Reengineered tRNA and uses thereof

Abstract

The present application relates to the field of biotechnology and the field of RNA technology. In particular, the present application relates to an engineered tRNA, isolated nucleic acid molecules expressing the same, compositions comprising the same, delivery compositions and host cells. The present application also relates to their use in the manufacture of a medicament and a formulation.

Description

Technical Field

[0001] This application relates to the fields of biotechnology and RNA technology. Specifically, this application relates to a modified tRNA, an isolated nucleic acid molecule expressing the tRNA, a composition containing the tRNA, a delivery composition, and a host cell. This application also relates to their use in the preparation of pharmaceuticals and formulations. Background Technology

[0002] Nonsense mutations are genetic mutations caused by single-base substitutions in the coding region of mRNA. These substitutions convert a codon encoding a normal amino acid into a stop codon, resulting in a premature termination codon (PTC) and premature termination of protein translation. According to the Human Gene Mutation Database (HGMD), nonsense mutations account for more than 20% of disease-related single nucleotide mutations and as much as 11% of all mutations causing human genetic diseases.

[0003] Nonsense mutations are associated with a variety of genetic diseases and can cause severe disease phenotypes. For example, nonsense mutations in the α-L-iduronidase (IDUA) gene can lead to Hurler syndrome, a mucopolysaccharidosis caused by lysosomal abnormalities that prevents the metabolism of glycosaminoglycans, resulting in toxic effects and damage and dysfunction in multiple systems of the body. In patients with cystic fibrosis, nonsense mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene account for approximately 10%. Defects in this CFTR gene lead to ion transport disorders, the accumulation of thick mucus in epithelial cells, and consequently, chronic inflammation and irreversible lung damage. Another example is spinal muscular atrophy (SMA). Nonsense mutations in the Survivalmotor neuron 1 (SMN1) gene impair the structure and function of motor neurons, manifesting as progressive muscle weakness and atrophy, and patients exhibit a high mortality rate in infancy. Besides genetic diseases, nonsense mutations can also occur in certain cancer-related genes, such as the tumor suppressor gene p53 (TP53), leading to its dysfunction. Therefore, exploring strategies to suppress nonsense mutations is extremely important for the treatment of various diseases.

[0004] Given its significant harm to patients' 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 translation-read-through inducing drugs to interfere with ribosome recognition of PTCs, using repressive tRNAs to recognize PTCs and incorporate specific amino acids, applying DNA editing strategies to correct mutations in gene sequences, and employing 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 play an important role in nonsense mutation suppression. Researchers have designed gRNAs that enable ADAR to target and edit the A protein in the PTC (protocol site), converting the UGA or UAG codon to the UGG codon, and then incorporating tryptophan at the PTC site, thereby restoring the expression of the full-length protein. However, since ADAR can only achieve A-to-G editing, this strategy can only insert tryptophan at the PTC site. For some critical PTC sites that cannot tolerate missense mutations, further exploration of other RNA editing strategies capable of precisely inserting specific amino acids is needed to achieve more effective repair.

[0006] Besides ADAR-based RNA editing strategies, researchers are also exploring other RNA editing strategies to achieve broader nonsense mutation repair. Among them, the U-to-Ψ editing strategy, due to its unique base modification characteristics and endogenous targeted modification mechanism, is gradually becoming an important direction in nonsense mutation research. In 2011, Yu's laboratory first discovered that pseudouridine modification of PTC could inhibit nonsense mutations and restore the expression of full-length proteins in in vitro experiments and yeast cells. This discovery sparked researchers' enthusiasm for exploring the inhibition of nonsense mutations by pseudouridine modification.

[0007] To avoid delivery difficulties and immunogenicity caused by exogenous protein overexpression, researchers first considered using the endogenous pseudouridine mechanism for targeted modification. To this end, they systematically explored the catalytic mechanism of H / ACAbox snoRNPs in modified rRNA and snRNA in a yeast system. The study found that pseudouridine modification requires three core sequence and structural elements: the stability of the pseudouridine pocket and hairpin structure of the snoRNA, a fixed distance of 14–15 nt between the target uridine and the H / ACA box, and the base pairing strength between the pseudouridine pocket sequence and the target sequence. Furthermore, the researchers also explored the mechanism by which pseudouridine modification of PTCs inhibits nonsense mutations. They proposed that pseudouridine modification can affect the codon-anticodon interaction at the PTC site, making the binding affinity of certain tRNAs to the PTC site stronger than that of the releasing factor, thereby inhibiting translation termination and incorporating specific amino acids. By identifying the amino acids incorporated into pseudouridine-containing PTC sites in yeast cells, researchers found that the ΨAA and ΨAG codons primarily incorporate threonine or serine, while the ΨGA codon primarily incorporates phenylalanine or tyrosine. This result further suggests that pseudouridine-containing can influence the coding rules at PTC sites in a specific way, thereby enabling the precise incorporation of certain amino acids into the PTC site.

[0008] Overall, the H / ACA box snoRNP-based U-to-Ψ editing system is a highly flexible strategy for suppressing nonsense mutations. Its flexibility in targeting sequence pairing and diversity in amino acid incorporation enable it to play a significant role in the treatment of a wide range of nonsense mutation-related diseases. Furthermore, this strategy regulates only endogenous transcripts without affecting 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 nonsense mutation disease scenarios, have significant scientific and practical value.

[0009] Prior to this, the applicant developed a novel programmable RNA pseudouridine editing system—RESTART. By synergistically utilizing the endogenous targeting modification mechanism of pseudouridine and the regulatory properties of stop codons, it achieved precise pseudouridine modification at PTC sites, promoting PTC readthrough and restoring full-length functional protein expression, thereby efficiently and specifically repairing nonsense mutations. Building upon this, multiple generations of systems (RESTART v1, v2, v3, and v3-mini) were developed. These systems, by modifying snoRNA to guide endogenous snoRNP editing of target sites, further improved readthrough efficiency by increasing the content of endogenous near-homologous tRNA (nc-tRNA). However, the editing efficiency of these systems still has significant room for improvement. Therefore, it is urgent to optimize the design of components within the system and regulate the expression levels of endogenous proteins to comprehensively improve modification and readthrough levels, develop novel RNA editing systems, and mediate nonsense mutation repair. Summary of the Invention

[0010] This application optimizes and modifies the previously studied RESTART pseudouridine modification system, providing a modified tRNA that can serve as an nc-tRNA for PTC sites, significantly improving the efficiency of PTC recognition and decoding. Furthermore, this modified tRNA is suitable for multiple iterations of the RESTART system and can be used in combination with other elements in the RESTART system (e.g., gsnoRNA) to significantly improve PTC readthrough efficiency.

[0011] Therefore, in a first aspect, this application provides a method for inhibiting premature stop codons (PTCs) in target RNA in host cells, wherein the method includes introducing the following components into the host cell:

[0012] (1) an engineered guide small nucleolar RNA (gsnoRNA) or a nucleic acid molecule for expressing said gsnoRNA; and

[0013] (2) The modified tRNA or the nucleic acid molecule used to express the modified tRNA;

[0014] The modified tRNA, compared to the wild-type tRNA, contains mutations in the first, second, and / or third base pairs in the T stem from the 5' to the 3' direction, and has base pairs that are different from the corresponding base pairs in the wild-type tRNA.

[0015] In some embodiments, the methods, modified tRNAs, and compositions provided herein include modifications to target RNAs (e.g., mRNAs) in eukaryotic organisms (e.g., mammalian cells, such as human cells). In some aspects, the host cell can be a cell from any organ, such as skin, lung, heart, kidney, liver, pancreas, intestine, muscle, glands, eye, brain, blood, etc. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a human cell. The host cell can be located in vitro or in vivo. In some embodiments, the host cell is an ex vivo cell.

[0016] One advantage of the methods, modified tRNAs, and compositions provided herein is that they can be used both in situ in living organisms and in cells cultured in vitro. In some embodiments, the host cells are treated in vitro and then introduced into a living organism (e.g., reintroduced into their original source organism).

[0017] The methods, modified tRNAs, and compositions presented in this article can also be used to read PTCs or re-encode Ψ-modified codons within so-called organoid cells. Organoids can be considered three-dimensional, in vitro-derived tissues, but driven by specific conditions to produce individual tissues. In a therapeutic setting, organoids can be synthesized in vitro and reintroduced into patients as autologous material, making them less susceptible to rejection than normal transplantation.

[0018] In some embodiments, the host cell has a gene mutation. The mutation may be heterozygous or homozygous. In some embodiments, the methods, modified tRNAs, and compositions provided herein can be used to modify point mutations (e.g., for reading through point-mutated PTCs, or for recoding point mutations in sense codons). In some embodiments, when a human subject has a PTC-related disease, the methods, gsnoRNAs, and compositions provided herein are suitable for modifying RNA sequences in cells, tissues, or organs associated with the subject's (e.g., a human subject's) disease state.

[0019] The method of this application can effectively restore the coding information of mRNA without changing the DNA sequence of the host cell, so that the PTC site can be decoded, significantly improving the reading efficiency of PTC, thereby expressing the complete protein.

[0020] In some implementations, the method of this application is highly specific and does not lead to pseudouridineization and readthrough of normal stop codons.

[0021] In some implementations, PTC is caused by mutations in sense codons. In some implementations, the above method can significantly improve PTC readthrough efficiency. In some implementations, the PTC readthrough efficiency of the above method is at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, or at least 20-fold higher than that of other methods that do not use the modified tRNA described above.

[0022] When using the method of this application, the guide sequence in the gsnoRNA introduced into the host cell pairs complementaryly with the bases of the target mRNA, causing the uridine residue U in the PTC to enter the pseudouridine pocket formed by the stem-loop structure of the gsnoRNA. The gsnoRNA recruits endogenous and / or exogenously introduced DKC1, NOP10, NHP2, and GAR1 proteins to assemble into the RNP complex. The uridine residue located in the pseudouridine pocket can enter the catalytic center of DKC1, thereby catalyzing the modification of the uridine residue U into the pseudouridine residue Ψ. Furthermore, the modified tRNA introduced into the host cell binds to the Ψ-modified PTC and is translated into amino acids, achieving read-through of the PTC site.

[0023] In the method of this application, any suitable method can be used to introduce the modified tRNA into the host cell. Furthermore, regardless of the modified tRNA introduction method, the method can suppress premature stop codons (PTCs) in the target RNA of the host cell, significantly improving the readthrough efficiency of PTCs.

[0024] In some embodiments, the modified tRNA is introduced into the host cell via direct delivery. In other embodiments, the modified tRNA is introduced into the host cell by transfecting the host cell with a vector containing a nucleotide sequence expressing the modified tRNA.

[0025] T-stem modification

[0026] In some embodiments, the wild-type tRNA is selected from tRNA-R-UCU, tRNA-R-UCG, tRNA-G-UCC, tRNA-S-UGA, tRNA-W-CCA, tRNA-C-GCA, tRNA-Q-CUG, tRNA-K-CUU, tRNA-E-CUC, tRNA-S-CGA, tRNA-L-CAA, tRNA-Y-AUA, tRNA-Y-GUA, tRNA-Q-UUG, tRNA-K-UUU, and tRNA-E-UUC.

[0027] In some embodiments, the wild-type tRNA has or includes a nucleotide sequence as shown in any one of SEQ ID NO: 158-161 and SEQ ID NO: 187-270.

[0028] In some embodiments, the wild-type tRNA is selected from tRNA-R-UCU, tRNA-Q-CUG, tRNA-Q-UUG, and tRNA-W-CCA.

[0029] In some embodiments, the wild-type tRNA has or contains a nucleotide sequence as shown in any one of SEQ ID NO: 158-161.

[0030] In some implementations, in the tRNA standard numbering system, the positions of the first, second, and third base pairs are 49:65, 50:64, and 51:63, respectively.

[0031] In some implementations, the first, second, and third base pairs follow the Watson-Crick pairing principle or the wobble pairing principle, respectively.

[0032] In some embodiments, the first base pair, the second base pair, and / or the third base pair are each independently selected from: AU, UA, GC, CG, GU, and UG.

[0033] In some embodiments, the wild-type tRNA is tRNA-R-UCU, wherein the first, second, and third base pairs of the wild-type tRNA in the T-stem from the 5' to the 3' direction are CG, CG, and GC, respectively; the modified tRNA has the following mutations: the first base pair is mutated to AU, GU, or GC, and / or the second base pair is mutated to UA.

[0034] In some embodiments, the modified tRNA has or contains a nucleotide sequence as shown in any one of SEQ ID NO: 1-5.

[0035] In some embodiments, the wild-type tRNA is tRNA-W-CCA, wherein the first, second, and third base pairs of the wild-type tRNA in the T-stem from the 5' to the 3' direction are GU, CG, and GC, respectively; the modified tRNA has the following mutations: the first base pair is mutated to GC, AU, or CG, or, and / or, the third base pair is mutated to UG.

[0036] In some embodiments, the modified tRNA has or contains a nucleotide sequence as shown in any one of SEQ ID NO: 8-11.

[0037] In some embodiments, the wild-type tRNA is tRNA-Q-CUG, wherein the first, second, and third base pairs of the wild-type tRNA in the T-stem from the 5' to the 3' direction are CG, CG, and GC, respectively; the modified tRNA has the following mutations: the first base pair is mutated to UA, GC, or AU; the second base pair is mutated to UA, UG, or GU; and / or, the third base pair is mutated to CG.

[0038] In some embodiments, the modified tRNA has or includes a nucleotide sequence as shown in any one of SEQ ID NO: 12, 13, 18, 27-29, 31, 32, 34-36 and 38.

[0039] In some embodiments, the wild-type tRNA is tRNA-Q-UUG, wherein the first, second, and third base pairs of the wild-type tRNA in the T-stem from the 5' to the 3' direction are CG, CG, and GC, respectively; the modified tRNA has the following mutations: the first base pair is mutated to GC or AU, the second base pair is mutated to UA, UG, or GU, and / or, the third base pair is mutated to UA, GU, or CG.

[0040] In some embodiments, the modified tRNA has or contains nucleotide sequences as shown in any one of SEQ ID NO: 41, 43-50.

[0041] In some implementations, the modified tRNA significantly improves the readthrough efficiency of PTC.

[0042] In some embodiments, the wild-type tRNA is tRNA-R-UCU, wherein the first, second, and third base pairs of the wild-type tRNA in the T-stem from the 5' to the 3' direction are CG, CG, and GC, respectively; and the first base pair of the modified tRNA is mutated to GU.

[0043] In some embodiments, the modified tRNA has or contains a nucleotide sequence as shown in SEQ ID NO: 2.

[0044] In some embodiments, the wild-type tRNA is tRNA-W-CCA, wherein the first, second, and third base pairs of the wild-type tRNA in the T-stem from the 5' to the 3' direction are GU, CG, and GC, respectively; and the first base pair of the modified tRNA is mutated to AU.

[0045] In some embodiments, the modified tRNA has or contains a nucleotide sequence as shown in SEQ ID NO: 9.

[0046] In some embodiments, the wild-type tRNA is tRNA-Q-CUG, wherein the first, second, and third base pairs in the T-stem from the 5' to the 3' direction are CG, CG, and GC, respectively; the modified tRNA has the following mutations:

[0047] a) The second base pair mutates to UG, and the third base pair mutates to CG;

[0048] b) The first base pair mutates to GC, the second base pair mutates to UG, and the third base pair mutates to CG;

[0049] c) The first base pair mutates to AU, the second base pair mutates to UG, and the third base pair mutates to CG; or

[0050] d) The first base pair mutates to GC, the second base pair mutates to GU, and the third base pair mutates to CG.

[0051] In some embodiments, the modified tRNA has or contains nucleotide sequences as shown in any one of SEQ ID NO: 18, 35, 36 and 38.

[0052] In some embodiments, the wild-type tRNA is tRNA-Q-UUG, wherein the first, second, and third base pairs in the T-stem from the 5' to the 3' direction are CG, CG, and GC, respectively; the modified tRNA has the following mutations:

[0053] a) The second base pair mutates to UA, and the third base pair mutates to GU;

[0054] b) The first base pair mutates to GC, the second base pair mutates to UG, and the third base pair mutates to CG; or

[0055] c) The first base pair mutates to AU, the second base pair mutates to UG, and the third base pair mutates to CG.

[0056] In some embodiments, the modified tRNA has or contains nucleotide sequences as shown in any one of SEQ ID NO: 43, 48 and 49.

[0057] AC stem modification

[0058] In addition to the modification of the T-stem, this application also constructs modified tRNAs by modifying the AC stem (anticodon stem).

[0059] In some implementations, the modified tRNA has a reduced number of base pairs in the AC stem from 5 to 4 compared to the wild-type tRNA.

[0060] In some embodiments, the modified tRNA includes both T-stem modification and AC-stem modification as described above.

[0061] In some embodiments, the wild-type tRNA is selected from tRNA-R-UCU, tRNA-R-UCG, tRNA-G-UCC, tRNA-S-UGA, tRNA-W-CCA, tRNA-C-GCA, tRNA-Q-CUG, tRNA-K-CUU, tRNA-E-CUC, tRNA-S-CGA, tRNA-L-CAA, tRNA-Y-AUA, tRNA-Y-GUA, tRNA-Q-UUG, tRNA-K-UUU, and tRNA-E-UUC.

[0062] In some embodiments, the wild-type tRNA has or includes a nucleotide sequence as shown in any one of SEQ ID NO: 158-161 and SEQ ID NO: 187-270.

[0063] In some implementations, the wild-type tRNA is tRNA-W-CCA.

[0064] In some embodiments, the wild-type tRNA has or contains a nucleotide sequence as shown in SEQ ID NO: 159.

[0065] In some embodiments, the base pairs are paired according to the Watson-Crick pairing principle or the wobble pairing principle.

[0066] In some embodiments, the base pairs are selected from: AU, UA, GC, CG, GU, and UG.

[0067] In some implementations, in the tRNA standard numbering system, the modified tRNA corresponds to the base mispairing at position 27:43 of the wild-type tRNA.

[0068] In some implementations, in the tRNA standard numbering system, the modified tRNA corresponds to the wild-type tRNA at position 27:43, where the bases do not conform to the Watson-Crick pairing rule or the wobble pairing rule.

[0069] In some embodiments, the modified tRNA has or contains a nucleotide sequence as shown in SEQ ID NO: 6 or 7.

[0070] In some implementations, the modified tRNA significantly improves the readability of PTC, for example, by at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0071] In some implementations, the PTC is a UGA or a UAG.

[0072] nc-tRNA

[0073] nc-tRNA, or near-cognate tRNA, has an anticodon that has a single base mismatch or wobble pair with a Ψ-modified codon in mRNA (such as ΨAA, ΨAG, ΨGA) (the remaining positions are Watson-Crick pairs). It recognizes and decodes the codon into a specific amino acid through non-canonical base pairing.

[0074] In some implementations, the modified tRNA is a near-homologous transfer RNA (nc-tRNA) of the PTC.

[0075] In some implementations, the modified tRNA decodes the PTC into amino acids, thereby inhibiting the PTC.

[0076] In some embodiments, the nucleic acid molecule expressing the modified tRNA contains 1, 2, 3, 4 or more copies of the nucleotide sequence encoding the modified tRNA.

[0077] In some implementations, the modified tRNA is modified (e.g., psiU, m...). 1 A、m 1 G or m 5 C modifier).

[0078] In some embodiments, the modification includes one or more modifications to the mature tRNA. In some embodiments, the modification includes one or more modifications selected from the group consisting of psiU, m 1 A、m 1 G and m 5 C. In some embodiments, modified tRNA significantly increases PTC readthrough efficiency compared to unmodified tRNA of the same type. In some embodiments, the modified tRNA contains 5' phosphorylation. In some embodiments, the modified tRNA is a mature tRNA without introns.

[0079] In some implementations, the modified tRNA does not contain intron sequences.

[0080] In some implementations, the modified tRNA contains intron sequences.

[0081] In some embodiments, PTC is a UGA codon, wherein the Ψ-modified PTC is decoded to arginine or tryptophan. In some embodiments, the Ψ-modified PTC is decoded to arginine at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% of the time.

[0082] In some embodiments, PTC is a UAG codon, wherein Ψ-modified PTC is decoded as glutamine, leucine, or tyrosine. In some embodiments, nc-tRNA is tRNA-Q-CUG, tRNA-L-CAA, tRNA-Y-AUA, and / or tRNA-Y-GUA.

[0083] In some embodiments, PTC is a UAA codon, where Ψ-modified PTC is decoded as glutamine or tyrosine. In some embodiments, nc-tRNA is tRNA-Q-UUG, tRNA-Y-AUA, and / or tRNA-Y-GUA.

[0084] In some embodiments, the PTC is caused by a nonsense mutation in the codon encoding arginine. In some embodiments, the PTC is the UGA codon, and the nc-tRNA is tRNA-R-UCU, which decodes the Ψ-modified PTC into arginine.

[0085] In some embodiments, the PTC is caused by a nonsense mutation in the codon encoding glutamine. In some embodiments, the PTC is caused by a nonsense mutation in the codon encoding glutamine, and the nc-tRNA is tRNA-Q-CUG or tRNA-Q-UUG, which decodes the Ψ-modified PTC into glutamine.

[0086] The twenty common amino acids referred to herein are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0087] gsnoRNA

[0088] There are two types of natural snoRNAs and scaRNAs: one is a C / D type structure, and the other is an H / ACA type structure. The H / ACA type structure can recruit the DKC1 protein. Therefore, in this paper, the gsnoRNA described can recruit the DKC1 protein in this host cell and modify the target uridine residues in the target RNA into pseudouridine residues.

[0089] In some embodiments, the gsnoRNA comprises: at least one guide sequence and a scaffold sequence; wherein the guide sequence hybridizes with a sequence of target uridine residues (U) containing PTC in the target RNA; the scaffold sequence is derived from naturally occurring H / ACA type snoRNA and / or naturally occurring H / ACA type scaRNA.

[0090] In some embodiments, the naturally occurring 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.

[0091] In some embodiments, the naturally occurring H / ACA-type scaRNA is selected from the group consisting of scaRNA11 and scaRNA15.

[0092] In this paper, the "guide sequence" is the portion of the gsnoRNA responsible for target recognition, consisting of a nucleotide sequence complementary to a specific region in the target RNA (such as a PTC sequence containing the target uridine residue). The guide sequence pairs complementaryly with the target mRNA bases, allowing the uridine residue U (e.g., uridine residue U in the PTC) to enter the pseudouridine pocket formed by the gsnoRNA stem-loop structure. The uridine residue U in the pseudouridine pocket can then enter the catalytic center of the DKC1 enzyme, thereby achieving editing.

[0093] In some embodiments, the guiding sequence is located in the stem region of the first hairpin structure, or in the stem region of the second hairpin structure, or in the stem regions of both the first and second hairpin structures.

[0094] In some embodiments, the gsnoRNA comprises a first guide sequence and a second guide sequence, wherein 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.

[0095] In some implementations, the guide sequence in each hairpin structure is divided into a first part sequence and a second part sequence, with the first part sequence located on the 5' end side of its stem and the second part sequence located on the 3' end side of its stem.

[0096] In some embodiments, the guide sequence is not limited to a specific length and sequence, as long as it can hybridize with a sequence containing a target uridine residue (U) of PTC in the target RNA. In some embodiments, 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.

[0097] In some implementations, the guide sequence is fully or highly complementary to a specific region of the target RNA containing the target uridine residue (such as a PTC sequence). For example, the number of bases complementary to this specific region in the guide sequence is at least 70%, at least 80%, at least 90%, or 100% of the total number of bases in the guide sequence to ensure hybridization specificity and efficiency.

[0098] In some embodiments, the gsnoRNA recruits the DKC1 protein to modify the target uridine residue in the PTC of the target RNA into a pseudouridine residue (Ψ).

[0099] In some embodiments, the gsnoRNA hybridizes with the target RNA and recruits the DKC1 protein, and modifies the target uridine residue (U) of the PTC contained in the target RNA to a pseudouridine residue (Ψ).

[0100] In some embodiments, to improve in vivo stability and delivery efficiency, the gsnoRNA may contain one or more modified (e.g., chemically modified) nucleotides. In some embodiments, one or more nucleotides of the gsnoRNA contain a 2'-O-methyl (2'-OMe) modification. In some embodiments, the gsnoRNA contains one or more phosphate thioester (PS) nucleotide bonds. In some embodiments, the gsnoRNA contains a 5' cap modification (e.g., m... 7 (G-hat modification).

[0101] DKC1 protein

[0102] DKC1 protein is a highly conserved pseudouridine synthase. It is responsible for catalyzing the conversion of uridine residues in RNA to pseudouridine residues.

[0103] In some embodiments, the method further includes introducing a nucleic acid molecule encoding the DKC1 protein into the host cell.

[0104] In some embodiments, the DKC1 protein is overexpressed in the host cells.

[0105] In some embodiments, the DKC1 protein is a naturally occurring DKC1 subtype that has cytoplasmic localization in the host cell.

[0106] In some embodiments, the DKC1 is selected from human DKC1 protein iso1, human DKC1 protein iso3, or any combination thereof.

[0107] In some embodiments, the amino acid sequence of the DKC1 protein is shown in SEQ ID NO: 164 or SEQ ID NO: 163.

[0108] In some embodiments, the gsnoRNA recruits core proteins (DKC1, NHP2, GAR1, and NOP10) and forms snoRNA with the core proteins to modify the target uridine residues in the target RNA into pseudouridine residues (Ψ).

[0109] It is understood that the DKC1 protein can be endogenously expressed DKC1 iso1 and / or DKC1 iso3, or exogenously expressed DKC1 iso1 and / or DKC1 iso3. In some embodiments, the DKC1 protein recruited by the gsnoRNA comprises: endogenous DKC1 protein of the host cell, and / or exogenous DKC1 protein of the host cell.

[0110] In some embodiments, the DKC1 protein comprises an amino acid sequence having 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% identity with the sequence in SEQ ID NO: 164 or 163.

[0111] Based on codon degeneracy known in the art, in some embodiments, the nucleotide sequence encoding the DKC1 protein (e.g., DKC1 iso1 or DKC1 iso3) can be substituted according to codon degeneracy. In some embodiments, the nucleotide sequence encoding the DKC1 protein (e.g., DKC1 iso1 or DKC1 iso3) is codon-optimized.

[0112] Modified tRNA

[0113] On the other hand, this application provides a modified tRNA, wherein, compared with wild-type tRNA, the modified tRNA contains mutations in the first, second, and / or third base pairs in the T stem from the 5' to the 3' direction, and has base pairs that are different from the corresponding base pairs in the wild-type tRNA.

[0114] In some implementations, in the tRNA standard numbering system, the positions of the first, second, and third base pairs are 49:65, 50:64, and 51:63, respectively.

[0115] In some implementations, the first, second, and third base pairs follow the Watson-Crick pairing principle or the wobble pairing principle, respectively.

[0116] In some embodiments, the first base pair, the second base pair, and / or the third base pair are each independently selected from: AU, UA, GC, CG, GU, and UG.

[0117] In some embodiments, the wild-type tRNA is selected from tRNA-R-UCU, tRNA-R-UCG, tRNA-G-UCC, tRNA-S-UGA, tRNA-W-CCA, tRNA-C-GCA, tRNA-Q-CUG, tRNA-K-CUU, tRNA-E-CUC, tRNA-S-CGA, tRNA-L-CAA, tRNA-Y-AUA, tRNA-Y-GUA, tRNA-Q-UUG, tRNA-K-UUU, and tRNA-E-UUC.

[0118] In some embodiments, the wild-type tRNA has or includes a nucleotide sequence as shown in any one of SEQ ID NO: 158-161 and SEQ ID NO: 187-270.

[0119] In some embodiments, the wild-type tRNA is selected from tRNA-R-UCU, tRNA-Q-CUG, tRNA-Q-UUG, and tRNA-W-CCA.

[0120] In some embodiments, the wild-type tRNA has or contains a nucleotide sequence as shown in any one of SEQ ID NO: 158-161.

[0121] In some embodiments, the modified tRNA has or includes nucleotide sequences as shown in any one of SEQ ID NO: 1-5, SEQ ID NO: 8-11, SEQ ID NO: 12, 13, 18, 27-29, 31, 32, 34-36, 38, SEQ ID NO: 41, 43-50.

[0122] In some embodiments, the modified tRNA has or includes a nucleotide sequence as shown in any one of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 35, 36, 38, SEQ ID NO: 43, 48 and 49.

[0123] isolated nucleic acid molecules

[0124] On the other hand, this application provides isolated nucleic acid molecules containing nucleotide sequences for expressing the modified tRNA as described above.

[0125] In some implementations, the isolated nucleic acid molecule is DNA.

[0126] Composition

[0127] In another aspect, this application provides compositions comprising the modified tRNA as described above or the isolated nucleic acid molecule as described above.

[0128] In some embodiments, the composition further comprises engineered guide small nucleolar RNA (gsnoRNA) or a nucleic acid molecule for expressing said gsnoRNA.

[0129] In some embodiments, the composition further comprises a DKC1 protein (e.g., DKC1 iso1, DKC1 iso3) or a nucleic acid molecule encoding the DKC1 protein.

[0130] In some embodiments, the composition further comprises gsnoRNA or a nucleic acid molecule for expressing the gsnoRNA, and DKC1 protein or a nucleic acid molecule encoding the DKC1 protein.

[0131] In some embodiments, the composition comprises: the isolated nucleic acid molecule as described above, the nucleic acid for expressing the modified tRNA, and the nucleic acid molecule encoding the DKC1 protein, wherein the nucleic acid or nucleic acid molecule is present in the same or different vectors.

[0132] In some embodiments, the DKC1 protein has the characteristics described in the first aspect.

[0133] In some embodiments, the gsnoRNA has the characteristics described in the first aspect.

[0134] Delivery composition

[0135] The modified tRNA described in this paper can be directly delivered into host cells after in vitro transcription and can be delivered by any method known in the art.

[0136] Such methods include, but are not limited to, electroporation, lipid transfection, nuclear transfection, microinjection, acoustic hole effect, gene gun, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendritic transfection, heat shock transfection, magnetic transfection, puncture transfection, optical transfection, reagent-enhanced nucleic acid uptake, and delivery via liposomes, immunoliposomes, viral particles, artificial viruses, etc.

[0137] Therefore, in another aspect, this application provides a delivery composition comprising: a delivery vector, and one or more selected from the following: a modified tRNA as described above, an isolated nucleic acid molecule as described above, or a composition as described above.

[0138] In some implementations, the delivery carrier is a particle.

[0139] In some embodiments, the delivery vector is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, gene guns, or viral vectors (e.g., replication-defective retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses).

[0140] In some embodiments, the delivery composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0141] As used herein, the term “pharmaceuticalally acceptable carrier and / or excipient” means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives.

[0142] host cells

[0143] On the other hand, this application provides a host cell comprising the modified tRNA as described above, the isolated nucleic acid molecule as described above, the composition as described above, or the delivery composition as described above.

[0144] Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., Escherichia coli cells), 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, etc.).

[0145] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0146] In some implementations, the host cell is a mammalian cell (e.g., a human cell).

[0147] In some implementations, the host cell contains a premature stop codon (PTC) mutated gene.

[0148] use

[0149] On the other hand, this application provides the use of the modified tRNA as described above, the isolated nucleic acid molecule as described above, the composition as described above, the delivery composition as described above, or the host cell as described above in the preparation of a formulation for inhibiting premature stop codons (PTCs) in target RNA in a host cell.

[0150] In some embodiments, the formulation decodes the PTC into amino acids, thereby inhibiting the PTC.

[0151] In some embodiments, the uridine residue (U) in the PTC is a pseudouridine residue (Ψ), and the PTC is an Ψ-modified PTC.

[0152] In some implementations, the host cell is a mammalian cell (e.g., a human cell).

[0153] On the other hand, this application provides the use of the modified tRNA as described above, the isolated nucleic acid molecule as described above, the composition as described above, the delivery composition as described above, or the host cell as described above in the preparation of a medicine for treating a subject with a disease and / or symptoms caused or resulting from a premature stop codon (PTC) mutation.

[0154] In some embodiments, the disease is selected from cystic fibrosis, spinal muscular atrophy, fructose intolerance, dilated cardiomyopathy, Heller syndrome, alpha-1-antitrypsin (A1AT) deficiency, Parkinson's disease, Alzheimer's disease, albinism, amyotrophic lateral sclerosis, asthma, β-thalassemia, Cadasil syndrome, and Charcot-Marie-Tooth disease. Diseases including chronic obstructive pulmonary disease (COPD), distal spinal muscular atrophy (DSMA), Duchenne muscular dystrophy, dystrophic epidermolysis bullosa, epidermolysis bullosa, Fabry disease, Leiden factor V-related disorders, familial adenomatous polyposis, galactosemia, Gaucher disease, glucose-6-phosphate dehydrogenase, hemophilia, hereditary hemochromatosis, Huntington's disease, inflammatory bowel disease (IBD), hereditary polyagglutination syndrome, Lesch-Nair syndrome, Lynch syndrome, Marfan syndrome, mucopolysaccharidosis, muscular dystrophy, and type I and II myotonic dystrophy. Neurofibromatosis, Niemann-Pick disease types A, B, and C, NY-esol-related cancers, Boytz-Yage syndrome, phenylketonuria, Pomper's disease, primary ciliary body disease, prothrombin mutation-related diseases (such as prothrombin G20210A mutation), pulmonary hypertension, (autosomal dominant) retinitis pigmentosa, Sandhoff's disease, severe combined immunodeficiency syndrome (SCID), sickle cell anemia, Staggart disease, Tay-Sachs disease, Usher syndrome, X-linked immunodeficiency, Sturge-Weber syndrome, or any combination thereof.

[0155] In some implementations, the subject is a mammal (e.g., human, cynomolgus monkey, mouse).

[0156] In some embodiments, the disease is fructose intolerance. In some embodiments, the PTC mutation is a nonsense mutation in the nucleotide encoding amino acid 148 of ALDOB (fructose diphosphate aldolase B).

[0157] In some embodiments, the disease is cystic fibrosis. In some embodiments, the PTC mutation is a nonsense mutation in the nucleotide encoding amino acid 553 of the CFTR (cystic fibrosis transmembrane conduction regulator).

[0158] In some embodiments, the disease is dilated cardiomyopathy. In some embodiments, the PTC mutation is a nonsense mutation in the nucleotide encoding amino acid 225 of LMNA (lamin A / C).

[0159] Preparation method

[0160] On the other hand, this application provides a method for preparing the modified tRNA or the composition or the delivery composition as described above, characterized in that the method comprises culturing host cells as described above under conditions that allow nucleic acid and protein expression, and recovering the modified tRNA or the composition or the delivery composition from the cultured host cell culture.

[0161] Terminology Definition

[0162] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0163] As used herein, the term "tRNA" stands for transfer RNA, also known as transport RNA or transfer RNA. tRNA typically has 70-90 nucleotides, with an amino acid attached to its 3' end. The secondary structure of tRNA can be found in [link to tRNA documentation]. Figure 1 a. Arranged in a cloverleaf model. The secondary structure of tRNA includes four stem structures and three loop structures. The four stem structures are the T-stem, D-stem, AC-stem, and CCA-stem. Specifically, the T-stem (T arm), also called the TΨC-stem (TΨC arm), connects the T loop (also called the TΨC loop); the D-stem connects the D loop; the AC-stem, or anticodon stem, connects the anticodon loop containing the anticodon; and the CCA-stem, or amino acid acceptor arm.

[0164] As used herein, the term “nearly homologous tRNA (nc-tRNA)” refers to a tRNA molecule whose anticodon has a single base mismatch or wobble pair with a Ψ-modified codon in mRNA (such as ΨAA, ΨAG, ΨGA) (the remaining positions are Watson-Crick pairs), and that the anticodon is recognized and decoded as a specific amino acid through non-canonical base pairing.

[0165] By decoding nc-tRNA, the premature stop codon is recoded as Arg, Gln, Trp, or Tyr, depending on the stop codon sequence and the type of nc-tRNA. This maintains or partially maintains protein folding and function, achieving full-length protein restoration.

[0166] For example, for the ΨGA codon, the nc-tRNA anticodon UCU (e.g., tRNA-R-UCU) is decoded as arginine (R). For example, for the ΨAG codon, the nc-tRNA anticodon CUG (e.g., tRNA-Q-CUG) is decoded as glutamine (Q). For example, for the ΨAA codon, the nc-tRNA anticodon UUG (e.g., tRNA-Q-UUG) is decoded as glutamine (Q).

[0167] As used in this article, the term "Premature Termination Codon (PTC)" refers to a premature termination signal formed in the coding region of mRNA due to a nonsense mutation, leading to premature termination of translation and the production of truncated and often nonfunctional proteins. PTCs are caused by point mutations in sense codons and are associated with various genetic diseases, such as cystic fibrosis, Heller syndrome, and Duchenne muscular dystrophy. Therefore, PTCs are a core target for therapeutic intervention in many diseases. When target uridine residues are modified by pseudouridineization, they are transformed into "pseudouridine residues" (Ψ), forming ΨAA, ΨAG, or ΨGA codons.

[0168] As used herein, the terms “wild-type,” “wild,” or “natural” are used interchangeably. When these terms are used to describe nucleic acid molecules, peptides, or proteins, they indicate that the nucleic acid molecule, peptide, or protein exists in nature, is found in nature, and has not undergone any artificial modification or processing. As used herein, wild-type tRNA refers to naturally occurring, biologically active tRNA.

[0169] As used in this article, the term "corresponding position" refers to the amino acid position at the same position in two sequences being compared when performing optimal alignment, i.e., when aligning two sequences to obtain the highest percentage of identity.

[0170] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit is present (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For instance, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match).

[0171] As used herein, the terms "standard numbering system for tRNA" and "tRNA Numbering System" are used interchangeably. The standard numbering system for tRNA is a high-level nomenclature specification for tRNAs, used to standardize the location of each nucleotide on the tRNA molecule (e.g., D-stem, AC-stem, T-stem, variable stem, CCA-stem) to overcome sequence differences and species boundaries, enabling precise comparison and study of the structure and function of all mature tRNAs. Specifically, starting from the 5' end, the nucleotides are numbered sequentially according to the D-stem, AC-stem, T-stem, and CCA-stem sequence, regardless of the actual length. The first nucleotide (5' end) of the mature tRNA molecule is fixedly numbered 1, and the numbers are sequentially incremented until the last nucleotide (usually around position 76, with a common range of 74-76). This numbering is continuous and completely disregards whether the tRNA contains additional 5' leader sequences, 3' tail sequences, or introns in the initial transcript.

[0172] The standard numbering system for tRNA follows these principles: (1) Constant site principle: Functional site numbers are absolutely fixed. Regardless of the tRNA origin, position 34 is always the first base of the anticodon (the swing site), positions 54, 55, and 56 are always the T, Ψ, and C of the T stem, and positions 74-76 are always the CCA tail. (2) Intron numbering rule: Introns in precursor tRNA do not occupy integer numbers. Introns are located between two exon numbers (e.g., between 37 and 38), and the bases inside are labeled with "+1, +2, +3…". This ensures that the numbering of mature tRNA is always a consecutive integer. (3) Length variation rule: The variable loop (V loop) is the only region that allows for large variations in length. If the V loop is longer, the extra bases are numbered using extended numbers with decimal points (e.g., 44.1, 44.2…) or inserted letters (e.g., 47A, 47B…).

[0173] The standard numbering system for tRNAs was established by Holley et al. (in the 1970s) and later improved by Sprinnzl et al. It is a genome-wide numbering system based on three-dimensional structure (L-shape) and functional sites. The standard numbering system for tRNA can be found in, for example, HOLLEY, RW et al. “STRUCTURE OF A RIBONUCLEIC ACID.” Science (NewYork, NY) vol. 147,3664 (1965): 1462-5. doi:10.1126 / science.147.3664.1462; Gauss, DH et al. “Compilation of tRNA sequences.” Nucleic acids research vol. 6,1 (1979): r1-r19; Sprinzl, M et al. “Compilation of tRNA sequences and sequences of tRNA genes.” Nucleic acids research vol. 26,1 (1998): 148-53. doi:10.1093 / nar / 26.1.148, the contents of which are incorporated herein by reference.

[0174] As used herein, the term “guide small nucleolar RNA (gsnoRNA)” refers to an engineered non-coding RNA molecule. gsnoRNA can specifically hybridize with specific sequences in target RNA (such as target uridine residues in premature stop codons PTC) and recruit pseudouridine synthase complexes (e.g., complexes containing DKC1 protein) to the target site, thereby catalyzing the conversion of target uridine residues into pseudouridine residues (Ψ).

[0175] As used herein, the term "DKC1 protein" is a highly conserved pseudouridine synthase. It is responsible for catalyzing the conversion of uridine residues in RNA to pseudouridine residues. In this application, the DKC1 protein is recruited by gsnoRNA to a target RNA site for specific pseudouridine modification.

[0176] As used herein, the term "DKC1 iso1" refers to an isotype of the DKC1 protein, which is primarily located in the cell nucleus. Its accession number is DKC1 iso1: NP_001354.1.

[0177] As used herein, the term "DKC1 iso3" refers to an isotype of the DKC1 protein. Specifically, it is a splicing variant resulting from the retention of intron 12. This is because the retention of intron 12 produces a new stop codon, thus causing premature termination of translation, resulting in DKC1 iso3. This process leads to the absence of the C-terminal nuclear localization signal (NLS), and therefore DKC1 iso3 is primarily localized in the cytoplasm rather than the nucleus. In native endogenous mRNA expression, the expression level of DKC1 iso1 is much higher than that of DKC1 iso3. The accession number for DKC1 iso3 is: NP_001275676.1.

[0178] Beneficial effects of the invention

[0179] This application optimizes and modifies the previously studied RESTART pseudouridine modification system, providing a modified tRNA that can serve as an nc-tRNA for PTC sites, significantly improving the efficiency of PTC recognition and decoding. Furthermore, this modified tRNA is suitable for multiple iterations of the RESTART system and can be used in combination with other elements in the RESTART system (e.g., gsnoRNA) to significantly improve PTC readthrough efficiency.

[0180] For example, when applied to decoding pseudouridine-modified PTCs, systems containing this modified tRNA exhibit significantly improved decoding efficiency, enabling more efficient recovery of the mRNA's coding information and enhancing PTC readthrough efficiency, thereby allowing for the expression of the complete protein. Therefore, this modified tRNA holds significant importance and has broad application prospects in the treatment of PTC mutation-related diseases.

[0181] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0182] Figure 1 The secondary structure of tRNA and the mechanism by which this application inhibits PTC are illustrated schematically.

[0183] Figure 1Figure a shows the secondary structure of tRNA. The secondary structure of tRNA includes four stem structures and three loop structures. The four stem structures are the T-stem, D-stem, AC-stem, and CCA-stem. The T-stem (T arm) is also called the TΨC-stem (TΨC arm). The position of the first base pair from the 5' to the 3' direction of the T-stem is shown in the figure; the positions of the second and third base pairs are deduced accordingly.

[0184] Figure 1 b shows that when a PTC is present in mRNA, mRNA translation terminates prematurely, resulting in a truncated protein. In the method for inhibiting PTC in this application, the guide sequence in gsnoRNA pairs complementaryly with the bases of the target mRNA, allowing the uridine residue U in the PTC to enter the catalytic center formed by the stem-loop structure of gsnoRNA; gsnoRNA recruits DKC1, NOP10, NHP2, and GAR1 proteins to assemble into an RNP complex, catalyzing the modification of the uridine residue U into a pseudouridine residue Ψ. Furthermore, the Ψ-modified PTC can be decoded and translated into amino acids by the nc-tRNA of this application, achieving readthrough of the PTC site, thereby restoring the production of the full-length protein. In some embodiments, the DKC1 protein is supplied endogenously by the cell.

[0185] Figure 1 c further demonstrates the decoding function of nc-tRNA. Specifically, gsnoRNA binds to the PTC target site and recruits the RNP complex, modifying the uridine residue U of the PTC (UGA, UAG, or UAA) with a pseudouridine residue Ψ, resulting in Ψ-modified PTC (ΨGA, ΨAG, or ΨAA). nc-tRNA then binds to and decodes the Ψ-modified PTC site, translating it into amino acids, thereby achieving full-length readout of the PTC and restoring the expression of the full-length protein.

[0186] Figure 2 The results show that Near-cognate tRNA modification enhances the effectiveness of the RESTART system. Figure 2 The diagram shows the structure of near-cognate tRNA. The yellow part is the T-arm structure, and the part marked by the red box is the core region that has been modified. The type of base pairs in this region has a significant impact on the binding force between aa-tRNA and EF-Tu. Figure 2 b shows the ΔΔG values ​​for amino acids (aa) and tRNA, used to assess the binding affinity of aa-tRNA to EF-Tu. Figure 2 c display Figure 2 The variation pattern of ΔΔG values ​​of tRNA corresponding to the base pair types in the yellow part of section a. Figure 2d shows a schematic diagram of the validation of the effect of near-cognate tRNA, including the mcherry-PTC-EGFP reporter system, gsnoRNA targeting the PTC site, and nc-tRNA before and after modification; DKC1iso3 protein is expressed through additional plasmids. Figure 2 e shows the readability of tRNA-R-UCU before and after modification in a UGA reporter system expressing DKC1 iso3. Figure 2 f shows the readability of tRNA-R-UCU before and after modification in a UGA reporter system that does not express DKC1 iso3. Figure 2 g shows the readability of tRNA-W-CCA before and after modification in a UGA reporter system expressing DKC1 iso3. Figure 2 h shows the readthrough efficiency of tRNA-W-CCA before and after modification in a UAG reporter system expressing DKC1 iso3. Figure 2 i shows the readthrough efficiency of tRNA-Q-CUG before and after modification in a UAG reporter system expressing DKC1 iso3. Figure 2 j shows the readability of tRNA-Q-UUG before and after modification in a UAA reporter system expressing DKC1 iso3. Figure 2 k shows the readthrough efficiency of tRNA-R-UCU before and after modification in disease-related gene sequences expressing DKC1 iso3.

[0187] Figure 3 This study demonstrates two methods to enhance the expression level of endogenous DKC1 iso3. The upper pathway involves designing antisense oligonucleotides (ASOs) targeting the SR protein binding site to induce the retention of intron 12 in DKC1 pre-mRNA, thereby terminating the translation of the stop codon formed in intron 12 and yielding DKC1 iso3. The lower pathway involves introducing a small RNA (RTM) to induce 3'-trans-splicing between the RTM and intracellular transcripts, resulting in the DKC1 iso3 transcript, thus yielding DKC1iso3.

[0188] Figure 4This demonstrates the experimental design for validating RNA exon editing to enhance the expression level of endogenous DKC1 iso3. Two nucleic acid molecules were designed: the first molecule contained a nucleotide sequence encoding a 5' truncated GFP protein at the 5' end of the DKC1-iso3 transcript, mimicking the intracellular DKC1 iso1 transcript; the second molecule contained a nucleotide sequence encoding a 3' truncated GFP protein. Furthermore, the second molecule, acting as an RTM, was designed to target the DKC1 intron11 sequence of the first molecule and undergo 3' trans splicing with it. Therefore, the second molecule also contained the required BD sequence, spacer sequence, PPT sequence 3' splicing site, and target linker sequence (in this figure, the target linker sequence is exemplified by the IRES sequence and the nucleotide sequence encoding the DsRED protein). Thus, if 3' trans splicing occurs, a complete GFP transcript is generated, producing the GFP protein and emitting fluorescence; the higher the efficiency of the 3' trans splicing, the stronger the fluorescence.

[0189] Figure 5 RNA exon editing shows that it improves RESTART efficiency. Figure 5 a shows that BD sequences were set at different positions of the intron 11 of DKC1, and RTM sequences were designed to restore full-length GFP expression in order to screen for suitable target sequences. Figure 5 b displays the shorter spacer and PPT sequences in the RTM. A spacer is a spacer sequence with a conserved branch point; a PPT is a polypyrimidine region. Figure 5 c shows how shortened spacers and PPT sequences are used to screen for longer BD sequences near the selected BD target sequences. Figure 5 d shows the effect of the filtered RTM (BD36) in RESTART readthrough using the UGA system that does not express DKC1 iso3. Figure 5 e shows the expression of newly generated DKC1-iso3 after RTM expression.

[0190] Figure 6 This diagram illustrates a modification of gsnoRNA. The scaffold sequence (blue) of gsnoRNA contains two stem-loop structures: a first hairpin structure near the 5' end and a second hairpin structure near the 3' end. From 5' to 3', the scaffold sequence sequentially includes: a first hairpin structure near the 5' stem-loop (composed of a first stem and a first loop), a hinge structure (containing an "H box"), a second hairpin structure near the 3' stem-loop (composed of a second stem and a second loop), and a tail structure (containing an "ACA box").

[0191] The guide sequence (green) can be located in the first stem and / or the second stem. The CAB box (red) can be located in the first loop and / or the second loop. The CTE sequence (orange) can be attached to the 5' end and / or the 3' end of the gsnoRNA scaffold sequence. In a specific implementation, as shown in this figure, the first guide sequence is located in the first stem, the second guide sequence is located in the second stem, the CAB box is located in the first loop, and the CTE sequence is attached to the 3' end of the gsnoRNA scaffold sequence.

[0192] Figure 7 The introduction of the CAB box improves the read efficiency of the RESTART system. Figure 7 a and Figure 7 b shows that directly using scaRNA as the gsnoRNA backbone can achieve significant readability in UGA reporter systems with both underexpression (7a) and overexpression (7b) of DKC1 iso3. Figure 7 c shows the RESTART readthrough efficiency before and after directly replacing the loop containing the CAB box of a known scaRNA with gACA19 in a UGA reporter system that does not express DKC1 iso3. Figure 7 d. Select high-efficiency CAB boxes and test their impact on readability improvement in a UGA reporting system that overexpresses DKC1 iso3. Figure 7 e shows the effect of adding the CAB box to other snoRNA backbones in a UGA reporter system overexpressing DKC1-iso3. Figure 7 f shows the effect of adding 5' stem-loop and 3' stem-loop to gsnoRNA and adding CAB boxes to both ends on readthrough in a UGA reporter system that does not express DKC1 iso3. Figure 7 g shows a schematic diagram of the CAB box of scaRNA (U85) and the addition of a functional inactivation mutant to gsnoRNA. Figure 7 h shows the CAB box of scaRNA (U85) and the readability test of the inactivation mutant added to gsnoRNA. Figure 7 i shows the effect of adding CAB box on RESTART v1, RESTART v2, and RESTART v3 before and after testing in a disease scenario with CFTR-R553X nonsense mutation. Figure 7 j shows the effects of adding the CAB box on RESTART v1, RESTART v2, and RESTART v3 before and after testing in a disease scenario with the LMNA-R225X nonsense mutation. Figure 7k shows the CABbox effect on other H(ACA)-containing RNAs tested in a UGA reporter system that does not express DKC1 iso3. HTR is human telomerase RNA; BIO is a specific stemloop result on AluRNA with a CAB box.

[0193] Figure 8 The introduction of CTE shows that it improves the read efficiency of the RESTART system. Figure 8 a shows the structure of SRV CTE and mutants (M36CTE and ΔCTE). Figure 8 b and Figure 8 c shows the effect of adding CTE to the 5' and 3' ends of gsnoRNA and linkers of different lengths on RESTART readthrough in the UGA reporter system with non-expression (8a) and overexpression (8b) DKC1 iso3. Figure 8 d and 8e show the effects of CTE and functionally inactivating mutations on RESTART readthrough tested in the UGA reporting system for DKC1 iso3 under non-expression (8d) and overexpression (8e). Figure 8 f and 8g tests were used to test the interaction between CTE and functionally inactivating mutant CTE and helicase. Figure 8 h shows the effect of different CTEs and CTE mutants on RESTART readthrough in a UGA reporter system overexpressing DKC1-iso3.

[0194] Sequence information

[0195] Information on a portion of the sequence involved in this invention is provided below.

[0196] SEQ ID NO: 1~50 exemplarily describes the modified tRNA of this application, wherein the underlined sequence is the nucleotide sequence of the first, second and third base pairs of the T stem of the modified tRNA from the 5' direction to the 3' direction.

[0197] SEQ ID NO:158~161 and SEQ ID NO:187~270 exemplarily describe the nucleotide sequences of wild-type tRNA. Detailed Implementation

[0198] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it). Unless otherwise specified, the experiments and methods described in the embodiments are generally carried out in accordance with conventional methods well known in the art and described in various references.

[0199] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0200] Example 1. Preparation of components in the RESTART system

[0201] 1. Plasmid preparation

[0202] The molecular clones constructed in this study mainly include two types: PTCreporter plasmid and DKC1 isoform3 (DKC1 iso3) plasmid using pLenti-CMV-MCS-BSD as vector, and gsnoRNA, nc-tRNA and 3'trans RTM plasmid using Pcg2.0-BFP as vector.

[0203] Plasmids using pLenti-CMV-MCS-BSD and pAAV as vectors were primarily recombined via Gibson sequencing. The DKC1 iso3 gene sequence was amplified from HEK293T cDNA, and the disease reporter gene sequence was obtained from a human cDNA library from Peking University. Nonsense mutations in the disease reporter gene were induced by PCR primers, and the ligation sequence between mCherry and EGFP was adjusted via primer amplification. Specifically, this study used the TransStart FastPfu DNA polymerase kit to target and amplify the target fragment and vector backbone. The target fragment was obtained by agarose gel electrophoresis and purified using a universal DNA purification and recovery kit. After purification, homologous recombination ligation of the fragment and vector backbone was performed using NEBuilder® HiFi DNA Assembly Master Mix, and the ligation product was transformed into Trans T1 competent cells. Plasmids were extracted using the EndoFree Mini Plasmid Kit II and identified by Sanger sequencing.

[0204] The construction of gsnoRNA and nc-tRNA plasmids using Pcg2.0-BFP as vectors mainly involves two steps: primer bridging to construct the target sequence and Golden gate restriction enzyme ligation. First, using the TransStart FastPfu DNApolymerase kit, the designed gsnoRNA, nc-tRNA, or 3' trans RTM sequence was constructed via overlapping PCR with four primers (one forward primer and three reverse primers). Golden gate restriction enzyme ligation sites were then constructed at both ends of the sequence. The presence of Golden gate restriction enzyme ligation sites on the Pcg2.0-BFP backbone, along with the Bsd toxin protein in the middle of these ligation sites, prevents empty vector plasmids that fail to undergo restriction enzyme digestion from surviving during transformation. The product obtained by primer bridging was recovered using a universal DNA purification and recovery kit. 40 ng of the purified product and 20 ng of Pcg2.0-BFP plasmid were placed together in an enzyme digestion and ligation system containing the cutting enzymes BSMBI and T4 ligase (which also contains DTT and ATP) for reaction. The reaction product was then transformed into Trans T1 competent cells, and the plasmid was extracted for Sanger sequencing identification.

[0205] 2. Cell transfection and data analysis

[0206] HEK293T cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2. At cell passage, cells were washed with PBS, digested with 0.25% trypsin, and incubated at 37°C for 2 minutes. The trypsin was then neutralized with FBS-containing medium. After centrifugation at 600 rpm for 3 minutes, cells were counted and plated. The cell line was negative for mycoplasma contamination.

[0207] 20–24 hours before staining, inoculate cells at a concentration of 2 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / well in 24-well plates. Plasmids for transfection were extracted using a mini-prep kit, and plasmid concentration was quantified using Nanodrop. The target plasmid and target RNA were transfected using Lipofectamine LTXwith PLUS reagent, following the recommended transfection protocol. The medium was changed 24 hours after transfection, and cell function was assessed at 48 or 72 hours.

[0208] To evaluate the efficiency of PTC readthrough in the fluorescence reporter system, cells were imaged using an ImageXpress® Micro 4 high-content imaging system (Molecular Devices LLC, Sunnyvale, CA) 48–72 hours post-transfection. Sixteen images from different sites in the same well were captured under a 10x microscope and then automatically analyzed using MetaXpress software. The percentage of EGFP-positive cells was calculated by dividing the number of EGFP-positive cells in the fluorescence image by the number of BFP / mCherry-positive cells in the corresponding image, and then normalized using data from the positive control. EGFP fluorescence intensity was calculated by multiplying the EGFP intensity per cell by the number of EGFP-positive cells in the fluorescence image, and then normalized using data from the positive control. The mean of the 16 images was considered an independent replicate, with each set of fluorescence analysis data having 2–3 biological replicates. Data are presented as the mean of 2 replicates or the mean ± standard deviation of 3 replicates.

[0209] 3. RNA extraction and modification detection

[0210] Discard the culture medium from the target cells in a clean bench, wash once with PBS, and aspirate dry. Add TRIzolreagent to the cells and aspirate 10 times until homogeneous. Transfer to an EP tube and incubate for 5 minutes. Add Chloroform (one-fifth the volume of TRIzolreagent) and vortex vigorously for 15 seconds. Incubate at room temperature for 15 minutes, then centrifuge at 12,000 rpm for 15 minutes at 4°C. Transfer the supernatant to a clean EP tube, add an equal volume of Isopropanol, mix by inverting, and incubate at -20°C for at least 1 hour. Centrifuge at 12,000 rpm for 30 minutes at 4°C. Discard the supernatant, wash twice with 1 mL of 75% ethanol solution, discard the supernatant again, centrifuge once empty, and incubate at room temperature for 10 minutes with the cap open. Once the RNA precipitate changes from white to clear, dissolve it in a certain amount of RNase-free water and determine the concentration using Nanodrop.

[0211] DNase was added to the target RNA to remove remaining genomic and plasmid fragments. After the reaction, the RNA sample was repurified. A labeling reaction solution was prepared: 85% K₂SO₃ / 15% NaHSO₃ solution was mixed with 100 mM Hydroquinone at a ratio of 100:1. 1 μg of purified RNA was mixed with 50 μL of the reaction solution and reacted at 70°C for 5 hours. After the reaction, the RNA was desalted and purified using a Micro Bio-spin 6 column, and then an equal volume of 1 M Tris-HCl [pH 9.0] was added and reacted at 75°C for 30 minutes. The labeled RNA sample was then purified. Reverse transcription was performed using a Maxima HMinus RT enzyme reaction system to obtain cDNA samples. End-specific PCR primers containing a ~130 nt sequence of the target site were designed. Library adapter sequences were added to the 5′ end of the primers, and specific amplification was performed using NEBNext Q5 Hot Start HiFi PCRMaster Mix. After amplification, PCR amplification was performed using Illumina primers, and sequencing adapter sequences were added to both sides of the target fragment. After the amplification reaction, the target DNA fragment was purified using AMPure XP beads, the concentration was determined, and it was identified using a 4150 microarray. Finally, next-generation sequencing analysis was performed.

[0212] RESTART v1-v3, v3 mini system

[0213] Prior to this, the applicant had developed multiple generations of RESTART systems, which contain different components and can achieve precise pseudouracil modification of PTC sites, promote PTC readthrough and restore full-length functional protein expression, thereby efficiently and specifically repairing nonsense mutations.

[0214] RESTART v1: The RESTART v1 system modifies the pseudouridine pocket of human snoRNA by designing a guide sequence, enabling the modified snoRNA to precisely bind to the target PTC site, resulting in gsnoRNA. Furthermore, this gsnoRNA recruits endogenous DKC1 iso1 to assemble snoRNPs for pseudouridine modification, achieving efficient PTC readthrough. In other words, the core component of the RESTART v1 system is gsnoRNA with a modified guide sequence. By introducing gsnoRNA into cells, endogenous DKC1 iso1 is recruited to perform pseudouridine modification of the PTC site in the target RNA.

[0215] RESTART v2: In the RESTART v2 system, DKC1 iso3 (Iso3) was found to enhance the modification efficiency of the RESTART system. Since DKC1 iso3 is a product of DKC1 aberrant splicing, cells under natural conditions generally express DKC1 iso1, expressing only a small amount of DKC1 iso3. Therefore, the RESTART v2 system, by overexpressing the catalytic enzyme DKC1 iso3, increased the modification efficiency of the RESTART system by 2-fold and the readthrough efficiency by 2–5-fold. In other words, the core components of the RESTART v2 system are gsnoRNA and DKC1 iso3. By introducing gsnoRNA and DKC1 iso3 into cells, pseudouracil modification of the PTC sites in the target RNA is achieved.

[0216] RESTART v3: The RESTART v3 system reveals that nc-tRNAs at PTC sites play a crucial role in decoding pseudouracil-modified PTC sites. Although these nc-tRNAs are naturally present, their expression levels in cells are low or they do not target PTC sites, thus requiring artificial introduction and overexpression. The RESTART v3 system significantly improves the readthrough efficiency of the RESTART system (1.3–8 times higher than RESTART v2) by overexpressing nc-tRNA, and also enhances the accuracy of amino acid incorporation at PTC sites, enabling approximately 50% of disease-related nonsense mutations to be accurately repaired to their original amino acids. In other words, the core components of the RESTART v3 system are gsnoRNA, DKC1 iso3, and nc-tRNA. By introducing gsnoRNA, DKC1 iso3, and nc-tRNA into cells, pseudouracil modification and readthrough of PTC sites in target RNA are achieved.

[0217] RESTART v3-mini: To simplify the RESTART v3 system and improve delivery flexibility, the RESTART v3-mini system delivers only gsnoRNA and nc-tRNA, without additional expression of DKC1 iso3. That is, the core components of the RESTART v3-mini system are gsnoRNA and nc-tRNA. By introducing gsnoRNA and nc-tRNA into the cell, pseudouracil modification and readthrough of the PTC site in the target RNA are performed.

[0218] In summary, it can be seen that the individual components of the RESTART system can function effectively, either individually or in combination. For example, gsnoRNA can be delivered alone to improve PTC readout efficiency, while other components required for modification rely solely on endogenous sources (similar to the RESTART v1 system). Similarly, with the introduction of gsnoRNA, DKC1 iso3 can be overexpressed alone, or nc-tRNA can be delivered alone. nc-tRNA plays a crucial role in the decoding process following pseudouracil modification at the PTC site. Even without delivering the complete RESTART system, the RESTART system can be assembled in the cell using endogenous components to achieve PTC readout; a schematic diagram can be found in [reference needed]. Figure 1 b and Figure 1 c.

[0219] Construction and application of the new RESTART system

[0220] Furthermore, this application modifies the nc-tRNA in the previous RESTART system by modifying its T stem and anticodon stem to improve the efficiency of recognizing and decoding Ψ-modified PTCs, thereby improving its final readthrough nonsense mutation efficiency, and using it in the future fourth-generation RESTART system (i.e., RESTART V4).

[0221] Specifically, in the RESTART system of this application, the gsnoRNA, nc-tRNA, and 3' trans-RTM components are expressed using the Pcg2.0-BFP (purchased from Addgene) backbone plasmid, while the DKC1 iso3 protein component is expressed using the pLenti-CMV-MCS-BSD backbone plasmid. These constructed vectors can be used to assemble the RESTART system in cells to further evaluate its reading function in disease-related mRNAs containing nonsense mutations. In some embodiments, the constructed vector containing the nucleotide sequences encoding gsnoRNA, nc-tRNA, 3' trans-RTM, and DKC1 iso3 protein can be directly transfected into cells to express the desired RNA or protein, thereby assembling a new RESTART system containing the modified nc-tRNA of this application in cells.

[0222] During the construction process, we introduced disease-related reporter genes carrying early stop codons via co-transfection and evaluated the reading efficiency and functional recovery effect of the RESTART system on target mRNA by using changes in the fluorescence expression level of the reporter system. In other words, a PTC disease model can be constructed by introducing a reporter system into cells, and the aforementioned vector can be introduced to detect the pseudouridine editing efficiency and PTC readthrough recovery effect of the RESTART system.

[0223] Alternatively, after obtaining the components of the system, it can be further constructed into a delivery system suitable for in vivo administration. Specific methods include integrating the system into a lentivirus or adeno-associated virus vector for delivery via in vitro transduction or in vivo injection; or using lipid nanoparticles to encapsulate the system's RNA components for targeted delivery via intravenous or local injection, etc.

[0224] Example 2. Optimization and modification of Near-cognate tRNA

[0225] 1. Optimization and modification of the T-stem of nc-tRNA

[0226] 1.1 Modification Principle

[0227] Near-cognate tRNA (nc-tRNA) is a crucial element in the RESTART editing system that mediates the translation and readout of PTC sites, and its decoding speed during translation has a key impact on readout efficiency. To improve the recognition ability of nc-tRNA for pseudouridine-modified PTCs, we first focused on sequence optimization of its T-stem region, improving decoding kinetics by regulating the binding strength of tRNA to elongation factors. Figure 1 a and Figure 2a). The binding stability of aa-tRNA to elongation factor is characterized by the change in free energy (ΔΔG). A lower free energy difference (ΔΔG) indicates higher binding stability, while a higher ΔΔG value indicates poorer stability.

[0228] Specifically, the ΔΔG value is characterized by the sum of the values ​​of ΔΔG(aa) and ΔΔG(tRNA). A value that is too high or too low is detrimental to the decoding process of aa-tRNA. Figure 2 b). Based on three key bases in T-stem ( Figure 2 a) Regarding the regulatory mechanism of ΔΔG, we systematically modified and functionally validated nc-tRNA. Figure 2 c, Figure 2 d). The three key base pairs are the first, second, and third base pairs in the stem structure of the T-stem. Each base pair has a different ΔΔG (tRNA) when using different specific base pairings (classical pairing or wobble pairing). By changing the specific base pairings, the ΔΔG value can be modulated, thereby improving the decoding speed.

[0229] Furthermore, we investigated the recognition efficiency of all different tRNAs for stop codons and found that the four tRNAs with the closest homologous codons tRNA-R-UCU, tRNA-Q-CUG, tRNA-Q-UUG, and tRNA-W-CCA had the highest decoding efficiency for PTCs. Therefore, we selected these four tRNAs for optimization.

[0230] 1.2 Description of Modification Sites

[0231] When using different subclasses of tRNA, the specific base positions of the three key base pairs in the tRNA are different, but in the standard numbering system of tRNA, the positions of these three base pairs are positions 49:65, 50:64, and 51:63.

[0232] Specifically, the unified tRNA numbering system, or standard tRNA numbering system, is a high-level nomenclature standard based on three-dimensional structure (L-shape) and functional sites, established by Holley et al. (1970s) and later refined by Sprinnzl et al. It is used to standardize the location of each nucleotide (e.g., D stem, AC stem, T stem, variable stem, CCA stem) on the tRNA molecule to overcome sequence differences and species boundaries, enabling precise comparison and study of the structure and function of all mature tRNAs. Specifically, starting from the 5' end, numbering follows the order of D stem, AC stem, T stem, CCA stem, regardless of the actual length. The first nucleotide (5' end) of the mature tRNA molecule is fixedly numbered 1, and the numbers are sequentially increased until the last nucleotide (usually around position 76, with a common range of 74-76). This numbering is continuous, completely disregarding whether the tRNA contains additional 5' leader sequences, 3' tail sequences, or introns in the initial transcript. The standard tRNA numbering system follows these principles:

[0233] (1) Constant site principle: The functional site numbering is absolutely fixed. Regardless of the source of tRNA, the 34th position is always the first base of the anticodon (the swing site), the 54th, 55th, and 56th positions are always the T, Ψ, and C of the T stem, and the 74th-76th positions are always the CCA tail.

[0234] (2) Intron numbering rules: Introns in precursor tRNA do not occupy integer numbers. Introns are located between two exon numbers (e.g., between 37 and 38), and the bases inside are labeled with "+1, +2, +3...". This ensures that the numbers of mature tRNA are always consecutive integers.

[0235] (3) Length variation rules: The variable ring (V ring) is the only region that allows for large variations in length. If the V ring is longer, the extra bases are numbered with an extension of the decimal point (e.g., 44.1, 44.2…) or inserted letters (e.g., 47A, 47B…).

[0236] Taking the tRNAs of this application as examples, for tRNA-W-CCA (SEQ ID NO: 159), tRNA-Q-CUG (SEQ ID NO: 160), and tRNA-Q-UUG (SEQ ID NO: 161), the base positions of these three base pairs in the tRNA are 48:64, 49:63, and 50:62, respectively. For tRNA-R-UCU (SEQ ID NO: 158), the base positions are 61:77, 62:76, and 63:75, respectively. tRNA-R-UCU contains an intron sequence, therefore its base position differs from other tRNAs without introns. However, the numbering in the standard tRNA numbering system does not change due to the presence or absence of introns, so the positions remain 49:65, 50:64, and 51:63.

[0237] 1.3 Description of tRNA isotypes

[0238] tRNAs have various isoforms, and even those carrying the same amino acid can differ significantly in structure, sequence, and processing. The tRNA-R-UCU sequence used in this application is R-UCU-1, which carries an intron structure. Compared to other tRNA isoforms without introns, it undergoes pre-tRNA intron splicing and the addition of endogenous base modifications. This application is the first to use the intron-containing isoform R-UCU-1 for modification, demonstrating that the above modification principles can function in pre-processed tRNA, and that this modification does not affect the tRNA processing. Besides the difference in intron structure, R-UCU-1 and R-UCU-3 have significant sequence differences (e.g., in the CCA stem and T stem).

[0239] 1.4 T-stem modification experiment

[0240] First, we modified the near-cognate tRNA targeting the UGA site—tRNA-R-UCU (SEQ ID NO:158)—to obtain different mutants as shown in SEQ ID NO: 1–5. Taking tRNA-R-UCU as an example, this name indicates that the anticodon sequence of this tRNA is UCU (3'-UCU-5'), and the transported amino acid is arginine (R). The results showed that the PTC reading efficiency of the modified tRNA-R-UCU as shown in SEQ ID NO: 1–5 was superior to or significantly superior to the unmodified tRNA, with the modified tRNA-R-UCU as shown in SEQ ID NO: 2 exhibiting the best performance. Therefore, sequence modification of T-stem can effectively improve the reading efficiency of the RESTART v3 system, with an improvement level of approximately 50%. Figure 2 e).

[0241] Furthermore, we also evaluated the effect of tRNA modification on the efficiency of RESTARTv3-mini in the absence of DKC1 iso3 overexpression. The results showed that the modified tRNA still significantly improved its readthrough efficiency, exhibiting the same improving trend as RESTART v3. Figure 2 f). Therefore, we believe that the T-stem modification strategy of tRNA can stably improve the readthrough efficiency of pseudouridine-modified PTCs.

[0242] In addition, we also modified another near-cognate tRNA targeting the UGA site—tRNA-W-CCA (SEQ ID NO: 159)—to obtain different mutants as shown in SEQ ID NO: 8~11. tRNA-W-CCA indicates that the anticodon sequence of this tRNA is CCA (3'-CCA-5'), and the transported amino acid is tryptophan (W). The results show that this modification strategy can still achieve an effective efficiency improvement in tRNA-W-CCA, with an improvement of ~25% ( Figure 2 g). Subsequently, we also systematically modified three near-cognate tRNAs targeting UAG and UAA—tRNA-W-CCA (SEQ ID NO: 159), tRNA-Q-CUG (SEQ ID NO: 160), and tRNA-Q-UUG (SEQ ID NO: 161), and the resulting mutant nucleotide sequences are shown in SEQ ID NO: 8~11, SEQ ID NO: 12~40, and SEQ ID NO: 41~50, respectively. The results show that this modification strategy can also stably improve the effect of RESTART v3 at UAG and UAA sites, with an efficiency improvement of ~40-80%, confirming that T-stem optimization is applicable to different types of nc-tRNAs ( Figure 2(h-2j). Among them, the PTC readthrough efficiency of the modified tRNA-W-CCA shown in SEQ ID NO: 8~11 is better or significantly better than that of the unmodified tRNA; the PTC readthrough efficiency of the modified tRNA-Q-CUG shown in SEQ ID NO: 12, 13, 18, 27~29, 31, 32, 34~36 and 38 is better or significantly better than that of the unmodified tRNA, with the modified tRNA-Q-CUG shown in SEQ ID NO: 18, 35, 36 and 38 showing the best effect; the PTC readthrough efficiency of the modified tRNA-Q-UUG shown in SEQ ID NO: 41, 43~50 is better or significantly better than that of the unmodified tRNA, with the modified tRNA-Q-UUG shown in SEQ ID NO: 43, 48 and 49 showing the best effect.

[0243] Based on the above results, we believe that this tRNA modification mode has good application value, and we will use tRNA-R-UCU-T2 (SEQ ID NO: 2), tRNA-Q-CUG-T7.3 (SEQ ID NO: 36) and tRNA-Q-UUG-T7.3 (SEQ ID NO: 49) for subsequent RESTART tool design.

[0244] 2. Optimization of the anticodon stem of nc-tRNA

[0245] In addition to the T-stem, we also adjusted the codon recognition precision of tRNA by reducing the number of base pairs in the anticodon stem from 5 to 4 (so that the bases at positions 27:43 no longer pair; the site description here uses the standard tRNA numbering system), thereby enhancing its ability to recognize pseudouridine-treated PTCs. We modified tRNA-W-CCA (SEQ ID NO: 159) to obtain mutants as shown in SEQ ID NO: 6 and 7, and tested the effect of this modification at the UGA and UAG sites, respectively. The results showed that modification of the anticodon stem also improved the effect of nc-tRNA in the RESTART v3 system, increasing the reading efficiency by 25-50%, indicating that anticodon stem regulation can be used as a complementary strategy to further improve decoding performance. Figure 2 g, Figure 2 h).

[0246] Finally, we evaluated the application potential of modified nc-tRNA in the context of disease-related genes. The results showed that the readthrough efficiency of the modified RESTART v3 was significantly improved, with an improvement of 50-80% (…). Figure 2 k).

[0247] Example 3. Enhancing endogenous DKC1 iso3 expression levels via ASO / RNA exon editing

[0248] DKC1 iso3 originates from aberrant splicing of DKC1 pre-mRNA. Specifically, the retention of intron 12 in the mRNA results in the formation of a stop codon, leading to premature translation termination and the generation of a protein isotype lacking the C-terminal NLS signal. Next, we attempted to increase the expression level of endogenous DKC1 iso3 protein using small RNAs to further enhance the pseudouridineization level at the PTC site. Figure 3 ).

[0249] 5.1 Enhancing DKC1 iso3 expression levels via ASO

[0250] Based on this, we designed antisense oligonucleotides (ASOs) targeting the SR protein binding site to induce intron 12 retention in DKC1pre-mRNA. Figure 3 The binding sites of SR proteins can be predicted using relevant websites. For example, ASOs can be designed by predicting the binding site of the SRp40 protein (serine-arginine-rich (SR) protein). The SRp40 protein is encoded by Uniprot as Q13243. Experimental results show that this strategy successfully increased the expression level of endogenous DKC1 iso3 by 4–5 times, laying the foundation for subsequent enhancement of pseudouridine modification efficiency.

[0251] 5.2 Enhancing DKC1 iso3 expression levels via the 3'-trans-splicing pathway

[0252] In addition, we also attempted to use RNA exon editing technology (3'-trans-splicing) to obtain transcripts of the DKC1-iso3 CDS sequence by introducing a new splicing pathway through small RNA, in order to increase the expression of DKC1-iso3 and thus increase the level of pseudouridineization. Figure 3 ).

[0253] We first designed targeting sequences at different positions on the intron 11 of DKC1, and then used a truncated GFP reporter gene to screen for suitable target sites. If a 3' end substitution could be achieved, the GFP would exhibit fluorescence (…). Figure 4We optimized the efficiency of 3'-trans-splicing by using a truncated GFP reporter gene system as a substitute. To achieve direct delivery in the form of small RNA, we strictly controlled the length of the RTM (RNA trans-splicing molecules) to within 200 nt.

[0254] The RTM sequence, from the 5' end to the 3' end, sequentially includes the BD sequence of the target intron sequence, the spacer sequence which acts as a connector, the PPT sequence which plays an important role in the RTM, the 3' splicing sequence (2 bp), and the target connector sequence. In this embodiment, the target connector sequence is a fragment formed by connecting the EXON12 sequence of DKC1 to four bases of INTRON12, as shown in SEQ ID NO: 186, totaling 108 bp. Therefore, when designing the RTM, the total length of the BD sequence, spacer sequence, and PPT sequence must be controlled within 90 nt. Furthermore, we selected the Spacer+PPT sequence studied in the current literature. Specifically, the spacer sequence is shown in SEQ ID NO: 148, and the PPT sequence is shown in SEQ ID NO: 162, totaling 52 bp. Considering the length limitation of the RTM, the BD sequence was designed with only 38 bases initially, and the sequences of each BD are shown in SEQ ID NO: 112~142.

[0255] Then, we constructed an RTM containing these BDs using a truncated GFP reporter gene system ( Figure 4 Cellular validation was performed, and fluorescence was recorded to reflect the efficiency of 3' trans-splicing. We obtained data from... Figure 5 As can be seen, the relative fluorescence intensity increased to varying degrees in all experimental groups, with BD22 and BD32 showing higher efficiency, increasing the relative fluorescence intensity by 30%. Both are located near the 5' splicing site (5'ss) of intron 11. This indicates that the design of this targeting site and RTM can effectively improve the splicing of the 3'-trans-splicing pathway, thereby increasing the expression level of intact EGFP.

[0256] To extend the BD sequence and improve efficiency, we attempted to shorten the spacer sequence (a sequence containing a conserved branching site) connecting BD and 3'EFP, as well as the PPT (polypyrimidine tract) sequence. Specifically, we first prepared shortened spacer variants spacer1-7, the sequences of which are shown in SEQ ID NO: 149-155, respectively; and then prepared a shortened PPT variant mPPT, the sequence of which is shown in SEQ ID NO: 156. Further, we constructed spacer+PPT elements by combining mPPT with the original spacer sequence and spacer1-7, respectively, and experimentally verified the effect. The sequence of the spacer+PPT element constructed by combining mPPT with spacer7 is shown in SEQ ID NO: 157.

[0257] The results showed that appropriately shortening the spacer and PPT sequences had virtually no effect on the 3'-trans-splicing effect. Specifically, the combination of spacer7 and mPPT (SEQ ID NO: 157) could shorten the sequence by 16 bases without affecting the 3'-trans-splicing effect at all. Figure 5 b).

[0258] Therefore, we chose the combination of spacer7 and mPPT to further construct the RTM, which increased the BD sequence by 16 bases (from 38 bases to 54 bases). Based on the currently best-performing BD32, we extended the BD sequence, as shown in SEQ ID NO: 143~147. Thus, we further constructed the RTM and followed... Figure 4 The truncated GFP reporter gene system shown was used in experiments. From Figure 5 As can be seen from c, compared with the original BD32, each extended BD sequence can improve the efficiency of 3'-trans-splicing. BD36, with 8 bases added to each end, has the highest efficiency, which can increase the efficiency of 3'-trans-splicing from about 30% to nearly 40%.

[0259] Thus, we obtained an optimized RTM structure, which includes a combination of BD36 (54bp), spacer7, and mPPT. Specifically, from the 5' end to the 3' end, it contains BD36 (SEQ ID NO: 146), spacer7 (SEQ ID NO: 155), mPPT (SEQ ID NO: 156), a 3' splice sequence, and a target connection sequence (SEQ ID NO: 186).

[0260] Finally, we validated the efficacy of this RTM. Co-expressing this RTM with RESTART v3-mini (delivering only gsnoRNA and tRNA, without additional expression of DKC1 iso3 protein) in PTC disease model cells, if 3'-trans-splicing occurred, a transcript containing only the CDS sequence of DKC1 iso3 would be generated. The results showed that after RTM addition, the expected 3'-trans-splicing occurred, DKC1 iso3 expression was significantly increased, and RESTART readthrough efficiency improved by approximately 30%. Figure 5 d), and can clearly produce the product after 3'-trans-splicing ( Figure 5 e).

[0261] As can be seen, by delivering the RTM sequence based on RESTART v3-mini and utilizing the intracellular 3'-trans-splicing pathway, the expression level of endogenous DKC1 iso3 protein can be effectively increased, thereby further improving the pseudouridineization efficiency of the system and improving the PTC readthrough level.

[0262] Furthermore, we experimented with various specific spacer sequences, PPT sequences, and BD sequences to construct multiple RTM sequences, all of which improved 3'-trans-splicing efficiency to varying degrees, thereby enhancing the expression level of endogenous DKC1 iso3 protein. Additionally, by optimizing the length of the spacer and PPT sequences, we designed a longer BD sequence RTM, further improving 3'-trans-splicing efficiency, thus increasing the expression level of endogenous DKC1 iso3 protein, and improving the system's pseudouridineization efficiency and PTC readthrough level.

[0263] Example 4. Modification of the localization sequence of snoRNA

[0264] Figure 6 The basic structure of the modified gsnoRNA is shown. In this paper, we provide examples of several unmodified gsnoRNA sequences (guide sequences are marked with X). n The remaining sequences (which are scaffold sequences) are ACA19, ACA36, ACA24, ACA5, and ACA14a, and their nucleotide sequences are shown in SEQ ID NO: 165~169, respectively.

[0265] The RNA pseudouridine modification system mainly consists of snoRNA and snoRNPs formed by four core proteins: DKC1, NHP2, GAR1, and NOP10, which perform pseudouridine modification. Figure 1(b) We attempted to improve the efficiency of pseudouridine modification by modifying snoRNA to increase the assembly rate of snoRNP. The sequences of the core proteins mentioned above 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 CAB box elements on the hairpin loop of natural scaRNAs (e.g., scaRNA11, scaRNA14, scaRNA15, scaRNA85 (i.e., U85)), AluRNA, and htrRNA into snoRNAs to form gsnoRNAs. The gsnoRNA in this embodiment contains two stem-loop structures, also known as hairpin structures, which, from 5' to 3', sequentially include: a first stem near the 5' end of the stem-loop structure, a first loop near the 5' end of the stem-loop structure, a second loop near the 3' end of the stem-loop structure, and a second stem near the 3' end of the 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.

[0266] CAB boxes possess a conserved sequence "X1X2AG", where the AGs at positions 3 and 4 are highly conserved, while the first two can vary. For example, conserved sequences for CAB box elements can include AAAAG, GAAG, UAAG, UGAG, UCAG, CGAG, AUAG, GCAG, CUAG, CAAG, and AGAG. htrRNA is a Human Telomerase RNA, whose 3' hairpin contains a CAB box. AluRNA is a non-coding RNA belonging to the Alu elements expressed by intron sequences, and its 3' hairpin contains a CAB box.

[0267] This embodiment adds the CAB box in the following two ways:

[0268] 1. Construct gsnoRNA directly using scaRNA (containing CAB box) as the backbone.

[0269] We directly used natural scaRNA as the backbone of gsnoRNA, replacing its targeting sequence with a sequence targeting ALDOB-W148X to construct two gsnoRNAs (using natural scaRNA14 and scaRNA15 as backbones, with the replaced gsnoRNA sequences shown in SEQ ID NO: 52 and SEQ ID NO: 53, respectively). ALDOB-W148X refers to a PTC disease model gene in which the 148th leucine (W, corresponding to the codon UGG) in the protein expressed by the ALDOB (fructose diphosphate aldolase B) gene is mutated to a stop codon (UAG). The ALDOB database ID is NP_000026.2.

[0270] The results showed that scaRNA, as a gsnoRNA backbone, had a high readability level in the RESTART v1 system (i.e., only gsnoRNA was introduced into the host cell), and this readability level was higher than that of gsnoRNA (gACA19, whose sequence is SEQ ID NO: 51) constructed using ACA19 as a snoRNA backbone. Figure 7 a-7b).

[0271] 2. Introduce a CAB box derived from scaRNA into gsnoRNA.

[0272] To improve the versatility of the CAB box in different scenarios, we directly added the CAB box or the entire loop sequence containing the CAB box from the scaRNA to the previously optimized gACA19. Depending on the scaRNA used, we constructed different gsnoRNAs as shown in SEQ ID NO: 54~72.

[0273] from Figure 7 As can be seen from c, this study screened CAB boxes on almost all scaRNAs. 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 reading efficiency by about 50%.

[0274] We then tested the CABboxes of some scaRNAs in a UGA reporter gene system overexpressing DKC1 iso3. The results showed that these CABboxes derived from different scaRNAs could improve readthrough efficiency, and the gsnoRNA (SEQ ID NO: 54) constructed using the CABbox derived from U85 had the highest relative efficiency. Figure 7 d).

[0275] Next, we investigated the universality of the CAB box on different gsnoRNAs. The CAB box of U85 was linked to different gsnoRNAs, and the resulting gsnoRNA sequences are shown in SEQ ID NO: 170~175. Figure 7 As can be seen, the CAB box of U85 has a 30%-50% improvement in readability across different gsnoRNAs.

[0276] Next, 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 adding CAB at different positions on gsnoRNA. We constructed gsnoRNAs with CAB boxes added near the 5' stem-loop structure, near the 3' stem-loop structure, and in both positions (sequences shown in SEQ ID NO: 73~75, respectively). The results showed that adding the CAB box to all three positions significantly improved pseudouridineization efficiency and enhanced readability. Furthermore, adding the CAB box to the loop of the 5' stem-loop structure of the CAB box yielded the best results. Figure 7 f).

[0277] 3. Introduce an inactivated CAB box into gsnoRNA

[0278] Furthermore, to verify that the improved readthrough is directly related to the function of the CAB box, we compared the effects of a normal CAB box and a CAB box with a functionally inactivating mutation (i.e., a conserved sequence that does not conform to X1X2AG) on readthrough, and constructed gsnoRNAs (SEQ ID NO: 76 and SEQ ID NO: 77) containing CAB boxes with functionally inactivating mutations. Figure 7 As seen in g and 7h, a functional CAB box significantly improves readability, while a CAB box with a functional inactivation mutation has little or no effect on readability, or even slightly reduces it. These results indicate that the CAB box does indeed improve the readability of the RESTART system.

[0279] Then, to enhance the application prospects of the CAB box, we tested its effectiveness using different versions of the RESTART system in two nonsense mutation disease scenarios: CFTR-R553X and LMNA-R225X. CFTR stands for Cystic fibrosis transmembrane conductance regulator. R553X means that the arginine at position 553 (R, codon CGA) has been mutated to a stop codon (UGA), and its NCBI number is NP_000483.3. LMNA (Lamin A / C) is a protein encoded by the human gene LMNA, belonging to the lamin family. R225X means that the arginine at position 225 (R, codon CGA) has been mutated to a stop codon (UGA), and its NCBI number is NP_001393912.1.

[0280] The results showed that ( Figure 7 (i and 7j) In all RESTART systems, the use of the CAB box effectively improves readthrough efficiency, with particularly significant effects in the RESTART v3mini and RESTART v3 systems. That is, whether the gsnoRNA linking the CAB box is used alone in the RESTARTv1 system, or in combination with DKC1 iso3 and / or nc-tRNA in the RESTART v1, v3, and v3 mini systems, this gsnoRNA can further enhance the pseudouracil modification level and readthrough efficiency of PTC.

[0281] 4. Introduce HTR or Alu-derived CAB boxes into gsnoRNA.

[0282] Finally, to explore the universality of the CAB box, we tested its effectiveness on CAB boxes carried on other non-scaRNAs. The study found ( Figure 7Adding the CAB box from the HTR directly to the 5' stem loop (5HTR) and 3' stem loop (3HTR) of gsnoRNA, respectively, resulted in gsnoRNAs (SEQ ID NO: 78 and SEQ ID NO: 79) with significantly improved readability. Furthermore, replacing half of the gsnoRNA backbone with the corresponding sequence containing the CAB box in the HTR (SEQ ID NO: 80 and SEQ ID NO: 81), both 5' and 3' end substitutions, showed higher readability than gsnoRNAs without the CAB box. Replacing half of the 5' end of the gsnoRNA backbone with the corresponding sequence containing the CAB box in the HTR yielded significantly better results. Similarly, adding the CAB box from AluRNA to gnoRNA (SEQ ID NO: 82 and SEQ ID NO: 83) also showed some improvement, with addition at the 5' end being more effective than at the 3' end. Both 5' and 3' end additions significantly improved readability compared to gsnoRNAs without the CAB box. Since the CAB boxes on different AluRNAs are highly similar, it can be expected that the CAB boxes on other AluRNAs will also produce similar effects.

[0283] In summary, regardless of the source of the CAB box (e.g., scaRNA, Alu, or hTR) or the connection position of the CAB box (5' end, 3' end, or 5' and 3' ends), gsnoRNAs carrying CAB boxes can significantly improve the pseudouridine esterification efficiency and PTC readthrough level of the RESTART system (v1-v3, v3 mini). Furthermore, while retaining targeting capabilities, directly using scaRNA as the backbone, or replacing a portion of the gsnoRNA sequence entirely with scaRNA, Alu, or hTR containing a CAB box, can also achieve the function of gsnoRNA, significantly improving the pseudouridine esterification efficiency and PTC readthrough level of the RESTART system. This also provides new possibilities for the selection and design of "gsnoRNA" or "guide RNA." That is, the key to recruiting the DKC1 enzyme lies in retaining the H / ACA structure; we can choose different small RNAs as the basic backbone for modification and design, or even design from scratch, to provide guide RNA with both "DKC1 enzyme recruitment capability" and "target sequence guidance capability."

[0284] Example 5. Design of CTE sequences for snoRNA

[0285] This embodiment attempts to open the structure of the substrate mRNA by linking a CTE sequence to gsnoRNA, thereby enhancing the targeting efficiency of snoRNA. The structure of the CTE is shown below. Figure 8 a.

[0286] 1. Introducing SRV-derived CTE into gsnoRNA

[0287] We selected the most commonly used sno-ACA19 to construct gsnoRNA (SEQ ID NO: 84). Furthermore, we linked the CTE element (SEQ ID NO: 176) of type D retrovirus (SRV) to the 5' and 3' ends of gsnoRNA (SEQ ID NO: 85 and SEQ ID NO: 86), respectively. The results showed that regardless of whether DKC1 iso3 was overexpressed, the reading efficiency was improved by 30%-80%. Figure 8 (b-8c), where connecting at the 3' end is more effective than connecting at the 5' end.

[0288] Next, we added linkers of different lengths between the gsnoRNA and the CTE. We experimented with the effects of linker sequences of 4-8 bp length attached to the 5' or 3' end, respectively. The experimental 4-8 bp linker sequences were UCUA, UCUAU, UCUAUC, UCUAUCU, and UCUAUCU, and the resulting gsnoRNA sequences are shown in SEQ ID NO: 87-96. The results showed that adding a linker at either the 5' or 3' end improved reading efficiency to some extent. The highest reading efficiency was observed when the linker was 6 bp and the CTE was attached to the 3' end of the gsnoRNA, reaching approximately twice the original efficiency. Figure 8 b-8c).

[0289] 2. Introducing inactivated CTE into gsnoRNA

[0290] Next, we verified that the improved readthrough efficiency was indeed caused by the function of CTE by adding the inactivated CTE mutant to the gsnoRNA. The sequence of the gsnoRNA containing the native CTE is shown in SEQ ID NO: 97, and the sequences of the gsnoRNA containing the inactivated CTE mutant are shown in SEQ ID NO: 98 and SEQ ID NO: 99. Figure 8As shown in d-8e, regardless of whether DKC1 iso3 is overexpressed, ligation with a normally functioning CTE significantly improves readability, while ligation with a functionally mutated CTE has virtually no effect on readability. These results indicate that the improved readability after CTE ligation is indeed related to CTE function. Then, to verify that gsnoRNA ligation with a CTE improves readability, we performed RIP experiments to analyze the interaction between helicase and ligated normal and inactivated CTEs. Figure 8 As can be seen from f-8g, only gsnoRNAs linked to normal CTEs can interact with helicase and be significantly enriched. These results further demonstrate that gsnoRNAs linked to CTEs can improve readthrough efficiency by interacting with helicase.

[0291] 3. Introducing MPMV-derived CTE into gsnoRNA

[0292] Finally, we attempted to find smaller CTEs to link into gsnoRNA to achieve gsnoRNA delivery as a small RNA. Mason Fischer monkey virus (MPMV) also has CTE elements; the nucleotide sequence of an MPMV CTE element is shown in SEQ ID NO:177. Furthermore, it has been reported that a truncated half-length MPMV CTE is sufficient to achieve the same function as a full-length MPMV CTE. Therefore, we explored the role of SRV CTE and MPMV CTE, as well as their corresponding truncated mutants, in readthrough.

[0293] The sequences of gsnoRNAs constructed using natural CTEs and truncated CTEs derived from SRV are shown in SEQ ID NO: 100 and SEQ ID NO: 101-102, respectively; the sequences of gsnoRNAs constructed using natural CTEs and truncated CTEs derived from MPMV are shown in SEQ ID NO: 103 and SEQ ID NO: 104-111, respectively. Figure 8 As can be seen, in the UGA reporter gene system without DKC1 iso3 overexpression, MPMV CTE and its partially truncated mutants can achieve similar or even higher reading efficiency improvements to SRVCTE. Among them, the SRV CTE truncated mutants SRV CTE-m1, SRV CTE-m2 and the MPMV CTE truncated mutants 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 PTC reading efficiency. The sequences of these CTE element truncated mutants are shown in SEQ ID NO: 178~185, respectively.

[0294] In summary, this application provides a modified tRNA by mutating the base pairs of the T stem and AC stem of the tRNA. Specifically, the modified tRNA's T stem contains base pairs different from the wild type and / or the number of paired base pairs in the AC stem is reduced. This modification improves the efficiency of nc-tRNA in recognizing and decoding Ψ-modified PTCs, thereby enhancing the efficiency of the RESTART system and enabling its use in the future fourth-generation RESTART system (i.e., RESTART V4). Furthermore, the modified tRNA can be combined with other RESTART components to significantly improve the system's PTC readthrough efficiency.

[0295] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A method for inhibiting premature stop codons (PTCs) in target RNAs in host cells, wherein, The method includes introducing the following components into the host cell: (1) an engineered guide small nucleolar RNA (gsnoRNA) or a nucleic acid molecule for expressing said gsnoRNA; and (2) The modified tRNA or the nucleic acid molecule used to express the modified tRNA; The modified tRNA, compared to the wild-type tRNA, contains mutations in the first, second, and / or third base pairs in the T stem from the 5' to the 3' direction, and has base pairs that are different from the corresponding base pairs in the wild-type tRNA.

2. The method according to claim 1, wherein, The wild-type tRNA is selected from tRNA-R-UCU, tRNA-R-UCG, tRNA-G-UCC, tRNA-S-UGA, tRNA-W-CCA, tRNA-C-GCA, tRNA-Q-CUG, tRNA-K-CUU, tRNA-E-CUC, tRNA-S-CGA, tRNA-L-CAA, tRNA-Y-AUA, tRNA-Y-GUA, tRNA-Q-UUG, tRNA-K-UUU, and tRNA-E-UUC; Preferably, the wild-type tRNA has or includes a nucleotide sequence as shown in any one of SEQ ID NO: 158-161 and SEQ ID NO: 187-270; Preferably, the wild-type tRNA is selected from tRNA-R-UCU, tRNA-Q-CUG, tRNA-Q-UUG, and tRNA-W-CCA; Preferably, the wild-type tRNA has or contains a nucleotide sequence as shown in any one of SEQ ID NO: 158-161.

3. The method according to claim 1 or 2, wherein, In the tRNA standard numbering system, the positions of the first, second, and third base pairs are 49:65, 50:64, and 51:63, respectively. Preferably, the first base pair, the second base pair, and the third base pair follow the Watson-Crick pairing principle or the wobble pairing principle, respectively. Preferably, the first base pair, the second base pair, and / or the third base pair are each independently selected from: AU, UA, GC, CG, GU, and UG.

4. The method according to any one of claims 1-3, wherein, The wild-type tRNA is tRNA-R-UCU, wherein the first, second, and third base pairs in the T-stem from the 5' to the 3' direction are CG, CG, and GC, respectively; the modified tRNA has the following mutations: the first base pair is mutated to AU, GU, or GC, and / or the second base pair is mutated to UA; Preferably, the modified tRNA has or contains a nucleotide sequence as shown in any one of SEQ ID NO: 1 to 5.

5. The method according to any one of claims 1-3, wherein, The wild-type tRNA is tRNA-W-CCA, and the first, second, and third base pairs of the wild-type tRNA in the T-stem from the 5' to the 3' direction are GU, CG, and GC, respectively; the modified tRNA has the following mutations: the first base pair is mutated to GC, AU, or CG, or, and / or, the third base pair is mutated to UG; Preferably, the modified tRNA has or contains a nucleotide sequence as shown in any one of SEQ ID NO: 8-11.

6. The method according to any one of claims 1-3, wherein, The wild-type tRNA is tRNA-Q-CUG, wherein the first, second, and third base pairs in the T-stem from the 5' to the 3' direction are CG, CG, and GC, respectively; the modified tRNA has the following mutations: the first base pair is mutated to UA, GC, or AU; the second base pair is mutated to UA, UG, or GU; and / or, the third base pair is mutated to CG. Preferably, the modified tRNA has or includes a nucleotide sequence as shown in any one of SEQ ID NO: 12, 13, 18, 27-29, 31, 32, 34-36 and 38.

7. The method according to any one of claims 1-3, wherein, The wild-type tRNA is tRNA-Q-UUG, wherein the first, second, and third base pairs in the T-stem from the 5' to the 3' direction are CG, CG, and GC, respectively; the modified tRNA has the following mutations: the first base pair is mutated to GC or AU, the second base pair is mutated to UA, UG, or GU, and / or the third base pair is mutated to UA, GU, or CG; Preferably, the modified tRNA has or contains a nucleotide sequence as shown in any one of SEQ ID NO: 41, 43-50.

8. The method according to any one of claims 1-7, wherein, The gsnoRNA has one or more of the following technical features: (i) The gsnoRNA comprises: at least one guide sequence and a scaffold sequence; wherein the guide sequence hybridizes with a sequence of target uridine residues (U) containing PTC in the target RNA; the scaffold sequence is derived from natural H / ACA type snoRNA and / or natural H / ACA type scaRNA; (ii) The gsnoRNA recruits the DKC1 protein to modify the target uridine residue in the PTC of the target RNA into a pseudouridine residue (Ψ). (iii) The gsnoRNA hybridizes with the target RNA and recruits the DKC1 protein, and modifies the target uridine residue (U) of the PTC contained in the target RNA into a pseudouridine residue (Ψ).

9. The method according to any one of claims 1-8, wherein, The method further includes introducing a nucleic acid molecule encoding the DKC1 protein into the host cell; Preferably, the DKC1 protein is overexpressed in the host cells; Preferably, the DKC1 protein is a naturally occurring DKC1 subtype that has cytoplasmic localization in the host cell; Preferably, the DKC1 is selected from human DKC1 protein iso1, human DKC1 protein iso3, or any combination thereof; Preferably, the amino acid sequence of the DKC1 protein is as shown in SEQ ID NO: 164 or SEQ ID NO:

163.

10. The method according to any one of claims 1-9, wherein, The method has one or more features selected from the following: (i) The modified tRNA is a near-homologous transfer RNA (nc-tRNA) of the PTC; (ii) The modified tRNA decodes the PTC into amino acids, thereby inhibiting the PTC; and (iii) The modified tRNA is modified (e.g., psiU, m 1 A、m 1 G or m 5 C modifier).

11. A modified tRNA, wherein, Compared with the wild-type tRNA, the modified tRNA contains mutations in the first, second, and / or third base pairs in the T stem from the 5' to the 3' direction, and has base pairs that are different from the corresponding base pairs in the wild-type tRNA. Preferably, in the tRNA standard numbering system, the positions of the first base pair, the second base pair, and the third base pair are: position 49:65, position 50:64, and position 51:63, respectively. Preferably, the first base pair, the second base pair, and the third base pair follow the Watson-Crick pairing principle or the wobble pairing principle, respectively. Preferably, the first base pair, the second base pair, and / or the third base pair are each independently selected from: AU, UA, GC, CG, GU, and UG.

12. The modified tRNA according to claim 11, wherein the wild-type tRNA is selected from tRNA-R-UCU, tRNA-R-UCG, tRNA-G-UCC, tRNA-S-UGA, tRNA-W-CCA, tRNA-C-GCA, tRNA-Q-CUG, tRNA-K-CUU, tRNA-E-CUC, tRNA-S-CGA, tRNA-L-CAA, tRNA-Y-AUA, tRNA-Y-GUA, tRNA-Q-UUG, tRNA-K-UUU, and tRNA-E-UUC; Preferably, the wild-type tRNA has or includes a nucleotide sequence as shown in any one of SEQ ID NO: 158-161 and SEQ ID NO: 187-270; Preferably, the wild-type tRNA is selected from tRNA-R-UCU, tRNA-Q-CUG, tRNA-Q-UUG, and tRNA-W-CCA; Preferably, the wild-type tRNA has or contains a nucleotide sequence as shown in any one of SEQ ID NO: 158-161; Preferably, the modified tRNA has or includes the nucleotide sequence shown in any one of SEQ ID NO: 1~5, SEQ ID NO: 8~11, SEQ ID NO: 12, 13, 18, 27~29, 31, 32, 34~36, 38, SEQ ID NO: 41, 43~50.

13. An isolated nucleic acid molecule, said isolated nucleic acid molecule comprising a nucleotide sequence for expressing the modified tRNA of claim 11 or 12; Preferably, the isolated nucleic acid molecule is DNA.

14. A composition comprising the modified tRNA of claim 11 or 12 or the isolated nucleic acid molecule of claim 13; Preferably, the composition further comprises engineered guide small nucleolar RNA (gsnoRNA) or a nucleic acid molecule for expressing the gsnoRNA; Preferably, the composition further comprises a DKC1 protein (e.g., DKC1 iso1, DKC1 iso3) or a nucleic acid molecule encoding the DKC1 protein; Preferably, the composition further comprises gsnoRNA or a nucleic acid molecule for expressing the gsnoRNA, and DKC1 protein or a nucleic acid molecule encoding the DKC1 protein.

15. A delivery composition comprising: a delivery vector, and one or more of the following: the modified tRNA of claim 11 or 12, the isolated nucleic acid molecule of claim 13, or the composition of claim 14; Preferably, the delivery carrier is a particle; Preferably, the delivery vector is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, gene guns, or viral vectors (e.g., replication-defective retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses).

16. A host cell comprising the modified tRNA of claim 11 or 12, the isolated nucleic acid molecule of claim 13, the composition of claim 14, or the delivery composition of claim 15; Preferably, the host cell is a prokaryotic cell or a eukaryotic cell; Preferably, the host cell is a mammalian cell (e.g., human). Preferably, the host cell contains a premature stop codon (PTC) mutant gene.

17. Use of the modified tRNA of claim 11 or 12, the isolated nucleic acid molecule of claim 13, the composition of claim 14, the delivery composition of claim 15, or the host cell of claim 16 in the preparation of a formulation for inhibiting premature stop codons (PTCs) in target RNA in host cells. Preferably, the formulation decodes the PTC into amino acids, thereby inhibiting the PTC; Preferably, the uridine residue (U) in the PTC is a pseudouridine residue (Ψ), and the PTC is a Ψ-modified PTC; Preferably, the host cell is a mammalian cell (e.g., a human cell).

18. Use of the modified tRNA of claim 11 or 12, the isolated nucleic acid molecule of claim 13, the composition of claim 14, the delivery composition of claim 15, or the host cell of claim 16 in the preparation of a medicament for treating a subject with a disease and / or symptoms caused or resulting from a premature stop codon (PTC) mutation; Preferably, the disease is selected from cystic fibrosis, spinal muscular atrophy, fructose intolerance, dilated cardiomyopathy, Heller syndrome, alpha-1-antitrypsin (A1AT) deficiency, Parkinson's disease, Alzheimer's disease, albinism, amyotrophic lateral sclerosis, asthma, β-thalassemia, Cadasil syndrome, Charcot-Marie-Tooth disease, chronic obstructive pulmonary disease (COPD), distal spinal muscular atrophy (DSMA), Duchenne muscular dystrophy, dystrophic epidermolysis bullosa, epidermolysis bullosa, Fabry disease, Leiden factor V-related disorders, familial adenomatous polyposis, galactosemia, Gaucher's disease, glucose-6-phosphate dehydrogenase, hemophilia, hereditary hemochromatosis, and Hunt syndrome. Huntington's disease, inflammatory bowel disease (IBD), hereditary polyagglutination syndrome, Lesch-Nair syndrome, Lynch syndrome, Marfan syndrome, mucopolysaccharidosis, muscular dystrophy, myotonic dystrophy type I and II, neurofibromatosis, Niemann-Pick disease type A, B and C, NY-esol-related cancers, Boytz-Yage syndrome, phenylketonuria, Pomper's disease, primary ciliary body disease, prothrombin mutation-related diseases (such as prothrombin G20210A mutation), pulmonary hypertension, (autosomal dominant) retinitis pigmentosa, Sandhoff's disease, severe combined immunodeficiency syndrome (SCID), sickle cell anemia, Staggart disease, Tay-Sachs disease, Usher syndrome, X-linked immunodeficiency, Sturge-Weber syndrome, or any combination thereof; Preferably, the subject is a mammal (e.g., human, cynomolgus monkey, mouse). Preferably, the disease is fructose intolerance; preferably, the PTC mutation is a nonsense mutation in the nucleotide encoding the 148th amino acid of ALDOB (fructose diphosphate aldolase B); Preferably, the disease is cystic fibrosis; preferably, the PTC mutation is a nonsense mutation of the nucleotide encoding amino acid 553 of the CFTR (cystic fibrosis transmembrane conduction regulator); Preferably, the disease is dilated cardiomyopathy; preferably, the PTC mutation is a nonsense mutation in the nucleotide encoding amino acid 225 of LMNA (lamin A / C).

Images