TRNA binding protein assisted termination codon readthrough system and application thereof

Abstract

The invention belongs to the technical field of biological medicine and gene therapy, and particularly relates to a tRNA binding protein assisted termination codon readthrough system and application thereof. The tRNA binding protein assisted termination codon reading-through system disclosed by the invention comprises inhibitory tRNA and binding protein thereof, wherein the inhibitory tRNA contains 70 pairs of G3: U, can be recognized by an endogenous alanyl-tRNA synthetase (AlaRS) of a cell, and is loaded with alanine; the tRNA binding protein is specifically bound with any structure, except for a receptor arm, of the corresponding tRNA, and can be specifically bound with a tRNA binding structural domain of pyrrole lysyl-tRNA synthetase. According to the system, alanine can be introduced in the translation process of a target gene carrying nonsense mutation, so that full-length translation of protein is recovered. The system can be applied to functional recovery and treatment research of various genetic diseases caused by the early termination codon.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and gene therapy technology, specifically relating to a tRNA-binding protein-assisted stop codon reading system and its application. Background Technology

[0002] Premature termination codons (PTCs) are a common type of gene mutation in molecular genetics. They occur when a normal amino acid codon in a gene's coding sequence is replaced by a stop codon (such as UAG, UAA, or UGA), leading to premature termination of messenger RNA (mRNA) translation. This premature translation interruption causes the target protein to lose its normal function due to structural truncation, resulting in a series of serious genetic diseases. Premature termination codon disorders, such as Duchenne muscular dystrophy (DMD), mucopolysaccharidosis type I (MPS I, IDUA gene defect), and Alport syndrome (COL4a5-related nephropathy), severely impact patients' quality of life and survival. Statistics show that approximately 11% of known pathogenic gene mutations are of the premature termination codon type, a proportion even more pronounced in rare disease populations, highlighting the urgent need to develop safe and effective molecular repair therapies.

[0003] To address protein function loss caused by premature stop codon mutations, the scientific community has proposed various "readthrough therapies" to restore the expression of functional proteins. The earliest approach involved inducing readthrough with small molecule drugs, such as aminoglycoside antibiotics (e.g., G418, Gentamicin) and Ataluren. This strategy interferes with the ribosomal translation termination process, inserting an "incorrect" amino acid at the PTC to synthesize full-length or near-full-length proteins, partially alleviating clinical symptoms. However, chemically induced readthrough therapies often suffer from low readthrough efficiency and high nonspecificity, easily leading to misreading of normal stop codons across the entire genome. Long-term use also presents significant toxic side effects, and the efficacy is highly individualized, making it difficult to meet the needs of precision medicine for hereditary diseases.

[0004] Advances in molecular biology techniques have driven targeted reading strategies based on tRNA engineering. Orthogonal tRNA / exogenous synthase systems (such as pylRS / tRNA pairs) are among the most important current reading techniques. This system introduces an exogenous aminoacyl-tRNA synthetase (such as pylRS) and its specifically recognized orthogonal tRNA, along with exogenous non-natural amino acids (such as UAA), to directionally insert specific amino acids into the stop codon, achieving highly specific reading. Theoretically, this approach can be flexibly adjusted through genetic engineering, exhibiting strong targeting and low misread rates. However, its practical application is limited by the complexity of the delivery system, the high immunogenicity of the exogenous enzyme, the difficulty of in vivo delivery of non-natural amino acids, and its long-term safety and clinical translation prospects.

[0005] sup-tRNA therapy modifies the anticodon of natural tRNA to directly recognize PTCs, relying solely on endogenous aminoacyl-tRNA synthetases for aminoacylation and delivery. This method has a low delivery burden and low immune risk, but it faces several limitations in mammals: First, the translation termination efficiency of stop codons is highly dominated by termination factors (such as eRF1), and sup-tRNAs have limited ability to compete with them, resulting in generally low readthrough efficiency. Second, after modification of the anticodon region, some tRNAs exhibit reduced binding affinity to endogenous enzymes, leading to insufficient aminoacylation efficiency and difficulty in achieving sustained and adequate protein restoration. Furthermore, the nonsense-mediated mRNA degradation (NMD) mechanism activated by PTCs further reduces the amount of mRNA to be repaired, further restricting protein expression. Currently, sup-tRNA therapy is mostly in the in vitro or short-term mouse model validation stage, and systematic long-term safety and efficacy data have not yet been reported.

[0006] With the increasing demand for precision medicine and gene therapy, tRNA engineering technology itself faces numerous challenges. For example, how to ensure that repressive tRNAs, even after anticodon modification, can still be efficiently recognized and loaded with amino acids by endogenous aminoacyl-tRNA synthetases, while maintaining structural stability and high translation efficiency; how to improve the delivery, expression, and stability of engineered tRNAs in vivo, preventing cellular clearance or degradation; and how to further enhance the competitive ability of tRNAs against termination factors, enabling more efficient and specific readout in vivo. Furthermore, applying these systems to different stop codons (UAG, UAA, UGA) to achieve broad-spectrum, precise repair targeting different types of diseases and mutation sites also places higher demands on molecular design and platform development.

[0007] Current readthrough technologies still face a series of limitations. Drug-induced readthrough is generally limited by low specificity, significant toxic side effects, and the potential for systemic safety hazards with long-term use. Orthogonal tRNA / exogenous enzyme systems rely on the supplementation of non-natural amino acids and the delivery of exogenous proteins, resulting in complex operation, limited readthrough efficiency, and high synthesis and delivery costs that further restrict their large-scale clinical application. While the sup-tRNA method is theoretically simple, its readthrough efficiency and site specificity are difficult to achieve due to insufficient competition between endogenous enzymes and termination factors in vivo. Currently, there is no method that can both avoid drug and non-natural amino acid-related toxicity and delivery barriers in mammalian cells and in vivo environments, and achieve "in-situ, programmable, natural amino acid" specific insertion at premature stop codons, truly meeting the needs of personalized precision repair and protein function restoration. Therefore, developing a functional readthrough translation system that does not rely on non-natural amino acids, requires no exogenous synthetic enzymes, uses natural amino acids as substrates, and can achieve safe and efficient readthrough translation at PTC sites has become a core technical challenge that urgently needs to be overcome in this field. Summary of the Invention

[0008] To address the problems existing in the prior art, the purpose of this invention is to design and provide a tRNA-binding protein-assisted stop codon reading system and its application.

[0009] The present invention is implemented using the following technical solutions: The first aspect of this invention provides a tRNA-binding protein-assisted stop codon reading system, the system comprising: Repressive tRNA: Contains the G3:U70 pair, which can be recognized by endogenous alanyl-tRNA synthetase and loaded with alanine. Its anticodon is mutated in a directed manner to recognize at least one of the premature stop codons UAG, UGA or UAA. tRNA-binding proteins: capable of specifically binding to the repressive tRNA domains other than the receptor arm.

[0010] Furthermore, the anticodon sequence of the repressive tRNA is selected from at least one of CUA, UCA, or UUA, which respectively recognize the stop codons UAG, UGA, or UAA.

[0011] Furthermore, the repressive tRNA is modified with D stem / loop, T stem / loop or anticodon stem, and its corresponding nucleotide sequence is shown in SEQ ID NO.1-107.

[0012] Furthermore, the tRNA-binding protein specifically binds to repressive tRNAs modified with G3:U70, and these repressive tRNAs, after being loaded with alanine via host endogenous AlaRS, are able to recognize premature stop codons.

[0013] Furthermore, the tRNA-binding protein can specifically bind to the tRNA-binding domain of pyrrolidone-lysyl-tRNA synthetase.

[0014] Furthermore, the binding domain of the pyrrolidone-lysyl-tRNA synthetase, in its full length or variant form, is derived from MbPylRS, MmPylRS, or their homologous sequences, with the corresponding nucleotide sequences shown in SEQ ID NO.108-122.

[0015] Furthermore, the system is delivered into mammalian cells via a plasmid vector.

[0016] Furthermore, the system can be used in conjunction with at least one of a reporter gene module, a regulatory element, or a promoter.

[0017] A second aspect of the present invention provides the application of the aforementioned readout system in the preparation of medicaments for treating diseases caused by premature stop codons resulting from gene stop codon mutations.

[0018] Furthermore, the gene stop codon mutation forming premature stop codon-related diseases is selected from one or more of the following: Duchenne muscular dystrophy, mucopolysaccharidosis type I, Allport syndrome, or familial adenomatous polyposis.

[0019] The third aspect of this invention provides the application of the aforementioned readout system in the visualization evaluation and regulation of readout efficiency of the inhibitory tRNA-tRNA binding protein system in vivo and in vitro.

[0020] The present invention has the following beneficial effects: (1) No need to introduce exogenous synthases or non-natural amino acids, avoiding potential immunogenicity and toxicity risks: Only the modified tRNA molecule and tRNA binding protein need to be delivered, without the need to introduce exogenous aminoacyl-tRNA synthases or non-natural amino acids, which simplifies the treatment system and reduces the risk of immunogenicity and toxic side effects.

[0021] (2) High endogenous recognition efficiency, significantly improving readability and protein expression recovery level: By optimizing the tRNA binding protein sequence and optimizing the tRNA structure to enhance the binding ability with endogenous synthases, the competitiveness of the termination codon site and termination factor is improved, achieving efficient protein synthesis recovery at the cellular level.

[0022] (3) tRNA binding proteins synergistically enhance tRNA stability, correct folding, and ribosome entry efficiency within cells; (4) The system is highly scalable and can be flexibly adapted to different stop codons and disease models; (5) Significant in vivo validation results: Functional protein recovery and phenotypic improvement were achieved in various PTC mouse models such as DMD, Col4a5 and IDUA, which are suitable for the precise treatment of various stop codon mutation-related genetic diseases.

[0023] (6) Easy to platformize and secondary development: tRNA sequence, structure and delivery method can be modularly designed, which facilitates customized development and optimization for different diseases or individual differences. Attached Figure Description

[0024] Figure 1 This is a schematic diagram illustrating the restoration of functional protein expression in PTC disease using codon redistribution through a functional read-through translation system. Figure 2 This is a schematic diagram of the experimental procedure for quantitatively analyzing the readability of an orthogonal translation system using flow cytometry, and a quantitative calculation formula for the readability of the orthogonal translation system. Figure 3 This is a graph showing the readability results of tRNA-binding proteins optimized by deletions to terminate codon mutation readthrough. Figure 4 This is a graph showing the optimized readability of tRNA-binding protein with single amino acid mutations 1-45 (A) and 46-90 (B) for stop codon mutation readthrough. Figure 5 This is a mutation information diagram of a single amino acid mutant of a tRNA-binding protein used to terminate codon mutation readthrough; Figure 6 This is a diagram showing the optimized results of the repressive tRNA-1 mutants 44 (A), 45-86 (B), and 87-107 (C) used to terminate codon mutation readthrough. Figure 7 The graphs show the reading efficiency of the Dystrophin protein subunit gene with TAA nonsense mutation, the reading efficiency of the stop codon reading system that recognizes TAA, TGA, and TAG; the reading efficiency of the COL4A5 protein subunit gene with TGA nonsense mutation; and the reading efficiency of the APC protein subunit gene with TAG nonsense mutation. Figure 8 The images show the immunoblotting results (A) and grayscale analysis of Dystrophin protein expression in myocardial tissue with a TAG stop codon mutation in Dmd mice using a read-through translation system, the immunoblotting results (B) and grayscale analysis of Dystrophin protein expression in diaphragmatic muscle tissue, and the results of the treadmill test (E). Figure 9The images show the serum IDUA enzyme activity (A), urine GAG ​​(B), Micro-CT schematic image (C), zygomatic arch width statistics (D), cardiac tissue GAG ​​(E) and enzyme activity (G), liver tissue GAG ​​(F) and enzyme activity (H), liver tissue LAMP1 protein immunoblotting results (I) and grayscale analysis expression statistics (K), and cardiac tissue LAMP1 protein immunoblotting results (J) and grayscale analysis expression statistics (L) in IDUA mice. Figure 10 The results of reading out TAG stop codon mutations in Col4a5 mice using a read-through translation system are as follows: urine albumin-to-creatinine ratio (A), endpoint serum creatinine (B), endpoint serum urea nitrogen (C), tissue Marson trichrome staining results (D), and statistical analysis of the stained areas (E). Detailed Implementation

[0025] To facilitate understanding and implementation of the present invention by those skilled in the art, the technical solution of the present invention will now be further described in detail with reference to the embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any equivalent modifications or improvements made to the present invention should fall within the protection scope of the present invention.

[0026] In this invention, unless otherwise specified, all units of measurement are parts by mass or conventional biological units of measurement, and all materials, reagents, and equipment used are commercially available or prepared in a conventional manner. Unless otherwise specified, the experimental methods used in the following examples are conventional methods commonly used in the art.

[0027] Example 1: Construction of a dual-fluorescent report system for evaluating read-through translation systems (1) Construction and acquisition of dual fluorescent reporter plasmids From mushroom coral ( mushroom coralThe red fluorescent protein gene *mCherry* was used as an internal control. A 2A peptide derived from *Thosea asigna* virus was used, with T2A as the linker fragment. During translation, a break occurs in the 2A peptide, prematurely releasing the first half of the synthesized peptide chain. The 2A peptide can cause the peptide chain translated from an open reading frame (ORF) to split into several independent peptide chains. An enhanced green fluorescent protein (EGFP) without the transcription start codon ATG, derived from wild-type green fluorescent protein (wt-GFP), was linked to the T2A fragment. The DNA encoding amino acid 190 of EGFP was mutated to TAG, TGA, and TAA, respectively, and constructed into the pEGFP plasmid backbone to obtain dual fluorescent reporter plasmids pEGFP-CMV-mCherry-T2A-EGFP190TAG, pEGFP-CMV-mCherry-T2A-EGFP190TGA, and pEGFP-CMV-mCherry-T2A-EGFP190TAA. By denormalizing the fluorescence intensity of EGFP using the fluorescence brightness of mCherry, the reading efficiency of TAG, TGA, and TAA functional read-through translation systems can be evaluated, respectively.

[0028] The nucleotide sequence of mCherry-T2A-EGFP190TAG is shown in SEQ ID NO. 123, the nucleotide sequence of mCherry-T2A-EGFP190TGA is shown in SEQ ID NO. 124, and the nucleotide sequence of mCherry-T2A-EGFP190TAA is shown in SEQ ID NO. 125.

[0029] (2) Construction and acquisition of functional read-through translation system plasmids The tRNA-binding protein was expressed using the CMV promoter, and the repressive tRNA was expressed using the U6 promoter. Both sets of elements were cloned into the same pCMV plasmid backbone to obtain a read-through system plasmid. The repressive tRNA was then replaced with a repressive tRNA paired with Amber / Ochre / Opal and co-expressed with a universal tRNA-binding protein cloned downstream of the CMV promoter.

[0030] Example 2: Evaluation of functional read-through translation system in mammalian cell line HEK 293T (1) Culture and passage of HEK 293T cell line HEK 293T cells were cultured in complete medium (DMEN, Gibco, 8120106; 10% fetal bovine serum, ExCellBio, FSP500; 1% penicillin / streptomycin) until confluence reached 80% or higher. The original medium was then discarded, and the cells were gently washed twice with pre-warmed 1x PBS to remove residual medium, serum, and antibiotics. The cells were then digested with 0.25% trypsin (Biological Industry, 03-050-1B) containing 0.02% EDTA and collected into 15mL centrifuge tubes. The cells were centrifuged at 200rcf for 3 minutes, the supernatant was discarded, and the cell pellet was resuspended in 1mL of medium. Cells were then aliquoted into new culture dishes as needed, medium was added, and the mixture was stirred using the cross-hatching method. The cells were then cultured further.

[0031] (2) HEK 293T cell line transfected with dual fluorescent reporter system Collect and resuspend cells according to the steps in (1) above, take 15 μL for cell counting, and then use 1.8 x 10 μL of the solution. 5 HEK 293T cells were distributed into 24-well plates. On the second day, when the cell confluence in each well reached 50%-70%, the functional read-through translation system plasmid to be tested and its corresponding stop codon mutation dual-fluorescent reporter plasmid were co-transfected into HEK 293T cells at a 1:1 (g:g) ratio using PEI reagent, following the manufacturer's instructions. Six hours after transfection, the culture medium was replaced with complete culture medium, and the cells were cultured for an extended period.

[0032] (3) Quantitative analysis of the readability of functional read-through translation systems using flow cytometry. Forty-eight hours later, HEK 293T cells in 24-well plates were digested and collected into flow cytometry tubes according to the steps in (1) above. The fluorescence intensity of EGFP and mCherry fluorescent proteins was measured using a flow cytometer (Beckman Coulter, CytoFlex S). The fluorescence intensity of EGFP was measured using the FITC channel, and the fluorescence intensity of mCherry was measured using the PE channel. First, the flow cytometry program was divided into partitions using blank HEK 293T cells, HEK 293T cells transfected only with EGFP, HEK 293T cells transfected only with mCherry, and HEK 293T cells transfected with mCherry-T2A-EGFP. Based on the flow cytometry results, the fluorescence intensity of EGFP was normalized using mCherry fluorescence intensity as an internal control, and the reading efficiency of the functional read-through translation system was quantitatively analyzed.

[0033] A schematic diagram of the tRNA-binding protein-assisted stop codon readthrough system for redistributing codons to restore functional protein expression in PTC disease is shown below. Figure 1 As shown, premature stop codon disease is caused by nonsense mutations. Prematurely terminated mRNAs terminate prematurely during translation into proteins, resulting in nonfunctional truncated proteins and disrupting cellular homeostasis. The tRNA-binding protein-assisted stop codon reading system recognizes repressive tRNAs, adding their native amino acids. The repressive tRNA with the native amino acids decodes the nonsense-mutated codon on the prematurely terminated mRNA, introducing the native amino acid into the nonsense mutation site during translation, thereby restoring the expression of the functional, full-length active protein and achieving disease remission.

[0034] A schematic diagram of the experimental procedure for quantitatively analyzing the readability of a functional fluent translation system using flow cytometry, and the quantitative calculation formula for the functional fluent translation system are shown below. Figure 2 As shown in the figure, the quantitative results of the read-through efficiency of the read-through translation system in the HEK 293T cell line are as follows. Figure 3 , Figure 4 and Figure 6 As shown in the figure, the functional readthrough translation systems can all achieve readthrough with varying efficiencies within mammalian cells, indicating that the functional readthrough translation systems of this invention can all restore the expression of functional proteins in PTC disease within mammalian cells.

[0035] Example 3: Sequence optimization of tRNA-binding protein for stop codon mutation readthrough (1) Construction and acquisition of functional read-through translation system plasmids with optimized tRNA-binding protein sequences A series of optimized tRNA-binding proteins (involving 15 deletion mutants and 90 single-amino acid mutants in this patent) were expressed using the CMV promoter, and the same repressive tRNA was expressed using the U6 promoter. Both sets of elements were cloned into the same pCMV plasmid backbone, resulting in 105 functional readthrough system plasmids. The nucleotide sequences of the deletion mutants of the tRNA-binding proteins are shown in SEQ ID NO. 108-122, and the specific mutation information for the single-amino acid mutants is provided in [link to relevant documentation]. Figure 5 .

[0036] (2) Culture and passage of HEK 293T cell line The steps are as described in Example 2 (1).

[0037] (3) HEK 293T cell line transfected with dual fluorescent reporter system Collect and resuspend cells according to the steps in (2) above, take 15 μL for cell counting, and then add 1.8 x 10 μL of the solution. 5HEK 293T cells were distributed into 24-well plates. On the second day, when the cell confluence in each well reached 50%-70%, the functional read-through translation system plasmid to be tested and its corresponding stop codon mutation dual-fluorescent reporter plasmid were co-transfected into HEK 293T cells at a 1:1 (g:g) ratio using PEI reagent, following the manufacturer's instructions. Six hours after transfection, the culture medium was replaced with complete culture medium, and the cells were cultured for an extended period.

[0038] (4) Quantitative analysis of the readthrough efficiency of the optimized tRNA-binding protein readthrough system using flow cytometry. Forty-eight hours later, HEK 293T cells in 24-well plates were digested and collected into flow cytometry tubes according to the steps in (2) above. The fluorescence intensity of EGFP and mCherry fluorescent proteins was measured using a flow cytometer (Beckman Coulter, CytoFlex S). The method was the same as in Example 2 (3). The results are as follows. Figure 3 , Figure 4 As shown, compared to the unoptimized system, the readthrough efficiency of the readthrough system with the optimized tRNA-binding protein TD103 was increased by 1.61 times. After single-amino acid site-directed mutagenesis, mutants such as TD103-mut-28 showed an increasing trend.

[0039] Example 4: Optimization and functional identification of repressive tRNA for stop codon mutation readthrough (1) Construction and acquisition of functional read-through translation system plasmids with optimized repressive tRNA sequences The optimized tRNA-binding protein TD103 was expressed using the CMV promoter, and a series of optimized repressive tRNAs (107 mutants in this patent, nucleotide sequences shown in SEQ ID NO. 1~107) were expressed using the U6 promoter. These mutants were derived from separate sequence optimization of each part of the tRNA to enhance its affinity for endogenous AlaRS and its aminoacylation efficiency. Both sets of elements were cloned into the same pCMV plasmid backbone, resulting in 107 functional readthrough system plasmids. The nucleotide sequences of the tRNA-binding proteins are shown in SEQ ID NO. 108-122.

[0040] (2) Culture and passage of HEK 293T cell line The steps are as described in Example 2 (1).

[0041] (3) HEK 293T cell line transfected with dual fluorescent reporter system Collect and resuspend cells according to the steps in (2) above, take 15 μL for cell counting, and then add 1.8 x 10 μL of the solution. 5HEK 293T cells were distributed into 24-well plates. On the second day, when the cell confluence in each well reached 50%-70%, the functional read-through translation system plasmid to be tested and its corresponding stop codon mutation dual-fluorescent reporter plasmid were co-transfected into HEK 293T cells at a 1:1 (g:g) ratio using PEI reagent, following the manufacturer's instructions. Six hours after transfection, the culture medium was replaced with complete culture medium, and the cells were cultured for an extended period.

[0042] (4) Quantitative analysis of the readability of the functional readability translation system using flow cytometry. Forty-eight hours later, HEK 293T cells in 24-well plates were digested and collected into flow cytometry tubes according to the steps in (2) above. The fluorescence intensity of EGFP and mCherry fluorescent proteins was measured using a flow cytometer (Beckman Coulter, CytoFlex S). The method was the same as in Example 2 (3). The results are as follows. Figure 6 As shown, compared to the unoptimized system, the readthrough efficiency of the system with the optimized repressive tRNA was increased by 1.56 times, reaching 61.2% of that of EGFP-WT.

[0043] Example 5: Evaluation of the readthrough efficiency of an orthogonal translation system for a specific protein in the HEK 293T cell line (1) Constructing dual fluorescent reporter plasmids for specific proteins The functional proteins Dystrophin, COL4A5, and APC from sex-linked recessive disease DMD, sex-linked dominant disease AS, and autosomal dominant disease FAP were selected as validation targets. Subunits with nonsense mutations were selected for each protein: amino acids 832-1156 of Dystrophin (sequence shown in SEQ ID NO. 126), where the glutamine codon at position 995 was mutated to TAA; amino acids 344-602 of COL4A5 (sequence shown in SEQ ID NO. 127), where the arginine codon at position 471 was mutated to TGA; and amino acids 766-942 of APC (sequence shown in SEQ ID NO. 128), where the leucine codon at position 850 was mutated to TAG, and the glutamate codon at position 851 was mutated to TAG. The genes encoding these protein subunits were linked downstream of the mCherry gene and upstream of the EGFP gene (which does not contain a transcription start codon), with the T2A term, where the EGFP gene did not carry a nonsense mutation. This yields a specific protein dual-fluorescent reporter plasmid that can be used to evaluate the readability of orthogonal translation systems.

[0044] (2) Transfection of HEK 293T cell line Following the steps in Example 2(2), a dual fluorescent reporter plasmid of a specific protein in a ratio of 1:1 (g:g) and the corresponding orthogonal translation system plasmid were co-transfected into HEK 293T cells.

[0045] The readthrough efficiency of the orthogonal translation system for a specific protein was quantitatively analyzed using flow cytometry. The readthrough efficiency was analyzed according to the steps in Example 2(3). The flowchart and results of the readthrough efficiency determination of the TAA-recognizing translation system for the Dystrophin protein subunit gene with nonsense mutation are shown below. Figure 7 The results showed that the readthrough strength of Dystrophin reached 17% of that of the wild type, indicating that the orthogonal translation system in this invention can restore the full-length expression of functional proteins in TAA-PTC mutant diseases. The flowchart and results of the readthrough efficiency determination of the TGA-recognizing readthrough translation system for the COL4A5 protein subunit with nonsense mutations are shown below. Figure 7 The results showed that the average readthrough strength of COL4A5 reached 50% of that of the wild type, indicating that the readthrough translation system in this invention can restore the full-length expression of functional proteins in TGA-PTC mutant diseases. The flowchart and results of the TAG-recognizing readthrough translation system for measuring the readthrough efficiency of APC protein subunit genes with nonsense mutations are shown below. Figure 7 The results showed that the average readthrough strength of APC reached 70% of that of wild type, indicating that the readthrough translation system in this invention can restore the full-length expression of functional proteins with TAG-PTC mutation disease.

[0046] Example 6: Evaluation of readthrough efficiency of orthogonal systems in Dystrophin-TAG mutant mice (1) Constructing pAAV-cis-transgene plasmid with a functional read-through translation system The tRNA-binding protein was expressed using the tMCK promoter, and the orthogonal tRNA was expressed using the U6 promoter. The two sets of elements were cloned into the same vector and constructed into the adeno-associated virus pAAV-cis-transgene plasmid vector using the preferred read-through translation system TD103 / tRNA-47-CUA, thereby obtaining the adeno-associated virus transgenic plasmid.

[0047] (2) Packaging of adeno-associated virus The plasmids required for AAV packaging, pAAV-2 / 9, pAAV-Helper, and the plasmid pAAV-cis-TD103 / tRNA-47-CUA with a functional read-through translation system, were extracted using an endotoxin-free plasmid extraction kit (Qiagen, 12362). AAV packaging was performed step-by-step according to the AAV production procedure (https: / / www.addgene.org / protocols / aav-production-hek293-cells / ) and AAV purification procedure (https: / / www.addgene.org / protocols / aav-purification-iodixanol-gradient-ultracentrifugation / ) provided on addgene.org to obtain AAV with a read-through translation system. AAV was then quantified using SDS-PAGE. Analysis of the results showed that the final concentration of AAV with a read-through translation system was 5.2 × 10⁻⁶. 13 μg / mL.

[0048] (3) Delivery of a functional read-through translation system to Dmd-TAG disease mice via AAV delivery The quantified AAV was diluted in 50 μL of PBS solution and injected into the experimental group mice (6 mice aged 4 weeks) at a dose of 1.0E12 / 1.5E12 / 2.0E12 vg per mouse. The control group mice (4 mice aged 4 weeks) were injected with an equal volume of PBS solution without AAV. First, the Dmd-TAG mutant mice were placed in the plexiglass chamber of a respiratory anesthesia machine (Reward, R550) and anesthesia was induced by introducing isoflurane (McLean, 26675-46-7) gas. After the mice were fully anesthetized, each mouse was removed one by one, placed on its right side facing up on a heating pad, and pressure was applied to make the mouse's right eyeball protrude from the eye socket. The insulin needle containing the drug was inserted into the inner canthus at an angle of about 30° downwards, with the needle tip pointing downwards along the edge of the eyeball until the needle tip was at the bottom of the eye. Then the injection was injected slowly and steadily. After the injection was completed, the needle was slowly withdrawn. The injection was completed one mouse at a time.

[0049] (4) Mouse treadmill experiment Eight weeks after injection, mice in the experimental and control groups underwent treadmill exercise capacity testing. Mice were placed on a treadmill track with an initial speed of 5 m / min, which was increased uniformly by 2 m / min after 2 minutes, until exhaustion (continuous contact with the electrical stimulation barrier for more than 10 seconds) at 11 m / min. The maximum running distance and time to exhaustion for each group were recorded. The results are as follows: Figure 8As shown in E, the time to exhaustion in the experimental group was longer than that in the control group, indicating that the functional read-through translation system effectively improved the exercise endurance of the mice.

[0050] (5) Collection and processing of mouse tissue samples Eight weeks after injection, mice in both the experimental and control groups were euthanized, and their hearts, diaphragms, tibialis anterior muscles, gastrocnemius muscles, and quadriceps femoris muscles were collected. A portion of the muscle tissue was fixed in 4% paraformaldehyde at 4°C for at least 24 hours, embedded in paraffin blocks for sectioning and subsequent HE staining; the other portion was added to RIPA lysis buffer and homogenized in a tissue homogenizer, followed by centrifugation at 8000 rf for 10 min at 4°C, and the supernatant protein liquid was collected.

[0051] (6) Protein immunoblotting analysis Total protein was extracted from heart and diaphragm tissues. A portion of the total protein solution was added to protein loading buffer and analyzed by SDS-PAGE. After membrane transfer and blocking with 5% skim milk for 1 hour, the membrane was washed with TBST and incubated overnight at 4°C with anti-Dystrophin antibody (Sigma, D8168) and anti-vinculin antibody (Proteintech, 66305-1-Ig). After washing with TSBT and incubation with secondary antibody for 1 hour, color development was performed. Western blot results are shown below. Figure 8 A, Figure 8 B, Figure 8 C and Figure 8 As shown in D, the experimental group recovered full-length expression of Dystrophin protein. Compared with the control group, which had no expression, the experimental group recovered full-length Dystrophin protein expression, which increased to about 20% of that in wild-type mice.

[0052] The results above show that the experimental group had significant improvements in protein and behavioral levels compared with the control group, indicating that the AAV delivery system for functional read-through translation into DMD mice can effectively restore the expression of functional proteins and improve their motor ability.

[0053] Example 7: Evaluation of readthrough efficiency of orthogonal systems in IDUA-TAG mutant mice (1) Constructing pAAV-cis-transgene plasmid with a functional read-through translation system The tRNA-binding protein was expressed using the CB6 promoter, and the orthogonal tRNA was expressed using the U6 promoter. The two sets of elements were cloned into the same vector and constructed into the adeno-associated virus pAAV-cis-transgene plasmid vector using the preferred read-through translation system TD103 / tRNA-47-CUA, thereby obtaining the adeno-associated virus transgenic plasmid.

[0054] (2) Packaging of adeno-associated virus The steps are as described in Example 5 (2), and the final AAV concentration obtained with the read-through translation system is 5.7*10. 13 μg / mL.

[0055] (3) Delivery of a functional read-through translation system to IDUA-TAG disease mice via AAV delivery The quantified AAV was diluted in 50 μL of PBS solution and injected into the experimental group mice (4 weeks old, 4 mice) at a dose of 1.5 E12 vg per mouse. The control group mice (4 weeks old, 4 mice) were injected with an equal volume of AAV-free PBS solution. The method was the same as in Example 5.

[0056] (4) Detection of serum IDUA enzyme activity and urine GAG ​​content Four weeks after delivery of the read-through system, orbital blood samples were collected from mice in both the control group (IDUA-PBS) and the treatment group (IDUA-AAV). Serum was then separated by centrifugation. The catalytic activity of IDUA enzyme in the serum was measured using a fluorescent substrate method. Results are as follows: Figure 9 As shown in Figure A, the serum IDUA enzyme activity in the treatment group was significantly higher than that in the control group, recovering to approximately 15%–25% of the activity in wild-type mice, suggesting that the AAV delivery readout system effectively functions in vivo. Simultaneously, metabolic excretion was assessed in both groups of mice. Urine samples were collected after 4 hours, and the urinary glycosaminoglycan (GAG) content was measured using a Blyscan-sulfated Glycos aminoglycan (sGAG) assay kit (Biocolor, B3000). The results are as follows: Figure 9 As shown in Figure B, the urinary GAG levels in the treatment group decreased significantly, by approximately 40% to 50% compared to the control group, indicating that the urinary system can effectively reduce GAG ​​accumulation in the body and alleviate MPS I-related metabolic abnormalities.

[0057] (5) Skeletal improvement assessment (micro-CT) Eighteen weeks after delivery, mice in both the control and treatment groups underwent skull micro-CT scans to reconstruct three-dimensional models and measure the width of the zygomatic arch. Results are as follows: Figure 9 C Figure 9 As shown in Figure D, the control group mice exhibited typical MPS I skeletal abnormalities, such as widening of the zygomatic arch; the treatment group mice showed significant improvement in skull structure, with a significant reduction in the width of the zygomatic arch and an overall morphology close to normal, indicating that the treatment has a significant therapeutic effect at the skeletal level.

[0058] (6) Analysis of tissue IDUA enzyme activity and GAG content After 18 weeks of treatment, mice in both groups were euthanized, and tissues from major organs (including liver, heart, spleen, and kidneys) were collected. The tissues were homogenized, and IDUA enzyme activity was measured using a fluorescent substrate assay. Results Figure 9 E, Figure 9 As shown in Figure F, the activity of IDUA enzyme in the liver and heart of mice in the treatment group was significantly higher than that in the control group, with recovery levels reaching 10%–30% of those in wild-type mice, with the most significant recovery observed in cardiac enzyme activity. Furthermore, the results of tissue GAG ​​content determination are shown in Figure 9. Figure 9 As shown in Figure H, the liver and heart GAG levels in the treatment group decreased significantly, by approximately 35% to 55% compared to the control group, indicating that the storage of polysaccharides in the body was effectively alleviated, demonstrating a significant metabolic recovery effect.

[0059] (7) Lysosomal stress assessment (LAMP1 expression) To assess the improvement in lysosomal function after treatment, LAMP1 protein levels in liver and heart tissues were detected using Western blot with anti-LAMP1 (Ratanti-LAMP1, BD Pharmingen, RUO-553792). WB results and quantitative analysis results are as follows: Figure 9 I~ Figure 9 As shown in Figure L, LAMP1 protein was significantly upregulated in the liver and heart tissues of control mice, indicating an abnormally high lysosomal load. LAMP1 expression was significantly decreased in the treatment group, approaching wild-type levels, suggesting that the positive inductively coupled transmissive system (AAV) delivery can alleviate lysosomal stress and improve the typical lysosomal swelling and accumulation phenotype of MPS I mice.

[0060] Example 8: Evaluation of reading efficiency of functional fluent translation system in Col4a5-TAG mutant mice (1) Constructing pAAV-cis-transgene plasmid with a functional read-through translation system The steps are as described in Example 7 (1).

[0061] (2) Packaging of adeno-associated virus The steps are as described in Example 7 (2).

[0062] (3) Delivery of a functional read-through translation system to Col4a5-TAG disease mice via AAV delivery The quantified AAV was diluted in 50 μL of PBS solution and injected into the experimental group mice (4 weeks old, 7 mice) at a dose of 1.5 E12 vg per mouse. The control group mice (4 weeks old, 5 mice) were injected with an equal volume of AAV-free PBS solution. The method was the same as in Example 4.

[0063] (4) Measurement of albumin and creatinine in mouse urine After the start of treatment, 4-hour urine samples were collected from mice in both the experimental and control groups every 3 weeks. Mice were placed individually in a homemade urine collector, and urine samples were collected over 4 hours. After recording the total volume, 0.5 mL of urine was centrifuged at 3000×g for 10 minutes at 4°C to remove insoluble precipitates. The supernatant was collected and stored at -80°C for later analysis. The concentration of albumin in urine was determined using a urinary microalbumin assay kit (immunoturbidimetric method) (Nanjing Jiancheng, E038-1-1), and the urinary creatinine level was determined using a urinary creatinine assay kit (sarcosine oxidase method) (Nanjing Jiancheng, C011-2-1). The urinary albumin / creatinine ratio (UACR) was calculated based on the measured albumin and creatinine values. Results are as follows: Figure 10 As shown in Figure A, the albumin-to-UACR ratio in the experimental group mice increased slowly with the progression of treatment, and was significantly lower than that in the control group by week 12, indicating that albuminuria symptoms were significantly relieved, suggesting that the functional read-through translation system has a sustained protective effect on improving glomerular filtration function. To maintain data comparability, all batch tests were performed under the same conditions and included in the quality control of standards.

[0064] (5) Collection and processing of mouse serum and tissue samples Twenty-three weeks after injection, mice in both the experimental and control groups were euthanized, and serum samples and kidneys were collected. Serum samples were analyzed using a biochemical analyzer to determine the levels of serum creatinine and blood urea nitrogen. The experimental results are as follows: Figure 10 B, Figure 10 As shown in Figure C, the treatment group and the PBS group showed significant differences, more closely resembling the level in wild-type mice, suggesting that the read-through translation system delayed disease progression. For kidney samples, they were fixed in 4% paraformaldehyde at 4°C for at least 24 hours, embedded in paraffin blocks for sectioning and subsequent Masson's trichrome (MT) staining.

[0065] (6) Analysis of Masson's Trichrome (MT) staining of kidney tissue Kidney samples fixed with 4% paraformaldehyde were sent to the company for paraffin embedding, tissue sectioning, and MT staining. The experimental results are shown in the figure below. Figure 10 D and Figure 10 As shown in E, the MT staining results of the sections show that, compared with the control group, the experimental group had a lower degree of glomerular fibrosis and a basically normal basement membrane morphology. This indicates that the read-through translation system can restore the full-length expression of TAG-PTC mutation-related disease functional proteins in mammalian somatic cells through AAV delivery, thereby alleviating disease symptoms.

[0066] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since it corresponds to the method disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to in the method section.

[0067] The functional read-through translation system provided by this invention and its application in restoring functional protein expression in PTC disease via codon redistribution have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of this invention. The descriptions of the embodiments above are merely for the purpose of helping to understand the method and core ideas of this invention. It should be noted that those skilled in the art can make various improvements and modifications to this invention without departing from its principles, and these improvements and modifications also fall within the protection scope of the claims of this invention.

Claims

1. A tRNA-binding protein-assisted stop codon reading system, characterized in that, The system includes: Repressive tRNA: Contains the G3:U70 pair, which can be recognized by endogenous alanyl-tRNA synthetase and loaded with alanine. Its anticodon is mutated in a directed manner to recognize at least one of the premature stop codons UAG, UGA or UAA. tRNA-binding proteins: capable of specifically binding to the repressive tRNA domains other than the receptor arm.

2. The reading system as described in claim 1, characterized in that, The anticodon sequence of the repressive tRNA is selected from at least one of CUA, UCA, or UUA, and corresponds to the stop codons UAG, UGA, or UAA, respectively.

3. The reading system as described in claim 1, characterized in that, The repressive tRNA is modified with D stem / loop, T stem / loop or anticodon stem, and its corresponding nucleotide sequence is shown in SEQ ID NO.1-107.

4. The reading system as described in claim 1, characterized in that, The tRNA-binding protein specifically binds to repressive tRNAs modified with G3:U70. These repressive tRNAs, after being loaded with alanine via host endogenous AlaRS, are able to recognize premature stop codons.

5. The reading system as described in claim 1, characterized in that, The tRNA-binding protein can specifically bind to the tRNA-binding domain of pyrrolidone-lysyl-tRNA synthetase.

6. The reading system as described in claim 5, characterized in that, The binding domain of the pyrrolidone-lysyl-tRNA synthetase, in its full length or variant form, is derived from MbPylRS, MmPylRS, or their homologous sequences, with the corresponding nucleotide sequences shown in SEQ ID NO. 108-122.

7. The reading system as described in claim 1, characterized in that, The system is delivered into mammalian cells via a plasmid vector.

8. The use of the read-through system as described in any one of claims 1-7 in the preparation of a medicament for treating diseases caused by premature stop codons resulting from gene stop codon mutations.

9. The application as described in claim 8, characterized in that, The diseases associated with premature stop codons resulting from gene stop codon mutations are selected from one or more of the following: Duchenne muscular dystrophy, mucopolysaccharidosis type I, Allport syndrome, or familial adenomatous polyposis.

10. The application of the readthrough system as described in any one of claims 1-7 in the visualization evaluation and regulation of the readthrough efficiency of the inhibitory tRNA-tRNA binding protein system in vivo and in vitro.

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