNA oligonucleotides. Structure of gsnoRNA prepared by in vitro transcription. [Figure 15C] 1 shows delivery of RESTART by RNA oligonucleotides. Structure of gsnoRNA prepared by in vitro transcription. [Figure 15D] 1 shows delivery of RESTART by RNA oligonucleotides.Bar plot showing the relative percentage of EGFP positive cells transfected with the indicated gsnoRNA constructs, in vitro transcribed gsnoRNA oligonucleotides, or chemically synthesized gsnoRNA oligonucleotides. [Figure 15E] 1 shows delivery of RESTART by RNA oligonucleotides. Structure of chemically synthesized gsnoRNA oligonucleotides. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0092] The present application provides methods and compositions for editing a target RNA in a host cell, comprising introducing an engineered guide small nucleolar RNA (gsnoRNA) into the host cell, wherein the gsnoRNA comprises a guide sequence that hybridizes to a sequence comprising a target uridine residue in the target RNA, and wherein the gsnoRNA recruits DKC1 protein to modify the target uridine residue in the target RNA to a pseudouridine residue. In some embodiments, the gsnoRNA is an engineered gsnoRNA comprising one or more mutations compared to a wild-type H / ACA scaffold. In some embodiments, the one or more mutations increase the editing efficiency of the gsnoRNA. In some embodiments, the method further comprises increasing the cellular level of DKC1 protein with cytoplasmic localization, thereby increasing the editing efficiency of the gsnoRNA / DKC1 protein complex. In some embodiments, the methods and compositions provided herein can be used to edit premature stop codons (PTCs) in target gene mRNA, thereby suppressing nonsense-mediated decay of the mRNA and promoting translation of full-length proteins. In some embodiments, the methods disclosed herein can be used to treat a disease associated with a PTC in a target gene.

[0093] In some aspects, the present disclosure provides engineered gsnoRNAs and gsnoRNA scaffolds, or nucleic acid molecules encoding gsnoRNAs. In some embodiments, the engineered gsnoRNA scaffolds are based on the wild-type H / ACA snoRNA scaffolds identified by the inventors as having higher editing efficiency compared to other scaffolds. In some embodiments, the engineered gsnoRNA scaffolds contain mutations that increase editing efficiency.

[0094] The methods and compositions described in this application are based at least in part on the unexpected discovery that expression of a cytoplasmically localized DKC1 isoform (e.g., human DKC1 isoform 3) significantly increases the editing efficiency of target RNA using the gsnoRNA / DKC1 system. In one embodiment, the inventors recognized that the editing efficiency of gsnoRNA can be increased by introducing an exogenous DKC1 isoform with a cytoplasmic localization. In another embodiment, the inventors identified truncated and deleted variants of the DKC1 protein that can be used to increase the editing efficiency of gsnoRNA.

[0095] In some aspects, provided herein are nucleic acid constructs encoding gsnoRNAs for use in accordance with the methods described herein. In some embodiments, the inventors have identified promoter and construct configurations for gsnoRNA expression that provide increased efficiency of gsnoRNA editing.

[0096] I. Definition Terms are used herein as follows and as generally used in the art, unless otherwise defined.

[0097] The terms "polynucleotide," "nucleic acid," "nucleotide sequence," and "nucleic acid sequence" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0098] 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 and wobble base pairing. Percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing and wobble base pairing) with a second nucleic acid (e.g., about 5, 6, 7, 8, 9, 10 out of 10 are about 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). "Fully complementary" means that all consecutive residues of a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. "Substantially complementary," as used herein, refers to a degree of complementarity that is any one of at least about 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.

[0099] References to "hybridization" typically refer to specific hybridization and exclude non-specific hybridization. Specific hybridization can occur under experimental conditions selected using techniques well known in the art to ensure that the majority of stable interactions between the probe and the target are ensured when the probe and the target have at least 70%, preferably at least 80%, more preferably at least 90% sequence identity.

[0100] The term "mismatch" is used herein to refer to opposing nucleotides in a double-stranded RNA complex that do not form perfect base pairs according to the Watson-Crick and wobble base pairing rules. Mismatched nucleotides are GA, CA, UC, AA, GG, CC, and UU pairs. Wobble base pairs are GU, IU, IA, and IC base pairs.

[0101] The present disclosure provides multiple types of polynucleotide- or polypeptide-based compositions, including variants and derivatives. These include, for example, substitution, insertion, deletion, and covalent variants and derivatives. The term "derivative" is synonymous with the term "variant" and generally refers to a molecule that is modified and / or changed in any way compared to a reference or starting molecule.

[0102] Thus, polynucleotides encoding peptides or polypeptides containing substitutions, insertions and / or additions, deletions, and covalent modifications with respect to a reference sequence (particularly the polypeptide sequences disclosed herein) are included within the scope of this disclosure. For example, sequence tags or amino acids (such as one or more lysines) can be added to the peptide sequence (e.g., at the N-terminus or C-terminus). Sequence tags can be used for peptide detection, purification, or localization. Lysines can be used to increase peptide solubility or enable biotinylation. Alternatively, amino acid residues located at the carboxy- and amino-terminal regions of the amino acid sequence of a peptide or protein can be optionally deleted to provide a truncated sequence. Certain amino acids (e.g., C- or N-terminal residues) can alternatively be deleted depending on the use of the sequence (e.g., expression of the sequence as part of a larger sequence that is soluble or linked to a solid support).

[0103] The term "identity" refers to the overall relationship between polymer molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules), and / or between polypeptide molecules. Calculation of the percent identity of two polynucleic acid sequences can be accomplished, for example, by aligning the two sequences for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced into one or both of the first and second nucleic acid sequences, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. If a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.For example, the percent identity between two nucleic acid sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991, each of which is incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 1989, 4:11-17) incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. The percent identity between two nucleic acid sequences can alternatively be determined using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix. Commonly used methods for determining percent identity between sequences include, but are not limited to, those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988), which is incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs.Exemplary computer software for determining homology between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1), 387(1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol., 215, 403(1990)).

[0104] With respect to the polypeptide sequences identified herein, "percent (%) amino acid sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the polypeptide being compared, after alignment of the sequences, taking into account any conservative substitutions as part of sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the scope of those skilled in the art, for example, using publicly available computer software (such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), or MUSCLE software). Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, % amino acid sequence identity values ​​are generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004, each of which is incorporated herein by reference in their entirety and for all purposes).

[0105] The terms "non-naturally occurring" or "engineered" are used interchangeably and indicate the involvement of human hand. The terms, when referring to a nucleic acid molecule or polypeptide, mean that the nucleic acid molecule or polypeptide contains at least one modification (e.g., at least one mutation (such as a substitution, insertion, or deletion) or at least one non-naturally occurring chemical modification) compared to a naturally occurring nucleic acid molecule or polypeptide, or is at least substantially free of at least one other component with which it is naturally associated and found in nature.

[0106] The term "wild-type" as used herein with reference to an ACA scaffold sequence refers to the sequence of a naturally occurring box H / ACA small nucleolar RNA.

[0107] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (and into mRNA or other RNA transcripts, etc.) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as the "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0108] The term "polypeptide" or "peptide" is used herein to encompass all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins, and modified proteins, including, but not limited to, glycoproteins, and all other types of modified proteins, such as proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, PEGylation, biotinylation, and the like.

[0109] The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredients contained therein to be effective and does not contain additional components that are unacceptably toxic to the subject to which the formulation will be administered.

[0110] "Pharmaceutically acceptable carrier" refers to one or more components in a pharmaceutical formulation other than the active ingredient that are non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizing substances, cryoprotectants, tonicity agents, preservatives, and combinations thereof. Pharmaceutically acceptable carriers or excipients preferably meet the required standards of toxicological and manufacturing testing, and / or are included in the Inactive Ingredient Guide drawn up by the U.S. Food and Drug Administration or other state / federal governments, or are listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias for use in mammals (more specifically, in humans).

[0111] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, which contain information about the indications, usage, dosage, administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic products.

[0112] An "article of manufacture" is any product (e.g., package or container) or kit that contains at least one reagent, such as a pharmaceutical agent for the treatment of a disease or condition (e.g., coronavirus infection) or a probe for specifically detecting a biomarker described herein. In certain embodiments, the product or kit is promoted, distributed, or sold as a unit for performance of a method described herein.

[0113] It is understood that embodiments described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0114] Reference herein to "about" a value or parameter encompasses (and describes) a variation about that value or parameter per se. For example, a description that refers to "about X" includes the description of "X."

[0115] As used herein, a reference to "is not" a value or parameter generally means and describes "other than" the value or parameter. For example, a method is not used to treat disease type X means that the method is used to treat diseases other than type X.

[0116] The term "about X to Y" used in this specification has the same meaning as "about X to about Y".

[0117] As used in this specification and the appended claims, the singular forms "a," "an," or "the" include plural referents unless the context clearly dictates otherwise.

[0118] The term "and / or," when used herein in phrases such as "A and / or B," is intended to encompass both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or," when used herein in phrases such as "A, B, and / or C," is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0119] II. Compositions and Systems In some aspects, there is provided herein an engineered gsnoRNA comprising a guide sequence that hybridizes to a sequence comprising a target uridine residue in a target RNA in a host cell, wherein the gsnoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4-6, 9-12, 15-19, 22-36, and 177-179, and wherein the gsnoRNA is capable of recruiting DKC1 protein in a host cell to modify a target uridine residue in the target RNA to a pseudouridine residue. In some embodiments, the gsnoRNA comprises one or more nucleosides with a 2'-OMe or 2'-MOE modification. In some embodiments, the engineered gsnoRNA comprises no more than 10, no more than 8, no more than 6, or no more than 4 chemically modified nucleosides. In some embodiments, the engineered gsnoRNA comprises one or more phosphorothioate internucleoside linkages. In some embodiments, the gsnoRNA comprises no more than 10, no more than 9, no more than 8, or no more than 6 phosphorothioate internucleoside linkages. In some embodiments, the gsnoRNA contains a 5' cap modification, such as 7-methylguanosine (m 7 G) cap modification). In some embodiments, the 5' cap modification comprises m 7 The G(5')ppp(5')G cap analog is introduced by in vitro transcription.

[0120] In some embodiments, engineered gsnoRNAs are produced by in vitro transcription. In some embodiments, engineered gsnoRNAs produced by in vitro transcription are full length gsnoRNAs (e.g., including a 3' hairpin, a 5' hairpin, an H box, and an ACA box). In some embodiments, engineered gsnoRNAs produced by in vitro transcription are 5' cap modified (e.g., 7-methylguanosine (m 7 G) Cap modification).

[0121] In some embodiments, the engineered gsnoRNA comprises a single hairpin and an H box, but no ACA box. In some embodiments, the engineered gsnoRNA comprises the sequence of SEQ ID NO: 179. In some embodiments, the engineered gsnoRNA comprises a single hairpin and an ACA box, but no H box. In some embodiments, the engineered gsnoRNA comprises the sequence of SEQ ID NO: 180. In some embodiments, the gsnoRNA comprises one or more nucleosides with 2'-OMe or 2'-MOE modifications. In some embodiments, the engineered gsnoRNA comprises no more than 10, no more than 8, no more than 6, or no more than 4 chemically modified nucleosides. In some embodiments, the engineered gsnoRNA comprises one or more phosphorothioate internucleoside linkages. In some embodiments, the gsnoRNA comprises no more than 10, no more than 9, no more than 8, or no more than 6 phosphorothioate internucleoside linkages.

[0122] In some aspects, there is provided herein an engineered gsnoRNA comprising a guide sequence that hybridizes to a sequence comprising a target uridine residue in a target RNA in a host cell, said gsnoRNA comprising a scaffold sequence derived from wild type ACA2b or ACA36, said gsnoRNA being capable of recruiting DKC1 protein in a host cell to modify a target uridine residue in a target RNA to a pseudouridine residue. In some embodiments, the gsnoRNA comprises a scaffold sequence derived from the sequence of SEQ ID NO: 11 or 12. In some embodiments, the gsnoRNA comprises 1, 2, 3, or 4 substitution, deletion, and / or insertion mutations compared to SEQ ID NO: 11 or 12. In some embodiments, the gsnoRNA comprises one or more nucleosides with a 2'-OMe or 2'-MOE modification. In some embodiments, the engineered gsnoRNA comprises no more than 10, no more than 8, no more than 6, or no more than 4 chemically modified nucleosides. In some embodiments, the engineered gsnoRNA contains one or more phosphorothioate internucleoside linkages. In some embodiments, the gsnoRNA contains 10 or less, 9 or less, 8 or less, or 6 or less phosphorothioate internucleoside linkages. In some embodiments, the gsnoRNA contains 5' cap modifications (e.g., 7-methylguanosine (m 7 G) cap modification). In some embodiments, the 5' cap modification comprises m 7 The G(5')ppp(5')G cap analog is introduced by in vitro transcription.

[0123] In some embodiments, provided herein is an isolated nucleic acid molecule comprising a sequence encoding a gsnoRNA provided herein. In some embodiments, the gsnoRNA comprises a scaffold sequence derived from a wild-type H / ACA-snoRNA selected from the group consisting of ACA19, ACA2b, ACA36, ACA44, ACA27, E2, ACA3, and ACA17. In some embodiments, the gsnoRNA comprises a scaffold sequence derived from a wild-type H / ACA-snoRNA selected from the group consisting of ACA2b, ACA36, ACA44, ACA27, E2, ACA3, and ACA17. In some embodiments, the gsnoRNA comprises a scaffold sequence derived from ACA36. In some embodiments, the gsnoRNA comprises a scaffold sequence derived from ACA2b. In some embodiments, the gsnoRNA comprises a scaffold sequence derived from ACA19. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a substance that promotes expression of isoform 3 of the DKC1 protein (e.g., a splice-switching antisense oligonucleotide (ASO), where the ASO promotes expression of the DKC1 protein, an endogenous DKC1 isoform with cytoplasmic localization in the host cell). In some embodiments, the nucleic acid molecule further comprises a sequence encoding a DKC1 isoform or DKC1 protein variant, where the isoform or variant has cytoplasmic localization. Exemplary DKC1 proteins are described below in Section II A.

[0124] In some aspects, an engineered RNA editing system is provided herein, comprising: (a) a gsnoRNA (such as any one of the gsnoRNAs described in Section II B below) or a nucleic acid molecule encoding a gsnoRNA, comprising a guide sequence that hybridizes to a sequence that comprises a target uridine residue in a target RNA in a host cell; and (b) a DKC1 protein (such as any one of the gsnoRNAs described in Section II A below) or a nucleic acid molecule encoding a DKC1 protein, wherein the gsnoRNA is capable of recruiting the DKC1 protein to modify the target uridine residue in the target RNA to a pseudouridine residue. In some embodiments, the DKC1 protein is a DKC1 isoform with cytoplasmic localization.

[0125] In some embodiments, provided herein is a host cell comprising any of the gsnoRNAs, nucleic acid constructs / molecules, or engineered RNA editing systems described herein.

[0126] In some embodiments, provided herein are kits for editing target RNA in a host cell comprising any of the gsnoRNAs, nucleic acid constructs / molecules, or engineered RNA editing systems described herein.

[0127] A. DKC1 protein The present application provides, in some embodiments, engineered DKC1 proteins or nucleic acid constructs encoding DKC1 proteins.

[0128] Dyskerin (DKC1) is a highly conserved multifunctional protein that acts as an RNA-guided pseudouridine synthase and directs the enzymatic conversion of specific uridines to pseudouridines. DKC1 is concentrated in the nucleolus and Cajal bodies (CBs), where it associates with three other highly conserved proteins (Nop10, Nhp2, Gar1) to form a tetramer that can enter into the composition of different nuclear RNPs that perform important biological functions. Within the nucleolus, the tetramer associates with H / ACA small nucleolar RNAs (snoRNAs) to form H / ACA snoRNPs that regulate rRNA processing and pseudouridylate RNA targets by snoRNA-guided base complementation. Within the CBs, the tetramer associates with CB-specific small RNAs (scaRNAs) to form scaRNPs that direct the pseudouridylation of spliceosomal snoRNAs.

[0129] There are two DKC1 isoforms in human cells. DKC1 isoform 1 is the canonical DKC1 form that contains bipartite N- and C-terminal nuclear localization signals (NLS). DKC1 isoform 3 is an alternative splicing variant that is produced by retention of intron 12 and lacks the C-terminal NLS (Figure 9A). The endogenous mRNA expression level of isoform 1 is approximately 20-fold higher than that of isoform 3. 5 Surprisingly, the inventors found that increasing the level of DKC1 isoform 3 promoted gsnoRNA-guided targeted pseudouridylation editing efficiency (e.g., editing efficiency of target mRNA).

[0130] In some embodiments, the composition of the present disclosure comprises a nucleic acid construct for expression of DKC1 protein. In some embodiments, the composition of the present disclosure comprises a DKC1 protein (e.g., a DKC1 protein in a complex with gsnoRNA). In some embodiments, the DKC1 protein is isoform 3 of mammalian DKC1 protein. In some embodiments, the DKC1 protein is homologous to isoform 3 of human DKC1 protein. In some embodiments, the DKC1 protein has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to isoform 3 of human DKC1 protein. In some embodiments, the DKC1 protein is isoform 3 of human DKC1 protein. In some embodiments, the DKC1 protein comprises an amino acid sequence having at least 85% (e.g., at least any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:2. The sequences of full length DKC1 (isoform 1) and isoform 3 DKC1 are shown in Table 1 below.

[0131] In some embodiments, the DKC1 protein is part of a ribonucleoprotein (RNP) complex that associates with gsnoRNA. In some embodiments, the ribonucleoprotein complex includes NOP10, GAR1, and / or NHP2.

[0132] In some embodiments, truncated DKC1 protein variants and nucleic acid constructs encoding same are provided herein. In some embodiments, the DKC1 protein comprises a deletion of a nuclear localization signal (NLS) compared to a wild-type DKC1 protein of the same species. In some embodiments, the DKC1 protein comprises a deletion of amino acid residues 9-21 of DKC1 isoform 3, where the amino acid numbering is based on SEQ ID NO: 2. In some embodiments, the DKC1 protein comprises amino acid residues 22-420 of DKC1 isoform 3, where the amino acid numbering is based on SEQ ID NO: 2. In some embodiments, the DKC1 protein comprises amino acid residues 35-420 of DKC1 isoform 3, where the amino acid numbering is based on SEQ ID NO: 2. In some embodiments, the DKC1 protein comprises amino acid residues 41-420 of DKC1 isoform 3, where the amino acid numbering is based on SEQ ID NO: 2. The DKC1 sequence in SEQ ID NO:2 is isoform 3 of human DKC1, but a person skilled in the art would understand how to generate corresponding truncated and deletion variants of homologous DKC1 proteins based on sequence alignments (e.g., corresponding deletion / truncation variants of DKC1 proteins from other mammalian species).

[0133] In some embodiments, the DKC1 protein comprises an amino acid sequence having at least 85% (e.g., at least any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 85. In some embodiments, the DKC1 protein comprises an amino acid sequence having at least 85% (e.g., at least any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 86. In some embodiments, the DKC1 protein comprises an amino acid sequence having at least 85% (e.g., at least any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 87. In some embodiments, the DKC1 protein comprises an amino acid sequence having at least 85% (e.g., at least any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:88. [Table 1-1] [Table 1-2] [Table 1-3]

[0134] In some embodiments, amino acid sequence variants of the DKC1 protein provided herein are contemplated. For example, it may be desirable to improve the stability and / or other biological properties of DKC1 (e.g., of the catalytic domain of DKC1) or of its interactions with other proteins in a ribonucleoprotein complex. The structures of DKC1 and other proteins in a ribonucleoprotein complex are described, for example, in Rashid et al. (Molecular Cell (2006) 21(2): 249-260) and Czekay et al. (Front. Microbiol. (2021) 12: 654370), the contents of which are incorporated herein by reference in their entirety. Amino acid sequence variants of the DKC1 protein can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the target binding moiety or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions into and / or substitutions of residues within the amino acid sequence of the target binding moiety. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct retains the desired characteristics.

[0135] In some embodiments, DKC1 protein variants having one or more amino acid substitutions are provided. Amino acid substitutions can be introduced into the DKC1 protein and the products screened for a desired activity.

[0136] Conservative substitutions are shown in Table A below. [Table 5]

[0137] Amino acids can be grouped into different classes according to common side chain properties. a. Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; b. Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c. Acidic: Asp, Glu; d. Basic: His, Lys, Arg; e. Residues that affect chain orientation: Gly, Pro; f. Aromatics: Trp, Tyr, Phe.

[0138] Non-conservative substitutions would involve exchanging a member of one of these classes for another class.

[0139] Fragments of the naturally occurring DKC1 protein or functional variants thereof, as well as fusion proteins comprising a heterologous amino acid sequence, for example at the N-terminus, C-terminus, or internal position of the DKC1 fragment, are also contemplated.

[0140] B. Nucleic Acid Constructs and Engineered gsnoRNAs In some embodiments, engineered gsnoRNAs based on H / ACA snoRNAs are provided herein. In some embodiments, the gsnoRNA comprises a single guide sequence. In some embodiments, the gsnoRNA comprises two guide sequences. In some embodiments, the engineered gsnoRNA comprises more than two (e.g., 3, 4, 5, 6, or more) guide sequences. For example, the H / ACA snoRNA contains two hairpins followed by an H box motif and an ACA box motif. In some embodiments, both hairpins of the engineered gsnoRNAs provided herein contain guide sequences capable of targeting a target pseudouridylation site. In other embodiments, only one hairpin of the engineered gsnoRNA contains a guide sequence capable of targeting a target pseudouridylation site. Exemplary engineered gsnoRNA sequences are provided in Tables 2 and 3 below.

[0141] In some aspects, the gsnoRNA disclosed herein are synthetic oligonucleotides and can be synthesized according to methods known in the art. In some embodiments, the gsnoRNA according to the present disclosure is an oligoribonucleotide (full RNA). However, in some embodiments, the gsnoRNA of the present disclosure can comprise DNA. In some embodiments, the gsnoRNA can be expressed in situ, for example from a plasmid or viral vector, especially when it consists only of nucleotides or linkages that can be expressed in a biological system.

[0142] In some embodiments, the gsnoRNA comprises a scaffold sequence derived from a wild type H / ACA-snoRNA selected from the group consisting of ACA2b and ACA36. In some embodiments, the editing efficiency of the gsnoRNA derived from the wild type H / ACA scaffold is at least 5% (e.g. between about 5%-15% or 5-10%) in mammalian cells (e.g. in human cells such as HEK293T cells). In some embodiments, the gsnoRNA comprises a scaffold sequence derived from ACA2b. In some embodiments, the gsnoRNA comprises a scaffold sequence derived from ACA36. In some embodiments, the gsnoRNA comprises a scaffold sequence derived from ACA19.

[0143] In some embodiments, engineered gsnoRNAs and engineered gsnoRNA scaffolds derived from wild type H / ACA-snoRNAs (e.g. from ACA2b, ACA36 or ACA19) are disclosed herein, where the gsnoRNA is capable of modifying a PTC in a protein-encoding RNA, said modification resulting in the expression of a full-length protein. In some embodiments, the engineered gsnoRNA is capable of causing expression of a full-length protein in a host cell at at least 4% (e.g. at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10%) of the expression level of the full-length protein without a premature stop codon. In some embodiments, the engineered gsnoRNA is capable of causing expression of a full-length protein, the expression of the protein being detectable without enrichment (e.g. without enrichment by immunoprecipitation). In some embodiments, the protein is detected via a tag (e.g. via a fluorescent tag). In some embodiments, the protein is detected by immunostaining according to methods known in the art. In some embodiments, the engineered gsnoRNA is capable of causing expression of the full length protein in at least 20% of the host cells (e.g. at least 25%, at least 30% or at least 35%, at least 40%, at least 45%, or at least 50% of the host cells).

[0144] In some embodiments, the gsnoRNA comprises one or more guide sequences, each located in a region corresponding to a hairpin structure of the wild-type H / ACA-snoRNA. In some embodiments, the gsnoRNA comprises one or more guide sequences located in a hairpin structure at the 3'-terminal end of the wild-type H / ACA-snoRNA. In some embodiments, the gsnoRNA comprises one or more guide sequences located in a hairpin structure at the 5'-terminal end of the wild-type H / ACA-snoRNA.

[0145] In some embodiments, the gsnoRNA comprises one or more mutations (e.g. substitutions, insertions, and / or deletions) in one or more hairpin structures (e.g. 3' and / or 5' hairpin structures) of wild type ACA19. In some embodiments, the gsnoRNA comprises one or more mutations that change the distance between the nucleotide residue in the guide region that hybridizes to the target uridine and the H / ACA box compared to the wild type scaffold. In some embodiments, the one or more mutations comprise an insertion or deletion of one or more nucleotide residues. In some embodiments, the engineered gsnoRNA comprises 14 nucleotides between the nucleotide residue in the guide region that hybridizes to the target uridine and the H / ACA box. In some embodiments, the engineered gsnoRNA comprises 15 nucleotides between the nucleotide residue in the guide region that hybridizes to the target uridine and the H / ACA box. In some embodiments, the mutation increases the efficiency of pseudouridylation (eg, the efficiency of PTC readthrough) by at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, or 1.6-fold compared to the wild-type scaffold.

[0146] In some embodiments, the one or more mutations comprise a substitution in a small poly-U sequence (e.g., a sequence of 4 or more or 5 or more consecutive uridine (U) residues). In some embodiments, the one or more mutations comprise altering the small poly-U sequence to include no more than two consecutive U residues. In some embodiments, the one or more mutations comprise a single base mutation in a "UUUU" sequence. In some embodiments, the mutation is a "UUCU" or "UGUU" mutation. In some embodiments, the mutated poly-U sequence is located in a loop region of the gsnoRNA scaffold. In some embodiments, the engineered gsnoRNA comprises the sequence of SEQ ID NO: 49 or 50. In some embodiments, the engineered gsnoRNA comprises the sequence of SEQ ID NO: 15 or 16. In some embodiments, the mutations increase the efficiency of pseudouridylation (e.g., the efficiency of PTC read-through) by at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, or 1.6-fold compared to the wild-type scaffold.

[0147] In some embodiments, the one or more mutations include mutations that increase the openness of the guide region compared to the guide region of the wild type scaffold. In some embodiments, the one or more mutations reduce the base pairing probability of one or more residues in the guide region of the gsnoRNA scaffold (e.g., the 5' guide region of the gACA19 scaffold). In some embodiments, the one or more mutations include an insertion of one or more nucleotides. In some embodiments, the one or more mutations include the addition of a CU after residue 8, numbering according to SEQ ID NO: 37. In some embodiments, the engineered gsnoRNA is the gsnoRNA of SEQ ID NO: 53. The predicted secondary structure of gACA19-5addCU (SEQ ID NO: 53) is shown in Figure 5D. In some embodiments, the mutations increase the efficiency of pseudouridylation (e.g., the efficiency of PTC readthrough) by at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, or 1.6-fold compared to the wild type scaffold.

[0148] In some embodiments, the one or more mutations are selected from the group consisting of a substitution of residues 26-29 with UUCU, a substitution of residues 26-29 with UGUU, the addition of a G to the 3' hairpin structure after residue 115, and the addition of a dinucleotide sequence (XX, e.g., CU) to the 5' hairpin after residue 8, where X is a nucleotide selected from A, U and C and G, and the numbering is according to SEQ ID NO:37.

[0149] In some embodiments, the gsnoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 17-19 and 22-29.

[0150] In some embodiments, the gsnoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4-6, 9-12, 15-19, 22-36, and 177-179.

[0151] In some embodiments, the gsnoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15-19.

[0152] In some embodiments, the gsoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 20-21 and 145-150.

[0153] In some embodiments the gsnoRNA is a disease targeting gsnoRNA (eg any of the gsnoRNA sequences provided in Table 4).

[0154] In some embodiments, the gsnoRNA comprises one or more chemically modified nucleosides and / or internucleoside linkages. In some embodiments, the gsnoRNA comprises one or more nucleosides with 2'O-methyl (2'-OMe) or 2'-O-methoxyethyl (2'-MOE) modifications. In some embodiments, almost the entirety of a gsnoRNA according to the present disclosure may be chemically modified, for example by providing nucleotides with 2'-O-methylated sugar moieties (2'-OMe) and / or nucleotides with 2'-O-methoxyethyl sugar moieties (2'-MOE). In some embodiments, the gsnoRNA comprises no more than 20, no more than 15, no more than 10, no more than 8, no more than 6, or no more than 4 2'-OMe or 2'-MOE modifications. In some embodiments, the gsnoRNA comprises from about 2 to about 6 2'-OMe or 2'-MOE modifications. In some embodiments, the gsnoRNA comprises about 4 2'-OMe or 2'-MOE modifications. In some embodiments, the gsnoRNA comprises 5 or fewer modified sugars. In some embodiments, the gsnoRNA comprises 2 nucleosides comprising a modified sugar moiety (e.g., 2'-OMe) at the 5' end of the gsnoRNA and 2 nucleosides comprising a modified sugar moiety (e.g., 2'-OMe) at the 3' end. In some embodiments, the gsnoRNA comprises 4, 3, or 2 or fewer nucleosides comprising a modified sugar moiety (e.g., 2'-OMe) at the 5' end of the gsnoRNA and 4, 3, or 2 or fewer nucleosides comprising a modified sugar moiety (e.g., 2'-OMe) at the 3' end. In some embodiments, the gsnoRNA comprises one or more phosphorothioate internucleoside linkages. In some embodiments, the gsnoRNA comprises 20 or fewer, 15 or fewer, 10 or fewer, 8 or fewer, or 6 or fewer phosphorothioate linkages. In some embodiments, the gsnoRNA comprises about 2 to about 10 phosphorothioate linkages. In some embodiments, the gsnoRNA comprises about six phosphorothioate linkages.In some embodiments, the gsnoRNA comprises about 3 phosphorothioate linkages at the 5' end and about 3 phosphorothioate linkages at the 3' end of the gsnoRNA. In some embodiments, the gsnoRNA comprises no more than 5, 4, or 3 phosphorothioate linkages at the 5' end and no more than 5, 4, or 3 phosphorothioate linkages at the 3' end of the gsnoRNA. Example 7 provides results demonstrating that a limited number of modifications are sufficient for stability and function of gsnoRNA oligonucleotides.

[0155] In some embodiments, the gsnoRNA comprises one or more chemically modified nucleosides and / or internucleoside linkages. In some embodiments, the gsnoRNA comprises one or more nucleosides with 2'O-methyl (2'-OMe) or 2'-O-methoxyethyl (2'-MOE) modifications. In some embodiments, almost the entire gsnoRNA according to the present disclosure may be chemically modified, for example by providing nucleotides with 2'-O-methylated sugar moieties (2'-OMe) and / or nucleotides with 2'-O-methoxyethyl sugar moieties (2'-MOE). In some embodiments, the gsnoRNA comprises a 5' hairpin, an H box (consensus sequence ANANNA), a 3' hairpin, and an ACA box (consensus sequence ANA). In some embodiments, the gsnoRNA comprises a single hairpin and an H box (referred to herein as gH5 or rH5 for the 5' half of the gsnoRNA coding sequence or gsnoRNA oligonucleotide, respectively) and lacks an ACA box. In some embodiments, the gsnoRNA comprises a single hairpin and an ACA box (referred to herein as gH3 or rH3 for the 3' half of the gsnoRNA coding sequence or the gsnoRNA oligonucleotide, respectively) and lacks an H box. In some embodiments, the gsnoRNA comprising a single hairpin is 60-70 nucleotides in length. In some embodiments, the gsnoRNA comprising a single hairpin is about 65 nucleotides in length.

[0156] In some embodiments, the gsnoRNA is prepared by in vitro transcription. In some embodiments, the gsnoRNA prepared by in vitro transcription comprises any one of SEQ ID NOs: 4-6, 9-12, 15-19, 22-36. In some embodiments, the gsnoRNA prepared by in vitro transcription comprises a 5' cap modification or a 5' hairpin (e.g., of a U6+U27 expression cassette). In some embodiments, the gsnoRNA prepared by in vitro transcription comprises a 5' cap modification. In some embodiments, the 5' cap modification is m 7 G modification (e.g., cap0 modification, cap1 modification, or cap2 modification) or m 6 A m 5' cap is a modification. Suitable methods for adding a 5' cap to an RNA oligonucleotide are described, for example, in U.S. Pat. No. 10,494,399, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the gsnoRNA further comprises a 3' hairpin (e.g., the gsnoRNA comprises any one of SEQ ID NOs: 4-6, 9-12, 15-19, and 22-36, and a 3' hairpin). In some embodiments, the gsnoRNA comprises a 5' cap modification and does not comprise a 3' hairpin (e.g., as shown in FIG. 15A). In some embodiments, the 5' cap modification is a 5' cap modification that ... 7 The G(5')ppp(5')G cap analog is introduced by in vitro transcription.

[0157] A variety of chemistries and modifications are known in the art of oligonucleotides and can be readily used in accordance with the present disclosure. The regular internucleoside linkages between nucleotides can be modified by monothioation or dithioation of the phosphodiester bond resulting in phosphorothioate or phosphorodithioate esters, respectively. Other modifications of the internucleoside linkages are possible, including amidated linkers and peptide linkers. In a preferred embodiment, the gsnoRNA of the present disclosure has 1, 2, 3, 4, 5, 6 or more phosphorothioate linkages between the most terminal nucleotides of the gsnoRNA (thus preferably at both the 5' and 3' ends), meaning that in the case of three phosphorothioate linkages, the resulting four nucleotides are linked. It will be understood by those skilled in the art that the number of such linkages can vary at each end depending on the target sequence or based on other aspects (such as toxicity). However, it is some embodiments of the present disclosure that the gsnoRNA comprises one or more PS linkages between any position in the terminal seven nucleotides.

[0158] The ribose sugar can be modified by substitution of the 2'-O moiety with lower alkyl (C1-4 such as 2'-OMe), alkenyl (C2-4), alkynyl (C2-4), methoxyethyl (2'-methoxyethoxy; or 2'-O-methoxyethyl; or 2'-MOE), or other substituents. In some embodiments, the 2'OH group substituent is a methyl group, a methoxyethyl group, or a 3,3'-dimethylallyl group. The latter is known for its property of improving the efficiency of hybridization while inhibiting nuclease sensitivity due to its bulkiness. Alternatively, locked nucleic acid sequences (LNA) can be applied, which contain an internal 2'-4' intramolecular bridge (usually a methylene bridge between the 2' oxygen and the 4' carbon) linkage of the ribose ring. Purine and / or pyrimidine nucleobases can be modified, for example, by amination or deamination of the heterocyclic ring to change their properties. Other modifications that may be present in the gsnoRNA of the present disclosure are 2'-F modified sugars, BNAs, and cEts. The exact chemistry and format will vary from oligonucleotide construct to oligonucleotide construct and application to application, and can be accomplished according to the desires and preferences of the skilled artisan.

[0159] Examples of chemical modifications in gsnoRNA of the present disclosure are modifications of the sugar moiety, including by bridging substituents within the sugar (ribose) moiety (e.g., as in LNA or locked nucleic acid, BNA, cEt, and the like), by replacement of the 2'-O atom with alkyl (e.g., 2'-O-methyl), alkynyl (2'-O-alkynyl), alkenyl (2'-O-alkenyl), alkoxyalkyl (e.g., 2'-O-methoxyethyl, 2'-MOE) groups, and the like, with lengths as specified above. In the context of the present disclosure, sugar "modification" also includes 2' deoxyribose (as in DNA). In addition, the phosphodiester groups of the backbone may be modified by thioation, dithioation, amidation, and the like, resulting in internucleoside linkages such as phosphorothioates, phosphorodithioates, phosphoramidates, and the like. The internucleoside linkages may be completely or partially replaced by peptide linkages, resulting in peptide nucleic acid sequences and the like. Alternatively or additionally, the nucleobases may be (de)modified by amination, resulting in inosine or 2'6'-diaminopurine and the like. A further modification may be methylation of C5 in the cytidine moiety of the nucleotide, reducing potential immunogenic properties known to be associated with CpG sequences.

[0160] In some embodiments, the gsnoRNA does not contain one or more chemically modified nucleosides and / or internucleoside linkages. In some embodiments, the gsnoRNA does not contain non-natural internucleoside linkages.

[0161] Mammalian H / ACA snoRNAs are generally embedded (located) within pre-mRNA intronic regions of protein-coding genes. During transcriptional elongation, several proteins with functional roles in pseudouridylation (such as NOP10, dyskerin (DKC1), or NHP2) bind to the nascent H / ACA snoRNA sequence. Following splicing, the guide RNA is processed via debranching and terminal nucleolytic processing, resulting in an RNA-protein complex called a "small nuclear ribonucleoprotein" (snRNP, or snRNP complex). Box H / ACA snoRNAs have no localization preference to the 5' or 3' end of the intron and can be present in small or very large introns, but are usually localized 60-90 nucleotides upstream of the 3' splice site, in contrast to box C / D snoRNAs, which are encoded in relatively small introns. It has been suggested by Kiss and Filipowicz (1995, Genes Dev 9(11):1411-1424) that a given snoRNA sequence can be excised from the intron region of any given actively spliced ​​mRNA and processed completely. To show that this snoRNA processing can occur independently of the host intron context, Kiss and Filipowicz artificially embedded several snoRNAs (III7a, U17b, and U19) into the second intron of the human β-globin gene and expressed the resulting vector in fibroblast-like cells. After transfection, they found that the snoRNA delivered by the artificial intron was properly processed from the human β-globin intron and the β-globin pre-mRNA was correctly spliced. Darzacq et al. (2002, EMBO J 21(11);2746-2756) determined that other guide RNAs could be inserted into the second intron of the human β-globin gene and delivered to mammalian cells via transfection using an expression vector under the control of the cytomegalovirus (CMV) promoter.

[0162] The inventors of the present application unexpectedly identified diverse host intron context-dependent effects on the pseudouridylation editing efficiency of different gsnoRNAs (as discussed in Example 1). For example, the inventors tested the PTC read-through efficiency of gsnoRNAs based on the wild-type ACA19 (embedded in the host intron of EIF3A), ACA-44 (embedded in the host intron of SNHG12), ACA27 (embedded in the host intron of RPL21) and E2 (embedded in the host intron of RPSA) host genes, as well as gsnoRNAs embedded in a non-host intron of the HBB gene (Figures 2A and 2B). Surprisingly, the inventors found that the editing efficiency of gsnoRNAs based on the E2 scaffold was lower when gE2 was embedded in the HBB intron compared to the host RPSA intron, whereas the editing efficiency of gACA19 was similar when embedded in the HBB intron compared to the host EIF3A intron. Based on this observation that host gene sequences have diverse effects on different gsnoRNAs, the inventors envisaged that directly expressing gsnoRNAs without host gene effects may further increase the efficiency of PTC read-through. Therefore, the inventors designed a series of gsnoRNA expression constructs in which the nucleic acid molecule encoding the gsnoRNA is not embedded in an intron. As discussed in Example 1, the inventors demonstrated that the pseudouridylation activity of gsnoRNAs not embedded in an intron is promoted in nucleic acid molecules encoding gsnoRNAs driven by hU6 promoter (type III RNA polymerase III promoter) and hU1 promoter (snRNA type RNA polymerase II promoter). Thus, in one aspect, a nucleic acid molecule encoding a gsnoRNA is provided herein, said nucleic acid molecule being under the control of a small RNA promoter (e.g. U6 promoter or U1 promoter). In some embodiments, the nucleic acid encoding the gsnoRNA is not embedded in an intron sequence.

[0163] In some embodiments, a nucleic acid construct encoding a gsnoRNA is provided herein. In some embodiments of the methods described herein, the method comprises introducing a nucleic acid molecule encoding a gsnoRNA into a host cell. In some embodiments, the nucleic acid molecule encoding the gsnoRNA is under the control of a small RNA promoter. In some embodiments, the small RNA promoter is a U6 promoter (transcribed by polymerase III) or a U1 promoter (transcribed by polymerase II). In some embodiments, expression of a gsnoRNA from a small RNA promoter according to the methods disclosed herein provides an increased pseudouridylation efficiency (e.g., increased PTC read-through efficiency) compared to the same gsnoRNA embedded in a host intron sequence or other intron sequence. In some embodiments, the pseudouridylation efficiency of a gsnoRNA expressed from a nucleic acid under the control of a small RNA promoter is 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, or 2-fold higher compared to the same gsnoRNA embedded in a host intron. Example 1 (Figures 1A-1E and Figures 2A-2C) provides results demonstrating the promotion of PTC readthrough by gsnoRNA expressed from a nucleic acid under the control of a small RNA promoter compared to gsnoRNA embedded in an intron.

[0164] In some embodiments, the nucleic acid molecule encoding the gsnoRNA is embedded in an intron sequence located between the first exon sequence and the second exon sequence. In some embodiments, the first exon sequence, the intron sequence, and the second exon sequence are derived from a naturally occurring gene. In some embodiments, the intron may contain additional nucleotides (besides the nucleic acid molecule of the present disclosure containing the guide region). Since the guide region is expressed from the intron sequence, such additional nucleotides may be selected to give the most efficient expression from the intron. In some embodiments, the exon A / intron / exon B sequence is present in a vector (such as a plasmid or viral vector). Such vectors can be used to deliver the exon-intron-exon sequence to a cell. Additional introns and exons may be present in such vectors. In some embodiments, the exon A sequence (upstream of the intron carrying the nucleic acid encoding the gsnoRNA (which is expressed post-transcriptionally)) comprises or consists of exon 1 of the human β-globin gene, and the exon B sequence (downstream of the intron carrying the nucleic acid encoding the gsnoRNA (which is expressed post-transcriptionally)) comprises or consists of exon 2 of the human β-globin gene. In some embodiments, the exon A sequence (upstream of the intron carrying the nucleic acid encoding the gsnoRNA (which is expressed post-transcriptionally)) comprises or consists of exon 2 of the human hemoglobin subunit β (HBB) gene, and the exon B sequence (downstream of the intron carrying the nucleic acid encoding the gsnoRNA (which is expressed post-transcriptionally)) comprises or consists of exon 3 of the human hemoglobin subunit β (HBB) gene. In some embodiments, the nucleic acid molecule encoding the gsnoRNA is embedded in an intron sequence between a first exon sequence and a second exon sequence, and the intron sequence, the first exon sequence, and the second exon sequence correspond to the sequence of a host gene carrying a naturally occurring snoRNA.In some embodiments, the construct comprising the intron-embedded gsnoRNA coding sequence is under the control of a CMV promoter.

[0165] In some embodiments, engineered gsnoRNAs targeting disease-associated PTCs are provided herein. In some embodiments, the engineered gsnoRNAs targeting disease-associated PTCs comprise one or more mutations to enhance the editing efficiency and / or expression of the gsnoRNA. In some embodiments, the engineered gsnoRNAs targeting disease-associated PTCs are selected from SEQ ID NOs: 71-84 (shown in Figures 14-15). Exemplary engineered gsnoRNA sequences targeting disease-associated PTCs are shown in Table 4 below.

[0166] In some embodiments, gsnoRNA may be administered in free form (or "naked" without the context of a vector) or may be delivered to cells by other means (such as liposomes or nanoparticles) or by the use of iontophoresis. In some embodiments, gsnoRNA may be administered in a ribonucleoprotein complex (e.g. in a complex with DKC1, HNP2, NOP10, and / or GAR1). In some embodiments, free gsnoRNA comprises one or more chemically modified nucleosides and / or internucleoside linkages as described above.

[0167] In some aspects, provided herein is a nucleic acid construct encoding DKC1 (e.g., any of the DKC1 proteins described in Section IIA above). In some embodiments of the methods described herein, the method comprises introducing a nucleic acid molecule encoding a DKC1 protein into a host cell. In some embodiments, the nucleic acid molecule comprises a promoter operably linked to a nucleotide sequence encoding DKC1. In some embodiments, the promoter is a pol II promoter. In some embodiments, the promoter is a CMV promoter.

[0168] As disclosed herein, a vector may carry DNA or RNA and is generally used to express the gsnoRNA constructs and / or DKC1 protein constructs of the present disclosure after processing in the cell into which the vector is introduced. This is generally via transcription of the DNA or RNA present in the vector. In some embodiments, the vector is a viral vector (which can be used to infect the target cell to be treated) or a plasmid, which can be introduced into the cell by various techniques known to those skilled in the art.

[0169] In some embodiments, the nucleic acid molecule encoding the DKC1 protein and / or the nucleic acid molecule encoding the gsnoRNA is present in a viral vector. In some embodiments, the method includes introducing into a host cell a vector (e.g., a plasmid or viral vector) comprising a first nucleic acid sequence encoding the DKC1 protein and a second nucleic acid sequence encoding the gsnoRNA. In some embodiments, the vector is an adeno-associated viral (AAV) vector.

[0170] Exemplary engineered ACA scaffold sequences are shown in Table 2 below. Guide sequences are shown and underlined as (Xn), where Xn is a sequence of X nucleotides of length n, where X is any of A, U, G, or C, and n is 4, 5, 6, 7, 8, 9, 10, 11, or 12. Guide sequences (Xn) can be modified to target gsnoRNA to desired target sites, as will be understood by those skilled in the art. In some embodiments, n is an integer of the length suitable for the guide region. In some embodiments, n is 4, 5, 6, 7, 8, or 9.

[0171] Exemplary engineered gsnoRNA sequences, including exemplary guide sequences, are shown in Table 3 below.

[0172] Exemplary engineered gsnoRNA sequences targeting exemplary disease-associated PTCs are shown in Table 4 below. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 4-1] [Table 4-2] [Table 4-3]

[0173] In one aspect, the inventors have found that the editing efficiency of gsnoRNA is surprisingly high when the gsnoRNA is encoded in tandem with its target RNA. Example 3 provides results demonstrating increased editing efficiency using a reporter construct that encodes a gsnoRNA and a target RNA in tandem.

[0174] Thus, in some aspects, provided herein is a nucleic acid molecule comprising a nucleotide sequence encoding a guide small nucleolar RNA (gsnoRNA) in tandem with a nucleotide sequence encoding a target RNA. In some embodiments, the nucleotide sequence encoding the gsnoRNA is driven by a U6 promoter or a U1 promoter. In some embodiments, the nucleotide sequence encoding the target RNA is driven by the same promoter or a different promoter. In some embodiments, the gsnoRNA encoded in tandem with the nucleotide sequence encoding the target RNA provides at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, or 2-fold more editing efficiency of the target RNA compared to the same gsnoRNA encoded in a nucleic acid molecule separate from the target RNA.

[0175] III. Method In some embodiments, a method for editing a target RNA in a host cell is provided herein, comprising introducing into the host cell an engineered guide small nucleolar RNA (gsnoRNA) and a nucleic acid molecule encoding a DKC1 protein, wherein the gsnoRNA comprises a guide sequence that hybridizes to a sequence comprising a target uridine residue in the target RNA, and the gsnoRNA recruits the DKC1 protein to modify the target uridine residue in the target RNA to a pseudouridine residue. In some embodiments, the DKC1 protein is a DKC1 isoform (e.g., isoform 3) with cytoplasmic localization. In some embodiments, the DKC1 protein is part of a ribonucleoprotein (RNP) complex that associates with the gsnoRNA. In some embodiments, the DKC1 protein comprises a deletion of a nuclear localization signal (NLS) compared to a wild-type DKC1 protein of the same species. In some embodiments, the DKC1 protein is a truncated or deleted DKC1 variant (such as any of the truncated or deleted variants described in Section IIA above). In some embodiments, the gsnoRNA recruits NOP10, GAR1, and NHP2 in the host cell.

[0176] In some embodiments, the method comprises introducing a nucleic acid (e.g., a nucleic acid vector) encoding a gsnoRNA into the cell. In other embodiments, the method comprises introducing a gsnoRNA oligonucleotide into the cell. In some embodiments, the gsnoRNA comprises a first hairpin and an H box, and a second hairpin and an ACA box. In some embodiments, the gsnoRNA is prepared by in vitro transcription. In some embodiments, the gsnoRNA prepared by in vitro transcription comprises any one of SEQ ID NOs: 4-6, 9-12, and 15-19, 22-36. In some embodiments, the gsnoRNA prepared by in vitro transcription comprises a 5' cap modification or a 5' hairpin (e.g., of a U6+U27 expression cassette). In some embodiments, the gsnoRNA prepared by in vitro transcription comprises a 5' cap modification. In some embodiments, the 5' cap modification is m 7 G modification (e.g., cap0 modification, cap1 modification, or cap2 modification) or m 6 A m modification. Suitable methods for adding a 5' cap to an RNA oligonucleotide are described, for example, in US Pat. No. 10,494,399, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the gsnoRNA further comprises a 3' hairpin (e.g., the gsnoRNA comprises any one of SEQ ID NOs: 4-6, 9-12, 15-19, and 22-36, and a 3' hairpin). In some embodiments, the gsnoRNA comprises a 5' cap modification and does not comprise a 3' hairpin (e.g., as shown in Figure 15A). In some embodiments, the in vitro transcribed gsnoRNA is capable of guiding targeted pseudouridylation in cells. In some embodiments, the 5' cap modification is m 7 The G(5')ppp(5')G cap analog is introduced by in vitro transcription.

[0177] In some embodiments, the method comprises introducing into the cell a nucleic acid (e.g. a nucleic acid vector) encoding a gsnoRNA half (e.g. comprising a single hairpin and an H box, or comprising a single hairpin and an ACA box). In other embodiments, the method comprises introducing into the cell a gsnoRNA half (e.g. comprising a single hairpin and an H box, or comprising a single hairpin and an ACA box). In some embodiments, the gsnoRNA comprises no more than 20, no more than 15, no more than 10, no more than 8, no more than 6, or no more than 4 2'-OMe or 2'-MOE modifications. In some embodiments, the gsnoRNA comprises from about 2 to about 6 2'-OMe or 2'-MOE modifications. In some embodiments, the gsnoRNA comprises about 4 2'-OMe or 2'-MOE modifications. In some embodiments, the gsnoRNA comprises no more than 5 modified sugars. In some embodiments, the gsnoRNA comprises two nucleosides that comprise a modified sugar moiety (e.g., 2'-OMe) at the 5' end of the gsnoRNA, and two nucleosides that comprise a modified sugar moiety (e.g., 2'-OMe) at the 3' end. In some embodiments, the gsnoRNA comprises no more than 4, 3, or 2 nucleosides that comprise a modified sugar moiety (e.g., 2'-OMe) at the 5' end of the gsnoRNA, and no more than 4, 3, or 2 nucleosides that comprise a modified sugar moiety (e.g., 2'-OMe) at the 3' end. In some embodiments, the gsnoRNA comprises one or more phosphorothioate internucleoside linkages. In some embodiments, the gsnoRNA comprises no more than 20, no more than 15, no more than 10, no more than 8, or no more than 6 phosphorothioate linkages. In some embodiments, the gsnoRNA comprises about 2 to about 10 phosphorothioate linkages. In some embodiments, the gsnoRNA comprises about 6 phosphorothioate linkages. In some embodiments the gsnoRNA comprises about three phosphorothioate linkages at the 5' end and about three phosphorothioate linkages at the 3' end of the gsnoRNA.In some embodiments, the gsnoRNA comprises no more than 5, 4, or 3 phosphorothioate linkages at the 5' end of the gsnoRNA and no more than 5, 4, or 3 phosphorothioate linkages at the 3' end. In some embodiments, the gsnoRNA comprises a 5' hairpin, an H box (consensus sequence ANANNA), a 3' hairpin, and an ACA box (consensus sequence ANA). In some embodiments, the gsnoRNA comprises a single hairpin and an H box (referred to herein as gH5 or rH5 for the 5' half of the gsnoRNA coding sequence or the gsnoRNA oligonucleotide, respectively) and lacks an ACA box. In some embodiments, the gsnoRNA comprises a single hairpin and an ACA box (referred to herein as gH3 or rH3 for the 3' half of the gsnoRNA coding sequence or the gsnoRNA oligonucleotide, respectively) and lacks an H box. In some embodiments, the gsnoRNA comprising a single hairpin is 60-70 nucleotides in length. In some embodiments, the gsnoRNA comprising a single hairpin is about 65 nucleotides in length.

[0178] The present disclosure is exemplified, but not limited to, by reversing the effects of nonsense stop mutations that normally lead to translation termination and mRNA degradation (via nonsense mediated decay, see below). In another embodiment, targeted pseudouridylation can act as a means to recode uridine-containing codons, for example as a means to modulate protein function via amino acid substitutions in critical protein regions (such as protein kinase active sites).

[0179] One of the consequences of mutations leading to PTCs in the coding sequence of a gene is a reduction in the levels of mRNA. This is due to a mechanism known as nonsense-mediated decay (NMD), a cellular surveillance mechanism that degrades abnormal mRNA transcripts and prevents incorrectly processed transcripts from being translated. It is estimated that one-third of genetic disorders are the result of mutations leading to PTCs (e.g., in CF, retinitis pigmentosa (RP), and β-thalassemia). In a normal scenario, exon-junction complexes (EJCs) are formed during splicing. Ribosomes then replace these EJCs during the first round of translation. On the other hand, when a PTC is located more than 50-54 nucleotides upstream of the last EJC, the NMD pathway is triggered by the formation of a termination complex consisting of EJC-associated NMD factors. This occurs during the first pioneer round of translation, when ribosomes co-locate with at least one EJC downstream of their location, which induces decapping and 5' to 3' exonuclease activity, as well as further tail deadenylation and 3' to 5' exonuclease-mediated transcript decay. Inhibition of this pathway in a gene-specific and sequence-specific manner is therefore important to address any disorders resulting from the aforementioned genetic disorders or similar mutations.

[0180] In some embodiments, methods are provided herein for recoding PTCs that result in increased levels of mRNA and translational read-through of the recoded mRNA into full-length protein. In some embodiments, the methods and compositions provided herein allow for PTC read-through of more than 4%, more than 5%, more than 10%, more than 12%, more than 15%, more than 20%, or more than 30%. In some embodiments, the methods and compositions provided herein allow for suppression of nonsense-mediated decay (NMD) by more than 10%, more than 12%, more than 15%, more than 20%, or more than 30%. PTC read-through can be assayed by assessing protein levels (either by directly quantifying protein expression or by assaying the activity of expressed proteins). Methods for assessing NMD suppression are also known in the art. For example, known NMD inhibition reporter assays (Zhang et al. 1998, RNA 4(7):801-815) can be used to assess NMD suppression, and translational read-through of genes carrying PTCs can also be assessed. As exemplified herein, a fluorescent reporter gene carrying a nonsense mutation was used as a target sequence. Without correction, this nonsense mutation leads to lower abundance of mRNA (as a result of NMD) and a shortened protein, resulting in the absence of fluorescent signal. As shown herein, the correction of the mutation through targeted pseudouridylation allows full-length protein to be translated from mRNA. Those skilled in the art will understand that the PTC region of the fluorescent reporter construct described herein can be replaced by other models or therapeutically relevant target RNAs of interest.

[0181] In some embodiments, provided herein is a method for recoding a PTC in a protein-encoding RNA, the method resulting in expression of a full-length protein in a host cell at least 4% (e.g., at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10%) of the expression level of the full-length protein without a premature stop codon. In some embodiments, the method results in expression of a full-length protein, and the expression of the protein is detectable without enrichment (e.g., without enrichment by immunoprecipitation). In some embodiments, the protein is detected via a tag (e.g., via a fluorescent tag). In some embodiments, the protein is detected by immunostaining according to methods known in the art. In some embodiments, the method results in expression of a full-length protein in at least 20% of the host cells (e.g., at least 25%, at least 30%, or at least 35%, at least 40%, at least 45%, or at least 50% of the host cells).

[0182] In some embodiments, provided herein is a method for treating, preventing, and / or blocking nonsense-mediated RNA decay of a target mRNA, comprising introducing into a host cell a guide small nucleolar RNA (gsnoRNA) and a nucleic acid molecule encoding a DKC1 protein, wherein the gsnoRNA comprises a guide sequence that hybridizes to a premature termination codon (PTC) sequence that comprises a target uridine residue in the target mRNA, and the gsnoRNA recruits the DKC1 protein to modify the target uridine residue in the target RNA to a pseudouridine residue, whereby pseudouridine of the target uridine promotes read-through of the PTC. In some embodiments, the DKC1 protein is a DKC1 isoform with cytoplasmic localization. In some embodiments, the DKC1 protein is part of a ribonucleoprotein (RNP) complex that associates with the gsnoRNA. In some embodiments, the DKC1 protein comprises a deletion of a nuclear localization signal (NLS) compared to a wild-type DKC1 protein of the same species. In some embodiments, the DKC1 protein is a truncated DKC1 variant or a deletion-containing DKC1 variant (such as any of the truncated or deletion variants described in Section IIA above).

[0183] In some embodiments, DKC1 protein is the endogenous protein of host cell.In some embodiments, DKC1 protein is the endogenous naturally expressed DKC1 isoform of host cell, and DKC1 isoform has cytoplasmic localization in host cell.In some embodiments, DKC1 protein corresponds to isoform 2 of human DKC1 protein.

[0184] In some embodiments, DKC1 and snoRNA can be delivered together into a cell (e.g., as part of a ribonucleoprotein (RNP) complex). In some embodiments, the snoRNP comprises a gsnoRNA as well as DKC1, NHP2, GAR1, and / or NOP10.

[0185] In some embodiments, a method for editing a target RNA in a host cell is provided herein, comprising introducing an engineered gsnoRNA into the host cell, the gsnoRNA comprising a guide sequence that hybridizes to a sequence comprising a target uridine residue in the target RNA (e.g., mRNA), the host cell expresses a DKC1 isoform with cytoplasmic localization, and the gsnoRNA recruits the DKC1 isoform to modify the target uridine residue in the target RNA to a pseudouridine residue. In some embodiments, the method comprises introducing a splice-switching antisense oligonucleotide (ASO) into the host cell, the ASO promoting the expression of DKC1 protein, which is an endogenous DKC1 isoform with cytoplasmic localization in the host cell.

[0186] Splice-switching antisense oligonucleotides (ASOs) alter splicing by directing splice site selection. Splice-switching ASOs can be used to regulate pre-mRNA splicing by binding to target pre-mRNA and blocking access of the splicing apparatus to specific splice sites, producing novel splice variants, correcting aberrant splicing, or manipulating alternative splicing. Methods for the design and delivery of splice-switching antisense oligonucleotides to cells are described, for example, in U.S. Patent Publications US20180334677 and US20120040917, U.S. Patent No. 10,190,117, and Disterer et al. Hum Gene Ther. 2014 July; 25(7):587-98, the contents of which are incorporated herein by reference in their entirety.

[0187] In some embodiments, the splice-switching ASO binds to the pre-mRNA of the DKC1 gene and directs splicing of DKC1 isoform 3. In some embodiments, introduction of the splice-switching ASO increases expression of DKC1 protein, an endogenous DKC1 isoform localized in the cytoplasm, in a host cell by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, or up to 10-fold compared to expression of the same isoform in the host cell in the absence of the ASO. In some embodiments, administration of the splice-switching ASO increases expression of DKC1 isoform 3 by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold compared to expression of DKC1 isoform 3 in a host cell in the absence of the ASO.

[0188] In some embodiments, splice-switching ASOs can be delivered via aptamers, reverse molecular sentinel nanoprobes, ASO-encapsulated liposomal DNA polycations, or ASO-encapsulated liposomal-protamine-hyluronic acid nanoparticles, and the like. Suitable methods for delivering aptamers can be found in Kotula, JW, et al., Aptamer-mediated delivery of splice-switching oligonucleotides to the nuclei of cancer cells. Nucleic Acid Ther, 2012.22(3):p.187-95, the contents of which are incorporated herein by reference in their entirety.

[0189] In some embodiments, a method for editing a target RNA in a host cell is provided herein, comprising introducing an engineered gsnoRNA into the host cell, wherein the gsnoRNA comprises a guide sequence that hybridizes to a sequence that comprises a target uridine residue in the target RNA, the gsnoRNA comprises a scaffold sequence derived from wild-type ACA19, ACA44, ACA27, E2, ACA3, ACA17, ACA2b, or ACA36, and the gsnoRNA recruits DKC1 protein in the host cell to modify the target uridine residue in the target RNA (e.g., mRNA) to a pseudouridine residue. In some embodiments, the gsnoRNA comprises a scaffold sequence derived from wild-type ACA2b or ACA36. In some embodiments, the DKC1 protein is a DKC1 isoform with cytoplasmic localization. In some embodiments, the DKC1 protein is part of a ribonucleoprotein (RNP) complex that associates with the gsnoRNA.

[0190] In some embodiments, provided herein is a method for editing a target RNA in a host cell, comprising introducing an engineered gsnoRNA into the host cell, wherein the gsnoRNA comprises a guide sequence that hybridizes to a sequence comprising a target uridine residue in the target RNA, wherein the gsnoRNA comprises a scaffold sequence derived from wild type ACA19, ACA44, ACA27, E2, ACA3, ACA17, ACA2b, or ACA36, and wherein the gsnoRNA recruits DKC1 protein in the host cell to modify the target uridine residue in the target RNA (e.g., mRNA) to a pseudouridine residue. The engineered gsnoRNA can be any one of the engineered gsnoRNAs described in section IIB. In some embodiments, the gsnoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4-6, 9-12, 15-19, 22-36, and 177-179. In some embodiments, the DKC1 protein is a DKC1 isoform with cytoplasmic localization. In some embodiments, the DKC1 protein is part of a ribonucleoprotein (RNP) complex that associates with gsnoRNA. In some embodiments, the DKC1 protein comprises a deletion of a nuclear localization signal (NLS) compared to a wild-type DKC1 protein of the same species. In some embodiments, the DKC1 protein is a truncated DKC1 variant or a DKC1 variant comprising a deletion (such as any of the truncated or deleted variants described in Section IIA above).

[0191] In some embodiments, provided herein is a method for editing a target RNA in a host cell, comprising introducing an engineered gsnoRNA into the host cell, wherein the gsnoRNA comprises a guide sequence that hybridizes to a sequence comprising a target uridine residue in the target RNA, the gsnoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4-6, 9-12, 15-19, 22-36, and 177-179, and the gsnoRNA recruits DKC1 protein in the host cell to modify the target uridine residue in the target RNA to a pseudouridine residue. In some embodiments, the DKC1 protein is a DKC1 isoform with cytoplasmic localization. In some embodiments, the DKC1 protein comprises a DKC1 protein fragment corresponding to amino acid residues 1-419 of the full-length human DKC1 protein, amino acid numbering according to SEQ ID NO: 1. In some embodiments, the DKC1 protein is part of a ribonucleoprotein (RNP) complex that associates with the gsnoRNA. In some embodiments, the DKC1 protein comprises a deletion of a nuclear localization signal (NLS) compared to a wild-type DKC1 protein of the same species, In some embodiments, the DKC1 protein is a truncated DKC1 variant or a DKC1 variant comprising a deletion (such as any of the truncated or deleted variants described in Section IIA above).

[0192] In some embodiments, the methods provided herein include introducing into a host cell a nucleic acid molecule comprising a nucleotide sequence encoding a guide small nucleolar RNA (gsnoRNA) in tandem with a nucleotide sequence encoding a target RNA. In some embodiments, the nucleotide sequence encoding the gsnoRNA is driven by a U6 promoter or a U1 promoter. In some embodiments, the nucleotide sequence encoding the target RNA is driven by the same promoter or a different promoter. In some embodiments, the gsnoRNA encoded in tandem with the nucleotide sequence encoding the target RNA provides at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, or 2-fold more editing efficiency of the target RNA compared to the same gsnoRNA encoded in a nucleic acid molecule separate from the target RNA.

[0193] In some embodiments, the methods provided herein comprise introducing a guide small nucleolar RNA (gsnoRNA) into an endogenous nucleic acid molecule of a host cell, the endogenous nucleic acid molecule comprising a nucleotide sequence encoding a target RNA. In some embodiments, the introduction comprises inserting a nucleotide sequence encoding a gsnoRNA into a region of the endogenous nucleic acid molecule adjacent, directly or indirectly, to a region encoding the target RNA. In some embodiments, the nucleotide sequence encoding the gsnoRNA is driven by a U6 promoter or a U1 promoter. Methods for inserting a nucleotide sequence into an endogenous nucleic acid molecule are known in the art and include guided nuclease (e.g., CRISPR / Cas) editing and homology directed repair. In some embodiments, a gsnoRNA inserted into a region of an endogenous nucleic acid molecule directly or indirectly adjacent to a nucleotide sequence encoding a target RNA provides at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, or 2-fold greater editing efficiency of the target RNA compared to the same gsnoRNA encoded in a nucleic acid molecule separate from the target RNA.

[0194] In some embodiments, the degree of recruitment and redirection of pseudouridylated entities present in cells can be regulated by the dosing and dosing regimen of gsnoRNA, as determined by the experimenter (e.g. in vitro) or by the clinician, usually in Phase I and / or Phase II clinical trials.

[0195] In some embodiments, the methods provided herein include modifying target RNA (e.g., mRNA) sequences in eukaryotic organisms (e.g., mammalian cells, such as metazoan cells or human cells). In some aspects, the methods and compositions provided herein can be used by cells from any organ (e.g., skin, lung, heart, kidney, liver, pancreas, intestine, muscle, gland, eye, brain, blood, and the like). Cells can be placed in vitro or in vivo. One advantage of the methods, compositions, systems, kits, and articles of manufacture of the present disclosure is that they can be used by cells in situ in living organisms, but also by cells in culture. In some embodiments, cells are treated ex vivo and then introduced into living organisms (e.g., reintroduced into the organism they originally came from). The methods, compositions, systems, kits, and articles of manufacture of the present disclosure can also be used to edit target RNA sequences in cells in so-called organoids. Organoids can be considered as three-dimensional in vitro derived tissues, but are made using specific conditions to generate individual isolated tissues (see, e.g., Lancaster and Knoblich. 2014, Science 345(6194):1247125). In a therapeutic setting, organoids are useful because they can be derived in vitro from the patient's cells and then reintroduced into the patient as autologous material, which is less likely to be rejected than a normal transplant. The cells to be treated will generally have a genetic mutation. The mutation can be heterozygous or homozygous. In some embodiments, the methods and compositions provided herein can be used to modify point mutations. In some embodiments, the methods and compositions provided herein are suitable for modifying sequences in cells, tissues, or organs involved in a disease state of a subject (e.g., a human subject), for example, when the human subject suffers from a disease associated with PTC.

[0196] The present disclosure provides methods that can be used to alter (pseudourylate) a target RNA sequence in a eukaryotic cell through the use of oligonucleotides (e.g., any of the gsnoRNAs described in Section IIB above, or any gsnoRNA based on an engineered scaffold described in Section IIB above) that can target the site to be edited and recruit an RNA editing protein (e.g., DKC1) to effect the editing reaction(s). In some embodiments, the DKC1 is endogenous DKC1. In some embodiments, the DKC1 is delivered exogenously. In some embodiments, the method includes increasing the relative proportion of DKC1 isoform 3 or DKC1 protein with cytoplasmic localization. The target RNA sequence can include a mutation, such as a point mutation (transition or transversion), that one wishes to correct or change. The target RNA can be any cellular or viral RNA sequence, but is usually a pre-mRNA or mRNA with protein coding function. In some embodiments, the target sequence is endogenous to the eukaryotic (e.g., mammalian, e.g., human) cell.

[0197] In some embodiments, the methods provided herein are suitable for promoting read-through of a PTC, where the PTC is an opal codon (UGA), an amber codon (UAG), or an ochre codon (UAA). In some embodiments, the PTC is an opal codon, and the method provides a read-through efficiency of at least 10%, at least 15%, at least 20%, or at least 25% compared to a control lacking the PTC, assayed as a percentage of protein expression or activity (e.g., fluorescence intensity). In some embodiments, the PTC is an amber codon (UAG), and the method provides a read-through efficiency of at least 2%, at least 5%, at least 10%, at least 12%, or at least 14% compared to a control lacking the PTC, assayed as a percentage of protein expression or activity (e.g., fluorescence intensity). In some embodiments, the methods result in cells expressing at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% detectable levels of full-length protein encoded by a target gene comprising a PTC.

[0198] In some embodiments, the target uridine in the target RNA is a premature stop codon in a protein-encoding sequence and the method results in expression of a full-length protein in the host cell that is at least 4% (e.g., at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, or more) of the expression level of the full-length protein without the premature stop codon.

[0199] In some embodiments, the target uridine in the target RNA is a premature stop codon in a protein-encoding sequence, the method results in expression of a full-length protein, and the expression of the protein is detectable without enrichment (e.g., without enrichment by immunoprecipitation). In some embodiments, the protein is detected via a tag (e.g., via a fluorescent tag). In some embodiments, the protein is detected by immunostaining according to methods known in the art.

[0200] In some embodiments, the target uridine in the target RNA is a premature stop codon in a protein-encoding sequence and the method results in expression of the full-length protein in at least 20% of the host cells (e.g., at least 25%, at least 30%, or at least 35%, at least 40%, at least 45%, at least 50%, or more percentages of the host cells).

[0201] Also provided is an engineered gsnoRNA composition or an engineered RNA editing system as described herein for use in any one of the methods described herein, such as a method of editing a target RNA or a method of treatment. Use of any one of the engineered gsnoRNA compositions or engineered RNA editing systems as described herein in the preparation of a medicament for the treatment of a disease or condition.

[0202] A. Treatment methods In some aspects, the methods provided herein include modifying a target RNA using a gsnoRNA that recruits DKC1 protein to modify the target RNA. In some embodiments, the gsnoRNA hybridizes to a target sequence that includes a target uridine residue, and the modification of the RNA includes modifying the target uridine to a pseudouridine.

[0203] In some embodiments, the target RNA is endogenous RNA of a cell (e.g., a eukaryotic cell such as a mammalian cell or a human cell). In some embodiments, the target RNA is RNA endogenously transcribed of the cell (e.g., transcribed from an endogenous nucleic acid sequence of the cell). In some embodiments, the target RNA is transcribed from a nucleic acid sequence introduced into the cell (e.g., RNA transcribed from an exogenously added nucleic acid molecule). In some embodiments, the target RNA is ribosomal RNA. In some embodiments, the target RNA is messenger RNA (mRNA).

[0204] In some embodiments, the sequence containing the target uridine in the target RNA is a stop codon, and modification of the target uridine to pseudouridine causes the stop codon to be translated as a coding codon. In some embodiments, the stop codon is a premature stop codon (PTC). In some embodiments, the PTC is associated with an inherited disease or condition. By using the means and methods of the present disclosure, conversion of the target uridine in such a PTC to pseudouridine then results in proper read-through of the reading frame during translation, thereby providing a (partially or fully) functional full-length protein.

[0205] In some embodiments, provided herein is a method of treating a disease or condition associated with a PTC in a target RNA in a subject, comprising editing a target RNA in a cell of the subject using any of the RNA editing methods described herein, wherein the gsnoRNA comprises a guide sequence that hybridizes to the PTC in the target RNA, and modification of a uridine residue in the PTC to a pseudouridine residue causes translational read-through of the PTC in the target RNA, thereby treating the disease or condition in the subject.

[0206] In some embodiments, a method of treating a disease or condition associated with a PTC in a target RNA in a subject comprises introducing an engineered gsnoRNA into a host cell of the subject, the gsnoRNA comprises a guide sequence that hybridizes to a PTC comprising a uridine residue in the target RNA, the gsnoRNA comprises a scaffold sequence derived from wild type ACA2b, ACA36, ACA44, ACA27, E2, ACA3, or ACA17, and the gsnoRNA recruits a DKC1 protein in the host cell to modify the target uridine residue in the target RNA to a pseudouridine residue. In some embodiments, the DKC1 protein is an endogenous DKC1 protein of the host cell. In some embodiments, the method further comprises introducing a nucleic acid encoding the DKC1 protein into the host cell. In some embodiments, the DKC1 protein has a cytoplasmic localization in the host cell. In some embodiments, the DKC1 protein comprises a DKC1 protein fragment corresponding to amino acid residues 41-420 of human DKC1 isoform 3 protein, where amino acid numbering is according to SEQ ID NO: 2. In some embodiments, the DKC1 protein comprises an amino acid sequence having at least 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity to SEQ ID NO: 88. In some embodiments, the DKC1 protein comprises the amino acid sequence of SEQ ID NO: 88.

[0207] In some embodiments, a method of treating a disease or condition associated with a PTC in a target RNA in a subject comprises introducing an engineered gsnoRNA into a host cell of the subject, the gsnoRNA comprising a guide sequence that hybridizes to a PTC comprising a target uridine residue in the target RNA, the gsnoRNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4-6, 9-12, 15-19, 22-36, and 177-179, and the gsnoRNA recruits a DKC1 protein in the host cell to modify the target uridine residue in the target RNA to a pseudouridine residue. In some embodiments, the DKC1 protein is an endogenous DKC1 protein of the host cell. In some embodiments, the method further comprises introducing a nucleic acid encoding the DKC1 protein into the host cell. In some embodiments, the DKC1 protein has a cytoplasmic localization in the host cell. In some embodiments, the DKC1 protein comprises a DKC1 protein fragment corresponding to amino acid residues 41-420 of human DKC1 isoform 3 protein, where amino acid numbering is according to SEQ ID NO: 2. In some embodiments, the DKC1 protein comprises an amino acid sequence having at least 85% (e.g., at least about any one of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity to SEQ ID NO: 88. In some embodiments, the DKC1 protein comprises the amino acid sequence of SEQ ID NO: 88.

[0208] In some embodiments, the gsnoRNA is half of a gsnoRNA (e.g. comprising a single hairpin and an H box, or a single hairpin and an ACA box). In some embodiments, the gsnoRNA comprises or consists of any one of the sequences set forth in SEQ ID NOs: 89-100 and 113-128, as shown in Figure 12B and Figure 13.

[0209] In some embodiments, a method of treating a disease or condition associated with a PTC in a target RNA in a subject comprises introducing an engineered gsnoRNA into a host cell of the subject, wherein the gsnoRNA comprises a sequence selected from SEQ ID NOs: 71-84. Exemplary engineered gsnoRNA sequences targeting uridine residues of exemplary disease-associated PTCs are shown in Table 4.

[0210] In some embodiments, a method for treating a disease or condition associated with a PTC in a target RNA in a subject comprises introducing (a) an engineered gsnoRNA and (b) a splice-switching antisense oligonucleotide (ASO) into a host cell of the subject, wherein the gsnoRNA comprises a guide sequence that hybridizes to a sequence comprising a target uridine residue in the target RNA, and the ASO promotes expression of DKC1 protein, an endogenous DKC1 isoform with cytoplasmic localization, in the host cell, and the gsnoRNA recruits the DKC1 protein to modify the target uridine residue in the target RNA to a pseudouridine residue. In some embodiments, the splice-switching ASO binds to the pre-mRNA of the DKC1 gene and directs splicing of DKC1 isoform 3. In some embodiments, introduction of a splice-switching ASO increases expression of DKC1 protein, an endogenous DKC1 isoform with cytoplasmic localization, in a host cell by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold compared to expression of the same isoform in a host cell in the absence of the ASO. In some embodiments, administration of a splice-switching ASO increases expression of DKC1 isoform 3 by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold compared to expression of DKC1 isoform 3 in a host cell in the absence of the ASO. In some embodiments, the gsnoRNA comprises a scaffold sequence derived from a wild type H / ACA-snoRNA selected from the group consisting of ACA19, ACA2b, ACA36, ACA44, ACA27, E2, ACA3, and ACA17. In some embodiments, the gsnoRNA comprises a scaffold sequence derived from ACA2b. In some embodiments, the gsnoRNA comprises a scaffold sequence derived from ACA36. In some embodiments, the gsnoRNA comprises a scaffold sequence derived from ACA19.In some embodiments, the gsnoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3 to 12, 15 to 19, 22 to 36, and 177 to 179. In some embodiments, the gsnoRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15 to 19.

[0211] In some embodiments, the disease or condition is cystic fibrosis, Hurler syndrome, alpha-1-antitrypsin (A1AT) deficiency, Parkinson's disease, Alzheimer's disease, albinism, amyotrophic lateral sclerosis, asthma, 8-thalassemia, Cadasil syndrome, Charcot-Marie-Tooth disease, chronic obstructive pulmonary disease (COPD), distal spinal muscular atrophy (DSMA), Duchenne / Becker muscular dystrophy, dystrophic epidermolysis bullosa, bullous epidermolysis, Fabry disease, factor V Leiden-related disorder, familial adenomatous polyposis, galactosemia, Gaucher disease, glucose-6-phosphate dehydrogenase, hemophilia, hereditary hemochromatosis, Hunter syndrome, Huntington's disease, inflammatory bowel disease (IBD), hereditary polyaggregation syndrome, syndrome, Leber's congenital amaurosis, Lesch-Nyhan syndrome, Lynch syndrome, Marfan syndrome, mucopolysaccharidoses, muscular dystrophies, myotonic dystrophy types I and II, neurofibromatosis, Niemann-Pick disease types A, B, and C, NY-esol-associated cancer, Peutz-Jeghers syndrome, phenylketonuria, Pompe disease, primary ciliary diseases, prothrombin mutation-associated disorders (such as prothrombin G20210A mutation), pulmonary hypertension (autosomal dominant), retinitis pigmentosa, Sandhoff disease, severe combined immunodeficiency syndrome (SCID), sickle cell anemia, spinal muscular atrophy, Stargardt disease, Tay-Sachs disease, Usher syndrome, X-linked immunodeficiency, Sturge-Weber syndrome, and cancer. Exemplary diseases or conditions associated with PTCs in target RNA are listed in the Human Gene Mutation Database (HGMDR (available at hgmd.cf.ac.uk)) and the ClinVar database (see Landrum et al. ClinVar: improvements to accessing data. Nucleic Acids Res. 2020;48(D1):D835-D844; available at ncbi.nlm.nih.gov / clinvar / intro). In some embodiments, threonine or serine is incorporated at the ΨAA codon and the ΨAG codon, and phenylalanine or tyrosine is incorporated at the ΨGA codon.

[0212] In some embodiments, the present disclosure provides for the use of a nucleic acid molecule (encoding an engineered gsnoRNA described herein) in the manufacture of a medicament for the treatment of one or more of the diseases listed herein. In some embodiments, an engineered gsnoRNA is provided herein for use in the treatment of cystic fibrosis (CF). Exemplary PTCs associated with CF are known in the art, for example as described in International Patent Publication WO2019191232, the contents of which are incorporated herein by reference in their entirety. Exemplary cystic fibrosis-associated PTC mutations include, but are not limited to, G542X (UGA), W1282X (UGA), R553X (UGA), R1162X (UGA), Y122X (UAA), W1089X, W846X, and W401X mutations, which can be modified into codons encoding amino acids via pseudouridylation, thereby allowing translation into full-length proteins. For example, it is well established in the art that instead of being regarded as a stop codon, both ΨAA and ΨAG codons are translated into serine or threonine, while ΨGA is translated into tyrosine or phenylalanine (Karijolich and Yu, 2011). In some embodiments, the host cell is an archaeal cell or a eukaryotic cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a human cell. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed ex vivo.

[0213] The disclosed method can be applied to suppress NMD of disease-associated PTCs and / or promote PTC read-through for a wide range of known disease-associated PTCs. There are numerous human diseases resulting from nonsense mutations in the respective disease genes. For example, Usher syndrome is an inherited retinal dystrophy (IRD) that is a major cause of concomitant hearing impairment and blindness. Nonsense mutations occur in 12% of Usher syndrome patients and have been described in different genes (such as the USH2A gene). Some Hurler syndrome patients, who suffer from skeletal abnormalities and cognitive dysfunction, carry nonsense mutations in the IDUA gene that prevent the production of functional full-length IDUA protein in these patients. A significant proportion of cystic fibrosis (CF) cases, a disease that affects the lungs and digestive system, are due to nonsense mutations in the CFTR gene. PTCs resulting from these nonsense mutations have been identified in the coding region at multiple different sites, each of which leads to a complete lack of functional full-length CFTR protein. Nonsense mutations are also found in several associated cancer genes in many cancer patients, resulting in a complete lack of full-length protein product. Given the deleterious role of nonsense mutations in gene expression and disease, nonsense suppression represents an attractive strategy and ultimate goal to combat these diseases.

[0214] C. Delivery to Target Cells In some embodiments, the methods provided herein include delivering (e.g., administering) a gsnoRNA and / or DKC1 protein, or a nucleic acid encoding a gsnoRNA and / or a nucleic acid encoding a DKC1 protein, to a host cell containing a target RNA. The amount, dosage, and dosing regimen of the administered gsnoRNA-encoding nucleic acid and / or DKC1 protein may vary depending on differences between cell types, the disease being treated, the target population, the mode of administration (e.g., systemic vs. local), the severity of the disease, and the acceptable level of side activity, but these can and should be assessed by trial and error during in vitro studies, in preclinical and clinical trials. Testing is particularly straightforward when the modified sequence leads to an easily detectable phenotypic change.

[0215] In some embodiments, the methods comprise delivering one or more nucleic acids (e.g., a gsnoRNA, or a nucleic acid encoding a gsnoRNA and / or a nucleic acid encoding a DKC1 protein) and / or a preformed gsnoRNA-protein complex (which may comprise a gsnoRNA, a DKC1 protein, a NOP10 protein, a GAR1 protein, and / or an NHP2 protein) to a cell (e.g., a mammalian or human cell). Exemplary intracellular delivery methods include, but are not limited to, viruses or virus-like agents; chemical-based transfection methods (such as those using calcium phosphate, dendrimers, liposomes, or cationic polymers such as DEAE dextran or polyethyleneimine); non-chemical methods (such as microinjection, electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, bacterial conjugation, delivery of plasmids or transposons); particle-based methods (such as using a gene gun, magnetofection or magnet-assisted transfection, particle bombardment, etc.); and hybrid methods (such as nucleofection). In some embodiments, the application further provides cells produced by such methods, and organisms (e.g., non-human mammals) that contain or are produced from such cells.

[0216] Methods of non-viral delivery of nucleic acid include lipofection, nucleofection, microinjection, particle bombardment, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid complexes, naked DNA, artificial virions, and drug-facilitated DNA uptake. Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., TRANSFECTAMINE™ and LIPOFECTAMIN®). In some embodiments, LIPOFECTAMINE® 2000 is used to transfect nucleic acid encoding gsnoRNA and / or DKC1 protein (e.g., nucleic acid vector encoding gsnoRNA and / or DKC1 protein).

[0217] One suitable testing technique involves delivering the nucleic acid molecule according to the present disclosure into a cell extract, cell line, or test organism, and then taking biopsy samples at various time points thereafter. The sequence of the target RNA can be assessed in the biopsy sample, and the percentage of cells with the modification can be easily tracked. Even after this test is performed once, the findings are retained and future deliveries can be performed without the need to then take a biopsy sample. The method of the present disclosure can thus include identifying the presence of the desired change in the target RNA sequence of the cell, thereby confirming that the target RNA sequence is modified. The change can be assessed on the protein level (length, glycosylation, function, or the like), or by some functional readout (such as (induced) current), for example, if the protein encoded by the target RNA sequence is an ion channel. In the case of CFTR function, Ussing chamber assays or NPD tests in mammals, including humans, are well known to those skilled in the art to assess restoration or gain of function.

[0218] After pseudouridylation occurs in a cell, the modified RNA may become diluted over time due to, for example, cell division, the limited half-life of the edited RNA, etc. Thus, in practical therapeutic terms, the methods of the present disclosure may involve repeated delivery of oligonucleotides until enough of the target RNA is modified to provide a tangible benefit to the patient and / or maintain the benefit over time.

[0219] In some embodiments, gsnoRNAs can be delivered to cells in the form of naked nucleic acids. Another way in which such constructs (gsnoRNAs and / or DKC1 proteins, or nucleic acids encoding gsnoRNAs and / or nucleic acids encoding DKC1 proteins) can be delivered to cells (either in vitro, ex vivo, or in vivo) is by the use of delivery vehicles (such as viral vectors).

[0220] Conventional virus-based systems for nucleic acid delivery include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex virus vectors. Integration in the host genome is possible by retroviral, lentiviral, and adeno-associated virus methods, often resulting in long-term expression of the inserted transgene. Additionally, high transduction efficiency has been observed in different cell types. The tropism of retroviruses can be altered by incorporating exogenous envelope proteins, increasing the potential target population of target cells. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing high viral titers. Retroviral vectors are composed of cis-acting long terminal repeats with the capacity to package exogenous sequences up to 6-10 kb. The minimal cis-acting LTRs are sufficient for replication and packaging of the vector, which is then used to integrate the nucleic acid into the target cell to provide persistent transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof. In applications where transient expression is preferred, adenovirus-based systems can be used. Adenovirus-based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. High titers and high levels of expression have been obtained with such vectors. This vector can be produced in large quantities in a relatively simple system.

[0221] Packaging cells are typically used to form viral particles capable of infecting host cells. Such cells include 293T cells, which package adenovirus, and ψ2 or PA317 cells, which package retrovirus. Viral vectors are usually produced by the production of cell lines that package nucleic acid vectors into viral particles. The vector typically contains the minimal viral sequences required for packaging and subsequent integration into the host, with other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. Missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically retain only the ITR sequences from the AAV genome, which are required for packaging and integration into the host genome. Viral DNA is packaged in the cell line, which contains a helper plasmid that encodes other AAV genes (i.e., rep and cap) but lacks the ITR sequences. The cell line can also be infected by adenovirus as a helper. Helper virus promotes the replication of AAV vector and the expression of AAV genes from helper plasmid.Helper plasmid is not packaged in significant amounts due to the lack of ITR sequence.Adenovirus contamination can be reduced, for example, by heat treatment, and adenovirus is more sensitive to heat treatment than AAV.

[0222] In some embodiments, the viral vector is based on adeno-associated virus (AAV). In some embodiments, the viral vector is, for example, a retroviral vector (such as lentiviral vectors and the like). Also, plasmids, artificial chromosomes, and plasmids usable for targeted homologous recombination and integration in the human genome of cells may be suitably applied for the delivery of gsnoRNA as described herein. In some embodiments, when gsnoRNA is delivered by a viral vector, it is in the form of an RNA transcript that includes the sequence of an oligonucleotide according to the present disclosure in a portion of the transcript. In some embodiments, the AAV vector according to the present disclosure is a recombinant AAV vector, and refers to an AAV vector that includes, encapsidated, a portion of the AAV genome that includes an exon-intron-exon sequence according to the present disclosure in a protein shell of a capsid protein derived from an AAV serotype. The portion of the AAV genome may contain an inverted terminal repeat (ITR) derived from an adeno-associated virus serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and others. The protein shell, which is composed of capsid proteins, can be derived from AAV serotypes such as AAV1, 2, 3, 4, 5, 6, 7, 8, 9, and others. The protein shell can also be named capsid protein shell. AAV vectors can be deleted of one or all wild-type AAV genes, but still contain functional ITR nucleic acid sequences. Functional ITR sequences are necessary for the replication, rescue, and packaging of AAV virions. ITR sequences can be wild-type sequences, or can have at least 80%, 85%, 90%, 95, or 100% sequence identity with wild-type sequences, or can be modified, for example, with nucleotide insertions, mutations, deletions, or substitutions, so long as they remain functional. In this context, functionality refers to the ability to direct packaging of the genome into a capsid shell and then allow expression in the infected host or target cell. In the context of the present disclosure, the capsid protein shell can be of a different serotype than the AAV vector genome ITR.An AAV vector according to the present disclosure may thus be composed of a capsid protein shell (i.e., an icosahedral capsid comprising the capsid proteins (VP1, VP2, and / or VP3) of one AAV serotype (e.g., AAV serotype 2), but the ITR sequences contained in the AAV2 vector may be of any of the AAV serotypes described above, including AAV2 vectors. Thus, an "AAV2 vector" comprises an AAV serotype 2 capsid protein shell, while, for example, an "AAV5 vector" comprises an AAV serotype 5 capsid protein shell, thereby either may encapsidate any AAV vector genomic ITRs according to the present disclosure. In some embodiments, a recombinant AAV vector according to the disclosure comprises an AAV serotype 2, 5, 8, or AAV serotype 9 capsid protein shell, and the AAV genome or ITRs present in said AAV vector are derived from AAV serotype 2, 5, 8, or AAV serotype 9; such AAV vectors are referred to as AAV2 / 2, AAV2 / 5, AAV2 / 8, AAV2 / 9, AAV5 / 2, AAV5 / 5, AAV5 / 8, AAV5 / 9, AAV8 / 2, AAV8 / 5, AAV8 / 8, AAV8 / 9, AAV9 / 2, AAV9 / 5, AAV9 / 8, or AAV9 / 9 vectors.

[0223] In some embodiments, a recombinant AAV vector according to the present disclosure comprises a capsid protein shell of AAV serotype 2 and the AAV genome or ITRs present in the vector are derived from AAV serotype 5; such a vector is referred to as an AAV2 / 5 vector. In some embodiments, a recombinant AAV vector according to the present disclosure comprises a capsid protein shell of AAV serotype 2 and the AAV genome or ITRs present in the vector are derived from AAV serotype 8; such a vector is referred to as an AAV2 / 8 vector. In some embodiments, a recombinant AAV vector according to the present disclosure comprises a capsid protein shell of AAV serotype 2 and the AAV genome or ITRs present in the vector are derived from AAV serotype 9; such a vector is referred to as an AAV2 / 9 vector. In some embodiments, a recombinant AAV vector according to the present disclosure comprises an AAV serotype 2 capsid protein shell, and the AAV genome or ITRs present in the vector are derived from AAV serotype 2; such vectors are referred to as AAV2 / 2 vectors. In some embodiments, a nucleic acid molecule carrying an exon-intron-guide RNA-intron-exon sequence according to the present disclosure represented by a selected nucleic acid sequence is inserted between the AAV genome or ITR sequences identified above, for example, between an expression construct comprising an expression regulatory element operably linked to a coding sequence and a 3' termination sequence. "AAV helper functions" generally refer to the corresponding AAV functions required for AAV replication and packaging that are provided to the AAV vector in trans. AAV helper functions complement AAV functions that are missing in the AAV vector but lack the AAV ITRs (which are provided by the AAV vector genome). AAV helper functions include the two major ORFs of AAV (i.e., the rep coding region and the cap coding region, or functionally substantially identical sequences thereof). The Rep and Cap regions are well known in the art. AAV helper functions can be provided on an AAV helper construct, which can be a plasmid.

[0224] Introduction of the helper construct into the host cell can occur, for example, by transformation, transfection, or transduction, prior to or simultaneously with the introduction of the AAV genome present in the AAV vector identified herein. Thus, the AAV helper construct of the present disclosure can be selected to produce the desired combination of serotypes, on the one hand, for the capsid protein shell of the AAV vector, and on the other hand, for the AAV genome present during replication and packaging of said AAV vector. An "AAV helper virus" provides additional functions required for AAV replication and packaging.

[0225] Suitable AAV helper viruses include adenovirus, herpes simplex virus (such as HSV types 1 and 2), and vaccinia virus. As described in US 6,531,456, additional functions provided by helper viruses can also be introduced into host cells via vectors. In some embodiments, the AAV genome present in a recombinant AAV vector according to the present disclosure does not include nucleotide sequences encoding viral proteins (such as the AAV rep (replication) or capsid genes). The AAV genome may further include marker or reporter genes (such as antibiotic resistance genes, genes encoding fluorescent proteins (e.g., gfp), or genes encoding chemically, enzymatically, or otherwise detectable and / or selectable products known in the art (e.g., lacZ, aph, etc.)). In some embodiments, the AAV vector according to the present disclosure is an AAV2 / 5 vector, an AAV2 / 8 vector, an AAV2 / 9 vector, or an AAV2 / 2 vector.

[0226] In some embodiments, gsnoRNA and DKC1 are delivered to cells as ribonucleoprotein complexes (e.g., complexes containing gsnoRNA, DKC1, NOP10, GAR1, and / or NHP2). Methods for intracellular delivery of proteins or protein complexes (such as preformed gsnoRNA-DKC1 / NOP10 / GAR1 / NHP2 complexes) include, but are not limited to, mechanical methods (such as microinjection, electroporation, and mechanical deformation of cells using microfluidic devices); carrier-based methods (such as cell-penetrating peptides (CPPs), virus-like particles, supercharged proteins, nanocarriers, supramolecular carrier-based delivery systems, and nanoparticle-stabilized nanocapsules). See, for example, Fu et al. Bioconjugate Chem. 2014, 25, 1602-1608. Some mechanical methods (such as microinjection and electroporation) can be invasive and low throughput. In some embodiments, ribonucleoprotein complexes are delivered into cells by inserting the complexes through the cell membrane while passing the cells through a microfluidic system (such as CELL SQUEEZE®) (see, e.g., U.S. Patent Application Publication No. 20140287509).

[0227] As described above, the introduction of the nucleic acid molecule according to the present disclosure into cells is accomplished by general methods known to those skilled in the art. After pseudouridylation, the effect readout (mutation of target RNA sequence) can be monitored through different techniques in an optional identification step. Thus, the identification step of whether the desired pseudouridylation of the target uridine has actually occurred generally depends on the position of the target uridine in the target RNA sequence and the effect caused by the presence of the uridine (point mutation, PTC). Thus, in some embodiments, depending on the final effect of the U to Ψ conversion, the identification step includes the assessment of the presence of functional, extended, full-length, and / or wild-type protein; the assessment of whether the splicing of the pre-mRNA has been altered by pseudouridylation; or the use of functional readout that the target RNA after pseudouridylation codes for a functional, full-length, extended, and / or wild-type protein. The functional assessment for each of the diseases mentioned herein will generally follow methods known to those skilled in the art.

[0228] The nucleic acid molecule (such as a gsnoRNA expression construct or a vector according to the present disclosure) is preferably administered in an aqueous solution (e.g., saline) or suspension, optionally including additives, excipients, other ingredients compatible with pharmaceutical use. Administration may be by injection or infusion, intranasal, oral inhalation (e.g., via nebulization), intravenous, subcutaneous, intradermal, intracranial, intravitreal, intramuscular, intratracheal, intraperitoneal, intrarectal, and the like. Administration may be in the form of a solid, powder, pill, or other form compatible with pharmaceutical use in humans. The present disclosure is particularly suitable for the treatment of genetic diseases (CF).

[0229] In some embodiments, the nucleic acid molecule (such as gsnoRNA, expression construct, or vector) can be delivered systemically. In some embodiments, the nucleic acid molecule (such as gsnoRNA, expression construct, or vector) can be delivered to cells or locally to tissues where the phenotype of the target sequence is observed. For example, mutations in CFTR cause CF to be found primarily in lung epithelial tissues, and therefore, in some embodiments involving CFTR target sequences, the oligonucleotide construct is delivered specifically and directly to the lungs. This can be conveniently achieved, typically through the use of a nebulizer, for example by inhalation of a powder or aerosol. In some embodiments, the nebulizer is a so-called vibrating mesh nebulizer, such as the PARI eFlow (Rapid) or the i-neb from Respironics. It is expected that inhalation delivery of oligonucleotide constructs according to the present disclosure can also efficiently target these cells and lead to the amelioration of gastrointestinal symptoms associated with CF in the case of CFTR gene targeting. In some diseases, the mucus layer shows an increased thickness, leading to a decrease in the absorption of pharmaceuticals through the lungs. One such disease is chronic bronchitis, and another example is CF. Various mucus normalizing substances are available, such as DNase, hypertonic saline, or mannitol, which is commercially available under the name Bronchitol. When mucus normalizing substances are used in combination with pseudouridinated oligonucleotide constructs (such as gsnoRNA constructs according to the present disclosure), they can increase the efficacy of pharmaceuticals. Thus, administration of an oligonucleotide construct according to the present disclosure to a subject (such as a human subject) can be combined with a mucus normalizing substance. In addition, administration of an oligonucleotide construct according to the present disclosure can be combined with administration of a small molecule (such as a potentiator compound (e.g., Kalydeco (ivacaftor; VX-770)), or a collector compound (e.g., VX-809 (lumacaftor) and / or VX-661)) for the treatment of CF.Alternatively, or in combination with a mucus normalizing agent, delivery in mucus-penetrating particles or nanoparticles may be applied for efficient delivery of, for example, pseudouridinated molecules to lung and intestinal epithelial cells. In some embodiments, administration of an oligonucleotide construct according to the present disclosure to a subject (such as a human subject) is combined with antibiotic treatment to reduce bacterial infection and symptoms of bacterial infection, such as mucus increase and / or biofilm formation. Antibiotics may be administered systemically or locally or both. For application in CF patients, an oligonucleotide construct according to the present disclosure, or a packaged or complexed oligonucleotide construct according to the present disclosure, is combined with any mucus normalizing agent (such as DNase, mannitol, hypertonic saline, and / or antibiotics) and / or small molecules for the treatment of CF (such as potentiator compounds (e.g., ivacaftor), or collector compounds (e.g., lumacaftor and / or VX-661)). To increase access to target cells, Bronchio-Alveolar Favage (BAF) can be applied to lavage the lungs prior to administration of oligonucleotides according to the present disclosure.

[0230] IV. Pharmaceutical Compositions, Kits, and Articles of Manufacture In some embodiments, provided herein is a pharmaceutical composition comprising any of the gsnoRNAs, nucleic acid constructs / molecules, or engineered RNA editing systems described herein and a pharma- ceutically acceptable carrier.

[0231] Pharmaceutical compositions may be prepared in the form of lyophilized formulations or aqueous solutions by mixing the therapeutic agents described herein having the desired purity with optional pharma- ceutically acceptable carriers, excipients, or stabilizing substances (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizing substances are non-toxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants (including ascorbic acid, methionine, vitamin E, sodium metabisulfite); preservatives, isotonicity substances (e.g., sodium chloride), stabilizing substances, metal complexes (e.g., Zn-protein complexes); chelating agents (such as EDTA and / or nonionic surfactants).

[0232] In some embodiments, the pharmaceutical composition is contained in a single-use vial (single-use sealed vial). In some embodiments, the pharmaceutical composition is contained in a multi-use vial. In some embodiments, the pharmaceutical composition is contained in bulk in a container. In some embodiments, the pharmaceutical composition is stored frozen.

[0233] In some embodiments, the pharmaceutical composition comprises a gsnoRNA. In other embodiments, the pharmaceutical composition comprises a nucleic acid construct (e.g. a vector such as a plasmid vector or a viral vector) encoding the gsnoRNA. In some embodiments, the pharmaceutical composition comprises free gsnoRNA ("naked" gsnoRNA) or conjugated to other components for uptake and / or intracellular trafficking (such as ligands for targeting). The gsnoRNA may be used in aqueous solution (typically a pharma- ceutically acceptable carrier and / or solvent) or may be formulated using transfection agents, liposomes, or nanoparticle forms (e.g. SNALP, LNP, and the like). Such formulations may include functional ligands to promote bioavailability and the like.

[0234] The present application further provides kits and articles of manufacture for use in any of the embodiments of the methods of treatment described herein. The kits and articles of manufacture may include any one of the formulations and pharmaceutical compositions described herein.

[0235] In some embodiments, a kit for editing a target RNA in a host cell is provided herein, comprising any of the gsnoRNA or nucleic acid molecules described in Section IIB. In some embodiments, the kit further comprises an agent for promoting expression of endogenous DKC1 isoform 3 in a host cell. In some embodiments, the kit comprises a splice-switching antisense oligonucleotide (ASO), which promotes expression of a DKC1 protein that is an endogenous DKC1 isoform with cytoplasmic localization in a host cell. In some embodiments, the kit further comprises a DKC1 protein or a nucleic acid encoding the DKC1 protein. In some embodiments, the DKC1 protein is a DKC1 isoform with cytoplasmic localization (e.g., isoform 3). In some embodiments, the DKC1 protein is part of a ribonucleoprotein (RNP) complex that associates with gsnoRNA. In some embodiments, the DKC1 protein comprises a deletion of a nuclear localization signal (NLS) compared to a wild-type DKC1 protein of the same species. In some embodiments, the DKC1 protein is a truncated DKC1 variant or a DKC1 variant containing a deletion (such as any of the truncated or deleted variants described in Section IIA above). In some embodiments, the kit further comprises instructions for editing the target RNA according to any of the methods described herein.

[0236] In some embodiments, a kit for editing a target RNA in a host cell is provided herein, the kit comprising an engineered RNA editing system, the engineered RNA editing system comprising (a) a gsnoRNA comprising a guide sequence that hybridizes to a sequence comprising a target uridine residue in a target RNA in a host cell, or a nucleic acid molecule encoding the gsnoRNA; and (b) a DKC1 protein, or a nucleic acid molecule encoding the DKC1 protein, the gsnoRNA being capable of recruiting the DKC1 protein to modify the target uridine residue in the target RNA to a pseudouridine residue. In some embodiments, the DKC1 protein is a DKC1 isoform with cytoplasmic localization. In some embodiments, the DKC1 protein is a DKC1 isoform (e.g., isoform 3) with cytoplasmic localization. In some embodiments, the DKC1 protein is part of a ribonucleoprotein (RNP) complex that associates with a gsnoRNA. In some embodiments, the DKC1 protein comprises a deletion of a nuclear localization signal (NLS) compared to a wild-type DKC1 protein of the same species. In some embodiments, the DKC1 protein is a truncated DKC1 variant or a DKC1 variant containing a deletion (such as any of the truncated or deleted variants described in Section IIA above). In some embodiments, the kit further comprises instructions for editing the target RNA according to any of the methods described herein.

[0237] The kit of the present disclosure is 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. The kit may optionally provide additional components, such as buffers and interpretive information. The present application therefore also provides articles of manufacture, including vials (such as sealed vials), bottles, jars, flexible packaging, and the like.

[0238] The instructions for use of the composition generally include information regarding dosages, dosing schedules, and routes of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. For example, kits may be provided that contain sufficient dosages of gsnoRNA and / or DKC1 protein, or nucleic acid molecules encoding gsnoRNA and / or DKC1 protein disclosed herein, to provide effective treatment of an individual or a number of individuals. Additionally, kits may be provided that contain sufficient dosages of gsnoRNA and / or DKC1 protein, or nucleic acid molecules encoding gsnoRNA and / or DKC1 protein, to allow multiple administrations to an individual. The kits also include multiple unit doses of the pharmaceutical composition and instructions for use, packaged in sufficient quantities for storage and use in pharmacies (e.g., hospital pharmacies and compounding pharmacies).

[0239] In some embodiments, the kit comprises a delivery system. The delivery system may be a unit dose delivery system. The delivery system for these various dosage forms may be a syringe, a dropper bottle, a plastic squeeze unit, an atomizer, a nebulizer, or a pharmaceutical aerosol, either in a unit dose package or in a multi-dose package. In some embodiments, a delivery system is provided for any one of the nucleic acid molecules encoding gsnoRNA and / or DKC1 protein or gsnoRNA and / or DKC1 protein described herein, including a nucleic acid molecule encoding gsnoRNA and / or DKC1 protein and a device for delivery of gsnoRNA and / or DKC1 protein, or a nucleic acid molecule encoding gsnoRNA and / or DKC1 protein.

[0240] All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. EXAMPLES

[0241] The present disclosure will be more fully understood by reference to the following examples. However, they should not be interpreted as limiting the scope of the present disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes in light thereof will be suggested to those skilled in the art and are encompassed within the spirit and scope of the present application and the scope of the appended claims.

[0242] Example 1. Utilization of engineered guide snoRNAs for readthrough of premature stop codons This example demonstrates the efficiency of pseudouridylation of target RNA by engineered guide snoRNAs (e.g., pseudouridylation of mRNA, as evidenced by pseudouridylation-dependent PTC readthrough) and provides a different expression system for the guide snoRNA.

[0243] To achieve site-specific pseudouridylation in mRNA in vivo, artificial guide snoRNAs (gsnoRNAs) were engineered to target specific mRNAs for modification (Figure 1A). H / ACA snoRNAs contain two hairpins followed by H-box and ACA-box motifs, and both hairpins of the engineered snoRNAs provided herein contain guide sequences capable of targeting PTC sites. To assess the efficiency of PTC read-through, a Venus reporter (reporter-1) was designed to express the Venus fluorescent reporter gene with an amber codon (TAG) inserted between the 154th and 155th amino acid codons to prematurely terminate Venus translation. Such a reporter allows the measurement of the efficiency of PTC read-through by monitoring the expression level of Venus. A positive control with a glycine codon (GGT) at the same position was included (Figure 1B). To assay PTC read-through as a result of modification of the corresponding stop codon by snoRNA-guided pseudouridylation, reporter-1 (VenusTAG) or control (Venus-GGT) was co-transfected with gsnoRNA expression constructs (gCtrl (SEQ ID NO: 14), gACA19 (SEQ ID NO: 37), gACA44 (SEQ ID NO: 38), gACA27 (SEQ ID NO: 39), gE2 (SEQ ID NO: 40), gACA19-S (SEQ ID NO: 41), or gACA19-L (SEQ ID NO: 42)) into HEK293T cells. The effect of PTC read-through was measured by a high content imaging system and quantified by comparison with the positive control group (Figure 1C, 1D). Relative Venus expression is reported as the percentage (%) of Venus detected compared to the control (Venus-GGT). These gsnoRNAs served as the first generation RESTART (RESTART v1).

[0244] The sequence of the control gsnoRNA (gCtrl) is provided below with the guide region underlined (SEQ ID NO: 14). [ka]

[0245] In the human genome, over 90% of snoRNA genes are encoded in pre-mRNA introns 1We first evaluated the effect of PTC read-through mediated by multiple gsnoRNAs located in host gene introns (RESTART v1.0). We first selected four endogenous snoRNAs with high expression levels in humans2, including ACA19, ACA44, ACA27, and E2 (in host genes EIF3A, SNHG12, RPL21, RPSA, respectively) (Figures 2A-2C and 3A-3F), and engineered gsnoRNAs based on these scaffolds to target Venus reporter PTCs. The host gene fragments containing snoRNAs were cloned into a construct driven by a CMV promoter. After co-transfection of reporter-1 with constructs expressing these gsnoRNAs (host-gCtrl; host-gACA19, host-gACA-44, host-gACA27, and host-gE2), evidence of PTC read-through indicated by Venus expression was observed, with 5.2% and 5.0% Venus positive cells (compared to the control Venus-GGT reporter) being detected in cells transfected with host-gACA19 and host-gE2, respectively, while the others showed negligible signals (Figure 2A). Venus expression was apparently sequence-dependent, since the control gsnoRNA (gCtrl) was unable to activate Venus expression. We found that gACA19 and gE2 scaffolds (which showed higher activity than the other scaffolds) were predicted to have more stable secondary structures than gACA44 and gACA27 (Figures 3A-3D), suggesting that the higher efficiency of target modification evidenced by PTC readthrough correlates with the stability of the secondary structure. To test the effect of the host gene sequence carrying the gsnoRNA on PTC readthrough efficiency, we performed further comparisons by cloning different gsnoRNAs into the intron between exon 2 and exon 3 of the hemoglobin subunit β (HBB) gene.gACA19 again showed the highest efficiency in mediating PTC readthrough for reporter-1 (relative Venus-positive cells: 7.3%), and gE2 showed the second highest efficiency (relative Venus-positive cells: 1.8%) (Figure 2B).

[0246] Based on our observation that there are diverse effects on gsnoRNAs with different host gene sequences (as shown in Figures 2A-2B), we assumed that directly expressing gsnoRNAs without host gene effects could further increase the efficiency of PTC read-through. Therefore, we designed a series of gsnoRNA expression constructs driven by hU6 (type III RNA polymerase III promoter) and hU1 (snRNA type RNA polymerase II promoter) promoters (RESTART v1.1) (Figures 1C-1D and Figure 2C) and co-transfected them with reporter-1 into HEK293T cells. The PTC read-through efficiency of hU6 promoter-driven gACA19 was increased by 1.9- and 1.3-fold compared to that of gACA19 embedded in the host gene intron and gACA19 embedded in the HBB intron, respectively (Figures 1C-1D and Figures 2A-2B). The efficiency of hU6 promoter-driven gsnoRNA is similar to that of hU1 promoter-driven gsnoRNA (Figure 2C). The effect of PTC read-through was further characterized by extending or shortening gsnoRNA. No obvious effect was observed from the extension of gACA19 (gACA19-L, 9 nucleotides at 5' and 9 nucleotides at 3'), whereas the shortened gACA19 (gACA19-S, 3 nucleotides at 5') reduced reporter-1 PTC read-through efficiency to 35% compared to full-length gACA19 (Figure 1D and Figure 2E-2F). Since gsnoRNA driven by small RNA promoters showed higher efficiency in mediating PTC read-through compared to intron-embedded gsnoRNA, we selected gsnoRNA driven by hU6 promoter to carry out subsequent analysis.

[0247] To determine whether endogenous DKC1 protein is responsible for the above observations, we performed RESTART v1.1 on HEK293T cells with stable knockdown of DKC1 (DKC1 KD) (Figure 1E). Although PTC readthrough was not observed for gsnoRNAs from DKC1-KD cells, these gsnoRNAs activated the expression of Venus in the control group (Figure 1F), supporting the role of endogenous DKC1 in mediating PTC readthrough of reporter-1 (Figure 1A). Taken together, these observations demonstrated that gsnoRNAs can induce PTC readthrough of targeted transcription.

[0248] Example 2: Optimization of gsnoRNA scaffold improves the efficiency of PTC readthrough To identify optimal gsnoRNA scaffolds, we used RNAfold as candidate scaffolds for further characterization (RESTART v1.2). 3 We selected five snoRNAs (gACA3, gACA17, gACA19, gACA2b, and gACA36) with stable secondary structures predicted by (Figure 4A and Figure 5A). We designed snoRNA expression constructs consisting of hU6 promoter-driven gsnoRNA and CMV promoter-driven BFP gene (used to normalize transfection efficiency) (Figure 4B). Among them, gACA36 and gACA2b showed the most efficient PTC read-through (relative Venus positive cells were 13.7% and 12.2%, respectively), surpassing gACA19 (Figure 4C-4D). gACA19 has a minimum free energy of -37.10 kcal / mol, gACA2b has a minimum free energy of -54.90 kcal / mol, and gACA36 has a minimum free energy of -43.50 kcal / mol. There appears to be no direct relationship between gsnoRNA scaffold stability and editing efficiency.

[0249] To investigate the role of the two hairpins of gsnoRNA, we introduced mutations in the 5' and 3' guide elements, respectively (Figure 4A, Figure 5A, and Figure 6A). The editing efficiency of gACA19 5' hairpin mutation (gACA19-5m) was comparable to that of gACA19, while gACA19-3m showed reduced efficiency (Figure 4E-4F). For gACA36, the editing efficiency of gACA36-3m was comparable to that of gACA36, while gACA36-5m showed negligible signal (Figure 6B). These results indicated that only one hairpin of gACA19 / gACA36 plays a major role, and two hairpins of gsnoRNA targeting the same site may not compete with each other.

[0250] Next, we sought to further improve the PTC read-through efficiency by engineering the gsnoRNA scaffold (RESTART v1.3) (Figures 4E-4F and 3-4). Considering that RNA polymerase III terminates transcription at a small poly-U stretch, we introduced single base mutations into the "UUUU" sequence in the apical loop of gACA19 (Figures 4A and 5C). Remarkably, both gACA19-UUCU and gACA19-UGUU showed improvement (Figures 4E-4F). Without being bound by theory, we recognized that altering the distance in the gsnoRNA hairpin such that the distance between the nucleotide in the guide region hybridizing to the target uridine and the H / ACA box was 14 nucleotides increased the editing efficiency of gsnoRNA. In one example, we inserted a single base after U115 of gACA19 such that the distance between the nucleotide in the guide region that hybridizes to the target uridine and the H / ACA box is 14 nucleotides (Figures 4A and 5D). gACA19-3addG increased the efficiency by 1.4-fold compared to unmodified gACA19 (Figures 4E-4F). Furthermore, without being bound by theory, we found that making the guide element of the gsnoRNA hairpin more open (e.g., decreasing the base pairing probability of the secondary structure in the guide region) can increase the editing efficiency of the gsnoRNA. To make the guide element more open, we inserted a dinucleotide after U8 in the 5' hairpin of gACA19 (Figures 4A and 5D). Remarkably, gACA19-5addCU increased the PTC readthrough efficiency by 60% (Figures 4E-4F). However, the engineered gACA36 scaffold did not further improve the efficiency of PTC readthrough (Figures 6A-6B). We also expressed two tandem gsnoRNAs combining optimized mutations of gACA19, but neither improved the efficiency further.

[0251] Example 3: Spatial proximity effect of gsnoRNA and target PTC sites We next asked whether the spatial proximity of gsnoRNA and target PTC site has an effect on the efficiency of PTC read-through. We designed two new reporters: (1) Reporter-2 contains a PTC site between the coding regions of mCherry and EGFP and was activated by gsnoRNA from RESTART v1.3. mCherry was utilized to normalize transfection efficiency. (2) In Reporter-3 (RESTART v1.4), gsnoRNA was arranged in tandem with a PTC reporter, which is the same PTC reporter as Reporter-2. GsnoRNA had comparable efficiency in suppressing PTC in both Reporter-2 and Reporter-1, indicating that gsnoRNA works on a different reporter. Unexpectedly, gsnoRNA increased the PTC read-through efficiency in Reporter-3 (relative EGFP positive cells: approx. 30%, approx. 2-fold compared to RESTART v1.3).

[0252] Example 4. RESTART enables PTC readthrough in multiple cell lines We tested RESTART v1.4 in four different cell lines originating from separate tissues, including three human and one mouse cell line. Efficient PTC read-through events were observed for all cell lines tested, suggesting that the disclosed gsnoRNA design is a versatile strategy to suppress PTCs in different mammalian cell types.

[0253] Example 5. Increasing DKC1 isoform 3 expression significantly improves PTC readthrough Notably, neither the combination of optimized mutations nor increasing gsnoRNA expression levels by transfection of a construct of two tandem gsnoRNAs further increased PTC read-through, suggesting that RESTART v1.3 provides gsnoRNAs with optimal structure and expression levels. Based on our recognition that the engineered gsnoRNAs of the present disclosure provided optimized gsnoRNA structure and expression levels, we suspected that enzyme levels and accessibility, rather than gsnoRNA stability and expression, might be the rate-limiting factor. DKC1 is involved in snoRNA-guided attachment of pseudouridine and the concomitant PTC read-through in RESTART (Figure 1A, 1F). There are two DKC1 isoforms in human cells. DKC1 isoform 1 is the canonical DKC1 form that contains bipartite N- and C-terminal nuclear localization signals (NLS). DKC1 isoform 3 is an alternative splicing variant that is produced by retention of intron 12 and lacks the C-terminal NLS (Figure 7A). The endogenous mRNA expression level of isoform 1 is approximately 20-fold higher than that of isoform 3. 4 .

[0254] First, we generated a cell line stably overexpressing DKC1 and transfected reporter-3 into the cells overexpressing DKC1 isoform 1 (Figures 7B-7C). Overexpression of DKC1 isoform 1 only slightly increased the relative percentage of EGFP-positive cells and the relative EGFP intensity by 1.2-fold and 1.3-fold, respectively, compared to those of control cells (Figures 7D-7F). Surprisingly, in isoform 3-overexpressing cells, the relative percentage of EGFP-positive cells and the relative EGFP intensity were greatly increased by 2.5-fold and 5.2-fold, respectively (Figures 7D-7F). These observations were further confirmed by cotransfecting reporter-1 and gsnoRNA constructs into cells stably overexpressing DKC1 (Figures 8A-8C). To further investigate the transient role of DKC1, reporter-3 was cotransfected together with the construct expressing DKC1. Again, transient overexpression of isoform 3 greatly increased PTC readthrough (Figure 8D). The N-terminal NLS of DKC1 isoform 3 was also deleted, and these truncations had similar PTC readthrough efficiency as isoform 3 (Figure 9). These unexpected results demonstrate that exogenous DKC1 isoform 3 can significantly improve the efficiency of PTC readthrough, achieving 61.4% EGFP positive cells (compared to the control reporter) and 13.2% EGFP intensity (compared to the control reporter). gsnoRNA and DKC1 isoform 3 served as the second generation RESTART (RESTART v2).

[0255] To further characterize RESTART, an additional set of reporter 3 was constructed to contain all three types of stop codons, and the resulting reporter constructs were transfected into HEK293T with and without exogenous DKC1 isoform 3 (Figure 7B). The efficiency of RESTART-mediated readthrough positively correlated with that of basal or drug-induced translational readthrough. 5, the opal codon (UGA) had the highest readthrough, followed by the amber codon (UAG) and then the ochre codon (UAA) (Figures 7G-7H and 10A-10D). For the UGA (opal) codon, the relative percentage of EGFP-positive cells and the relative EGFP intensity were 45.3% and 5.8% (RESTART v1.4), and 72.3% and 28.6% (RESTART v2), respectively. Meanwhile, the UAA codon showed negligible signal without exogenous DKC1 (RESTART v1.4), and 2.9% relative EGFP-positive cells and 0.2% relative EGFP intensity with DKC1 isoform 3 overexpression (RESTART v2) (Figures 7G-7H and 10A-10B). Increasing the amount of DKC1 isoform 3 expression construct improved PTC readthrough of UAA (ochre) codons to 14.8% relative EGFP-positive cells, but still only 25% and 19% compared to UAG (amber) and UGA (opal), respectively (Figures S10C-S10E). Taken together, RESTART promoted readthrough of all three nonsense codons.

[0256] Next, reporter-3 constructs for each of the three stop codons were individually cotransfected with 200 ng of the DKC1 isoform 3 expression construct into HEK293T cells. Targeted locus-specific pseudouridine modifications were analyzed using a qPCR-based method without radioisotope labeling. 6 The melting curves were detected by (Figure 7I and Figures 11A-11C) for all three stop codons (Figure 7I), while negligible changes were observed for the gCtrl group lacking the Ψ modification (Figure 11A). In contrast, the melting curve changes for the Ψ1045 site in 18S rRNA were comparable between the gACA19 and gCtrl groups (Figures 11A-11C). Collectively, these results demonstrate that gsnoRNA-guided pseudouridylation by DKC1 isoform 3 can efficiently promote readthrough of all three PTC codons.

[0257] Example 6. RESTART suppresses disease-associated PTC This example demonstrates the correction of disease-associated premature stop codons (PTCs) using RESTART. RNA-guided pseudouridylation of disease-associated PTCs by the RESTART system resulted in expression of full-length gene products. Furthermore, restoration of protein function using RESTART was demonstrated for the CFTR gene containing disease-associated PTCs. In the following examples, "X" indicates the stop codon mutation. The sequences of the gsnoRNAs tested are provided in Table 4.

[0258] A PTC disease reporter was constructed in which the disease gene containing the PTC site was followed by EGFP (as shown in Figure 12A). gACA19-based gsnoRNA and gACA36-based gsnoRNA were designed and tested targeting seven disease-associated nonsense mutations from six pathogenic genes (PEX7, SMN1, ALDOB, C8orf37, PCCB, and CBS) (Figures 12B-13). PTC read-through was achieved at all sites by co-expression of the gsnoRNA / PTC disease gene pair in HEK293T cells (RESTARTv1). 6.7% (ALDOB-W148X expressing cells), 25.2% (SMN1-W190X), 33.8% (PEX7-R232X), 1.7% (C8orf37-W185X), 38.8% (PCCB-R111X), 22.1% (CBS-C275X), and 8.0% (CBS-W390X) of EGFP-positive cells were detected compared to the positive control, respectively (Figure 14A). Next, PTC readthrough of disease genes was tested for RESTARTv2 (overexpression of DKC1 isoform 3) (Figure 14B). Overexpression of DKC1 isoform 3 (RESTARTv2) increased the relative percentage of EGFP-positive cells (indicating PTC readthrough) by about 2.8-fold on average compared to RESTARTv1. For cells expressing ALDOB-W148X and CBS-W390X, the relative percentage of EGFP-positive cells was greatly increased by overexpression of the three DKC1 isoforms (by 4.8- and 6.3-fold, respectively), as shown in Figures 14A-14B.

[0259] RESTART was further validated for inhibition of disease-associated PTCs LMNA-R225X (associated with familial dilated cardiomyopathy (DCM) with conduction defect (DCM-CD)), F9-Y22X and F9-G21X (associated with hemophilia B), ABCA4-R408X (associated with Stargardt disease), RS1-Y65X (associated with X-linked retinoschisis), and Rpe65-R44X (associated with Leber's congenital amaurosis), as shown in FIG. 14C.

[0260] Finally, restoration of protein function using RESTART was demonstrated for the CFTR CFTR (cystic fibrosis transmembrane conductance regulator) gene containing disease-associated PTCs. Mutations in CFTR cause the monogenic disease cystic fibrosis, which occurs in approximately 1:2500 live births in Caucasians. The ability of RESTART to repair CFTR R553X (CGA-TGA) and W1282X (TGG-TGA) PTC sites and restore protein function was tested by electrophysiology assays, the "gold standard" for the evaluation of functional rescue of CFTR. After delivery of RESTART, the function of CFTR containing PTCs could be rescued to approximately 30% of wild-type CFTR levels, demonstrating the therapeutic potential of RESTART in targeting certain monogenic diseases.

[0261] Example 7. Delivery of RESTART in a clinically relevant gsnoRNA format This example demonstrates the design and synthesis of functional oligonucleotides for gsnoRNA delivery to cells.

[0262] Full-length gsnoRNA oligonucleotides were prepared by in vitro transcription (IVT). To increase the stability of gsnoRNA oligonucleotides in cells, 5' cap modifications (m 7A 5' cap modification (G(5')ppp(5')G cap analog) was added to the gsnoRNA oligonucleotide. The 5' cap modification is not present in endogenous intronic snoRNA. As an example, a 5' cap modified full-length gACA19 oligonucleotide targeting reporter-2 (rACA19) was prepared by in vitro transcription (Figure 15A-C). Notably, rACA19 increased the efficiency of PTC read-through for both RESTARTv1 and RESTARTv2 compared to the gACA19 expression construct vector (Figure 15D; data shown as mean ± standard deviation).

[0263] As shown in Figure 15E, chemically synthesized rACA19 oligonucleotide halves with 2'-O methyl and phosphorothioate linkage modifications were prepared and tested for their ability to achieve efficient PTC read-through in cells ("P" indicates phosphorothioate linkage, "2'O-methyl" indicates 2'O-methyl modified nucleoside). gsnoRNA was delivered to cells by transfection.

[0264] Advantageously, gsnoRNA oligonucleotide halves facilitate chemical synthesis compared to full-length gsnoRNAs (approximately 130 nucleotides), which are too long to synthesize efficiently. Furthermore, rH5 and rH3 oligonucleotides were synthesized with only six phosphorothioate linkages and four 2'O-methyl modifications per oligonucleotide, indicating that a small number of modifications is sufficient to promote the stability and function of chemically synthesized gsnoRNA halves. The 5' hairpin construct (gH5, with H box) and the 3' hairpin construct (gH3, with ACA box) reduced the efficiency of PTC read-through compared to the gACA19 oligonucleotide prepared by IVT. However, both the rH5 and rH3 oligonucleotides (which have the same sequence as gH5 and gH3) exhibited efficiencies comparable to the full-length gACA19 construct (Figure 15D).

[0265] These results show that gsnoRNA can be effectively delivered to cells as full-length RNA oligonucleotides (e.g. with a 5' cap to increase stability) prepared by in vitro transcription, or as half of an oligonucleotide containing a 5' or 3' hairpin prepared by chemical synthesis. Furthermore, the data demonstrate that chemically synthesized rH3 or rH5 with only six phosphorothioate linkages and four 2'O-methyl modifications are stable and functional in cells. Advantageously, the use of chemically synthesized rH3 and rH5 oligonucleotides with fewer modifications can reduce the cost of preparing chemically synthesized oligonucleotides. The delivered RNA oligonucleotides may perform better than the same construct delivered to cells as a DNA vector encoding the same gsnoRNA construct.

[0266] References 1.Dieci, G., Preti, M. & Montanini, B.Eukaryotic snoRNAs: a paradigm for gene expression flexibility.Genomics 94,83-8 (2009).

[0267] 2. Jorjani, H. et al. An updated human snoRNAome. Nucleic Acids Res 44,5068-82 (2016).

[0268] 3. Gruber, AR, Lorenz, R., Bernhart, SH, Neubock, R. & Hofacker, IL The Vienna RNA websuite. Nucleic Acids Res 36, W70-4 (2008).

[0269] 4.Angrisani,A.,Turano,M.,Paparo,L.,Di Mauro,C.& Furia,M.A new human dyskerin isoform with cytoplasmic localization.Biochim Biophys Acta 1810,1361-8 (2011).

[0270] 5.Dabrowski,M.,Bukowy-Bieryllo,Z.& Zietkiewicz,E.Translational readthrough potential of natural termination codons in eukaryotes--The impact of RNA sequence.RNA Biol 12,950-8(2015).

[0271] 6.Lei,Z.& Yi,C.A Radiolabeling-Free,qPCR-Based Method for Locus-Specific Pseudouridine Detection.Angew Chem Int Ed Engl 56,14878-14882(2017).

Claims

**Claim 1** An RNA editing system comprising: (a) a gsn oRNA or a nucleic acid molecule encoding the gsn oRNA, the gsn oRNA comprising a guide sequence that hybridizes to a sequence containing a target uridine residue in a target RNA in a host cell, the gsn oRNA being capable of recruiting a DKC1 protein in the host cell to modify the target uridine residue in the target RNA to a pseudouridine residue; (b) a DKC1 protein or a nucleic acid molecule encoding the DKC1 protein. An RNA editing system comprising the above. **Claim 2** The RNA editing system according to claim 1, wherein the DKC1 protein comprises a DKC1 protein without an N-terminal NLS sequence and / or a C-terminal NLS sequence. **Claim 3** The RNA editing system according to claim 1, wherein the DKC1 protein is an endogenous DKC1 protein of the host cell. **Claim 4** The RNA editing system according to claim 2, wherein the DKC1 protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2 or 88. **Claim 5** The RNA editing system according to claim 2, wherein the DKC1 protein comprises a DKC1 protein fragment corresponding to amino acid residues 41 to 420 of the human DKC1 isoform 3 protein, wherein the amino acid numbering follows SEQ ID NO:

2. **Claim 6** The RNA editing system according to claim 2, wherein the DKC1 protein is the human DKC1 isoform 3 protein. **Claim 7** The RNA editing system according to claim 1, wherein the gsn oRNA comprises a scaffold sequence derived from the scaffold sequence of a wild-type H / ACA-snoRNA ("wild-type scaffold sequence"), the scaffold sequence comprising one or more mutations, additions and / or substitutions. **Claim 8** The RNA editing system according to claim 7, wherein the wild-type H / ACA-snoRNA is selected from the group consisting of ACA19, ACA36, ACA2b, ACA44, ACA27, E2, ACA3, and ACA17. **Claim 9** The RNA editing system according to claim 8, wherein the wild-type H / ACA-snoRNA is ACA19 or ACA36. **Claim 10**: The RNA editing system according to claim 7, wherein the scaffold array contains one or more substitution mutations in the nucleotides of the poly-U sequence within the wild-type scaffold array, and the poly-U sequence contains at least four consecutive U residues. **Claim 11**: The RNA editing system according to claim 10, wherein the one or more mutations are selected from the group consisting of substitution of residues 26-29 with UUCU, substitution of residues 26-29 with UGUU, addition of G to the 3' hairpin structure after residue 115, and addition of CU to the 5' hairpin after residue 8, where the numbering follows SEQ ID NO:

37. **Claim 12**: The RNA editing system according to claim 7, wherein the gsnRNA contains one or more mutations, additions, and / or deletions in a guide region that reduces the base pairing probability of one or more residues. **Claim 13**: The RNA editing system according to claim 7, wherein the gsnRNA contains one or more insertion or deletion mutations located between the position corresponding to the target uridine and the H / ACA box of the gsnRNA, such that the gsnRNA contains 14 or 15 nucleotides between the position corresponding to the target uridine and the H / ACA box. **Claim 14**: The RNA editing system according to claim 7, wherein the gsnRNA contains a scaffold array contained in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4-6, 9-12, 15-19, 22-36, 177-180, 37-70, 171-173, 71-84, and 181-192. **Claim 15**: The RNA editing system according to any one of claims 1-14, wherein the system contains a nucleic acid molecule encoding the DKC1 protein or a nucleic acid molecule encoding the gsnRNA. **Claim 16**: The RNA editing system according to any one of claims 1-14, wherein the system contains a nucleic acid molecule encoding the DKC1 protein and a nucleic acid molecule encoding the gsnRNA. **Claim 17**: The RNA editing system according to any one of claims 1-14, wherein the system contains a vector containing a first nucleic acid sequence encoding the DKC1 protein and a second nucleic acid sequence encoding the gsnRNA.

18. The RNA editing system according to claim 17, wherein the vector is a viral vector.

19. The RNA editing system according to claim 18, wherein the vector is an adeno-associated virus (AAV) vector.

20. The RNA editing system according to any one of claims 1 to 14, wherein the target RNA is mRNA.

21. Use of the RNA editing system according to any one of claims 1 to 14 for the manufacture of a medicament for the treatment of a disease or condition associated with a PTC in a target RNA.

22. A pharmaceutical composition for editing a target RNA in a host cell, comprising the RNA editing system according to any one of claims 1 to 14.

23. A pharmaceutical composition for use in treating a disease or condition associated with a PTC in a target RNA, comprising the RNA editing system according to any one of claims 1 to 14.

24. A kit for use in treating a disease or condition associated with a PTC in a target RNA, comprising the RNA editing system according to any one of claims 1 to 14.

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