Base editor system and application

Abstract

The invention belongs to the technical field of gene editing, and discloses a base editor system and application. According to the invention, a heterologous peptide sequence is inserted into IscB protein, the obtained peptide embedded IscB is fused with deaminase or glycosylase to construct a base editor, and DNA base editing is carried out. According to the base editor system, the size limitation is overcome, and single AAV delivery is achieved; the editing efficiency is obviously improved; the broad-spectrum applicability is realized; the system optimization is more flexible; the method has efficient editing potential in vivo, and the off-target effect is reduced. The invention provides an important new strategy for hyperlipemia treatment and provides a basis for gene therapy of other hereditary diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of gene editing, and particularly relates to a base editor system and its application. Background Art

[0002] Gene editing technology has broad application prospects in the field of biomedicine, especially in the treatment of genetic diseases. Currently, the CRISPR-Cas9 system is one of the most commonly used gene editing tools. The base editor (BE) formed by its combination with deaminase can achieve single-base precise editing without introducing DNA double-strand breaks, significantly reducing the off-target effect. Common base editors include cytosine base editors (CBE, which can achieve C→T conversion), adenine base editors (ABE, which can achieve A→G conversion), and glycosylase-mediated base editors developed in recent years (such as DAF-CBE for C→G conversion and DAF-TBE for T→G conversion).

[0003] However, the Cas9 protein is relatively large in size (about 1368 amino acids, with a molecular weight of about 160 kDa), resulting in the entire editing system after fusion with deaminase or glycosylase exceeding the packaging limit of the adeno-associated virus (AAV) vector (about 4.7 kb). It cannot be delivered by a single AAV and must be packaged separately in two AAVs, which severely limits its application efficiency in in vivo gene therapy. In addition, injecting multiple AAVs at one time increases the treatment cost, while also increasing the immunogenicity and off-target risk, and the delivery efficiency of the dual-AAV system is significantly lower than that of single-AAV delivery.

[0004] In recent years, researchers have discovered a protein IscB (496 amino acids) that is evolutionarily related to Cas9 but smaller in size. It can recognize and cleave target DNA under the guidance of non-coding RNA (ωRNA). In theory, the small size of IscB makes it an ideal candidate to overcome the size limitation of gene therapy delivery systems. However, the naturally occurring IscB protein has relatively low activity and poor cleavage efficiency in eukaryotic cells, limiting its practical application in gene editing. There is no report in the prior art on an efficient base editing system based on IscB. Conventional protein engineering methods (such as point mutations, truncations, etc.) have limited effects in improving the activity of IscB and often affect the stability and specificity of the protein. Moreover, existing editing efficiency optimization methods often only apply to specific types of base editing systems, lacking a general strategy that can broadly enhance the editing efficiency of different types of base editors. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide a base editor system and application. As a miniaturized, highly efficient, and single-AAV deliverable base editing platform, it provides a new technical path for clinical gene therapy.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] In the first aspect, the present invention provides a method for enhancing the efficiency of IscB-mediated DNA base editors, including the following steps:

[0008] (1) Insert a heterologous peptide sequence into the IscB protein to obtain peptide-embedded IscB;

[0009] (2) Fuse the peptide-embedded IscB with a deaminase or glycosylase to construct a base editor;

[0010] (3) Perform DNA base editing through the base editor.

[0011] As a preferred embodiment of the method of the present invention, the heterologous peptide includes gp41 peptide and / or GCN4 peptide; flexible linker peptides are added to both ends of the heterologous peptide sequence.

[0012] As a preferred embodiment of the method of the present invention, the insertion site includes at least one of the positions after the amino acids E48, S49, A50, E51, and E52 selected from the IscB protein.

[0013] As a preferred embodiment of the method of the present invention, the amino acid sequence of the IscB protein is as shown in SEQ ID NO:3.

[0014] As a preferred embodiment of the method of the present invention, the nucleotide sequence of the ωRNA used for DNA base editing is as shown in SEQ ID NO:5.

[0015] In the second aspect, the present invention provides a base editor system, in which a heterologous peptide is inserted into its nuclease, and the heterologous peptide includes gp41 peptide and / or GCN4 peptide; flexible linker peptides are added to both ends of the heterologous peptide sequence.

[0016] In the third aspect, the present invention provides an IscB base editor system, including an IscB protein and ωRNA; the amino acid sequence of the IscB protein is as shown in SEQ ID NO:3; the nucleotide sequence of the ωRNA used for DNA base editing is as shown in SEQ ID NO:5.

[0017] As a preferred embodiment of the IscB base editor system of the present invention, a heterologous peptide is inserted into the IscB protein; the heterologous peptide includes gp41 peptide and / or GCN4 peptide; flexible linker peptides are added to both ends of the heterologous peptide sequence.

[0018] As a further preferred embodiment of the IscB base editor system of the present invention, the insertion sites include at least one of the positions after the amino acids E48, S49, A50, E51, and E52 selected from the IscB protein.

[0019] As a preferred embodiment of the IscB base editor system of the present invention, the base editing system is any one of CBE, ABE, DAF-CBE, and DAF-TBE.

[0020] Fourthly, the present invention provides a tissue-specific gene editor system, in which a tissue-specific targeting peptide is embedded in the nuclease of the base editor system.

[0021] Fifthly, the present invention applies the IscB base editor system and the tissue-specific gene editor system to any one of the following fields:

[0022] i. Gene editing;

[0023] ii. Preparation of a preparation for reducing the immunogenicity of gene therapy;

[0024] iii. Preparation of a drug for treating and / or preventing hyperlipidemia;

[0025] v. Preparation of a gene therapy drug for treating and / or preventing genetic diseases.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] (1) Overcoming size limitations and achieving single AAV delivery: The base editing system developed by the present invention based on the small-sized IscB protein (about 700-800 amino acids) has a much smaller overall volume than the existing system based on Cas9 (about 1368 amino acids), enabling the entire editing system to be delivered by a single AAV vector, significantly improving the in vivo delivery efficiency, reducing the treatment cost and immunogenicity risk, and overcoming the limitation of the currently too large CRISPR-Cas9 system.

[0028] (2) Significantly improving the editing efficiency: Through an innovative peptide embedding strategy, the present invention significantly improves the editing efficiency of the IscB protein, transforming it from a state of insufficient activity into an efficient gene editing tool, showing an obvious synergistic effect in various base editing systems.

[0029] (3) Broad-spectrum applicability: The peptide insertion strategy of the present invention is applicable to various types of base editing systems (such as CBE, ABE, DAF-CBE, DAF-TBE, etc.), not limited to specific types of base conversions, providing a unified synergistic solution for various gene editing applications.

[0030] (4) Flexibility in system optimization: By screening different peptide sequences, insertion sites, and designs, the present invention provides multiple possibilities for system optimization and can be customized according to specific application requirements.

[0031] (5) High in vivo editing potential: The peptide-inserted IscB base editing system developed in the present invention exhibits significant editing efficiency, providing a good foundation for achieving effective in vivo gene editing and a new technical means for gene therapy.

[0032] (6) Reducing off-target effects: The present invention utilizes the precise base editing principle to avoid potential genomic instability caused by DNA double-strand breaks. At the same time, by improving the editing efficiency, it enables the use of lower doses of editing components to achieve the desired therapeutic effect, further reducing the potential off-target risk. Brief Description of the Drawings

[0033] Figure 1 Results of enhancing the base editing efficiency of peptide-inserted IscB. By inserting the gp41 peptide into IscB, the editing efficiency was significantly improved; Figure 1Among them, (a) Schematic diagram of the enIscB(D61A) base editing system. After the IscB protein undergoes the D61A mutation, it is transformed into a nickase and fused with a deaminase to form a complete editing system. This system is guided by ωRNA* to target specific DNA sequences and achieve single-base editing near the nick site. (b) Schematic diagram of the domain organization and peptide chimeric sites of the IscB protein. The numbers indicate different peptide insertion sites, and the region between 48-52 is located between the PLMP and RUVC domains and is the most effective peptide chimeric region. (c) Comparison of the editing efficiencies of the ABE system at the VEGFA-AS3 and HBG-sg2 targets. The heatmap shows the editing efficiencies of wild-type IscB-ABE and gp41 peptide chimeric variants at different sites, and the color depth represents the A→G conversion efficiency. The 48gp41 and 50gp41 variants show the highest editing efficiency. (d) Comparison of the editing efficiencies of the CBE system at the PCSK9-9 and PCSK9-C55 targets. The left and right heatmaps respectively show the C→T conversion efficiencies of different variants at the two targets, and the 48-gp41 and 50-gp41 variants perform the best. (e) Schematic diagram of the enIscB(D61A) base editing system. After the IscB protein undergoes the D61A mutation, it is transformed into a nickase and fused with a glycosylase to form a complete editing system. Performance of the TDG3-IscB system (T→G conversion) at the EMX-AS1 target. The data indicate that gp41 peptide chimerization can significantly improve the editing efficiency in multiple editing systems and targets. The figure shows the comparison of the efficiencies of CBE-gp41-enIscB(D61A) and CBE-enIscB(D61A) in cytosine base editing (C→T conversion), and the comparison of the efficiencies of enIscB-gp41-ABE and enIscB-ABE in adenine base editing (A→G conversion), showing that gp41 peptide insertion significantly improves the editing efficiency of various IscB base editing systems. Specifically, the editing efficiency of ABE-gp41-enIscB(D61A) is on average 2.417 times higher than that of ABE-enIscB(D61A); the VEGFA-AS3 site shows the most obvious efficiency improvement, increasing from 38% to 72%; although the basic editing efficiency of the CBE-gp41-enIscB(D61A) at the PCSK9-CS5 site is relatively high (72%), the editing at this site is still improved after peptide insertion, reaching 79%, increasing from 72% to 79%; the editing efficiency of DAF-TBE-gp41-enIscB(D61A) is on average 1.3 times higher than that of DAF-TBE-enIscB(D61A).

[0034] Figure 2Results of peptide insertion into IscB to enhance base editing efficiency; by inserting the GCN4 peptide into IscB, the editing efficiency was significantly improved; Figure 2 Among them, (a) Schematic diagram of the structure of the GCN4 peptide-chimeric ABE-enIscB(D61A) system, showing the arrangement of key components and functional domains. (b) Heat map of the editing efficiency of GCN4 peptide-chimeric variants at three different targets (VEGFA-AS2, VEGFA-AS3, and EMX1-AS1). The results showed that variants such as 49-GCN4, 50-GCN4, and 52-GCN4 performed excellently at multiple targets, confirming the broad applicability of the GCN4 peptide-chimeric strategy. It was shown that by inserting the GCN4 peptide into IscB, the editing efficiency was significantly improved. Among all sites, the editing efficiency of ABE-GCN4-enIscB(D61A) was 1.6 times higher than that of ABE-enIscB(D61A). The comparison of the editing efficiency between ABE-GCN4-enIscB(D61A) and ABE-enIscB(D61A) was shown in the figure, indicating that the insertion of the GCN4 peptide could also significantly improve the editing efficiency of the IscB base editing system, confirming the generality of the peptide insertion strategy. Detailed implementation manners

[0035] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0036] Unless otherwise specified, the test methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0037] Example 1: Method for enhancing the editing efficiency of the IscB-CBE system by peptide insertion strategy

[0038] (1) Optimization of IscB protein design

[0039] First, the variant enIscB (amino acid sequence shown in SEQ ID NO: 2) obtained by evolutionary modification of the wild-type IscB protein (amino acid sequence shown in SEQ ID NO: 1) was used. On this basis, a D61A mutation was introduced at the 61st amino acid to create an IscB variant enIscB(D61A) (amino acid sequence shown in SEQ ID NO: 3) with nickase activity, which could generate a nick on the single strand of DNA rather than a double-strand break, which was crucial for the subsequent construction of base editors. At the same time, the truncated variant ωRNA* (base sequence shown in SEQ ID NO: 5) of the non-coding guide RNA (ωRNA, base sequence shown in SEQ ID NO: 4) was used.

[0040] (2) Construction of CBE-enIscB(D61A) fusion protein

[0041] The optimized enIscB-D61A was fused with the human deaminase hAPOBEC3A to construct a cytosine base editor (CBE-enIscB(D61A)). The fusion strategy was as follows: the N-terminus was the hAPOBEC3A deaminase with optimized catalytic domain (the amino acid sequence is shown in SEQ ID NO:6), the middle was a flexible linker peptide (SGSETPGTSESATPES), and the C-terminus was the optimized enIscB(D61A) protein sequence.

[0042] Since the IscB protein (496 amino acids) is significantly smaller than Cas9 (about 1368 amino acids), the coding sequence length of the entire CBE-enIscB(D61A) fusion protein is much smaller than the AAV packaging limit (4.7 kb), providing the possibility for single AAV delivery.

[0043] (3) Design of peptide insertion strategy

[0044] To further improve the editing efficiency of CBE-enIscB(D61A), the present invention innovatively proposed a "peptide insertion" strategy. A method for inserting short peptide sequences into specific regions of the CBE-enIscB(D61A) fusion protein was designed, including:

[0045] i. Selection of candidate peptides: gp41 (HIV envelope protein-derived peptide, KNEQELLELDKWASL) was selected as the candidate insertion peptide.

[0046] ii. Determination of insertion sites: Through structural analysis, suitable sites for insertion in the enIscB(D61A) protein were determined.

[0047] iii. Insertion method: The gp41 peptide sequence was inserted at the selected sites.

[0048] The finally obtained inserted CBE-enIscB(D61A) was named CBE-gp41-enIscB(D61A) (the amino acid sequence is shown in SEQ ID NO:7).

[0049] (4) Evaluation of the editing efficiency of the CBE-enIscB(D61A) system

[0050] To evaluate the effect of the peptide insertion strategy on the editing efficiency, the following experiments were carried out:

[0051] 1) Plasmid construction

[0052] The plasmid DNA sequences and PCR primer sequences used are shown in Table 1.

[0053] Table 1 Sequence Information

[0054]

[0055] Human codon-optimized enIscB(D61A) and ωRNA* were synthesized by Genewiz Suzhou Co., Ltd. and cloned into BE3 (Addgene #203811) and pxz-U6-CMV-GFP (self-constructed, nucleotide sequence shown in SEQ ID NO: 8) plasmids, respectively. For human genome editing, enIscB(D61A) and sgRNA were cloned into different plasmids. CMV-driven enIscB(D61A) was constructed at the C-terminus of the hAPOBEC3A protein in the BE3 plasmid, while the sgRNA driven by the U6 promoter was cloned into the pxz-U6-CMV-GFP plasmid. On this basis, for the construction of relevant plasmids, enIscB(D61A) in the base editing tool was inserted with GCN4 or gp41, and 2×Phanta Max Master Mix (Dye Plus) (Vazyme) was used for PCR and 2X MultiF Seamless Assembly Mix (RK21020, ABclonal) was used for fragment assembly. Briefly, a pair of oligonucleotides was slowly cooled from 95 °C to room temperature and annealed, and then ligated into linearized BE3 or pxz-U6-CMV-GFP. The gRNA target oligonucleotides were synthesized by Wuhan Kingcare Biotechnology Co., Ltd.

[0056] 2) Mammalian cell culture and transfection

[0057] The human embryonic kidney cell line HEK293T (ATCC CRL-3216) was cultured in DMEM (Gibco, 11965-118) supplemented with 10% fetal bovine serum (Gibco, 16000-044) and 1% penicillin-streptomycin (100×, Gibco, 15140-163) at 37 °C in a 5% CO2 atmosphere. Cells were seeded in 24-well plates and transiently transfected at a confluence of approximately 70%. Plasmids were extracted using the TIANprep Mini Plasmid kit (TIANGEN, DP103-03). Transient transfection was performed using Hieff Liposomal transfection reagent (Yeasen, no.40802ES08) according to the manufacturer's protocol. For BE experiments, cells were co-transfected with 1 μg of plasmid expressing CBE-enIscB(D61A) or CBE-gp41-enIscB(D61A) and 1.0 μg of plasmid expressing sgRNA. The medium was changed 12 hours after transfection.

[0058] 3) FACS Analysis and Genomic DNA Extraction from Mammalian Cells

[0059] Approximately 60 - 72 hours after transfection, GFP-positive cells were sorted using a BD FACS Aria III flow cytometer. Approximately 30,000 sorted cells were lysed in 20 μL of QuickExtract DNA Extraction Solution 1.0 (Lucigen, QE0905T), and the operation was performed according to the manufacturer's manual.

[0060] 4) Analysis of Editing Efficiency of Sanger Sequencing Data Using EditR

[0061] The PCR products were sequenced by Sanger sequencing technology (Hecegene, Wuhan). Subsequently, the online EditR tool (https: / / moriaritylab.shinyapps.io / editr_v10 / ) was used to quantify the Sanger sequencing data to obtain the base editing efficiency. The editing efficiencies of all Sanger sequencing data are listed in Table 2 - 9 (in the following tables, "A", "T", "C" are bases, and the numbers are the positions of the bases corresponding to the sgRNA). The editing efficiency was further integrated into the editing window. All experiments were performed by transfecting plasmids in HEK293T cells, ensuring the consistency and comparability of the results.

[0062] Table 2 Sanger Sequencing Data

[0063]

[0064]

[0065] Table 3 Sanger Sequencing Data

[0066] PCSK9-CS5 C3 C4 C8 C10 C12 C14 CBE-enIscB(D61A) 6 59 6 72 3 2 48-GP41 4 62 6 79 4 3 49-GP41 3 53 6 69 3 2 50-GP41 3 62 7 76 4 2 51-GP41 3 35 6 39 3 2 52-GP41 5 46 5 61 2 2 124-GP41 6 41 5 61 3 3 126-GP41 2 6 3 6 0 1 128-GP41 4 3 4 3 2 2 145-GP41 7 9 5 2 8 2 150-GP41 1 10 4 11 2 2 198-GP41 4 12 8 13 4 3 201-GP41 0 14 4 11 2 2 298-GP41 5 0 6 4 4 3 350-GP41 3 5 4 5 2 1 354-GP41 5 21 3 30 5 3 355-GP41 5 17 2 19 6 2 359-GP41 1 0 3 0 0 0 360-GP41 8 26 2 39 2 0 382-GP41 5 3 3 11 2 1 404-GP41 5 0 3 0 3 3 436-GP41 4 28 4 38 2 0 438-GP41 3 2 4 2 0 0 444-GP41 4 10 4 11 3 3

[0067] Table 4 Sanger Sequencing Data

[0068]

[0069]

[0070] Table 5 Sanger Sequencing Data

[0071] HBG-sg2 A4 A8 A10 A12 A14 A15 ABE8e-enIscB(D61A) 5 16 4 6 2 1 48gp41-ABE8e-enIscB(D61A) 12 25 10 13 0 4 50gp41-ABE8e-enIscB(D61A) 11 23 8 12 3 3 51gp41-ABE8e-enIscB(D61A) 4 6 3 1 3 3 52gp41-ABE8e-enIscB(D61A) 4 4 4 3 1 2 128gp41-ABE8e-enIscB(D61A) 5 6 5 4 3 3 350gp41-ABE8e-enIscB(D61A) 0 4 2 4 3 3 359gp41-ABE8e-enIscB(D61A) 1 3 2 3 2 3 444gp41-ABE8e-enIscB(D61A) 3 4 1 3 0 5

[0072] Table 6 Sanger Sequencing Data

[0073] EMX1-AS1 T3 T6 Untreated 2 4 TDG3-enIscB(D61A) 3 10 48gp41-TDG3-enIscB(D61A) 3 8 51gp41-TDG3-enIscB(D61A) 3 11 124gp41-TDG3-enIscB(D61A) 4 8 128gp41-TDG3-enIscB(D61A) 4 10 201gp41-TDG3-enIscB(D61A) 3 7 402gp41-TDG3-enIscB(D61A) 1 4 404gp41-TDG3-enIscB(D61A) 0 2 436gp41-TDG3-enIscB(D61A) 2 4 444gp41-TDG3-enIscB(D61A) 2 4

[0074] Table 7 Sanger Sequencing Data

[0075]

[0076]

[0077] Table 8 Sanger sequencing data

[0078]

[0079]

[0080] Table 9 Sanger sequencing data

[0081]

[0082]

[0083] Example 2: Method for enhancing the editing efficiency of the ABE-enIscB(D61A) system by peptide insertion strategy

[0084] (1) Construction of ABE-enIscB(D61A) fusion protein

[0085] To verify the universality of the peptide insertion strategy for different types of base editors, an adenine base editor (ABE-enIscB(D61A)) was constructed on the PB-ABE8e-P2A-mcherry vector (self-constructed, nucleotide sequence shown in SEQ ID NO:9). The specific construction method was as follows: the N-terminus was the TadA8e adenine deaminase (realizing A→G conversion); the middle was a flexible linker peptide (SGGSSGGSSGSETPGTSESATPESSGGSSGGS); the C-terminus was the optimized enIscB-D61A protein sequence (amino acid sequence shown in SEQ ID NO:3).

[0086] (2) Design of the peptide insertion strategy

[0087] Using the same principle and sites as in Example 1, the gp41 peptide was inserted into ABE-enIscB(D61A) to construct ABE-gp41-enIscB(D61A) (amino acid sequence shown in SEQ ID NO:10). This included inserting the gp41 peptide sequence at an appropriate site in the enIscB(D61A) protein while keeping the other parts of the structure unchanged.

[0088] (3) Evaluation of the editing efficiency of the ABE-enIscB(D61A) system

[0089] To evaluate the effect of the peptide insertion strategy on the editing efficiency, the following experiment was conducted:

[0090] Using the same cell culture, transfection, and analysis methods as in Example 1, the editing efficiency of the A→G editing target site of the target gene was tested by plasmid transfection in HEK293T cells. All experiments were carried out by transfecting plasmids in HEK293T cells, ensuring the comparability of the results with those of other examples.

[0091] The results showed that the editing efficiency of ABE-gp41-enIscB(D61A) was significantly higher than that of the original ABE-enIscB(D61A). The ABE-enIscB(D61A) system embedded with the gp41 peptide showed a significant improvement in editing efficiency, confirming the synergistic effect of the gp41 peptide embedding strategy on the ABE system.

[0092] Example 3: Method for enhancing more types of IscB base editing systems by peptide embedding strategy

[0093] (1) Construction of base editing systems

[0094] To further verify the universality of the peptide embedding strategy, various types of IscB base editing systems were constructed, including:

[0095] 1) DAF-TBE-enIscB(D61A) system:

[0096] The N-terminus is TDG thymine glycosylase (to achieve T→G conversion); the middle is a flexible linker peptide (SGGSSGGSSGSETPGTSESATPESSGGSSGGS); the C-terminus is the optimized IscB-D61A protein sequence. The amino acid sequence of DAF-TBE-enIscB(D61A) is shown in SEQ ID NO:11, and this system can achieve precise editing of thymine to guanine.

[0097] 2) Implementation of the gp41 peptide embedding strategy

[0098] Using the same principle and sites as in Example 1, the gp41 peptide was embedded in different enIscB(D61A) base editing systems.

[0099] 3) Evaluation of editing efficiency

[0100] Using the same experimental methods as in Examples 1 and 2, the efficiency of various editing systems was evaluated by plasmid transfection in HEK293T cells. As Figure 1 shown, after embedding the gp41 peptide in different types of IscB base editing systems, a significant improvement in editing efficiency was shown. All experiments were carried out in the same cell line and conditions, ensuring the consistency and reliability of the results.

[0101] These results confirm that the gp41 peptide embedding strategy can generally enhance the editing efficiency of various IscB base editing systems in HEK293T cells, indicating that this strategy has good generality.

[0102] Example 4: Evaluation of alternative peptide sequence (GCN4)

[0103] (1) Selection and embedding of alternative peptide sequence

[0104] To verify the flexibility of the peptide embedding strategy, another short peptide GCN4 (derived from yeast transcription factor, amino acid sequence: EELLSKNYHLENEVARLKK) was selected for testing:

[0105] The GCN4 peptide sequence was inserted at the appropriate site of enIscB (D61A) protein; for other details, refer to Example 2 to construct editing systems such as ABE-GCN4-enIscB (D61A).

[0106] (2) Comparison of editing efficiency

[0107] In HEK293T cells, the efficiency of the IscB base editing system embedded with GCN4 peptide was evaluated by transfecting plasmids. The same experimental conditions and analysis methods as in Example 1 were maintained to ensure the comparability of the results.

[0108] As Figure 2 shown, the IscB base editing system embedded with GCN4 peptide also showed a significant improvement in editing efficiency. These results obtained in HEK293T cells indicate that the enhancement effect of the peptide embedding strategy on editing efficiency does not depend on a specific peptide sequence and has broad applicability.

[0109] Example 5: Theoretical mechanism and feasibility analysis of the peptide embedding strategy to enhance IscB-mediated DNA base editor in PCSK9 targeted therapy

[0110] (1) PCSK9 as a therapeutic target for hyperlipidemia

[0111] Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a protease mainly secreted by the liver, and its core function is to regulate the metabolism of low-density lipoprotein receptor (LDLR). After PCSK9 binds to the extracellular domain of LDLR, it can prevent LDLR from cycling back to the cell surface and promote its degradation in the endolysosome, thereby reducing the number of LDLRs on the cell surface and leading to an increase in the level of low-density lipoprotein cholesterol (LDL-C) in the blood.

[0112] Epidemiological studies have shown that loss-of-function mutations of PCSK9 that occur naturally in the population are associated with significantly reduced LDL-C levels and cardiovascular disease risk. In particular, individuals carrying non-functional mutations of PCSK9 have a lifelong reduction in LDL-C levels of approximately 30%-40% and a reduction in the risk of coronary heart disease of approximately 80%-90%, with no obvious adverse health consequences. These natural experiments provide a strong theoretical basis for PCSK9 inhibition as a strategy for lipid-lowering and prevention and treatment of atherosclerosis.

[0113] Clinical studies of currently approved PCSK9 inhibitors (monoclonal antibodies such as evolocumab and alirocumab) have confirmed that blocking the interaction between PCSK9 and LDLR can reduce LDL-C levels by approximately 50%-60% and simultaneously significantly reduce the risk of cardiovascular events. These data further support that PCSK9 is an ideal therapeutic target for cardiovascular diseases.

[0114] However, existing PCSK9 inhibitors face some limitations: they require frequent (once every 2-4 weeks) subcutaneous injection, have high production costs, and some patients may produce drug-resistant antibodies. In addition, these drugs can only transiently block the action of circulating PCSK9, and the effect disappears rapidly once the drug is stopped. Therefore, developing gene editing strategies that can persistently inhibit PCSK9 production has obvious advantages.

[0115] (2) Peptide-embedded CBE-enIscB(D61A) system targets PCSK9

[0116] 1) Molecular basis for downregulating PCSK9 expression by introducing a stop codon

[0117] The peptide-embedded CBE-enIscB(D61A) system was used to modify the CAG codon (glutamine, Q) in the first exon of the PCSK9 gene to the TAG stop codon through a C→T conversion. Theoretically, this would lead to premature termination of protein translation, producing truncated non-functional PCSK9 protein fragments, thereby reducing the level of functional PCSK9, as follows:

[0118] i. Structural importance of the expected editing site: The target CAG codon is located within the catalytic domain of the PCSK9 protein, and premature termination of translation will prevent the formation of a functional catalytic domain;

[0119] ii. Molecular effect of the stop codon: The introduced TAG stop codon will activate the nonsense-mediated mRNA decay (NMD) pathway, further reducing the level of PCSK9 mRNA;

[0120] iii. Validation of existing gene knockout models: PCSK9 gene knockout mice showed a significant reduction in LDL-C levels, but no obvious adverse reactions, supporting this gene as a safe intervention target.

[0121] 2) Advantages of Base Editing over the Double-Strand Break Gene Editing CRISPR-Cas9 System

[0122] i. Reducing off-target risk: The CBE system does not rely on DNA double-strand breaks (DSBs), reducing the risks of insertions, deletions, and chromosomal rearrangements associated with DSBs;

[0123] ii. Higher precision: CBE can achieve single-base precise replacement, generating predictable gene modifications rather than random insertions and deletions;

[0124] iii. Lower cytotoxicity: Avoiding p53-dependent apoptosis activated by DSBs, increasing editing efficiency and reducing damage to cells.

[0125] 3) Molecular Mechanisms of the Peptide Insertion Strategy to Improve the Editing Efficiency of the CBE-enIscB(D61A) System

[0126] The peptide insertion strategy may improve editing efficiency by inserting functional peptides (such as gp41 or GCN4) at specific sites of the IscB protein through the following mechanisms:

[0127] i. Enhancing DNA binding ability: The inserted peptide may change the protein conformation and enhance the binding affinity of IscB to the target DNA;

[0128] ii. Optimizing protein folding and stability: Peptide insertion may stabilize the active conformation of the IscB protein and extend its half-life;

[0129] iii. Promoting protein-protein interactions: The inserted peptide may promote the interaction of IscB with cellular factors such as transcription and repair, enhancing the residence time at the target site;

[0130] iv. Improving nuclear localization: Peptide insertion may optimize the nuclear import signal and increase the number of proteins effectively entering the nucleus;

[0131] v. Optimizing spatial configuration: Peptide insertion may change the spatial arrangement between IscB and the deaminase, promoting the deaminase to contact the target DNA.

[0132] (3) Feasibility of Single AAV Delivery System and Liver Targeting

[0133] 1) Advantages of the Single AAV8 / 9 Delivery System

[0134] In the gene editing therapy of PCSK9, using a single AAV8 or AAV9 vector to deliver the peptide-embedded CBE-enIscB(D61A) system has the following advantages:

[0135] i. High liver tropism: Both AAV8 and AAV9 exhibit significant affinity for the liver. In particular, AAV8 can achieve up to 90% hepatocyte targeting, making it extremely suitable for editing the PCSK9 gene expressed in the liver.

[0136] ii. Long-term expression potential: AAV-mediated gene expression can persist for months to years without integration, making it very suitable for hyperlipidemia that requires long-term intervention.

[0137] iii. Clinical safety verification: Multiple AAV gene therapy products have been approved by the FDA (such as Luxturna, Zolgensma), with abundant safety data and a clear clinical translation path.

[0138] iv. Relatively low immunogenicity: Compared with adenovirus and lipid nanoparticles, AAV elicits a milder immune response, making it more suitable for in vivo delivery.

[0139] v. Advantage of single-vector delivery: Thanks to the relatively small molecular weight of the IscB protein (496 amino acids), the entire CBE-enIscB(D61A) system (including the ωRNA* expression cassette) can be encapsulated into a single AAV vector, with a payload capacity of approximately 4.7 kb.

[0140] 2) Special advantages of the IscB system compared to the Cas9 system in AAV delivery

[0141] The traditional SpCas9 protein (about 1,400 amino acids) is relatively large, and usually requires dual AAV vectors to deliver the complete CRISPR-Cas9 editing system, increasing the delivery complexity and cost. The IscB protein is only about 1 / 3 the size of SpCas9, and this key advantage enables it to:

[0142] i. Single AAV complete delivery: Package all components of the editing system (including CBE-enIscB(D61A) and ωRNA*) into a single AAV vector, simplifying the delivery strategy.

[0143] ii. Improve in vivo delivery efficiency: Single-vector delivery ensures that each transduced cell simultaneously receives all components of the system, improving the overall editing efficiency.

[0144] iii. Reduce production complexity and cost: Only one type of AAV vector needs to be produced, simplifying the GMP production process and reducing production costs.

[0145] iv. Reduce related immunogenicity: Reducing the virus load may reduce AAV-related immune responses.

[0146] v. Larger design space: There is still additional space in the single AAV vector to add liver-specific promoters (such as hAAT, ApoE) or regulatory elements.

[0147] 3) Physiological Advantages of Liver Gene Editing

[0148] i. Main production site of PCSK9: The liver is the main production site of PCSK9. Targeting the liver can directly block the main source;

[0149] i. High transduction efficiency: Hepatocytes have extremely high transduction efficiency for AAV8 / 9. The transduction rate of the adult human liver can reach 30 - 70%;

[0150] iii. Immune tolerance environment: The liver has a relatively immune - tolerant microenvironment, which may reduce the immune response to foreign proteins;

[0151] iv. Hepatocyte regeneration ability: The liver has good regenerative ability and strong adaptability to gene - editing - related stress;

[0152] v. Diverse administration routes: Liver targeting can be achieved by injecting through the peripheral vein, hepatic portal vein or hepatic artery, and the clinical operation is relatively simple.

[0153] (4) Clinical Transformation Potential and Expected Therapeutic Value

[0154] 1) Expected therapeutic effects

[0155] Based on the data of existing PCSK9 inhibitors and gene - knockout models, the therapeutic effects of the peptide - embedded CBE - enIscB(D61A) system targeting PCSK9 can be estimated as follows:

[0156] i. Degree of reduction of PCSK9 protein: According to the in - vitro editing efficiency of up to 70% in the foregoing examples, it is expected that the PCSK9 protein level will be reduced by about 60% - 70%;

[0157] ii. Degree of reduction of LDL - C: It is expected that the LDL - C level will be reduced by about 40% - 50%, and this effect is comparable to that of existing PCSK9 monoclonal antibodies;

[0158] iii. Reduction of the risk of cardiovascular events: Referring to the existing studies on PCSK9 inhibitors, it is expected to reduce the risk of cardiovascular events by 25% - 30%;

[0159] iv. Persistence of effect: Different from existing drugs that require repeated injections, the gene - editing effect mediated by AAV may last for several years, greatly reducing the treatment burden.

[0160] 2) Theoretical safety assessment

[0161] i. Long - term safety of PCSK9 inhibition: Epidemiological studies have shown that individuals carrying loss - of - function mutations in PCSK9 have no obvious health problems, supporting the safety of long - term inhibition of PCSK9;

[0162] ii. Off-target risk: Although peptide insertion may slightly increase the off-target rate, the off-target effects of the CBE system are still mainly limited to single-base mutations, and the risk is relatively controllable.

[0163] iii. AAV-related safety considerations: Non-integrating: AAV mainly exists in an episomal form, and the integration risk is extremely low; Known hepatotoxicity risk: High-dose AAV may cause transient elevation of transaminases, but it usually recovers on its own; Pre-existing neutralizing antibodies: Some people may have anti-AAV neutralizing antibodies, which can be avoided by screening; Extremely low cholesterol risk: Extremely low LDL-C levels (<15 - 20 mg / dL) may increase the risks of bleeding, cognitive dysfunction, etc., but based on the clinical data of PCSK9 inhibitors, this risk is extremely small.

[0164] 3) Analysis of potential advantaged populations

[0165] i. Patients with familial hypercholesterolemia (FH): These patients have insufficient response to conventional statin therapy and require more potent lipid-lowering strategies.

[0166] ii. Statin-intolerant patients: Approximately 7% - 29% of patients cannot tolerate full-dose statin therapy and require alternative lipid-lowering regimens.

[0167] iii. Patients at high risk of cardiovascular disease: Patients with multiple risk factors or who have experienced cardiovascular events require intensive lipid-lowering therapy.

[0168] iv. Patients with poor compliance: Patients who have difficulty adhering to long-term drug treatment can benefit from gene editing strategies that provide long-term benefits from a single treatment.

[0169] v. Patients with a heavy financial burden for existing PCSK9 antibody treatments: One-time gene editing treatment may reduce long-term medical costs.

[0170] (5) Analysis of application promotion and scalability

[0171] i. Other lipid metabolism targets: In addition to PCSK9, this system can be extended to other lipid-regulating genes such as ANGPTL3 and APOC3 to further optimize the lipid profile.

[0172] ii. Customized precision medicine: Personalized editing strategies can be designed according to the specific gene mutations of different patients to improve the precision of treatment.

[0173] iii. Other liver diseases: This technology platform can be extended to other monogenic liver diseases such as α1-antitrypsin deficiency and hereditary hemochromatosis.

[0174] In summary, the technical platform has demonstrated comprehensive feasibility from theoretical mechanisms to technical implementation. With the continuous optimization of gene editing and AAV delivery technologies, the peptide-embedded CBE-enIscB(D61A) system is expected to become an important new strategy for the treatment of hyperlipidemia and provide a reference for the gene therapy of other genetic diseases.

[0175] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for enhancing the efficiency of IscB-mediated DNA base editors, characterized in that, Comprising the following steps: (1) Inserting a heterologous peptide sequence into the IscB protein, namely peptide-embedded IscB; (2) Fusing the peptide-embedded IscB with a deaminase or glycosylase to construct a base editor; (3) Performing DNA base editing through the base editor.

2. The method according to claim 1, wherein The heterologous peptide includes gp41 peptide and / or GCN4 peptide.

3. The method according to claim 1, wherein The insertion site includes at least one of the positions after the amino acids selected from E48, S49, A50, E51, and E52 of the IscB protein.

4. The method according to claim 1, characterized in that, The amino acid sequence of the IscB protein is as shown in SEQ ID NO:

3.

5. The method according to claim 1, wherein The nucleotide sequence of ωRNA used for the DNA base editing is as shown in SEQ ID NO:

5.

6. A base editor system, characterized in that, A heterologous peptide is inserted into its nuclease, and the heterologous peptide includes gp41 peptide and / or GCN4 peptide; flexible linker peptides are added to both ends of the heterologous peptide sequence.

7. An IscB base editor system, characterized in that, Comprising an IscB protein and ωRNA; the amino acid sequence of the IscB protein is as shown in SEQ ID NO:3; the nucleotide sequence of ωRNA used for the DNA base editing is as shown in SEQ ID NO:

5.

8. The IscB base editor system according to claim 7, characterized in that, A heterologous peptide is inserted into the IscB protein; the heterologous peptide includes gp41 peptide and / or GCN4 peptide; flexible linker peptides are added to both ends of the heterologous peptide sequence.

9. The IscB base editor system according to claim 8, wherein The insertion site includes at least one of the positions after the amino acids selected from E48, S49, A50, E51, and E52 of the IscB protein.

10. The IscB base editor system according to claim 7, wherein The base editing system is any one of CBE, ABE, DAF-CBE, and DAF-TBE.

11. A tissue-specific gene editor system, characterized in that, A tissue-specific targeting peptide is embedded in the nuclease of the base editor system according to any one of claims 6-10.

12. Use of the IscB base editor system according to any one of claims 6-10 and the tissue-specific gene editor system according to claim 11 in any of the following fields: i. Gene editing; ii. Preparing a preparation for reducing the immunogenicity of gene therapy; iii. Preparing a drug for treating and / or preventing hyperlipidemia; v. Preparing a gene therapy drug for treating and / or preventing genetic diseases.

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