RNA single base editing system and method based on pseudouridine modification

Abstract

The present application relates to the fields of biotechnology and RNA editing. The invention relates to an RNA single base editing system and method based on pseudouridine modification. In particular, the present application relates to a method for inhibiting an early termination codon (PTC) in a target RNA in a host cell. The present application also relates to an engineered guide small nucleolus RNA (gsnoRNA), an isolated nucleic acid molecule comprising the same, a composition, and a host cell. The present application also relates to their use in the preparation of medicaments. According to the method provided by the invention, the efficiency of pseudouridine modification can be remarkably improved, so that the coding information of mRNA is recovered, the PTC site is decoded, and the read-through efficiency of PTC is remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology and the field of RNA editing. Specifically, the present application relates to a method for inhibiting a premature termination codon (PTC) in a target RNA in a host cell. The present application also relates to an engineered guide small nucleolar RNA (gsnoRNA), an isolated nucleic acid molecule comprising the same, a composition, a host cell. The present application also relates to their use in the manufacture of a medicament. BACKGROUND

[0002] Nonsense mutation is a genetic mutation caused by single base substitution in the coding region of mRNA, which converts a normal amino acid-encoding codon into a stop codon, resulting in a premature termination codon (PTC) and leading to premature termination of protein translation. According to the statistics of Human gene mutation database (HGMD), nonsense mutation accounts for more than 20% of disease-related single nucleotide mutations, and accounts for up to 11% of all mutations leading to human genetic diseases.

[0003] Nonsense mutation is associated with a variety of genetic diseases and can cause severe disease phenotypes. For example, nonsense mutation of Alpha-L-iduronidase (IDUA) gene can cause Hurler syndrome, a lysosomal abnormality caused mucopolysaccharidosis that causes glycosaminoglycans to be unable to be metabolized and produce toxic effects, thereby causing damage and dysfunction in multiple systems in the body. In patients with cystic fibrosis, the proportion of nonsense mutation of Cystic fibrosis transmembrane conductance regulator (CFTR) gene reaches ~10%, and the defect of CFTR gene can cause ion transport disorder, accumulation of thick mucus in epithelial cells, and further cause chronic inflammation and irreversible lung damage. Another example is spinal muscular atrophy, and nonsense mutation of Survival motor neuron 1 (SMN1) gene can damage the structure and function of motor neurons, showing progressive muscle weakness and muscle atrophy, and patients show high mortality in infancy. In addition to genetic diseases, nonsense mutation can also occur in some genes related to cancer, such as tumor protein p53 (TP53), and cause its dysfunction. Therefore, exploring strategies to inhibit nonsense mutation is extremely important for the treatment of a variety of diseases.

[0004] Given its significant harm to patient health, developing effective treatment strategies has become a research focus. Currently, scientists are exploring various methods to address the challenges posed by nonsense mutations, including using translational readthrough-inducing drugs to interfere with ribosome recognition of PTCs, using suppressive tRNAs to recognize PTCs and incorporate specific amino acids, applying DNA editing strategies to correct mutations in gene sequences, and using RNA editing strategies to restore the coding information of mRNA.

[0005] As one of the most widely studied RNA editing strategies, ADAR-based RNA editing tools also play an important role in nonsense mutation suppression. Researchers designed gRNAs to enable ADAR to target and edit A in PTCs, converting UGA codons or UAG codons to UGG codons and incorporating tryptophan at PTC sites, thereby restoring the expression of full-length proteins. However, since ADAR can only achieve A-to-G editing, this strategy can only insert tryptophan at PTC sites. For some critical PTC sites that cannot tolerate missense mutations, further exploration of other RNA editing strategies that can accurately insert specific amino acids is needed to achieve more effective repair.

[0006] In addition to ADAR-based RNA editing strategies, researchers are also exploring other RNA editing strategies in the hope of achieving more extensive nonsense mutation repair. Among them, the U-to-Ψ editing strategy has gradually become an important direction in nonsense mutation research due to its unique base modification characteristics and endogenous target modification mechanisms. In 2011, Yu's laboratory first discovered that pseudouridine modification of PTCs could suppress nonsense mutations in in vitro experiments and yeast cells, restoring the expression of full-length proteins. This discovery sparked researchers' enthusiasm for exploring pseudouridine modification to suppress nonsense mutations.

[0007] To avoid the delivery difficulty and immunogenicity caused by overexpression of exogenous proteins, researchers first considered using the endogenous pseudouridylation mechanism to achieve targeted modification. To this end, they systematically explored the catalytic mechanism of H / ACA box snoRNP that modifies rRNA and snRNA in a yeast system. The study found that pseudouridylation requires three core sequence and structural elements: the pseudouridylation pocket of snoRNA and the stability of the hairpin structure, the fixed distance of 14-15 nt between the target uridine and the H / ACA box, and the base pairing strength between the pseudouridylation pocket sequence and the target sequence. In addition, researchers also tried to explore the mechanism of pseudouridine modification PTC to suppress nonsense mutations. They proposed that pseudouridine modification can affect the interaction between the codon-anticodon pair at the PTC site, making some tRNAs bind to the PTC site stronger than the release factor, thereby inhibiting translation termination and incorporating specific amino acids. By identifying the amino acids incorporated at the pseudouridylation PTC site in yeast cells, researchers found that ΨAA and ΨAG codons mainly incorporate threonine or serine, while ΨGA codons mainly incorporate phenylalanine or tyrosine. This result further suggests that pseudouridylation can affect the coding rules at the PTC site in a specific way, thereby achieving precise incorporation of certain amino acids at the PTC site.

[0008] Overall, the U-to-Ψ editing system based on H / ACA box snoRNP is a highly flexible nonsense mutation suppression strategy. Its flexibility in targeting sequence pairing and diversity in amino acid incorporation enable it to play an important role in the treatment of a wide range of nonsense mutation-related diseases. In addition, this strategy only regulates endogenous transcripts and does not affect ribosome function, demonstrating higher safety and precision. Therefore, further development and optimization of the U-to-Ψ editing system and exploration of its effects in the context of nonsense mutation diseases are of great scientific significance and application value.

[0009] Prior to this, the applicant developed a new programmable RNA pseudouridylation editing system, RESTART, which achieved precise pseudouridine modification at the PTC site by synergistically utilizing the endogenous targeting modification mechanism of pseudouracil and the regulatory properties of the stop codon, promoting PTC readthrough and restoring full-length functional protein expression, thereby efficiently and specifically repairing nonsense mutations. On this basis, multiple generations of systems (RESTART v1, v2, v3, and v3-mini) were developed. However, the editing efficiency of these systems still has room for improvement, so it is necessary to optimize the design of the components in the system and regulate the expression level of endogenous proteins to improve the overall modification and readthrough level. Therefore, it is necessary to develop a new RNA editing system and mediate nonsense mutation repair. SUMMARY

[0010] The present application is based on the optimization of the previously studied RESTART pseudouridine modification system, and provides a modified gsnoRNA which can efficiently bind to the target site and improve the recruitment and assembly rate of pseudouridine synthetase DKC1 and NHP2, GAR1 and NOP10 core proteins, thereby significantly improving the efficiency of pseudouridine modification.

[0011] Therefore, in a first aspect, the present application provides a method for inhibiting a premature termination codon (PTC) in a target RNA in a host cell, characterized in that the method comprises: introducing an engineered guide small nucleolar RNA (gsnoRNA) or a nucleic acid for expressing the gsnoRNA into the host cell, wherein the gsnoRNA comprises: (i) at least one guide sequence, (ii) at least one CAB box and / or CTE (constitutive transport element) element, and (iii) a scaffold sequence; wherein, the guide sequence hybridizes to a sequence of a target uridine residue (U) containing the PTC in the target RNA; the CAB box is derived from the loop region of the stem-loop structure of a natural scaRNA, AluRNA or htrRNA (Human Telomerase RNA); the scaffold sequence is derived from a natural snoRNA of H / ACA type structure and / or a natural scaRNA of H / ACA type structure.

[0012] There are two types of natural snoRNAs and scaRNAs, one of which is C / D type structure and the other is H / ACA type structure, and the H / ACA type structure can recruit DKC1 protein. Therefore, in this context, the gsnoRNA can recruit DKC1 protein in the host cell and modify the target uridine residue in the target RNA into a pseudouridine residue.

[0013] In certain embodiments, the scaffold sequence is not limited to a specific length and sequence as long as it retains the ability of snoRNA or scaRNA and DKC1 protein interaction. In certain embodiments, the length of the scaffold sequence is 30-50 nt, 50-80 nt, 80-100 nt, 100-130 nt, 130-150 nt, 150-200 nt, or longer.

[0014] In certain embodiments, the scaffold sequence is an entire snoRNA derived from a natural H / ACA type structure (e.g., comprising two hairpins, an H box, an ACA box) or a portion thereof (e.g., comprising 1 hairpin, an H box, an ACA box). In some embodiments, the gsnoRNA comprises a single hairpin and an H box, but not an ACA box. In some embodiments, the gsnoRNA comprises a single hairpin and an ACA box, but not an H box.

[0015] In certain embodiments, the scaffold sequence is an entire scaRNA derived from a natural H / ACA type structure (e.g., comprising two hairpins, an H box, an ACA box) or a portion thereof (e.g., comprising 1 hairpin, an H box, an ACA box).

[0016] In certain embodiments, to enhance in vivo stability and delivery efficiency, a gsnoRNA can comprise one or more modified (e.g., chemically modified) nucleotides. In certain embodiments, one or more nucleotides of the gsnoRNA comprise a 2’-O-methyl (2’-OMe) modification. In some embodiments, the gsnoRNA comprises one or more phosphorothioate (PS) internucleoside linkages. In some embodiments, the gsnoRNA comprises a 5’ cap modification (e.g., a m7G cap modification).

[0017] In some embodiments, the methods, gsnoRNAs, and compositions provided herein comprise modifying a target RNA (e.g., mRNA) in a eukaryotic organism (e.g., a mammalian cell, such as a human cell). In some aspects, the host cell can be a cell from any organ, e.g., skin, lung, heart, kidney, liver, pancreas, intestine, muscle, gland, eye, brain, blood, etc. In certain embodiments, the host cell is a mammalian cell. In certain embodiments, the host cell is a human cell. The host cell can be located in vitro or in vivo. In certain embodiments, the host cell is an ex vivo cell.

[0018] An advantage of the methods, gsnoRNAs, and compositions provided herein is that they can be used both for in situ cells in a living organism and for cells in culture in vitro. In some embodiments, the host cell is treated in vitro and then introduced into a living organism (e.g., reintroduced into the organism from which they were originally derived).

[0019] The methods, gsnoRNAs, and compositions provided herein can also be used to readthrough PTCs or recode Ψ-modified codons in cells within so-called organoids. Organoids can be thought of as three-dimensional in vitro derived tissues, but are driven using specific conditions to produce individual tissues. In a therapeutic setting, organoids can be synthesized in vitro and reintroduced into a patient as autologous material, which is less likely to be rejected than normal transplants.

[0020] In some embodiments, the host cell has a genetic mutation. The mutation can be heterozygous or homozygous. In some embodiments, the methods, gsnoRNAs, and compositions provided herein can be used to modify a point mutation (e.g., to readthrough a PTC caused by a point mutation or to recode a point mutation in a sense codon). In some embodiments, the methods, gsnoRNAs, and compositions provided herein are suitable for modifying an RNA sequence in a cell, tissue, or organ related to a disease state of a subject (e.g., a human subject) when the human subject has a disease associated with a PTC.

[0021] In the methods of the application, the gsnoRNA can be introduced into the host cell using any suitable method. Also, the methods are capable of inhibiting a premature termination codon (PTC) in a target RNA in a host cell and significantly increasing the readthrough efficiency of the PTC under any method of introducing the gsnoRNA.

[0022] In certain embodiments, the gsnoRNA is introduced into the host cell by direct delivery. In certain embodiments, the gsnoRNA is introduced into the host cell by transfecting the host cell with a vector comprising a nucleotide sequence that expresses the gsnoRNA.

[0023] The methods of the application can effectively restore the coding information of an mRNA without changing the DNA sequence of the host cell, cause the PTC site to be decoded, and significantly increase the readthrough efficiency of the PTC, thereby expressing a complete protein.

[0024] In certain embodiments, the PTC is caused by a mutation in a sense codon. In certain embodiments, the methods described above are capable of significantly increasing the readthrough efficiency of the PTC. In certain embodiments, the readthrough efficiency of the PTC of the methods described above is at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, or at least 20-fold higher than the readthrough efficiency of the PTC of other methods that do not use the gsnoRNAs described above. For example, the methods described above of the application have a PTC readthrough efficiency that is at least 2-fold higher than RESTART v3 mini.

[0025] When the method of the application is used, the gsnoRNA is introduced into the host cell, the guide sequence in the gsnoRNA base pairs complementarily with the target mRNA, enabling the uridine residue U in the PTC to enter the pseudouridylation pocket formed by the stem-loop structure of the gsnoRNA; the gsnoRNA recruits the DKC1, NOP10, NHP2 and GAR1 proteins endogenously and / or exogenously introduced into the cell to assemble into an RNP complex, and the uridine residue in the pseudouridylation pocket is able to enter the catalytic center of DKC1, thereby enabling the catalysis of the modification of the uridine residue U into a pseudouridine residue Ψ. Further, the nc-tRNA endogenously and / or exogenously introduced into the cell binds to the Ψ-modified PTC and is translated into an amino acid, enabling the readthrough of the site of the PTC.

[0026] The gsnoRNA of the application can co-modify and decode the PTC with the DKC1 protein expressed endogenously and / or the nc-tRNA expressed endogenously.

[0027] In certain embodiments, the gsnoRNA of the application can be delivered alone to enable the readthrough of the PTC. In this case, the elements required for other modifications and decoding (e.g., DKC1 protein, nc-tRNA) are only provided by the cell endogenously. That is, the introduction of the engineered guide small nucleolar RNA (gsnoRNA) or the nucleic acid for expressing the gsnoRNA into the host cell is able to enable the suppression of the premature termination codon (PTC) in the target RNA in the host cell.

[0028] The gsnoRNA of the application can co-modify and decode the PTC with the DKC1 protein expressed exogenously and / or the nc-tRNA expressed exogenously.

[0029] In certain embodiments, a nucleotide sequence encoding the DKC1 protein can be delivered to enable the pseudouridine modification with the DKC1 protein expressed exogenously. In certain embodiments, the method further comprises introducing the nucleotide sequence encoding the DKC1 protein into the host cell.

[0030] In certain embodiments, a nucleotide sequence encoding the nc-tRNA or the nc-tRNA molecule can be delivered directly to enable the pseudouridine modification with the nc-tRNA expressed exogenously. In certain embodiments, the method further comprises introducing the nucleotide sequence encoding the nc-tRNA or the nc-tRNA into the host cell.

[0031] Therefore, in the method of the application, the source of the DKC1 protein and the nc-tRNA does not affect the implementation of the method of the application. For example, the DKC1 protein and the nc-tRNA can both be from the expression endogenously in the cell, can both be from the expression exogenously in the cell, or one can be from the expression endogenously in the cell and the other can be from the expression exogenously in the cell.

[0032] CAB box and scaffold sequence A CAB box (Cajal-body localization box) element is a short RNA motif that localizes to Cajal bodies.

[0033] In some embodiments, the CAB box has a sequence as shown below: X1X2AG; wherein each of X1and X2is independently selected from any one of A, U, C, G.

[0034] In some embodiments, the CAB box has a nucleotide sequence selected from the group consisting of UGAG, AAAG, GAAG, UAAG, UCAG, CGAG, AUAG, GCAG, CUAG, CAAG, and AGAG.

[0035] In some embodiments, the CAB box has a sequence of AGAG.

[0036] In some embodiments, the CAB box enhances the efficiency of assembly of the gsnoRNA with core proteins (e.g., DKC1, NOP10, NHP2, GAR1) and the ability to form a complete snoRNP (gsnoRNA, DKC1, NOP10, NHP2, and GAR1).

[0037] In some embodiments, the scaffold sequence comprises a first hairpin structure near the 5' end, and a second hairpin structure near the 3' end; and the CAB box is located in the loop of the first hairpin structure, or in the loop of the second hairpin structure, or in the loops of both the first and second hairpin structures.

[0038] In some embodiments, the CAB box is located in the loop of the first hairpin structure in the gsnoRNA.

[0039] In some embodiments, the gsnoRNA comprises a CAB box derived from a natural scaRNA, Alu RNA, or htrRNA, and a sequence in the natural scaRNA, Alu RNA, or htrRNA that comprises the entire loop region of the CAB box.

[0040] The CAB box can be introduced in different ways.

[0041] In one embodiment, in the engineered guide small nucleolar RNA (gsnoRNA), the CAB box can be directly introduced, i.e. the four nucleotide sequence of X1X2AG of the CAB box is directly introduced. In certain embodiments, the four nucleotide sequence of X1X2AG of the CAB box is inserted into the loop of the first hairpin structure, or into the loop of the second hairpin structure, or into the loops of both the first and the second hairpin structure in the gsnoRNA.

[0042] In such embodiments, the four nucleotide sequence of X1X2AG of the CAB box can be inserted between any two nucleotides that are in accordance with the above-mentioned positions, while maintaining the basic structure of the gsnoRNA.

[0043] In a second embodiment, in the engineered guide small nucleolar RNA (gsnoRNA), the entire loop sequence containing the CAB box can also be introduced. That is, the entire loop sequence containing the CAB box from the natural scaRNA, AluRNA or htrRNA from which the CAB box is derived is directly introduced. In certain embodiments, the entire loop sequence containing the CAB box from the natural scaRNA, AluRNA or htrRNA is inserted into the loop of the first hairpin structure, or into the loop of the second hairpin structure, or into the loops of both the first and the second hairpin structure in the gsnoRNA.

[0044] In such embodiments, the entire loop sequence containing the CAB box can be inserted between any two nucleotides that are in accordance with the above-mentioned positions, while maintaining the basic structure of the gsnoRNA.

[0045] In a third embodiment, in the engineered guide small nucleolar RNA (gsnoRNA), the entire or partial loop sequence of the scaffold sequence of the gsnoRNA can also be replaced with the entire loop sequence containing the CAB box. In certain embodiments, the entire loop sequence containing the CAB box from the natural scaRNA, AluRNA or htrRNA is used to replace the loop of the first hairpin structure, or to replace the loop of the second hairpin structure, or to replace the loops of both the first and the second hairpin structure in the gsnoRNA.

[0046] In such embodiments, the entire loop sequence of the first hairpin structure of the scaffold sequence of the gsnoRNA is replaced with the entire loop sequence containing the CAB box.

[0047] In certain embodiments, the natural scaRNA is selected from the group consisting of scaRNA1, scaRNA4, scaRNA5, scaRNA6, scaRNA8, scaRNA11, scaRNA12, scaRNA13, scaRNA14, scaRNA15, scaRNA16, scaRNA18, scaRNA20, scaRNA21, scaRNA22, scaRNA23, scaRNA26, scaRNA85 and / or scaRNA27.

[0048] In certain embodiments, the natural AluRNA is selected from the group consisting of AluACA2, AluACA5, AluACA7, AluACA8, AluACA9, AluACA13, AluACA15, AluACA17, AluACA21, AluACA24, AluACA43, AluACA48, AluACA91, AluACA97, AluACA177, AluACA208, AluACA214 and / or AluACA303.

[0049] In certain embodiments, the natural htrRNA is human telomerase RNA.

[0050] guide sequence A "guide sequence" is the part of a gsnoRNA responsible for target recognition, which consists of a stretch of nucleotide sequence that is complementary to a specific region in the target RNA (e.g. the PTC sequence containing the target uridine residue). The guide sequence base pairs complementarily with the target mRNA, allowing the uridine residue U (e.g. the uridine residue U in the PTC) to enter the pseudouridylated pocket formed by the stem loop structure of the gsnoRNA. The uridine residue U in the pseudouridylated pocket is able to enter the catalytic center of the DKC1 enzyme, thus enabling editing.

[0051] In certain embodiments, the guide sequence is located in the stem of the first hairpin structure, or in the stem of the second hairpin structure, or in the stem of both the first hairpin structure and the second hairpin structure.

[0052] In certain embodiments, the gsnoRNA comprises a first guide sequence and a second guide sequence, and the first guide sequence is located in the stem of the first hairpin structure and the second guide sequence is located in the stem of the second hairpin structure.

[0053] In some embodiments, the gsnoRNA comprises, in the 5' to 3' order, from 5' end to 3' end, a first portion of a first guide sequence, a loop region derived from a CAB box or a stem loop structure of a natural scaRNA, Alu RNA or htrRNA (Human Telomerase RNA), a second portion of the first guide sequence, a first portion of a second guide sequence, a second portion of the second guide sequence, connected by a scaffold sequence.

[0054] In some embodiments, the guide sequence in each hairpin structure is divided into a first portion and a second portion, and the first portion is located at the 5' end side of the stem where it is located, and the second portion is located at the 3' end side of the stem where it is located. That is, in a certain stem loop structure of the gsnoRNA, it comprises, in the 5' to 3' order, from 5' end to 3' end, a first portion of a first guide sequence, a loop region derived from a CAB box or a stem loop structure of a natural scaRNA, Alu RNA or htrRNA (Human Telomerase RNA), a second portion of the first guide sequence, connected by a scaffold sequence.

[0055] In some embodiments, the guide sequence is not limited to a specific length and sequence as long as it can hybridize to the sequence containing the target uridine residue (U) of the PTC in the target RNA. In some embodiments, the length of the guide sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nt or longer.

[0056] In some embodiments, the guide sequence is fully or highly complementary to the specific region containing the target uridine residue (such as the PTC sequence) in the target RNA. For example, the number of bases of the guide sequence that base-pair with the bases of the specific region accounts for at least 70%, at least 80%, at least 90%, or 100% of the total number of bases of the guide sequence, to ensure the specificity and efficiency of hybridization.

[0057] In some embodiments, the method of the present application has high specificity and does not cause false uridylation and read-through of normal stop codons.

[0058] In some embodiments, the natural H / ACA-type snoRNA is selected from the group consisting of ACA19, ACA2b, ACA36, ACA24, ACA5, ACA14a, ACA13, ACA20, ACA44, ACA27, E2, ACA3 and ACA17.

[0059] In certain embodiments, the natural H / ACA box snoRNA is selected from the group consisting of scaRNA 11 and scaRNA 15.

[0060] In certain embodiments, the gsnoRNA comprises a nucleotide sequence selected from the group consisting of any one of SEQ ID NO: 2~25, SEQ ID NO: 28~33, SEQ ID NO: 68~72, SEQ ID NO: 73~78, or SEQ ID NO: 50~61.

[0061] CTE element The CTE element is a segment of RNA element, which can also be referred to as Constitutive Transport Element. It is believed that the CTE sequence can interact with helicases to open the complex secondary structure of the target RNA and thus enhance the accessibility. In certain embodiments, the CTE element is capable of enhancing the binding of the gsnoRNA to the target RNA.

[0062] In certain embodiments, the CTE element is linked to the 5' end of the scaffold sequence of the gsnoRNA, or linked to the 3' end of the scaffold sequence of the gsnoRNA, or linked to both the 5' end and the 3' end of the scaffold sequence of the gsnoRNA.

[0063] In certain embodiments, the CTE element is a CTE element derived from a retrovirus or a truncation thereof, wherein the truncation retains or partially retains the function or activity of the CTE element from which it is derived. In certain embodiments, the CTE element is a CTE element derived from Mason Fisher virus (MPMV) or Simian retrovirus type D (SRV) or a truncation thereof, wherein the truncation retains or partially retains the function or activity of the CTE element from which it is derived.

[0064] In certain embodiments, the truncation retains or partially retains the secondary structure of the CTE element from which it is derived.

[0065] In certain embodiments, a truncation of the CTE element is used to link to the gsnoRNA in order to reduce the molecular weight of the gsnoRNA and to improve the efficiency of direct delivery of the RNA molecule to the host cell.

[0066] In certain embodiments, the sequence of the CTE element is set forth in SEQ ID NO: 79~88.

[0067] In certain embodiments, the gsnoRNA comprises, in the 5' to 3' order, a first portion of a first guide sequence, a CAB box derived from a natural scaRNA, Alu RNA or htrRNA (Human Telomerase RNA) or a loop region comprising a stem-loop structure of the CAB box, a second portion of the first guide sequence, a first portion of a second guide sequence, a second portion of the second guide sequence, and a CTE element, linked by a scaffold sequence.

[0068] In certain embodiments, the CTE element is linked to the scaffold sequence by a linker.

[0069] In certain embodiments, the linker is 4-12 bp in length (e.g., 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp).

[0070] In certain embodiments, the CTE element is linked to the 3' end of the scaffold sequence of the gsnoRNA and is linked by a linker of 4-6 bp or 6-8 bp.

[0071] In certain embodiments, the gsnoRNA comprises a sequence selected from the group consisting of SEQ ID NO: 2~25, SEQ ID NO: 28~33, SEQ ID NO: 68~72, SEQ ID NO: 73~78, or SEQ ID NO: 50~61.

[0072] DKC1 protein In certain embodiments, the gsnoRNA recruits core proteins (DKC1, NHP2, GAR1 and NOP10) and forms a snoRNA with the core proteins to modify the target uridine residue (U) in the target RNA to a pseudouridine residue (Ψ). In certain embodiments, the gsnoRNA recruits DKC1 protein to modify the target uridine residue (U) in the target RNA to a pseudouridine residue (Ψ).

[0073] It can be appreciated that the DKC1 protein can be DKC1 iso1 and / or DKC1 iso3 expressed endogenously by the cell or DKC1 iso1 and / or DKC1 iso3 expressed exogenously.

[0074] In certain embodiments, a gsnoRNA is introduced into the host cell, which hybridizes to the target RNA and recruits DKC1 protein, and modifies the target uridine residue (U) of the PTC contained in the target RNA to a pseudouridine residue (Ψ).

[0075] In certain embodiments, the gsnoRNA-recruited DKC1 protein comprises: an endogenous DKC1 protein of the host cell, and / or, an exogenous DKC1 protein of the host cell.

[0076] In certain embodiments, the method further comprises: introducing into the host cell a nucleic acid molecule encoding a DKC1 protein.

[0077] In certain embodiments, the DKC1 protein is overexpressed in the host cell.

[0078] In certain embodiments, the DKC1 protein is a naturally occurring DKC1 isoform having cytoplasmic localization in the host cell.

[0079] In certain embodiments, the DKC1 is selected from isoform 1 of human DKC1 protein (DKC1 iso1), isoform 3 of human DKC1 protein (DKC1 iso3), or any combination thereof.

[0080] In certain embodiments, the amino acid sequence of isoform 3 of human DKC1 protein (DKC1 iso3) is set forth in SEQ ID NO: 66.

[0081] In certain embodiments, the amino acid sequence of isoform 1 of human DKC1 protein (DKC1 iso1) is set forth in SEQ ID NO: 67.

[0082] In certain embodiments, the DKC1 protein comprises an amino acid sequence that is at least 85%, or at least 85%, or at least 88%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% identical to SEQ ID NO: 66 or 67.

[0083] In certain embodiments, a nucleotide sequence encoding a DKC1 protein (e.g., DKC1 iso1 or DKC1 iso3) can be substituted according to codon degeneracy, according to codon degeneracy known in the art. In certain embodiments, a nucleotide sequence encoding a DKC1 protein (e.g., DKC1 iso1 or DKC1 iso3) is codon-optimized.

[0084] nc-tRNA A nc-tRNA is a near-cognate tRNA (nc-tRNA) that recognizes and decodes a Ψ-modified codon (e.g., ΨAA, ΨAG, ΨGA) in a PTC via non-canonical base pairing. The anticodon of a nc-tRNA has a single base mismatch or wobble pairing with the Ψ-modified codon (the remaining positions are Watson-Crick paired), and recognizes and decodes the codon as a specific amino acid.

[0085] In some embodiments, the method further comprises:

[0086] In some embodiments, the method further comprises: introducing into the host cell a near-cognate transfer RNA (nc-tRNA) for the PTC or a nucleic acid molecule for expressing the nc-tRNA.

[0087] In some embodiments, the nucleic acid molecule for expressing the nc-tRNA comprises 1, 2, 3, 4, or more copies of a nucleotide sequence encoding the nc-tRNA.

[0088] In some embodiments, the nc-tRNA is a mature tRNA. In some embodiments, the nc-tRNA is a full tRNA comprising an intron.

[0089] In some embodiments, the target uridine residue in the PTC of the target RNA is modified to a pseudouridine residue to provide a Ψ-modified PTC, and then a nc-tRNA for the Ψ-modified PTC or a nucleic acid molecule for expressing the nc-tRNA is introduced into the host cell to decode the PTC as an amino acid, thereby inhibiting the PTC.

[0090] In some embodiments, the nc-tRNA is modified.

[0091] In some embodiments, the modification comprises one or more modifications of a mature tRNA. In some embodiments, the modification comprises one or more modifications selected from the group consisting of psiU, mlA, mlG, and m5C. In some embodiments, the modified tRNA significantly increases the read-through efficiency of a gsnoRNA as compared to an unmodified tRNA of the same type. In some embodiments, the modified nc-tRNA comprises 5’ phosphorylation. In some embodiments, the modified tRNA is a mature tRNA without an intron.

[0092] In certain embodiments, the nc-tRNA sequence is as set forth in SEQ ID NOs: 62-65 and SEQ ID NOs: 89-172.

[0093] In certain embodiments, the PTC is a UGA codon, wherein the Ψ-modified PTC is decoded as arginine or tryptophan. In certain embodiments, the Ψ-modified PTC is decoded as arginine at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% of the time.

[0094] In certain embodiments, the PTC is a UAG codon, wherein the Ψ-modified PTC is decoded as glutamine, leucine, or tyrosine. In certain embodiments, the nc-tRNA is tRNA-Q-CUG, tRNA-L-CAA, tRNA-Y-AUA, and / or tRNA-Y-GUA.

[0095] In certain embodiments, the PTC is a UAA codon, wherein the Ψ-modified PTC is decoded as glutamine or tyrosine. In some embodiments, the nc-tRNA is tRNA-Q-UUG, tRNA-Y-AUA, and / or tRNA-Y-GUA.

[0096] In certain embodiments, the PTC is caused by a nonsense mutation of a codon that encodes arginine. In certain embodiments, the PTC is a UGA codon, and the nc-tRNA is tRNA R UCU, which decodes the Ψ-modified PTC as arginine.

[0097] In certain embodiments, the PTC is caused by a nonsense mutation of a codon that encodes glutamine. In certain embodiments, the PTC is caused by a nonsense mutation of a codon that encodes glutamine, and the nc-tRNA is tRNA-Q-CUG or tRNA-Q-UUG, which decodes the Ψ-modified PTC as glutamine.

[0098] The writing of the twenty conventional amino acids referred to herein follows conventional usage. See, e.g., Immunology - A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in the present application, amino acids are generally represented by the single and three letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0099] gsnoRNA In another aspect, the present application provides an engineered guide small nucleolar RNA (gsnoRNA), characterized in that the gsnoRNA comprises: (i) at least one guide sequence, (ii) at least one CAB box and / or CTE (constitutive transport element) element, and (iii) a scaffold sequence; wherein, the guide sequence hybridizes to a sequence comprising a target uridine residue (U) of the PTC in the target RNA; the CAB box is derived from the loop region of the stem-loop structure of a natural scaRNA, Alu RNA or htrRNA (Human Telomerase RNA); the scaffold sequence is derived from a natural H / ACA box-type structured snoRNA and / or a natural H / ACA box-type structured scaRNA.

[0100] In certain embodiments, the scaffold sequence is not limited to a specific length and sequence, as long as it retains the ability to interact with snoRNA or scaRNA and DKC1 protein. In certain embodiments, the scaffold sequence is 30-50 nt, 50-80 nt, 80-100 nt, 100-130 nt, 130-150 nt, 150-200 nt, or longer in length.

[0101] In certain embodiments, the scaffold sequence is derived from a complete snoRNA of natural H / ACA box-type structure (e.g., comprising two hairpin structures, H box, ACA box) or a portion thereof (e.g., comprising one hairpin structure, H box, ACA box).

[0102] In certain embodiments, the scaffold sequence is derived from a complete scaRNA of natural H / ACA box-type structure (e.g., comprising two hairpin structures, H box, ACA box) or a portion thereof (e.g., comprising one hairpin structure, H box, ACA box).

[0103] In certain embodiments, the gsnoRNA comprises two stem loops, comprising in 5’ to 3’ order: a first stem proximal to the 5’ end stem loop structure, a first loop proximal to the 5’ end stem loop structure, a hinge structure (comprising an “H box”), a second stem proximal to the 3’ end stem loop structure, a second loop proximal to the 3’ end stem loop structure, a tail structure (comprising an “ACA box”). Wherein the guide sequence can be located in the first stem and / or the second stem, and the CAB box can be located in the first loop and / or the second loop.

[0104] In certain embodiments, to enhance in vivo stability and delivery efficiency, the gsnoRNA can comprise one or more modified (e.g., chemically modified) nucleotides. In certain embodiments, the ribose moiety of one or more nucleotides of the gsnoRNA comprises a 2'-0-methyl (2'-OMe) modification. In some embodiments, the gsnoRNA comprises one or more phosphorothioate (PS) internucleoside linkages. In some embodiments, the gsnoRNA comprises a 5' cap modification (e.g., a m7G cap modification). In certain embodiments, the modifications are concentrated at the 5' and 3' ends of the gsnoRNA to resist nuclease degradation without significantly affecting function.

[0105] In certain embodiments, the CAB cassette has a sequence as shown below: X1X2AG; wherein each of X1and X2is independently selected from any one of A, U, C, G.

[0106] In certain embodiments, the scaffold sequence comprises a first hairpin structure proximal to the 5' end, and a second hairpin structure proximal to the 3' end.

[0107] and the CAB cassette is located in the loop of the first hairpin structure, or in the loop of the second hairpin structure, or in the loops of both the first and second hairpin structures.

[0108] In certain embodiments, the guide sequence is located in the stem of the first hairpin structure, or in the stem of the second hairpin structure, or in the stems of both the first and second hairpin structures.

[0109] In certain embodiments, the CTE element is a CTE element derived from Mason Fisher monkey virus (MPMV) or simian retrovirus type D (SRV) or a truncation thereof.

[0110] In certain embodiments, the gsnoRNA comprises, in the 5' to 3' order, a first part of a first guide sequence, a CAB cassette derived from a natural scaRNA, Alu RNA or htrRNA (Human Telomerase RNA) or a loop of a stem-loop structure comprising the CAB cassette, a second part of the first guide sequence, a first part of a second guide sequence, a second part of the second guide sequence, and a CTE element, linked by a scaffold sequence.

[0111] In certain embodiments, the CAB cassette has the features as described in the first aspect.

[0112] In certain embodiments, the guide sequence has the features as described in the first aspect.

[0113] In certain embodiments, the scaffold sequence has the features as described in the first aspect.

[0114] In certain embodiments, the CTE element has the features as described in the first aspect.

[0115] Isolated nucleic acid molecule In another aspect, the present application provides an isolated nucleic acid molecule, characterized in that the isolated nucleic acid molecule comprises a nucleic acid sequence for expressing a gsnoRNA as described above.

[0116] In certain embodiments, the isolated nucleic acid molecule is DNA.

[0117] Composition In another aspect, the present application provides a composition, characterized in that the composition comprises: a gsnoRNA as described above or an isolated nucleic acid molecule as described above.

[0118] In certain embodiments, the composition comprises: a gsnoRNA as described above or an isolated nucleic acid molecule as described above; and a near-cognate transfer RNA (nc-tRNA) of a PTC comprised in the target sequence or a nucleic acid for expressing the nc-tRNA.

[0119] In certain embodiments, the composition comprises: a gsnoRNA as described above or an isolated nucleic acid molecule as described above; and a DKC1 protein or a nucleic acid molecule encoding a DKC1 protein.

[0120] In certain embodiments, the composition comprises: a gsnoRNA as described above or an isolated nucleic acid molecule as described above, a near-cognate transfer RNA (nc-tRNA) of a PTC comprised in the target sequence or a nucleic acid for expressing the nc-tRNA, and a DKC1 protein or a nucleic acid molecule encoding a DKC1 protein.

[0121] In certain embodiments, the composition comprises: an isolated nucleic acid molecule as described above, the nucleic acid for expressing the nc-tRNA and a nucleic acid molecule encoding a DKC1 protein, and the nucleic acids or nucleic acid molecules are present in the same or different vectors, respectively.

[0122] In certain embodiments, the DKC1 protein has the features as described in the first aspect.

[0123] In certain embodiments, the nc-tRNA has the features as described in the first aspect.

[0124] Delivery and delivery compositions The gsnoRNAs herein can be delivered directly into a host cell after synthesis by in vitro transcription, which can be delivered by any method known in the art.

[0125] Such methods include, but are not limited to, electroporation, lipofection, nucleofection, microinjection, sonoporation, biolistics, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, magnetic transfection, perforation transfection, optical transfection, reagent-enhanced nucleic acid uptake, and delivery via liposomes, immunoliposomes, viral particles, artificial virions, and the like.

[0126] Accordingly, in another aspect, the present application provides a delivery composition comprising a delivery vehicle, and one or more of: a gsnoRNA as described above or an isolated nucleic acid molecule as described above or a composition as described above.

[0127] In certain embodiments, the delivery vehicle is a particle.

[0128] In certain embodiments, the delivery vehicle is selected from the group consisting of a lipid particle, a sugar particle, a metal particle, a protein particle, a liposome, an exosome, a microvesicle, a biolistic particle, or a viral vector (e.g., a replication-defective retrovirus, a lentivirus, an adenovirus, or an adeno-associated virus).

[0129] In certain embodiments, the delivery composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0130] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is compatible, in pharmacological and / or physiological terms, with the subject and the active ingredient, is well known in the art, and includes, but is not limited to: pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives.

[0131] Host cells In another aspect, the present application provides a host cell characterized in that the host cell comprises a gsnoRNA as described above or an isolated nucleic acid molecule as described above or a composition as described above or a delivery composition as described above.

[0132] Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E. coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, and the like).

[0133] In certain embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0134] In certain embodiments, the host cell is a mammalian cell (e.g., human).

[0135] In certain embodiments, the host cell comprises a PTC (premature termination codon) mutant gene.

[0136] Methods of manufacture In another aspect, the application provides a method of manufacturing a gsnoRNA as described above or a composition as described above or a delivery composition as described above, characterized in that the method comprises culturing a host cell as described above under conditions that allow expression of nucleic acids and proteins, and recovering the gsnoRNA or the composition or the delivery composition from the cultured host cell culture.

[0137] Uses In another aspect, the application provides use of a gsnoRNA as described above or an isolated nucleic acid molecule as described above or a composition as described above or a delivery composition as described above or a host cell as described above in the manufacture of a medicament for target RNA editing or for inhibiting a premature termination codon (PTC) in a target RNA in a host cell.

[0138] In certain embodiments, the medicament is for modifying a uridine residue (U) in the target RNA to a pseudouridine residue (Ψ).

[0139] In certain embodiments, the host cell is a mammalian cell. In certain embodiments, the host cell is a human cell.

[0140] In another aspect, the application provides use of a gsnoRNA as described above or an isolated nucleic acid molecule as described above or a composition as described above or a host cell as described above or a delivery composition as described above in the manufacture of a pharmaceutical product, characterized in that the pharmaceutical product is for treating a disease and / or a symptom caused or induced by a PTC mutation in a subject.

[0141] In certain embodiments, the disease is selected from cystic fibrosis, spinal muscular atrophy, fructose intolerance, dilated cardiomyopathy, Hurler syndrome, alpha-1-antitrypsin (A1AT) deficiency, Parkinson's disease, Alzheimer's disease, albinism, amyotrophic lateral sclerosis, asthma, beta-thalassemia, Cadasil syndrome, Charcot-Marie-Tooth disease, chronic obstructive pulmonary disease (COPD), distal spinal muscular atrophy (DSMA), Duchenne-Belsian muscular dystrophy, dystrophic epidermolysis bullosa, epidermolysis bullosa, Fabry disease, Factor V Leiden-related disorders, familial adenomatous polyposis, galactosemia, Gaucher's disease, glucose-6-phosphate dehydrogenase, hemophilia, hereditary hematochromatosis, Hunter syndrome, Huntington's disease, inflammatory bowel disease (IBD), hereditary polyagglutination syndrome, Lesch-Nyhan syndrome, Lynch syndrome, Marfan syndrome, mucopolysaccharidosis, muscular dystrophy, myotonic dystrophy type I and type II, neurofibromatosis, Niemann-Pick disease types A, B, and C, NY-esol-related cancer, Pelizaeus-Merzbacher disease, phenylketonuria, Pompe disease, primary ciliary disease, prothrombin mutation-related diseases (such as prothrombin G20210A mutation), pulmonary arterial hypertension, (autosomal dominant) retinitis pigmentosa, Sandhoff disease, severe combined immunodeficiency syndrome (SCID), sickle cell anemia, Stargardt disease, Tay-Sachs disease, Usher syndrome, X-linked immunodeficiency, craniofacial angiomatosis (Sturge-Weber Syndrome), or any combination thereof.

[0142] In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human, a cynomolgus monkey, a mouse.

[0143] In certain embodiments, the disease is fructose intolerance. In certain embodiments, the PTC mutation is a nonsense mutation of a nucleotide encoding amino acid number 148 of ALDOB (fructose-bisphosphate aldolase B).

[0144] In certain embodiments, the disease is cystic fibrosis. In certain embodiments, the PTC mutation is a nonsense mutation of a nucleotide encoding amino acid number 553 of CFTR (cystic fibrosis transmembrane regulator).

[0145] In certain embodiments, the disease is dilated cardiomyopathy. In certain embodiments, the PTC mutation is a nonsense mutation of a nucleotide encoding amino acid number 225 of LMNA (lamin A / C).

[0146] Methods In another aspect, the present application provides a method of editing a target RNA in vitro or in vivo, characterized in that the method comprises contacting the target RNA with one or more of the gsnoRNA as described above, or the isolated nucleic acid molecule as described above, or the composition as described above, or the delivery composition as described above, under conditions suitable for the editing of the target RNA, thereby editing the target RNA.

[0147] In certain embodiments, the method modifies a uridine residue (U) in the target RNA to a pseudouridine residue (Ψ).

[0148] In certain embodiments, the method modifies one or more uridine residues in the target RNA to a pseudouridine residue.

[0149] It should be understood that the present application provides a generally applicable method of target RNA editing, which can be used to edit any uridine residue of a target RNA in vitro or in vivo. By designing a suitable guide sequence of the gsnoRNA, the uridine residue to be edited is introduced into the pseudouridylation pocket formed by the gsnoRNA, and under conditions suitable for the catalysis of pseudouridine synthase, the pseudouridine synthase is contacted with the target RNA and the gsnoRNA, and the uridine residue located in the pseudouridylation pocket can enter the catalytic center of DKC1 to achieve editing. In certain embodiments, the method does not involve the use of tRNA (e.g., nc-tRNA).

[0150] In certain embodiments, the target RNA is edited to suppress a premature termination codon (PTC) in the target RNA in a host cell.

[0151] Definitions of terms In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person skilled in the art. Also, the molecular genetic, nucleic acid chemical, chemical, molecular biological, biochemical, cell culture, microbiological, cell biological, genomic, and recombinant DNA procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present application, the definitions and explanations of related terms are provided as follows.

[0152] As used herein, the term "guide small nucleolar RNA (gsnoRNA)" refers to an engineered non-coding RNA molecule. The gsnoRNA is capable of specifically hybridizing to a specific sequence in the target RNA (such as a target uridine residue in a premature termination codon PTC), and recruiting a pseudouridine synthase complex (e.g., a complex comprising a DKC1 protein) to the target site, thereby catalyzing the conversion of the target uridine residue to a pseudouridine residue (Ψ).

[0153] As used herein, the term "guide sequence" refers to the portion of a gsnoRNA responsible for target recognition, consisting of a stretch of nucleotide sequence that is complementary to a specific region in the target RNA (e.g., the PTC sequence containing the target uridine residue). This sequence hybridizes to the specific region in the target RNA through Watson-Crick base pairing.

[0154] As used herein, the term "CAB box" element is a short RNA motif that enables localization to Cajal bodies. Its sequence is a stretch of conserved sequence "X1X2AG", where the AG in the third and fourth positions is very conserved, and the first two positions can vary. For example, the CAB box element conserved sequence can be AAAG, GAAG, UAAG, UGAG, UCAG, CGAG, AUAG, GCAG, CUAG, CAAG, AGAG, etc. The CAB box is generally found in scaRNAs, and is also found in Alu RNAs and HTR (Human Telomerase RNA).

[0155] As used herein, the term "CTE element" is a stretch of RNA element, which can also be referred to as Constitutive Transport Element. Some studies suggest that the CTE sequence can interact with helicases to open complex secondary structures of the target RNA to enhance accessibility. The CTE element is found in Mason-Pfizer monkey virus (MPMV) or simian retrovirus type D (SRV), and functional homologous modules are also found in Rous sarcoma virus (RSV), avian leukosis virus (ALV). In certain embodiments, the CTE element used herein is the CTE functional homologous module described above.

[0156] As used herein, the term "snoRNA" (small nucleolar RNA) is a non-coding RNA. Depending on the structure, it can be divided into three types of C / D type, H / ACA type and MRP RNA type, the C / D type snoRNA can guide the 2'-O-ribosyl methylation modification of rRNA precursor, and the H / ACA type snoRNA can guide the pseudouridylation modification of rRNA precursor.

[0157] As used herein, the term "scaRNA" or small Cajal body-specific RNA is a non-coding RNA. It is structurally identical to a snoRNA (C / D or H / ACA type), but additionally carries a Cajal body localization signal, the CAB box. C / D type scaRNAs are able to direct 2'-O-ribose methylation modifications of rRNA precursors, and H / ACA type scaRNAs are able to direct pseudouridylation modifications of rRNA precursors.

[0158] As used herein, the term "H / ACA box" refers to a small RNA secondary structure. Specifically, the H / ACA box is a stem-loop structure, which is a type of hairpin structure. The H / ACA box comprises a stem structure and a loop structure. The stem structure is a double-stranded RNA structure, and the loop structure is a single-stranded RNA structure. The H / ACA box is located between the first hairpin and the second hairpin of the H / ACA structure. The H / ACA box is involved in the recruitment of the DKC1 protein. The DKC1, NOP10, NHP2, GAR1 tetrameric complex is recruited to the H / ACA structure, forms an RNP complex and catalyzes the modification of a uridine residue U to a pseudouridine residue.

[0159] As used herein, the term "scaffold sequence" refers to a sequence in a gsnoRNA other than the guide sequence, and the CAB box and / or the CTE (constitutive transport element) element. In certain embodiments, the scaffold sequence is derived from a snoRNA of a native H / ACA box. In certain embodiments, the scaffold sequence is derived from a scaRNA of a native H / ACA box. Thus, in a gsnoRNA, the scaffold sequence is responsible for maintaining the secondary structure of the RNA (e.g., the hairpin conformation) and mediating the interaction with core proteins (e.g., DKC1, NOP10, NHP2, GAR1). In certain embodiments, the scaffold sequence comprises, in the 5' to 3' direction, a first hairpin, a hinge structure comprising an "H box", a second hairpin, and a tail structure comprising an "ACA box". In certain embodiments, the scaffold sequence is used to link the CAB box, the guide sequence, and / or the CTE element. For example, in certain embodiments, the CAB box and the guide sequence are located in the first hairpin of the gsnoRNA, and the CTE element is located at the 3' end of the scaffold sequence, and the CAB box, the guide sequence, and the CTE element are linked together by the scaffold sequence and constitute the gsnoRNA of the present application.

[0160] As used herein, the term “Premature Termination Codon (PTC)” refers to a premature stop signal formed in the coding region of mRNA due to nonsense mutation of a gene, leading to premature termination of translation, resulting in a truncated and often non-functional protein. PTC is caused by point mutation of a sense codon, associated with a variety of genetic diseases, such as cystic fibrosis, Hurler syndrome, Duchenne muscular dystrophy, etc. Therefore, PTC is a core target for therapeutic intervention in a variety of diseases.

[0161] In this context, the specific uridine residue in the PTC targeted by the guide sequence is referred to as “target uridine residue”. When the target uridine residue is modified by pseudouridylation to “pseudouridine residue” (Ψ), forming ΨAA, ΨAG or ΨGA codon.

[0162] As used herein, the term “near-cognate tRNA (nc-tRNA)” refers to a tRNA molecule whose anticodon has a single base mismatch or wobble pairing with the Ψ-modified codon (such as ΨAA, ΨAG, ΨGA) in mRNA (with Watson-Crick pairing at the remaining positions), and recognizes and decodes the codon as a specific amino acid through non-canonical base pairing.

[0163] Through decoding by nc-tRNA, the premature termination codon is recoded as Arg, Gin or Trp / Tyr, depending on the termination codon sequence and nc-tRNA type. Thus, protein folding and function are maintained or partially maintained, achieving full-length protein restoration.

[0164] For example, for ΨGA codon, nc-tRNA anticodon UCU (such as tRNA-R-UCU) decodes as arginine (R). For example, for ΨAG codon, nc-tRNA anticodon CUG (such as tRNA-Q-CUG) decodes as glutamine (Q). For example, for ΨAA codon, nc-tRNA anticodon UUG (such as tRNA-Q-UUG) decodes as glutamine (Q).

[0165] As used herein, the term “DKC1 protein” is a highly conserved pseudouridine synthase. It is responsible for the catalysis of uridine residues to pseudouridine residues in RNA. In the invention, DKC1 protein is recruited to the target RNA site by gsnoRNA, performing specific pseudouridylation modification.

[0166] As used herein, the term “DKC1 iso1” is a subtype of DKC1 protein, which is mainly localized in the nucleus. Its accession number is referred to DKC1 iso1: NP_001354.1.

[0167] As used herein, the term "DKC1 iso3" is a subtype of DKC1 protein. Specifically, it is a splicing variant due to the retention of intron 12. This process results in the deletion of its C-terminal nuclear localization signal (NLS), thus DKC1 iso3 is mainly localized in the cytoplasm rather than the nucleus. In native cellular endogenous mRNA expression, the expression level of DKC1 iso1 is much higher than that of DKC1 iso3. DKC1 iso3 accession number see: NP_001275676.1.

[0168] Advantages of the invention The present application is based on the optimization and modification of the previously studied RESTART pseudouridine modification system, and provides a modified gsnoRNA which can efficiently bind to the target site and improve the recruitment and assembly rate of pseudouridine synthase DKC1 and NHP2, GAR1 and NOP10 core proteins, thereby significantly improving the efficiency of pseudouridine modification. Moreover, the modified gsnoRNA is suitable for various iterative RESTART systems, can efficiently recruit endogenous synthesis and exogenous expression of DKC1, and can also efficiently recruit different subtypes of DKC1 (such as DKC1 iso3 and DKC1 iso1). In addition, the modified gsnoRNA can also be used in combination with other elements (such as nc-tRNA) in the RESTART system, significantly improving the efficiency of pseudouridine modification.

[0169] For example, when applied to modify the uridine residues in PTC mutations, the system containing the gsnoRNA can significantly improve the efficiency of pseudouridine modification, thereby restoring the coding information of the mRNA, allowing the PTC site to be decoded, significantly improving the efficiency of PTC readthrough, and thus expressing a complete protein. Therefore, the modified gsnoRNA has important significance and broad application prospects in the treatment of diseases related to PTC mutations.

[0170] Embodiments of the present application will be described in detail below with reference to the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present application, and are not limiting the scope of the present application. According to the following detailed description of the preferred embodiments and the accompanying drawings, various objects and advantages of the present application will become apparent to those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0171] Figure 1A schematic diagram of one modification of the gsnoRNA of the present application. The scaffold sequence of the gsnoRNA (blue) comprises two stem loop structures, a first hairpin structure near the 5' end and a second hairpin structure near the 3' end. The scaffold sequence comprises, in the 5' to 3' direction: a first hairpin structure (comprising a first stem and a first loop) near the 5' end stem loop structure, a hinge structure (comprising a "H box"), a second hairpin structure (comprising a second stem and a second loop) near the 3' end stem loop structure, a tail structure (comprising an "ACA box").

[0172] Wherein a guide sequence (green) can be located in the first stem and / or the second stem. A CAB box (red) can be located in the first loop and / or the second loop. A CTE sequence (orange) can be attached to the 5' end and / or the 3' end of the scaffold sequence of the gsnoRNA.

[0173] In a particular embodiment, as shown in the present figure, a first guide sequence is located in the first stem, a second guide sequence is located in the second stem, a CAB box is located in the first loop, and a CTE sequence is attached to the 3' end of the scaffold sequence of the gsnoRNA.

[0174] Figure 2 A mechanism of the method of the present application to suppress PTC is schematically shown.

[0175] Figure 2 a shows that when a PTC occurs in an mRNA, the translation of the mRNA prematurely terminates, resulting in a protein truncation. When the method of the present application is used, the guide sequence in the gsnoRNA base pairs with the target mRNA, bringing the uridine residue U in the PTC into a catalytic center formed by the stem loop structure of the gsnoRNA; the gsnoRNA recruits DKC1, NOP10, NHP2 and GAR1 proteins to assemble an RNP complex that catalyzes the modification of the uridine residue U to a pseudouridine residue Ψ. Further, the Ψ-modified PTC can be decoded by an nc-tRNA to be translated into an amino acid, enabling the readthrough of the PTC site, thereby restoring the production of the full-length protein. In certain embodiments, the delivery of the gsnoRNA of the present application alone enables the readthrough of the PTC, and other elements required for the modification and decoding (e.g., DKC1 protein, nc-tRNA) are provided by the cell endogenously. In certain embodiments, the gsnoRNA of the present application co-modifies and decodes the PTC with an exogenously expressed DKC1 protein and / or an exogenously expressed nc-tRNA.

[0176] Figure 2bFurther shows the combination of PTC target of gsnoRNA and the recruitment of RNP complex, and the decoding function of nc-tRNA. Specifically, gsnoRNA binds to PTC target and recruits RNP complex, modifies the catalytic uridine residue U of PTC (UGA, UAG or UAA) to pseudouridine residue Ψ to obtain Ψ-modified PTC (ΨGA, ΨAG or ΨAA). nc-tRNA further binds to the Ψ-modified PTC site and decodes, translating the Ψ-modified PTC site into amino acids, thereby realizing the readthrough of PTC and restoring the expression of full-length protein.

[0177] Figure 3 The introduction of CAB box improves the readthrough efficiency of RESTART system. 3a-3b, Directly using scaRNA as the backbone of gsnoRNA can obtain significant readthrough efficiency in UGA reporter system without overexpression (a) and overexpression (b) of DKC1 iso3. 3c, In the UGA reporter system without overexpression of DKC1 iso3, test the replacement of the loop ring containing the CAB box of the known scaRNA directly to the RESTART readthrough efficiency before and after gACA19. 3d, Select the CAB box with higher efficiency to test the effect of CAB box on readthrough efficiency improvement in the UGA reporter system overexpressing DKC1 iso3. 3e, Test the effect of CAB box added to other snoRNA scaffolds in the UGA reporter system overexpressing DKC1-iso3. 3f, Compare the effects of CAB box added to the 5' stem-loop and 3' stem-loop of gsnoRNA and both ends on readthrough in the UGA reporter system without overexpression of DKC1 iso3. 3g, Schematic diagram of CAB box and functional inactivation mutants of scaRNA (U85) added to gsnoRNA. 3h, Test the readthrough effect of CAB box and functional inactivation mutants of scaRNA (U85) added to gsnoRNA. 3i, Test the effect of RESTART v1, RESTART v2 and RESTART v3 before and after adding CAB box in the disease scenario of CFTR-R553X nonsense mutation. 3j, Test the effect of RESTART v1, RESTART v2 and RESTART v3 before and after adding CAB box in the disease scenario of LMNA-R225X nonsense mutation. 3k, Test the effect of CAB box on other H(ACA)-containing RNAs in the UGA reporter system without overexpression of DKC1 iso3. HTR is human telomerase RNA (Human Telomerase RNA); BIO is a special stem loop result on AluRNA with CAB box.

[0178] Figure 4 Introduction of CTEs improves the read-through efficiency of the RESTART system. 4a, Structures of SRV CTE and mutants (M36 CTE and A CTE). 4b-4c, Testing the effects of adding CTEs at the 5’ end and 3’ end of gsnoRNA, respectively, and different lengths of linkers on RESTART read-through in UGA reporter systems without (a) and overexpressing (b) DKC1 iso3. 4d-4e, Testing the effects of comparing CTEs and functionally inactive mutants of CTEs on RESTART read-through in UGA reporter systems without (a) and overexpressing (b) DKC1 iso3. 4f-4g, Testing the interaction of CTEs with functionally inactive mutants of CTEs with helicase. 4h, Testing the effects of different CTEs and CTE mutants on RESTART read-through in UGA reporter systems overexpressing DKC1-iso3. DETAILED DESCRIPTION

[0179] Sequence Information Information of some sequences involved in the present application is provided as follows.

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188] Exemplary gsnoRNAs are shown in SEQ ID NOs: 68~72, wherein the guide sequence is represented by (Xn), Xn is a sequence of n nucleotides of X, wherein X is any one of A, U, G or C, and n is an integer suitable for the length of the guide sequence. In some embodiments, n is 4, 5, 6, 7, 8, 9, 10, 11 or 12. One of ordinary skill in the art would understand that the guide sequence (Xn) can be replaced to target the gsnoRNA to a desired target site.

[0189] SEQ ID NO: 4~33, SEQ ID NO: 68~72 and SEQ ID NO: 73~78, the underlined sequence is the sequence of the entire loop region of the CAB box derived from the natural scaRNA, AluRNA or htrRNA, and the underlined and italic sequence is the CAB box sequence.

[0190] The application will now be described with reference to the following examples which are intended to illustrate, but not limit, the application. The experiments and methods described in the examples were performed essentially according to conventional methods well known in the art and described in various references, unless specifically indicated otherwise.

[0191] In addition, the examples not specified in the specific conditions, according to the conventional conditions or manufacturer recommended conditions. The reagents or instruments used are not specified by the manufacturer, are conventional products can be obtained by the market. The skilled person in the art, the examples described by way of example of the application, and is not intended to limit the scope of the claimed invention. All the publications and other references mentioned herein are incorporated by reference in their entirety.

[0192] Example 1. Preparation of components in the RESTART system 1 Plasmid preparation The molecular clones constructed in this study mainly include two types: PTC reporter plasmid with pLenti-CMV-MCS-BSD as the vector and DKC1 isoform 3 (DKC1 iso3) plasmid with Pcg2.0-BFP as the vector, and gsnoRNA and nc-tRNA plasmid with Pcg2.0-BFP as the vector.

[0193] The plasmid with pLenti-CMV-MCS-BSD and pAAV as the vector was mainly recombined by Gibson's method. Among them, the gene sequence of DKC1 iso3 was amplified from HEK293T cDNA, the sequence of the disease reporter gene was obtained from the human cDNA library of Peking University, the nonsense mutation in the disease reporter gene was realized by PCR primer induced point mutation, and the connecting sequence between mCherry and EGFP was adjusted by primer amplification. Specifically, the target fragments and vector skeletons were amplified by TransStart FastPfu DNA polymerase kit, the target fragments were obtained by agarose gel electrophoresis, and purified by universal DNA purification recovery kit. After purification, the fragments and vector skeletons were connected by homologous recombination using NEBuilder® HiFi DNA Assembly Master Mix, and the connection product was transformed in Trans T1 competent cells. The plasmid was extracted using EndoFree Mini Plasmid Kit II and identified by Sanger sequencing.

[0194] The construction of gsnoRNA and nc-tRNA plasmids with Pcg2.0-BFP as the vector mainly includes two steps: primer bridging to construct the target sequence and Golden gate enzyme digestion and ligation. First, using TransStart FastPfu DNA polymerase kit, the designed gsnoRNA or nc-tRNA sequence was obtained by overlapping PCR of 4 primers (1 forward primer and 3 reverse primers), and Golden gate enzyme digestion and ligation sites were constructed at both ends of the sequence. Golden gate enzyme digestion and ligation sites exist on the backbone of Pcg2.0-BFP, and Bsd toxic protein exists in the middle of the enzyme digestion and ligation site, so that empty plasmids that fail to be digested cannot survive during transformation. The product obtained by primer bridging was recovered by universal DNA purification recovery kit, 40 ng of purified product and 20 ng of Pcg2.0-BFP plasmid were placed in the enzyme digestion and ligation system containing cutting enzyme BSMBI and T4 ligase (containing DTT and ATP) for reaction, and the reaction product was transformed in Trans T1 competent cells, and the plasmid was extracted for Sanger sequencing identification.

[0195] 2 Cell transfection and data analysis HEK293T cells were cultured at 37 °C, 5% CO2 in DMEM medium containing 10% FBS and 1% penicillin-streptomycin. When cells were passaged, cells were washed with PBS and treated with 0.25% trypsin and incubated at 37 °C for 2 min. Subsequently, trypsin was neutralized with medium containing FBS. After centrifugation at 600 rpm for 3 min, cells were counted and plated. The mycoplasma contamination of the cell line was negative.

[0196] Twenty to twenty-four hours before staining, cells were seeded in 24-well plates at a density of 2 x 105cells per well. Transfection of plasmids was performed by miniprep using the QIAprep® Miniprep Kit, and the plasmid concentration was quantified using a Nanodrop. The target plasmid and target RNA were transfected using Lipofectamine LTX with PLUS reagent reagent, and the transfection procedure was performed according to the recommended protocol of the transfection reagent. Twenty-four hours after transfection, the medium was changed, and 48 or 72 hours later, cell function was detected.

[0197] To evaluate the efficiency of PTC read-through in the fluorescent reporter system, cells were imaged 48-72 hours after transfection using an ImageXpress® Micro 4 high content imaging system (Molecular Devices LLC, Sunnyvale, CA). Sixteen images from different sites of the same well were captured under 10x microscopy, and then the images were analyzed automatically using MetaXpress software. The percentage of EGFP-positive cells was calculated by dividing the number of EGFP-positive cells in the fluorescent image by the number of BFP / mCherry-positive cells in the corresponding image, and then normalized with the data of the positive control. The EGFP fluorescence intensity was calculated by multiplying the EGFP intensity of each cell by the number of EGFP-positive cells in the fluorescent image, and then normalized with the data of the positive control. The average of 16 images was taken as one independent replicate, and each group of fluorescence analysis data had 2-3 biological replicates, and the data was presented as the average of 2 replicates or the average of 3 replicates ± standard deviation.

[0198] 3 RNA extraction and modification detection Discard the culture medium of the target cells in a clean bench, wash once with PBS and dry. Add TRIzol reagent to the cells, and blow and suck 10 times to make the solution uniform. Transfer to an EP tube and stand for 5 minutes, add chloroform (1 / 5 volume of TRIzol reagent) and shake vigorously for 15 seconds. After standing at room temperature for 15 minutes, centrifuge at 12,000 rpm in a 4°C centrifuge for 15 minutes. Take the supernatant and place it in a clean EP tube, add an equal volume of isopropanol, mix well, and place it in a -20°C refrigerator for more than 1 hour. Centrifuge at 12,000 rpm in a 4°C centrifuge for 30 minutes. Discard the supernatant, wash twice with 1 mL of 75% alcohol solution, discard the supernatant, and stand at room temperature for 10 minutes with the cap open. When the RNA precipitate changes from white to transparent, add a certain amount of RNase-free water to dissolve it, and measure the concentration with a Nanodrop.

[0199] Add DNAse to the target RNA for reaction to remove residual genomic and plasmid fragments in the RNA. After the reaction is complete, the RNA sample is purified again. Prepare a labeling reaction solution: mix 85% K2SO3 / 15% NaHSO3 solution with 100 mM hydroquinone at a ratio of 100:1. Take 1 μg of purified RNA and mix with 50 μL of the reaction solution, and react at 70°C for 5 hours. After the reaction, desalt and purify the sample using a Micro Bio-spin 6 chromatography column, then add an equal volume of 1 M Tris-HCl [pH 9.0] and react at 75°C for 30 minutes. After that, purify the labeled RNA sample. Use the Maxima HMinus RT enzyme reaction system for reverse transcription to obtain a cDNA sample. Design two specific PCR primers for the ~130 nt sequence at both ends of the target site, add a library adapter sequence to the 5' end of the primers, and use NEBNext Q5 Hot Start HiFi PCR Master Mix for specific amplification. After amplification, perform PCR amplification reaction with Illumina primers to add sequencing adapter sequences to both sides of the target fragment. After the amplification reaction is complete, purify the target DNA fragment using AMPure XP beads, determine the concentration, and perform 4150 chip identification. Finally, perform next-generation sequencing analysis.

[0200] RESTART v1-v3, v3 mini system Prior to this, the applicant has developed multiple generations of RESTART systems, which contain different components and can all achieve precise pseudouracil modification of PTC sites, promote PTC read-through and restore full-length functional protein expression, thereby efficiently and specifically repairing nonsense mutations.

[0201] RESTART v1: The RESTART v1 system improves the accuracy of snoRNA binding to the target PTC site by modifying the guide sequence and the pseudouridylation pocket of human snoRNA. The modified snoRNA, or gsnoRNA, recruits endogenous DKC1 iso1 to assemble snoRNP for pseudouridylation and effectively readthrough the PTC. The core element of the RESTART v1 system is the gsnoRNA with a modified guide sequence. The gsnoRNA is introduced into cells to recruit endogenous DKC1 iso1 for pseudouridine modification of the PTC site in the target RNA.

[0202] RESTART v2: The DKC1 iso3, a product of DKC1 abnormal splicing, is found to improve the modification efficiency of the RESTART system in the RESTART v2 system. Since DKC1 iso3 is expressed in small amounts in cells under natural conditions, the RESTART v2 system overexpresses the catalytic enzyme DKC1 iso3 to improve the modification efficiency of the RESTART system by 2-fold and the readthrough efficiency by 2-5-fold. The core element of the RESTART v2 system is the gsnoRNA and DKC1 iso3. The gsnoRNA and DKC1 iso3 are introduced into cells for pseudouridine modification of the PTC site in the target RNA.

[0203] RESTART v3: The nc-tRNA plays an important role in decoding the pseudouridine-modified PTC site in the RESTART v3 system. Although these nc-tRNAs naturally exist, their expression levels in cells are low or they do not target the PTC site, thus the need for artificial introduction and overexpression. The RESTART v3 system significantly improves the readthrough level of the RESTART system (1.3-8-fold higher than the RESTART v2) by overexpressing nc-tRNA, and also improves the accuracy of amino acid incorporation at the PTC site, enabling about 50% of disease-related nonsense mutation sites to be accurately repaired to the original amino acid. The core element of the RESTART v3 system is the gsnoRNA, DKC1 iso3, and nc-tRNA. The gsnoRNA, DKC1 iso3, and nc-tRNA are introduced into cells for pseudouridine modification and readthrough of the PTC site in the target RNA.

[0204] RESTART v3-mini: To simplify the RESTART v3 system and improve the flexibility of delivery, the RESTART v3-mini system only delivers gsnoRNA and nc-tRNA without additional expression of DKC1 iso3. That is, the core elements of the RESTART v3-mini system are gsnoRNA and nc-tRNA. By introducing gsnoRNA and nc-tRNA into cells, pseudouridine modification and readthrough of PTC sites in target RNA are performed.

[0205] In summary, it can also be found that each element in the RESTART system can function alone or in combination. For example, the delivery of gsnoRNA alone can improve the efficiency of PTC readthrough, and other elements required for modification are provided by the cell endogenously (similar to the RESTART v1 system). Similarly, in the presence of gsnoRNA, DKC1 iso3 can also be overexpressed alone, or nc-tRNA can also be delivered alone. Even without delivering the complete RESTART system, the endogenous elements in the cell can be used to assemble the RESTART system and achieve readthrough of PTC in the cell.

[0206] Construction and application of new RESTART system Further, the gsnoRNA in the previous RESTART system is modified in the present application. The efficiency of recognizing target sequences and recruiting RNP is increased by introducing CAB / CTE sequences, thereby improving the efficiency of ultimately readthrough nonsense mutations, and it will be used in the future fourth generation RESTART system (i.e. RESTART V4). Specifically, in the RESTART system of the present application, the gsnoRNA and nc-tRNA used are expressed from the Pcg2.0-BFP (purchased from Addgene) backbone plasmid, and the DKC1 iso3 protein component is expressed from the pLenti-CMV-MCS-BSD backbone plasmid. These constructed vectors can be used to assemble the RESTART system in cells in order to further evaluate its reading function in disease-related mRNA containing nonsense mutations. In some embodiments, the constructed vectors containing nucleotide sequences encoding gsnoRNA, nc-tRNA, and DKC1 iso3 protein can be directly transfected into cells to express the required RNA or protein, thereby assembling the new RESTART system containing the optimized gsnoRNA of the present application in the cells.

[0207] During the construction process, we introduce the disease-related reporter gene with premature stop codon by co-transfection, and evaluate the reading efficiency and functional recovery effect of the RESTART system on the target mRNA by the change of the fluorescence expression level of the reporter system. That is, by introducing the reporter system to constitute the PTC disease model in the cell, and introducing the above-mentioned vector, the efficiency of the RESTART system for pseudouridylation editing and the effect of restoring PTC readthrough can be detected.

[0208] After obtaining the components of the system, it can be further constructed into a delivery system suitable for in vivo administration. The specific method includes integrating the system into a lentivirus or adeno-associated virus vector for delivery by in vitro transduction or in vivo injection; or encapsulating the system RNA components with lipid nanoparticles for targeted delivery by intravenous or local injection, etc.

[0209] Example 2. Modification of the positioning sequence of snoRNA Figure 1 The basic structure of the modified gsnoRNA of the present application is shown. In this paper, we exemplarily list several sequences of the modified gsnoRNA of the present application (the guide sequence is represented by Xn, and the rest is the scaffold sequence), which are ACA19, ACA36, ACA24, ACA5 and ACA14a, and their nucleotide sequences are shown in SEQ ID NO: 68~72, respectively.

[0210] The RNA pseudouridylation modification system is mainly formed by snoRNP which is formed by snoRNA and four core proteins DKC1, NHP2, GAR1 and NOP10 for pseudouridine modification Figure 2). We tried to improve the efficiency of pseudouridine modification by engineering snoRNAs to increase the rate of assembly of snoRNPs. The sequences of the core proteins can be found in DKC1 (DKC1 iso1: NP_001354.1 or DKC1 iso3: NP_001275676.1), NHP2: NP_060308.1, GAR1: NP_061856.1, and NOP10: NP_061118.1. We introduced the CAB box element present in the loop of the hairpin of natural scaRNAs (e.g., scaRNA11, scaRNA14, scaRNA15, scaRNA85 (i.e., U85), Alu RNA, and htrRNA) into snoRNAs to form gsnoRNAs. The gsnoRNAs of the present application comprise two stem loop structures (also referred to as hairpin structures) comprising, in the 5’ to 3’ direction: a first stem proximal to the 5’ end stem loop structure, a first loop proximal to the 5’ end stem loop structure, a second loop proximal to the 3’ end stem loop structure, and a second stem proximal to the 3’ end stem loop structure. The guide sequence can be located in the first stem and / or the second stem, and the CAB box can be located in the first loop and / or the second loop.

[0211] The CAB box has a conserved sequence of “X1X2AG”, in which the AG of the third and fourth positions is very conserved, and the first two positions can vary. For example, the CAB box element conserved sequence can be AAAG, GAAG, UAAG, UGAG, UCAG, CGAG, AUAG, GCAG, CUAG, CAAG, AGAG, etc. The htrRNA is Human Telomerase RNA, and the hairpin at the 3’ end thereof has a CAB box. The Alu RNA is a type of non-coding RNA expressed in an Alu element of an intron, and the hairpin at the 3’ end thereof has a CAB box.

[0212] The CAB box is added in the following two ways in this embodiment: 1. Directly constructing gsnoRNAs using scaRNAs (containing CAB box) as the backbone We directly used the natural scaRNA as the backbone of gsnoRNA, replaced its targeting sequence with the sequence targeting ALDOB-W148X, and constructed two kinds of gsnoRNA (the replaced gsnoRNA sequences are shown as SEQ ID NO: 2 and SEQ ID NO: 3, using natural scaRNA14 and scaRNA15 as the backbone, respectively). Among them, ALDOB-W148X refers to a PTC disease model gene in which the 148th leucine (W, corresponding codon UGG) in the protein expressed by the ALDOB (fructose bisphosphate aldolase B) gene is changed into a stop codon (UAG) due to gene mutation. The database number of ALDOB is: NP_000026.2.

[0213] The results show that scaRNA as the backbone of gsnoRNA has a higher read-through level in the RESTART v1 system (i.e., only gsnoRNA is transferred into the host cell), and is higher than the read-through level of the gsnoRNA (gACA19, the sequence is SEQ ID NO: 1) constructed by us using ACA19 snoRNA as the backbone. Figure 3 a-3b).

[0214] 2 Introducing CAB box from scaRNA into gsnoRNA In order to improve the universality of CAB box in different scenarios, we directly added the CAB box or the entire loop sequence containing the CAB box in the scaRNA to the previously optimized gACA19, and constructed different gsnoRNAs as shown in SEQ ID NO: 4~22 according to the different scaRNAs used.

[0215] From Figure 3 It can be seen from c that almost all the CAB boxes on the scaRNAs in this paper are screened, and in the UGA reporter gene system that does not express DKC1 iso3 (only recruits endogenous DKC1 protein), the CAB boxes on most scaRNAs can improve the read-through efficiency by about 50%.

[0216] Then we tested the CAB boxes of some scaRNAs in the UGA reporter gene system that overexpresses DKC1 iso3. The results show that these CAB boxes derived from different scaRNAs can improve the read-through efficiency, and the gsnoRNA constructed using the CAB box derived from U85 (SEQ ID NO: 4) has the relatively highest efficiency (about 20% higher than gACA19). Figure 3 d).

[0217] Next, we explored the universality of CAB box on different gsnoRNAs. We connected the CAB box of U85 to different gsnoRNAs, and the sequences of the constructed gsnoRNAs are shown in SEQ ID NO: 73~78. From Figure 3 It can be seen that the CAB box of U85 can improve the readthrough efficiency of 30%-50% on different gsnoRNAs.

[0218] Then, since the CAB box is added to the loop of the stem-loop structure of gsnoRNA, and gsnoRNA has two stem-loop structures, we further explored the effect of CAB added to different positions of gsnoRNA. We constructed gsnoRNAs with CAB box added to the loop near the 5' end stem-loop structure, the loop near the 3' end stem-loop structure, and both loops (the sequences are shown in SEQ ID NO: 23~25, respectively). The research results showed that CAB box connected to the above three positions can significantly improve the pseudouridylation efficiency and increase the readthrough level. Moreover, the effect of CAB box added to the loop of the 5' end stem-loop structure is the best (f). Figure 3 f).

[0219] 3 Introducing inactive CAB box in gsnoRNA Further, in order to verify that the improvement of readthrough is directly related to the function of CAB box, we also compared the effect of normal CAB box and functionally inactive mutant CAB box (i.e., not consistent with the conserved sequence of XXAG) on readthrough. We constructed gsnoRNAs containing functionally inactive mutant CAB box (SEQ ID NO: 26 and SEQ ID NO: 27). From Figure 3 g and 3h can be seen that the normal function of CAB box can significantly improve the readthrough, while the functionally inactive mutant CAB box has little effect on the readthrough or even slightly reduces it. These results show that it is indeed the CAB box that improves the readthrough effect of the RESTART system.

[0220] Then, to improve the application prospect of CAB box, we tested the effect of CAB box in two nonsense mutation disease scenarios, CFTR-R553X and LMNA-R225X, using different versions of RESTART system. CFTR is the abbreviation of Cystic fibrosis transmembrane conductance regulator, and R553X means that the arginine (R, codon is CGA) at position 553 is mutated into a stop codon (here is UGA), and its NCBI number is NP_000483.3. LMNA is the abbreviation of Lamin A / C, which is a protein encoded by the human gene LMNA, belonging to the lamin family, and R225X means that the arginine (R, codon is CGA) at position 225 is mutated into a stop codon (here is UGA), and its NCBI number is NP_001393912.1.

[0221] The results show that (Fig. Figure 3 i and 3j), in each RESTART system, the use of CAB box can effectively improve the efficiency of read-through, and the effect is particularly significant in RESTART v3 mini and RESTART v3 systems. That is, whether the above gsnoRNA connected with CAB box is used alone in RESTART v1 system, or is used in combination with DKC1 iso3 and / or nc-tRNA in RESTART v1, v3 and v3 mini systems, the gsnoRNA can further improve the level of pseudouridine modification of PTC and the efficiency of read-through.

[0222] 4 Introducing CAB box of HTR or Alu origin into gsnoRNA Finally, to explore the universality of CAB box, we tested the effect of CAB box on other non-scaRNAs. It was found that (Fig. Figure 3k), directly adding the CAB box on the HTR to the 5' end stem loop (5HTR) and 3' end stem loop (3HTR) of the gsnoRNA, respectively, the constructed gsnoRNA (SEQ ID NO: 28 and SEQ ID NO: 29) both have a significant improvement effect; and replacing half of the gsnoRNA skeleton with the corresponding sequence of the HTR with the CAB box (SEQ ID NO: 30 and SEQ ID NO: 31), the 5' end replacement and 3' end replacement schemes are also higher than the gsnoRNA read-through efficiency without adding the CAB box, wherein the effect of replacing half of the 5' end of the gsnoRNA skeleton with the corresponding sequence of the HTR with the CAB box is significantly better. Similarly, adding the CAB box from the Alu RNA to the gnoRNA (SEQ ID NO: 32 and SEQ ID NO: 33) also has a certain improvement effect, and the effect of adding to the 5' end is better than that of adding to the 3' end. The effects of adding to the 5' end and the 3' end are both significantly higher than those of the gsnoRNA without adding the CAB box. Since the CAB boxes on different Alu RNAs are highly similar, it can be expected that the CAB boxes on other Alu RNAs can also produce similar effects.

[0223] In summary, regardless of the source of the CAB box (for example, scaRNA, Alu or hTR source) and the connection position of the CAB box (5' end, 3' end, or 5' end and 3' end), the gsnoRNA carrying the CAB box can significantly improve the pseudouridylation efficiency of the RESTART system (v1-v3, v3 mini) and significantly improve the PTC read-through level. Even in the case of retaining the targeting ability, directly using scaRNA as the skeleton, or replacing part of the sequence of the gsnoRNA with scaRNA, Alu or hTR with CAB box, the function of the gsnoRNA can also be achieved, the pseudouridylation efficiency of the RESTART system is significantly improved, and the PTC read-through level is significantly improved. This also provides new possibilities for the selection and design of "gsnoRNA" or "guide RNA", that is, the key to recruiting DKC1 enzyme lies in retaining the H / ACA structure, and we can choose different small RNAs as the basic skeleton for modification and design, or even design from scratch, to provide the "DKC1 enzyme recruitment ability" and "target sequence guiding ability" of the guide RNA.

[0224] Example 3. Design of CTE sequence of snoRNA This example attempts to open the structure of the substrate mRNA by connecting the CTE sequence to the gsnoRNA to enhance the targeting efficiency of the snoRNA. The structure of CTE is shown inFigure 4 a.

[0225] 1 Introduction of CTE from SRV into gsnoRNA We chose the most commonly used sno-ACA19 to construct gsnoRNA (SEQ ID NO: 34). Further, we connected the CTE element of the type D retrovirus (SRV) (SEQ ID NO: 79) to the 5' end and 3' end of the gsnoRNA (SEQ ID NO: 35 and SEQ ID NO: 36), respectively, and found that the read-through efficiency was improved by 30-80% regardless of whether DKC1 iso3 was overexpressed (b-4c), of which the effect of connecting to the 3' end was better than that of connecting to the 5' end. Figure 4 b-4c).

[0226] Then, we added different lengths of linkers between the gsnoRNA and the CTE, and we tested the effect of 4-8 bp linker sequences connecting to the 5' end or 3' end, with the 4-8 bp linker sequences being UCUA, UCUAU, UCUAUC, UCUAUCU, and UCUAUCU, respectively. The sequences of the constructed gsnoRNAs are shown in SEQ ID NO: 37-46. The results showed that adding a linker to the 5' end or 3' end could improve the read-through efficiency to some extent, and when the linker was 6 bp and the CTE was connected to the 3' end of the gsnoRNA, it had the highest read-through efficiency, which was about twice that of the original (b-4c). Figure 4 b-4c).

[0227] 2 Introduction of inactivated CTE into gsnoRNA Next, we added a functionally inactivated CTE mutant to the gsnoRNA to verify that it was indeed the function of the CTE that led to the improvement in read-through efficiency. The sequence of the gsnoRNA containing the natural CTE is shown in SEQ ID NO: 47, and the sequences of the gsnoRNAs containing the functionally inactivated CTE mutant are shown in SEQ ID NO: 48 and SEQ ID NO: 49. From Figure 4 d-4e), it can be seen that whether DKC1 iso3 was overexpressed or not, connecting a normally functioning CTE could significantly improve read-through, while connecting a functionally mutated CTE had little effect on read-through. These results indicate that the improvement in read-through after connecting the CTE is indeed related to the function of the CTE. Then, to verify the improvement in read-through after connecting the CTE to the gsnoRNA, we performed a RIP experiment to analyze the interaction of the CTE with the helicase. From Figure 4f-4g It can be seen that only the gsnoRNA with normal CTE connected can interact with helicase and be significantly enriched, which further proves that the gsnoRNA with CTE connected can improve the efficiency of read-through by interacting with helicase.

[0228] 3 Introducing CTE from MPMV in gsnoRNA Finally, we tried to find smaller CTE to connect in gsnoRNA to achieve the delivery of gsnoRNA in the form of small RNA. Mason-Pfizer monkey virus (MPMV) also has a CTE element, and the nucleotide sequence of MPMV CTE element is shown as SEQ ID NO: 80, and it has been reported that the half sequence of the truncated MPMV CTE is sufficient to achieve the same function as the full-length MPMV CTE, so we explored the effects of SRV CTE and MPMV CTE and their corresponding truncated mutants on read-through.

[0229] The sequences of the gsnoRNAs constructed using the natural CTE and CTE truncations from SRV are shown as SEQ ID NO: 50 and SEQ ID NO: 51~52, respectively; the sequences of the gsnoRNAs constructed using the natural CTE and CTE truncations from MPMV are shown as SEQ ID NO: 53 and SEQ ID NO: 54~61, respectively. From Figure 4 h It can be seen that in the UGA reporter system without overexpression of DKC1 iso3, MPMV CTE and its partial truncated mutants can achieve similar or even higher read-through efficiency than SRV CTE. Among them, SRV CTE truncations SRV CTE-m1, SRV CTE-m2 and MPMV CTE truncations MPMV CTE-m1, MPMV CTE-m2, MPMV CTE-m3, MPMV CTE-m4, MPMV CTE-m6 and MPMV CTE-m8 have particularly significant effects on improving the PTC read-through efficiency, and the sequences of these CTE element truncations are shown as SEQ ID NO: 81~88, respectively.

[0230] In summary, the present application improves the efficiency of the RESTART system by enhancing the recruitment and targeting ability of gsnoRNA, and uses it in the future fourth-generation RESTART system (i.e. RESTART V4). Specifically, we increase the CTE element connected to the gsnoRNA to enhance its ability to bind to target mRNA, and insert the CAB box element in the loop region of the gsnoRNA to improve its ability to recruit RNP. Moreover, the above modifications can be combined with other components of the RESTART system to significantly improve the PTC read-through efficiency of the system.

[0231] While the specific embodiments of the application have been described in detail, those skilled in the art will appreciate that various modifications and alterations to the details can be made within the scope of the application as disclosed in the teachings of the present application. The entire disclosure of the application is set out in the accompanying claims and any equivalents thereof.

Claims

1. A method for suppressing a premature termination codon (PTC) in a target RNA in a host cell, characterized in that, The method comprises: introducing into the host cell an engineered guide small nucleolar RNA (gsnoRNA) or a nucleic acid for expressing the gsnoRNA, wherein the gsnoRNA comprises: (i) at least one guide sequence, (ii) at least one CAB box and / or a constitutive transport element (CTE) element, and (iii) a scaffold sequence; wherein, the guide sequence hybridizes to a sequence of a target uridine residue (U) comprising the PTC in the target RNA; the CAB box is derived from a loop region of a stem-loop structure of a natural scaRNA, AluRNA or htrRNA (Human Telomerase RNA); the scaffold sequence is derived from a natural snoRNA of H / ACA type structure and / or a natural scaRNA of H / ACA type structure.

2. The method of claim 1, wherein, the CAB box has a sequence as shown below: X1X2AG; wherein X1and X2are each independently selected from any one of A, U, C, G.

3. The method of claim 1, wherein, the scaffold sequence comprises a first hairpin structure near the 5' end, and a second hairpin structure near the 3' end; and the CAB box is located in the loop of the first hairpin structure, or in the loop of the second hairpin structure, or in the loops of both the first and second hairpin structures.

4. The method of claim 3, wherein, the CAB box is located in the loop of the first hairpin structure in the gsnoRNA.

5. The method of claim 1, wherein, the guide sequence is located in the stem of the first hairpin structure, or in the stem of the second hairpin structure, or in the stems of both the first and second hairpin structures.

6. The method of claim 1, wherein, The method has one or more features selected from the following: (1) the gsnoRNA comprises a CAB box derived from a natural scaRNA, AluRNA or htrRNA, and a sequence of the entire loop region comprising the CAB box in the natural scaRNA, AluRNA or htrRNA; (2) the natural scaRNA is selected from scaRNA1, scaRNA4, scaRNA5, scaRNA6, scaRNA8, scaRNA11, scaRNA12, scaRNA13, scaRNA14, scaRNA15, scaRNA16, scaRNA18, scaRNA20, scaRNA21, scaRNA22, scaRNA23, scaRNA26, scaRNA85 and / or scaRNA27; (3) the natural Alu RNA is selected from the group consisting of AluACA2, AluACA5, AluACA7, AluACA8, AluACA9, AluACA13, AluACA15, AluACA17, AluACA21, AluACA24, AluACA43, AluACA48, AluACA91, AluACA97, AluACA177, AluACA208, AluACA214 and / or AluACA303; (4) the natural htrRNA is human telomerase RNA; (5) the natural H / ACA box snoRNA is selected from the group consisting of ACA19, ACA2b, ACA36, ACA24, ACA5, ACA14a, ACA13, ACA20, ACA44, ACA27, E2, ACA3 and ACA17; (6) the natural H / ACA box scaRNA is selected from the group consisting of scaRNA11 and scaRNA15; (7) the gsnoRNA comprises a first guide sequence and a second guide sequence, and the first guide sequence is located in the stem region of the first hairpin structure, and the second guide sequence is located in the stem region of the second hairpin structure; (8) the gsnoRNA comprises a nucleotide sequence selected from any one of SEQ ID NO: 2-25, SEQ ID NO: 28-33, SEQ ID NO: 68-72, SEQ ID NO: 73-78 or SEQ ID NO: 50-61.

7. The method of claim 1 wherein, The method has one or more features selected from the following: (1) the CTE element is connected to the 5' end of the scaffold sequence of the gsnoRNA, or connected to the 3' end of the scaffold sequence of the gsnoRNA, or connected to both the 5' end and the 3' end of the scaffold sequence of the gsnoRNA; (2) the CTE element is a CTE element derived from a retrovirus or a truncation thereof, wherein the truncation retains or partially retains the function or activity of the CTE element from which it is derived; (3) the CTE element is a CTE element derived from Mason Fisher monkey virus (MPMV) or simian retrovirus type D (SRV) or a truncation thereof, wherein the truncation retains or partially retains the function or activity of the CTE element from which it is derived; (4) the sequence of the CTE element is as shown in SEQ ID NO: 79-88; (5) the gsnoRNA comprises, in 5' to 3' order, a first portion of a first guide sequence, a CAB box derived from a natural scaRNA, Alu RNA or htrRNA (Human Telomerase RNA) or a loop region comprising a stem-loop structure of the CAB box, a second portion of the first guide sequence, a first portion of a second guide sequence, a second portion of the second guide sequence, and a CTE element, linked by a scaffold sequence.

8. The method of claim 1, wherein, The method has one or more features selected from the group consisting of: (1) the CTE element is linked to the scaffold sequence by a linker; (2) the CTE element is linked to the 3' end of the scaffold sequence of the gsnoRNA and is linked by a linker of 4-6 bp or 6-8 bp; (3) the gsnoRNA comprises a nucleotide sequence selected from any one of SEQ ID NOs: 2-25, 28-33, 68-72, 73-78, or 50-61.

9. The method of claim 1, wherein, The gsnoRNA recruits a DKC1 protein to modify the target uridine residue in the target RNA to a pseudouridine residue (Ψ); wherein the DKC1 protein recruited by the gsnoRNA comprises: an endogenous DKC1 protein of the host cell, and / or, an exogenous DKC1 protein of the host cell.

10. The method of claim 9, wherein, The method has one or more features selected from the group consisting of: (1) introducing into the host cell a gsnoRNA that hybridizes to the target RNA and recruits a DKC1 protein, and modifies a target uridine residue (U) of a PTC contained in the target RNA to a pseudouridine residue (Ψ); (2) the method further comprises: introducing into the host cell a nucleic acid molecule encoding a DKC1 protein; (3) the DKC1 protein is overexpressed in the host cell; (4) the DKC1 protein is a naturally occurring DKC1 isoform that has cytoplasmic localization in the host cell; (5) the DKC1 is selected from the group consisting of isoform 1 of human DKC1 protein (DKC1 iso1), isoform 3 of human DKC1 protein (DKC1 iso3), or any combination thereof; (6) the amino acid sequence of the DKC1 protein is set forth in SEQ ID NO: 66 or SEQ ID NO:

67.

11. The method of claim 1 wherein, The method further comprises: introducing into the host cell a near-cognate transfer RNA (nc-tRNA) of the PTC or a nucleic acid molecule for expressing the nc-tRNA.

12. The method of claim 11, wherein, The method has one or more features selected from the group consisting of: (1) modifying a target uridine residue in a PTC of the target RNA to a pseudouridine residue to provide a Ψ-modified PTC, and then introducing into the host cell an nc-tRNA of the Ψ-modified PTC or a nucleic acid molecule for expressing the nc-tRNA to decode the PTC into an amino acid, thereby suppressing the PTC; (2) the nc-tRNA is modified; (3) the nc-tRNA sequence is as shown in SEQ ID NO: 62~65 and SEQ ID NO: 89~172.

13. An engineered guide small nucleolar RNA (gsnoRNA) characterized in that, the gsnoRNA comprises: (i) at least 1 guide sequence, (ii) at least 1 CAB box and / or CTE (constitutive transport element) element, and (iii) a scaffold sequence; wherein, the guide sequence hybridizes to a sequence comprising a PTC-containing target uridine residue (U) in the target RNA; the CAB box is derived from a loop region of a stem-loop structure of a native scaRNA, AluRNA or htrRNA (Human Telomerase RNA); the scaffold sequence is derived from a native H / ACA box-type structured snoRNA and / or a native H / ACA box-type structured scaRNA.

14. The gsno RNA of claim 13, wherein, the gsnoRNA has one or more features selected from the following: (1) the CAB box has a sequence as shown in X1X2AG; wherein X1and X2are each independently selected from any one of A, U, C, G; (2) the scaffold sequence comprises a first hairpin structure proximal to the 5' end, and a second hairpin structure proximal to the 3' end; and the CAB box is located in the loop of the first hairpin structure, or in the loop of the second hairpin structure, or in the loops of both the first and the second hairpin structures; (3) the guide sequence is located in the stem of the first hairpin structure, or in the stem of the second hairpin structure, or in the stems of both the first and the second hairpin structures; (4) the CTE element is a CTE element derived from Mason Fisher virus (MPMV) or Simian retrovirus type D (SRV) or a truncation thereof; (5) the gsnoRNA comprises, in the order from 5' end to 3' end, a first part of a first guide sequence, a CAB box derived from a native scaRNA, AluRNA or htrRNA (Human Telomerase RNA) or a loop region of a stem-loop structure comprising the CAB box, a second part of the first guide sequence, a first part of a second guide sequence, a second part of the second guide sequence, and a CTE element, connected by a scaffold sequence.

15. An isolated nucleic acid molecule characterized in that, The isolated nucleic acid molecule comprises a nucleic acid sequence for expressing the gsnoRNA of claim 13 or 14.

16. A composition characterized in that, The composition comprises: the gsnoRNA of claim 13 or 14 or the isolated nucleic acid molecule of claim 15.

17. The composition of claim 16, wherein, The composition comprises: (a) the gsnoRNA of claim 13 or 14 or the isolated nucleic acid molecule of claim 15; and a near-cognate transport RNA (nc-tRNA) for a PTC contained in the target sequence or a nucleic acid for expressing the nc-tRNA. (b) the gsnoRNA of claim 13 or 14 or the isolated nucleic acid molecule of claim 15; and a DKC1 protein or a nucleic acid molecule encoding a DKC1 protein; or (c) the gsnoRNA of claim 13 or 14 or the isolated nucleic acid molecule of claim 15, a near-cognate transport RNA (nc-tRNA) of a PTC contained in the target sequence or a nucleic acid for expressing the nc-tRNA, and a DKC1 protein or a nucleic acid molecule encoding a DKC1 protein.

18. A delivery composition characterized in that, The delivery composition comprises a delivery vehicle, and one or more selected from the group consisting of the gsnoRNA of claim 13 or 14 or the isolated nucleic acid molecule of claim 15 or the composition of claim 16 or 17; wherein the delivery vehicle is a particle.

19. A host cell, characterized in that, The host cell comprises the gsnoRNA of claim 13 or 14 or the isolated nucleic acid molecule of claim 15 or the composition of claim 16 or 17 or the delivery composition of claim 18.

20. A method of preparing the gsno RNA of claim 13 or 14 or the composition of claim 16 or 17 or the delivery composition of claim 18, characterized in that, The method comprises culturing the host cell of claim 19 under conditions that allow expression of nucleic acids and proteins, and recovering the gsnoRNA or the composition or the delivery composition from the cultured host cell culture.

21. Use of the gsnoRNA of claim 13 or 14 or the isolated nucleic acid molecule of claim 15 or the composition of claim 16 or 17 or the delivery composition of claim 18 or the host cell of claim 19 in the manufacture of a medicament for target RNA editing or for inhibiting a premature termination codon (PTC) in a target RNA in a host cell.

22. Use of the gsno RNA of claim 13 or 14, or the isolated nucleic acid molecule of claim 15, or the composition of claim 16 or 17, or the delivery composition of claim 18, or the host cell of claim 19, for the manufacture of a pharmaceutical product, characterized in that, The pharmaceutical product is for treating a disease and / or a symptom caused or induced by a PTC mutation in a subject.

23. A method of editing a target RNA in vitro or in vivo, characterized in that, The method comprises contacting a target RNA with one or more of the gsnoRNA of claim 13 or 14 or the isolated nucleic acid molecule of claim 15 or the composition of claim 16 or 17 or the delivery composition of claim 18 under conditions suitable for performing target RNA editing, thereby editing the target RNA. The method comprises contacting a target RNA with one or more of the gsnoRNA of claim 13 or 14 or the isolated nucleic acid molecule of claim 15 or the composition of claim 16 or 17 or the delivery composition of claim 18 under conditions suitable for performing target RNA editing, thereby editing the target RNA.

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