A peroxisome-targeting chimera pextac and applications thereof

Abstract

This invention discloses a peroxisome-targeting chimeric compound, PexTAC, and its applications, belonging to the field of target protein degradation technology. The invention provides a peroxisome-targeting chimeric compound, PexTAC, with the general structural formula A-L-B, where A is the peroxisome-targeting module, L is the linker, and B is the target protein recognition module. PexTAC recruits target proteins to the peroxisome membrane and utilizes the endogenous E3 ubiquitin ligase SYVN1 for specific degradation via the proteasome pathway. This platform can not only target ROR1 to inhibit tumor growth but also induce ferroptosis through the bispecific PexTAC synergistic degradation of HSPA5 and GPX4. This invention overcomes the dependence of traditional PROTAC technology on limited E3 ligases, providing a novel modular degradation strategy for targeting "undruggable" proteins and organelle-related proteins.

Description

Technical Field

[0001] This invention belongs to the field of target protein degradation technology, specifically relating to a peroxisome-targeting chimera PexTAC and its applications. Background Technology

[0002] Targeted protein degradation (TPD) technology has become a cutting-edge direction in drug development due to its potential to target "undruggable" proteins that are difficult to target with traditional small molecules. However, current mainstream TPD technologies, such as PROTACs, still have significant limitations in design and application: their degradation function relies on recruiting target proteins to a specific class of E3 ubiquitin ligases. This strategy, which highly depends on a single degradation mechanism, greatly limits the design flexibility and development pathways of PROTAC molecules. In addition, the types of E3 ligases that can be effectively used for degradation are currently very limited (the most commonly used are only VHL and CRBN), further restricting the widespread application of this technology and making it difficult to effectively intervene in a large number of therapeutically potential targets. Although protein degradation technologies such as PROTAC and LYTAC have made significant breakthroughs in recent years, they still face a series of challenges. For example, PROTACs rely on a limited number of E3 ligands and their targeting ability for "undruggable" proteins is still insufficient; LYTACs depend on the expression level of specific receptors and have slow degradation kinetics. Therefore, there is an urgent need to develop a new degradation strategy that is not dependent on specific E3 ligands, has a higher degree of modularity, and has a wider range of applicability, in order to break through the current technological bottlenecks. Summary of the Invention

[0003] The purpose of this invention is to address the limitations in the field of targeted protein degradation, including the over-reliance on the paradigm of targeting proteins to specific E3 ubiquitin ligases to promote target protein degradation. Furthermore, only a few E3 ligases, such as VHL and CRBN, are currently widely used, severely limiting the range of degradable targets. We propose an alternative strategy: physically recruiting target proteins to peroxisomes and then using peroxisome localization to recruit endogenous E3 ligases for degradation. Through genome-wide CRISPR screening, we identified an E3 ligase, SYVN1, which had not previously been used for targeted protein degradation. Further systematic validation targeting 16 different membrane proteins on the peroxisome surface demonstrated that degradation is driven by peroxisome localization itself, independent of any specific anchor protein. Based on this mechanism, we established a modular PexTAC degradation platform. PexTAC achieved efficient degradation of the clinically relevant target ROR1 in vivo and significantly inhibited tumor growth. In addition, dual-target degradation of HSPA5 and GPX4 showed synergistic anti-tumor potential. This platform does not rely on small molecule ligands of target proteins, providing a new degradation pathway for "undruggable" targets that are difficult to intervene in by traditional methods, and expanding the application space of protein degradation technology at the organelle level.

[0004] This invention provides a peroxisome-targeting chimera, PexTAC, which is a chimera that recruits proteins to peroxisomes to achieve targeted degradation of the recruited proteins.

[0005] This invention provides a peroxisome-targeting chimeric PexTAC, which consists of a peroxisome-targeting module A, a target protein recognition module B, and a linker L, with L connecting A and B, and has the general formula ALB.

[0006] Further specifying, the peroxisome targeting module is a protein or polypeptide that targets the peroxisome; the target protein recognition module is a protein or polypeptide that can bind to the target protein; and the linker is a polypeptide linker or protein.

[0007] Further specifying, the peroxisome targeting module is a membrane protein of the peroxisome or a membrane localization sequence of the corresponding membrane protein.

[0008] Further specifying, the peroxisome targeting module consists of the peroxisome membrane proteins PEX2, PEX3, PEX5, PEX6, PEX7, PEX10, PEX11, PEX12, PEX13, PEX14, PEX16, PEX19, PEX26, PMP70, Fis1, PTS1, and PEX3N.

[0009] To be further specified, the target protein recognition module is a nanobody of the target protein.

[0010] Further specifying, the nanobodies for the target protein recognition module include the green fluorescent protein nanobodies GBP, ROR1 nanobodies NbROR1, GPX4 nanobodies NbGPX4, HSPA5 nanobodies NbHSPA5, and bispecific nanobodies NbHSPA5-NbGPX4 that simultaneously bind GPX4 and HSPA5.

[0011] The present invention provides a nucleic acid molecule characterized in that it encodes the PexTAC as described in any one of claims 1-7.

[0012] The present invention provides a recombinant plasmid or recombinant host cell containing the above-mentioned nucleic acid molecules.

[0013] This invention provides the application of the above-mentioned PexTAC, the nucleic acid molecule of claim 8, or a recombinant plasmid or recombinant host cell containing the nucleic acid molecule of claim 9 in the preparation of a targeted protein degradation drug.

[0014] Further, this is achieved through viral delivery of PexTAC or plasmid transfection expressing PexTAC.

[0015] Further, it refers to the use of PexTAC, delivered via a viral vector, that degrades ROR1, GPX4, or HSPA5 individually or simultaneously, in the preparation of drugs for treating diseases.

[0016] This invention provides the application of the above-mentioned PexTAC, the above-mentioned nucleic acid molecule, or recombinant plasmids or recombinant host cells containing the above-mentioned nucleic acid molecule in the preparation of drugs that inhibit cancer cell proliferation or induce programmed cell death.

[0017] This invention provides the use of the above-mentioned PexTAC, the above-mentioned nucleic acid molecule, or recombinant plasmids or recombinant host cells containing the above-mentioned nucleic acid molecule in the preparation of drugs for treating lung cancer, breast cancer, pancreatic cancer, hematologic malignancies, cervical cancer, ovarian cancer, liver cancer, and colorectal cancer.

[0018] Beneficial effects: one, 1. A novel targeted protein degradation platform is provided, which overcomes the limitation of traditional TPD technology that relies on direct targeting of E3 ligases and expands the application scope of TPD technology; 2. The mechanism of protein degradation driven by peroxisome localization was discovered for the first time, and the key E3 ligase SYVN1 was discovered, providing new targets and design paradigms for the field of protein degradation; 3. PexTAC has a highly modular nature, and can be quickly adapted to different target proteins by replacing the target protein recognition module (nanobody), achieving "plug and play" programmable degradation; 4. Bispecific PexTAC can achieve multi-target synergistic degradation, providing a new strategy for overcoming tumor heterogeneity and drug resistance; 5. AAV-based delivery systems have good in vivo safety and targeting, laying the foundation for the clinical translation of PexTAC; 6. The technology has a wide range of applications and can be used to treat various diseases such as tumors and neurodegenerative diseases. It can also be used as a tool for exploring protein function in basic research. two, 1. Tumor treatment: Single-target therapy: Targeting tumor-specific high-expression proteins (such as ROR1), construct a single-specific PexTAC to inhibit tumor cell proliferation and induce apoptosis by degrading the target protein; Synergistic therapy: Construct bispecific PexTACs (such as HSPA5 / GPX4 targeting) to synergistically promote cancer cell ferroptosis by simultaneously degrading key proteins in the ferroptosis pathway, thereby overcoming tumor drug resistance; 2. Treatment of other diseases: For neurodegenerative diseases and autoimmune diseases mediated by abnormally expressed proteins, corresponding PexTACs can be designed to achieve specific degradation of pathogenic proteins; 3. Basic Research Tool: PexTAC, as a programmable protein degradation tool, utilizes a newly discovered E3 ligase to achieve conditional degradation independent of classical E3, providing a new strategy for exploring the physiological functions of specific proteins in cell signaling pathways. First, PexTAC does not depend on specific E3 ligands; instead, it recruits endogenous E3 ubiquitin ligases through peroxisome localization, bypassing the bottleneck of E3 ligand shortage in PROTAC development. Second, by targeting 16 different membrane proteins on the peroxisome surface, we demonstrate that PexTAC's function is independent of specific anchor proteins, but is driven by the spatial event of "peroxisome localization," endowing it with high modularity and universality. Third, unlike LYTAC or AUTAC, PexTAC degrades via the ubiquitin-proteasome pathway, exhibiting faster kinetics and higher specificity. Finally, as a gene-encoded tool, PexTAC can achieve long-term, stable target protein inhibition via AAV delivery, complementing the short-term, reversible regulation of small molecule degraders such as PROTAC. These properties make PexTAC an important addition to the existing degradation toolkit.

[0020] III. The core principle of this invention is to initiate SYVN1-dependent ubiquitin-proteasome system (UPS)-mediated protein degradation by inducing proximity between the target protein and the peroxisome membrane. The specific mechanism is as follows: PexTAC targets the peroxisome membrane via a peroxisome targeting module (such as PEX3N) and specifically binds to the target protein via a target protein recognition module (nanobody). Once the target protein is localized to the peroxisome membrane, it recruits the endogenous E3 ubiquitin ligase SYVN1. SYVN1 catalyzes K48-linked ubiquitination of target proteins; The ubiquitinated target protein is recognized and degraded by the proteasome, and this process does not depend on the lysosome or autophagy pathway (the proteasome inhibitor MG132 can completely block the degradation, while the lysosome inhibitor BafA1 has no effect).

[0021] IV. Through CRISPR screening, siRNA knockdown, gene knockout (KO), and complementation experiments, it was confirmed that SYVN1 is the key E3 ligase mediating the degradation function of PexTAC, and all PexTACs based on different peroxisome targeting modules depend on SYVN1 to function.

[0022] 1. Broad applicability: PexTAC can efficiently degrade target proteins in a variety of cell lines (HeLa, A549, HepG2, Capan-1, MDA-MB-231, etc.) without being limited by cell type; 2. Highly efficient degradation activity: Through the regulation of the Tet-on system, PexTAC can achieve dose-dependent and time-dependent degradation of target proteins with significant degradation efficiency (such as EGFP, ROR1 and other proteins, the degradation rate can reach more than 70%). 3. High specificity: Quantitative proteomics analysis confirmed that PexTAC can specifically degrade the target protein without significantly affecting other intracellular proteins; 4. Synergistic therapeutic effect: Bispecific PexTACs (such as those that degrade HSPA5 and GPX4) can synergistically induce ferroptosis in cancer cells, and their cytotoxicity is significantly higher than that of single-target PexTACs; 5. In vivo therapeutic potential: ROR1-targeted PexTAC delivered by AAV significantly inhibited tumor growth in an A549 lung cancer xenograft mouse model, with no significant systemic toxicity (no significant change in mouse body weight). 6. Complementary technological advantages: Compared with existing TPD technologies, PexTAC does not rely on E3 ligase ligands, achieves degradation through physical recruitment, and acts on a novel degradation pathway localized to peroxisomes, which can target organelle-related target proteins that are difficult to reach by traditional TPD technologies.

[0023] V. 1. In vitro data: PexTAC can achieve efficient degradation of target proteins in a variety of cell lines (e.g., the degradation rate of ROR1 in A549 cells can reach more than 80%). DOX can regulate the expression of PexTAC, achieving dose-dependent (DOX concentration 0.01-0.5 μg·ml⁻¹) and time-dependent (DOX treatment 12-48 h) degradation of the target protein; Bispecific PexTAC (HSPA5 / GPX4 targeting) induced significantly higher ferrotoxicity in cancer cells than single-target PexTAC. 2. In vivo data: The tumor volume of mice treated with AAV-PexTAC was significantly smaller than that of the control group (AAV-PEX3N, PBS) (P<0.0001). The degradation efficiency of target proteins (such as ROR1) in tumor tissues is over 70%, and there is no obvious organ toxicity. Attached Figure Description

[0024] Figure 1. PexTAC targets and degrades EGFP protein in HeLa cells. a. Schematic diagram of the mechanism of action of PexTAC: The target protein (POI) is targeted to the peroxisome via a PEX3N-nanobody (Nb) chimera, thereby inducing its degradation; b. Laser confocal microscopy images show that the EGFP fluorescence intensity is significantly reduced in cells expressing EGFP and targeting PexTAC, while the EGFP fluorescence intensity remains unchanged in control cells (co-expressing PEX3N-mCherry or expressing EGFP alone). Western blot (WB) analysis further verifies the degradation effect of PexTAC on EGFP (bottom right).

[0025] Figure 2 Tet-on regulated PexTAC targets and degrades EGFP protein in HeLa cells. a. Schematic diagram of the tetracycline-induced (Tet-on) system: The addition of doxycycline (DOX) regulates the controllable expression of EGFP targeting PexTAC (PEX3N-mCherry-GBP); b. DOX induces EGFP degradation in a dose-dependent manner; c. DOX induces EGFP degradation in a time-dependent manner.

[0026] Figure 3 Tet-on-regulated PexTAC targets and degrades EGFP protein in various cell lines. Western blot analysis showed that PexTAC significantly induced EGFP degradation in multiple cell lines. A heatmap illustrates the statistical effects of PexTAC-induced EGFP degradation in multiple cell lines, with three independent replicates for each cell line.

[0027] Figure 4 Multiple PexTAC constructs target and degrade EGFP protein in HeLa cells. a. Schematic diagram of the construction of a series of novel EGFP-targeting PexTACs fused with different peroxisome-associated membrane proteins; b. Western blot analysis showed that PexTACs significantly induced EGFP degradation in live HeLa cells.

[0028] Figure 5 Multiple different PexTAC constructs targeted and degraded EGFP protein in A549 and HepG2 cells. ab. Western blotting analysis showed that PexTACs significantly induced EGFP degradation in A549 and HepG2 cells; c. A heatmap shows the statistical analysis of EGFP degradation induced by PexTACs in A549 and HepG2 cell lines, with three independent replicates for each cell line. (The x-axis of the heatmap represents different cell lines, the y-axis represents different PexTACs, and different colors represent different degradation effects; lighter colors indicate better degradation effects).

[0029] Figure 6. PexTAC degrades EGFP protein via the ubiquitin-proteasome pathway. a–c. Laser confocal microscopy showed that EGFP did not co-localize with lysosomal marker LAMP1 (a), autophagy marker LC3B (b), or p62 (c); d. Western blot analysis confirmed that PEX3N-mCherry-GBP-induced EGFP degradation could be inhibited by the proteasome inhibitor MG132, but was not affected by the lysosomal inhibitor BafA1, indicating that the degradation mechanism depended on UPS rather than the lysosomal-related pathway; e. Pulldown assays showed that EGFP underwent UPS-mediated K48 ubiquitination modification, rather than autophagy-related K63 ubiquitination modification. Figure 7Other PexTACs degrade EGFP protein via the ubiquitin-proteasome pathway. A series of PexTACs, including PEX3-mCherry-GBP, PEX2-mCherry-GBP, PEX10-mCherry-GBP, PEX12-mCherry-GBP, and PMP70-mCherry-GBP, induce EGFP degradation, all of which are consistently blocked by MG132 and unaffected by BafA1.

[0030] Figure 8. SYVN1 is a key E3 ubiquitin ligase in PexTAC-mediated degradation. a. Schematic diagram of CRISPR screening experiment, MOI: Multiplicity of Infection, FACS: Fluorescence-Activated Cell Sorting, NGS: Next-Generation Sequencing; b. NGS analysis identified several high-scoring proteins, among which SYVN1 was the only E3 ubiquitin ligase; c. siRNA-mediated SYVN1 knockdown inhibited the degradation of EGFP by PEX3N-GBP; d. SYVN1 gene knockout (KO) further confirmed that PEX3N-GBP-induced EGFP degradation was completely blocked; e. Re-expression of SYVN1 in SYVN1-KO cells restored the EGFP degradation activity of PEX3N-GBP; f. Expression of the inactive SYVN1 mutant (C329S) could not restore the degradation activity of PEX3N-GBP; g. K48 ubiquitination modification was detected only in KO cells with re-expression of SYVN1; h. Pull-down assay showed that in PEX3N-GBP... In the presence of EGFP, there is an interaction between EGFP and SYVN1.

[0031] Figure 9 SYVN1 is a key E3 ubiquitin ligase for the degradation mediated by other PexTACs. a. In SYVN1-KO cells, all other PexTACs failed to induce EGFP degradation; b. Statistical graph showing that none of the PexTACs induced EGFP degradation.

[0032] Figure 10PexTAC-ROR1 targets and degrades ROR1 protein in A549 cells. ab. Doxycycline (DOX)-induced PEX3N-mCherry-NbROR1 expression mediates endogenous ROR1 degradation in A549 lung cancer cells in a dose- and time-dependent manner; c. Western blot (WB) analysis showed that ROR1 degradation was inhibited by MG132 but not by BafA1; d. Quantitative proteomics analysis confirmed that ROR1 was specifically degraded in the whole proteome of A549 cells, and the volcano plot showed the relationship between log2-fold change and log10 P value.

[0033] Figure 11 PexTAC-ROR1 targets and degrades ROR1 protein in different cell lines. af. Western blot analysis showed that PEX3N-mCherry-NbROR1 mediated ROR1 degradation in different cell lines. g. A heatmap shows the statistical results of PexTAC-induced ROR1 degradation in multiple cell lines, with three independent replicates for each cell line.

[0034] Figure 12 PexTAC-ROR1-mediated ROR1 degradation induces apoptosis. a. ROR1 degradation leads to upregulation of the apoptosis marker Caspase-3; b. Flow cytometry analysis confirms that ROR1 degradation induces apoptosis 24 hours after degradation, with a more significant apoptotic effect at 48 hours.

[0035] Figure 13 PexTAC-ROR1-mediated ROR1 degradation inhibits lung cancer cell proliferation. a. Schematic diagram of the ROR1-RhoA-YAP signaling axis; b. Western blotting analysis showed that ROR1 degradation downregulated RhoA expression; c. Laser confocal microscopy images, line scan analysis, and nucleus / cytoplasm ratio analysis showed that ROR1 degradation promoted YAP translocation to the nucleus; d. EdU cell proliferation assays showed that ROR1 degradation inhibited A549 cell proliferation; e. ROR1 degradation downregulated p-AKT and p-ERK in the AKT-ERK proliferation axis, and the bar chart represents the statistical results of Western blotting.

[0036] Figure 14. Targeted degradation of ROR1 protein in A549 cells by various PexTACs-ROR1 constructs. Efficient ROR1 degradation was achieved in A549 cells by fusing different PexTACs with different peroxisome membrane-binding proteins (a, PEX2 fusion; b, PEX19 fusion; c, PMP70 fusion; and d, PEX11 fusion).

[0037] Figure 15 AAV6-PexTAC-ROR1 inhibits the growth of xenograft lung cancer tumors in vivo. a. Schematic diagram of the generation of the A549 xenograft mouse model: approximately 6 × 10⁻⁶. 6 A549 cells were dissolved in 0.1 ml PBS / Matrigel (1:1) solution and subcutaneously injected into the axillary region of BALB / c nude mice. AAV6 (1×10¹² vg·ml⁻¹, 20 μl) was injected into the tumor every 3 days. b. Tumor growth curves for each group of mice (PBS blank control, AAV6-PEX3N control, and AAV6 PEX3N-NbROR1 (PexTAC) group, n = 4 mice per group). c. Tumor growth curves for each group of mice; d. Body weight curves for each group of mice; e. Photographs of tumors removed 15 days after the first administration in each group; f. Statistical analysis of tumor weight in each group; g. After tumor dissection, tumor tissue was lysed, and Western blotting analysis of the lysate confirmed that ROR1 degradation was detected in tumors of mice treated with PexTAC only; h. IHC (Ki67) and H&E staining of tumor sections showed that PexTAC treatment induced apoptosis, while no obvious apoptotic features were detected in other treatment groups or major healthy organs.

[0038] Figure 16 PexTAC-HSPA5 targets and degrades HSPA5 protein in HeLa cells. PexTAC-HSPA5 effectively degrades HSPA5 in HeLa cells, and MG132 inhibits HSPA5 degradation, while BafA1 does not affect degradation. Figure 17 PexTAC-GPX4 targets and degrades GPX4 protein in HeLa cells. PexTAC-GPX4 effectively degrades GPX4 in HeLa cells, and MG132 inhibits GPX4 degradation, while BafA1 does not affect degradation. Figure 18 Bispecific PexTAC simultaneously targets and degrades HSPA5 and GPX4 proteins in HeLa cells. a. Schematic diagram of the HSPA5–GPX4 ferroptosis pathway; b. 0.5 ug / ml DOX-mediated expression of bispecific PexTAC (BsPexTAC) achieves simultaneous degradation of HSPA5 and GPX4 in HeLa cells. The bar chart represents the statistical analysis of HSPA5 and GPX4 degradation.

[0039] Figure 19Mechanism of bispecific PexTAC targeting and degrading HSPA5 and GPX4 proteins simultaneously in HeLa cells. a. Degradation of HSPA5 and GPX4 can be inhibited by the proteasome inhibitor MG132, but is not affected by the lysosomal inhibitor BafA1. The bar chart represents the statistical analysis of WB results.

[0040] Figure 20 Bispecific PexTAC promotes ferroptosis in cancer cells through a synergistic effect. CCK-8 assays confirmed that the ferroptosis inhibitor Fer-1 inhibits cell death, while the apoptosis inhibitor ZVAD has no such effect, indicating that the cell death mechanism is ferroptosis rather than apoptosis.

[0041] Figure 21 Bispecific PexTAC disrupts mitochondrial morphology through synergistic action. Laser confocal imaging was used to analyze mitochondrial morphology in cells co-expressing EGFP-mito (a mitochondrial marker) and PEX3N-mCherry (control), PEX3N-mCherry-NbHSPA5, PEX3N-mCherry-NbGPX4, PEX3N-mCherry-NbH5 / G4, or a blank control. Quantitative analysis of mitochondrial branch length and mitochondrial perimeter was also performed.

[0042] Figure 22 Bispecific PexTAC promotes lipid peroxidation through a synergistic effect. Lipid peroxidation analysis and quantification of the red / green fluorescence intensity ratio were performed using the BDPY 581 / 591C11 probe via laser confocal microscopy.

[0043] Figure 23 Bispecific PexTAC disrupts mitochondrial membrane potential through synergistic action. Mitochondrial membrane potential analysis and quantification of TMRE fluorescence intensity were performed using TMRE probes.

[0044] Figure 24 Bispecific PexTAC inhibits the growth of cervical cancer organoids through synergistic action. a. Schematic diagram of cervical cancer organoid formation; b. Bispecific PexTAC infects cervical cancer organoids with a virus. Western blotting analysis shows that bispecific PexTAC can effectively degrade HSPA5 and GPX4 proteins in cervical cancer organoids; c. Celltiter Glo 2.0 cell viability assay shows that bispecific PexTAC can effectively inhibit the growth of cervical cancer organoids.

[0045] Figure 25 PexTACs induce target proteins to peroxisomes, initiating SYVN1-dependent ubiquitin-proteasome system (UPS)-mediated protein degradation. Detailed Implementation

[0046] 1. Construction of xenograft tumor models and drug treatment Female BALB / c nude mice aged 4-6 weeks were purchased from Beijing Charles River Laboratory Animal Technology Co., Ltd. A549 cells were collected after reaching the logarithmic growth phase, washed with 10 ml PBS, and digested with 1 ml trypsin for 2-3 minutes until the cells were completely detached from the cell wall. The cells were resuspended in 3 ml of complete culture medium. The cell suspension was centrifuged at 1000 g for 5 minutes at 4°C, the supernatant was discarded, and the cell pellet was resuspended in a pre-chilled PBS / Matrix gel (Solarbio, #M8370) 1:1 (v / v) mixture, with a final cell density of approximately 5 × 10⁻⁶ cells / mL. 7 ml⁻¹. When constructing the A549 xenograft model, 0.1 ml of solution containing approximately 6 × 10⁻¹ ml of [a specific substance / component] was used. 6 A549 cells were subcutaneously injected into the axillae of BALB / c nude mice using a PBS / Matrix gel mixture. Tumors typically appeared within 1–2 weeks and continued to grow steadily. To evaluate the efficacy of AAV delivery of PexTAC, AAV6-PEX3N (1×10¹² vg·ml⁻¹, 20 μl), AAV6-PEX3N-NbROR1 (1×10¹² vg·ml⁻¹, 20 μl), or PBS were intratumorally injected into mice every 4 days for a total of 4 injections. Tumor size was measured daily using calipers, and tumor volume was calculated using the formula: V = (length × width²) / 2.

[0047] 2. Organoid generation and culture The organoid experiments were approved by the Clinical Research Ethics Committee of the Chengdu Tianfu Organoid Bank (No.: CTOB-2025-0611). All participants signed informed consent forms. Cryopreserved organoids were rapidly thawed in a 37°C water bath and transferred to pre-chilled MasterAim® cervical cancer organoid basal medium (AIMINGMED, #100-107), which was supplemented with MasterAim® cervical cancer organoid supplement (AIMINGMED, #100-108) at a ratio of 95:5 (v / v) and contained 0.1% (v / v) of MasterAim® major enhancer (1000×, AIMINGMED, #100-008). The medium was then centrifuged at 300×g for 5 minutes. After discarding the supernatant, the organoid pellet was resuspended in the aforementioned complete organoid medium. Gently mix the suspension with preheated MasterAim® matrix gel (AIMINGMED, #100-659) at a ratio of 1:2 (v / v), taking care to avoid air bubbles. Aliquot 50 μL of the mixture into the center of each well of a 24-well plate. Incubate the plate at 37°C and 5% CO2 for 5 minutes to allow the droplets to initially solidify, then invert and incubate for another 25 minutes under the same conditions to complete solidification. Once the gel dome has fully formed, add 500 μL of complete organoid culture medium to each well. Add 500 μL of calcium, magnesium, and phenol red-free DPBS (AIMINGMED, #100-184) to each of the outer 16 wells to maintain humidity during culture. Culture the organoids at 37°C and 5% CO2, changing the medium every two days. Monitor the morphology and growth of the organoids regularly using an inverted microscope, and passage them every 7–10 days.

[0048] 3. Lentiviral transduction of organoids Aspirate the organoid culture medium and dissociate the organoids into small clumps using MasterAim® rapid enzyme (1×) (AIMINGMED, #100-546) at 37°C for 5–10 minutes. Resuspend the resulting cell clumps in a transduction mixture containing organoid culture medium supplemented with MasterAim® major enhancer (1000×, AIMINGMED, #100-008), 8 μg / mL polybrene (Yeasen, #40804ES76), and concentrated lentivirus with a multiplicity of infection (MOI) of 50. Transduction is performed by centrifugation: the suspension is centrifuged at 350×g for 30 minutes at room temperature. After centrifugation, organoids in 1.5 mL EP tubes are incubated at 37°C and 5% CO2 for 10 hours, then embedded in matrix gel and re-inoculated into new 24-well plates. Two days after infection, transgene expression is induced by doxycycline treatment for 2 days.

[0049] 4. Cell viability was measured using CellTiter-Glo 2.0. The dissociated organoid aggregates prepared above were resuspended in a transduction mixture consisting of complete organoid culture medium supplemented with 8 μg / mL polybrene (Yeasen, #40804ES76) and concentrated lentivirus (MOI = 50). Transduction was performed by centrifugation: the suspension was centrifuged at 350×g for 30 minutes at room temperature. After centrifugation, the organoids were incubated at 37°C and 5% CO2 for 10 hours, and then seeded into 96-well plates at a density of 300-500 organoids per well, suspended in 5 μL of matrix gel, with a total volume of 100 μL of culture medium per well. Two days after infection, transgenic expression was induced by doxycycline treatment, followed by another 2 days of treatment. Subsequently, 100 μL of CellTiter-Glo 2.0 reagent (Chongqing Jinbo Pharmaceutical Co., Ltd., #KCM-6) was added to each well, and the matrix gel was mechanically disrupted. The mixture was vigorously mixed on a plate shaker for 5 minutes, incubated at room temperature for 25 minutes, and then the luminescence value was measured.

[0050] 5. Preparation of Lentivirals Using PEI MAX® (Polysciences, #49553-93-7) transfection reagent, lentiviral transfer plasmid, packaging plasmid psPAX2, and envelope plasmid pCMV-VSV-G were co-transfected into 293T cells (Pronosei Life Sciences Co., Ltd., #CL-0005) according to the manufacturer's operating procedures to generate lentiviral particles. Viral supernatants were collected at 48 and 72 hours post-transfection and combined for ultrafiltration concentration using a 100 kDa molecular weight cutoff (MWCO) ultrafiltration tube (Merck Millipore, #UFC910096). The concentrated lentiviral stock solution was frozen and thawed immediately before use.

[0051] 6. Experimental cells The cells used in this study included HeLa, HepG2, A549, Capan-1, MDA-MB-231, MCF-7, OVCAR3, and SK-BR-3 cells.

[0052] 7. Plasmid Construction The eukaryotic expression vectors used in this study were pmCherry-C1, pAAV-CMV, and Tet-on vectors. The base vector for the five plasmids, including pex3-mCherry-GBP, pex3-mCherry-NbROR1, pex3-mCherry-NbHSPA5, pex3-mCherry-NbGPX4, and pex3-mCherry-Nb-HSPA5-NbGPX4, was the pmCherry-C1 vector. Five plasmids were prepared by inserting PEX3, GBP, NbROR1, NbHSPA5, NbGPX4, and Nb-HSPA5-NbGPX4 fragments into the pmCherry-C1 vector. Five DOX-regulated plasmids, TRE-pex3-mCherry-GBP, TRE-pex3-mCherry-NbROR1, TRE-pex3-mCherry-NbHSPA5, TRE-pex3-mCherry-NbGPX4, and TRE-pex3-mCherry-Nb-HSPA5-NbGPX4, were prepared by inserting pex3-mCherry-GBP, pex3-mCherry-NbROR1, TRE-pex3-mCherry-NbHSPA5, TRE-pex3-mCherry-NbGPX4, and TRE-pex3-mCherry-Nb-HSPA5-NbGPX4 fragments into the Tet-on vector.

[0053] 7.1 Acquisition of the target fragment (1) Acquisition of target genes: Some target genes were synthesized by General Biotech Co., Ltd. (Anhui, China) and some target genes were purchased from the Miaoling plasmid sharing platform.

[0054] (2) PCR amplification of the target gene: The target fragment of the plasmid to be cloned is generally amplified by double enzyme digestion or by designing corresponding primers. In principle, the primers should not exceed 60 bp. After obtaining the primer sequence, the gene insertion fragment is amplified by polymerase chain reaction (PCR). The extension time is set according to the size of the target gene fragment. Generally, 1000 bp is amplified every 30 s. The PCR amplification system of the target gene fragment mainly includes the following components: 18 μL of HyPerFUsion™ DNA polymerases, 0.8 μL of forward primer, 0.8 μL of reverse primer, and 2 ng of template DNA. The polymerase chain reaction configuration is as follows: 0.8 μL of 10 μM forward primer, 0.8 μL of 10 μM reverse primer, and HyPerFUsion™ DNA polymerases. TMDNA polymerase 0.4 μL, 5x HyPerFUsion™ buffer 4 μL, 2.5 mMdNTPs 1.6 μL; the chain reaction system is as follows: template DNA 0.2 μL, forward primer 0.8 μL, reverse primer PCR 0.8 μL, HyPerFUsion 18 μL; the program settings are shown in Table 1.

[0055] Table 1 PCR program settings for the target fragment

[0056] 7.2 Vector double enzyme digestion After selecting the target plasmid as the construction vector, appropriate restriction enzyme sites are chosen for the target plasmid and double digestion is performed. The vector double digestion system is usually 20 μL, and the parameters of each component are shown in the table below: template DNA 2000 ng, restriction endonuclease A 1 μL, restriction endonuclease B 1 μL, CutSmart buffer 2 μL, ddH2O to 20 μL.

[0057] 7.3 DNA product ligation (1) Recombinase ligation: The DNA fragment, vector, and recombinase of the determined concentration were mixed in a 1.5 mL EP tube according to a certain calculated ratio. The tube was then centrifuged at 13500 rpm for 1 min to ensure thorough mixing of the components. The parameters of each component in the system are shown in the table below: vector 0.04 times the number of base pairs of the vector (ng), target fragment 0.02 times the number of base pairs of the fragment (ng), cloning mix 5 μL, ddH2O to 10 μL. The EP tube was then placed in a metal bath and ligated at 37℃ for 30 min. After ligation, the ligation product was stored on ice or at 4℃ for later use.

[0058] (2) T4 DNA ligase ligation: The T4 ligase system used in the experiment is usually 20 μL. The parameters of each component in the system are shown in the table below: vector 1 μL, target fragment 3C 载 V 载 M 片 / C 片 M 载 The following solutions were prepared: 1 μL T4 DNA Ligase, 1 μL T4 DNA Ligase buffer, 2 μL ddH2O, and 20 μL ddH2O. The vector and target fragment were generally mixed in a 3:1 ratio. Ligation times were 16 °C for 1 h, 18 °C for 1 h, and after ligation, the product was stored on ice or at 4 °C for later use.

[0059] 7.4 Identification of positive clones After transformation, colony PCR was used to identify positive clones. Appropriate forward and reverse primers were selected to amplify colonies of suitable size. The reaction ratio is shown in the table below: half the bacterial cell volume, 0.4 μL of forward primer, 0.4 μL of reverse primer, and 9.2 μL of 1.1x Taq Master Mix. This mixture was aliquoted into eight-tube strips at 9.5 μL each. Half of each pre-labeled colony was then added to the eight-tube strip using a pipette. The PCR program parameters are shown in Table 2. Positive clones were identified by agarose gel electrophoresis after PCR.

[0060] Table 2. Colony PCR Program Settings

[0061] 7. Cell Culture HeLa, HepG2, A549, Capan-1, MDA-MB-231, MCF-7, OVCAR3, and SK-BR-3 cells were cultured in proliferation medium using standard methods, with cells cultured in DMEM cell culture medium (containing 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin mixture) at 5% CO2 and 37 ℃.

[0062] 8. Cell transfection Transient transfection experiments are typically performed in Thermo Scientific 8-well cell culture plates (#155409) using Lipomaster 3000 transfection reagent (#TL301-01) from Nanjing Novizan Biotechnology Co., Ltd. 0.25 μg of DNA was dissolved in 12.5 μl of Gibco Opti-MEM medium (Life Technologies, #31985-062), and 0.5 μl of T3000 enhancer was added and gently pipetted to mix. 0.5 μl of Lipomaster 3000 transfection reagent was dissolved in 12.5 μl of Gibco Opti-MEM medium and mixed with the DNA mixture. The mixture was then added to a 2.5 × 10⁻⁶ well plate. 4 In 8-well plates containing adherent cells, each well contains 250 μl of complete DMEM medium. Cells are cultured at 37°C in a 5% CO2 incubator for approximately 2 hours, then the medium is replaced with preheated complete DMEM, and the cells are cultured at 37°C in a 5% CO2 incubator for at least 20 hours. When co-transfecting multiple plasmids, the DNA amount mentioned above refers to the total amount of plasmids used.

[0063] 9. Virus preparation and purification Recombinant adeno-associated virus (AAV) was prepared by transfecting AAV293T cells with a polyethyleneimine-mediated three-plasmid system.

[0064] 15-18 hours post-transfection, replace the culture medium containing the transfection reagent with serum-free DMEM medium. Collect the virus 4 days post-transfection: centrifuge the cell-medium mixture at 18000 g for 10 minutes at room temperature. Resuspend the cell pellet in 1 ml PBS, freeze-thaw five times, and centrifuge again at 18000 g for 20 minutes at room temperature. Combine the supernatant with the clear virus-containing medium and filter through a 0.45 μm sterile filter. Concentrate the virus solution using an Amicon® Ultra ultrafiltration column and wash twice with PBS. Aliquot the concentrated virus solution and store at -80°C for later use in mouse injection. Determine the viral titer using a linearized genomic plasmid as a standard.

[0065] 10. Mitochondrial membrane potential detection Tetramethylrhodamine ethyl ester (TMRE) accumulates in highly polarized mitochondria, and its fluorescence intensity is positively correlated with mitochondrial membrane potential (MMP). A mitochondrial membrane potential assay kit (containing TMRE) was purchased from Beyotime Biotechnology Co., Ltd. (#C2001S). After treatment, cells were incubated with 1× TMRE at 37°C in the dark for 30 minutes, washed twice with PBS, and resuspended in culture medium. Cells were observed using a Nikon A1 ECLIPSE Ti2 inverted laser confocal microscope within 1 hour, and fluorescence intensity was analyzed using ImageJ software.

[0066] 11. Detection of lipid peroxidation After seeding and treating the cells, 2 μM BDPY581 / 591 C11 (Beyotime Biotechnology Co., Ltd., #S0043S) medium was added, and the cells were incubated at 37°C for 20 minutes. After washing twice with PBS, the cells were observed using a Nikon A1 ECLIPSE Ti2 inverted laser confocal microscope within 1 hour. The level of lipid peroxidation was assessed by the decrease in the red / green fluorescence intensity ratio.

[0067] 12. Construction of CRISPR-Cas9 knockout cell lines The Cas9-sgRNA ribonucleoprotein (RNP) complex was electroporated into HeLa cells using a CUY21EDIT II electroporator to construct a SYVN1 CRISPR-Cas9 knockout cell line. Four sgRNAs targeting the SYVN1 gene (SYVN1 sgRNA1: CAATTCATAGGAGAAAGTGG; SYVN1 sgRNA2: TGGTCAGGTACACCACAGTG; SYVN1 sgRNA3: CCGCACGGCAGTGATGATGG; SYVN1 sgRNA4: ACCAGTCCAGCCCCAGCA) were mixed and electroporated. To verify the knockout effect of the SYVN1 gene, genomic DNA was extracted from the electroporated cells using the QuickExtract™ DNA extraction kit (Lucigen, #QE09050). The SYVN1 gene was amplified by PCR, and the results were verified by agarose gel electrophoresis and DNA sequencing. The sequencing primers for the SYVN1 gene are: forward primer CTCCTCCAACATTGCAGCTT (SEQ ID NO.1) and reverse primer CTTCCCTACTTACCTGACCTCT (SEQ ID NO.2).

[0068] 13. CRISPR-Cas9 Knockout Screening The gRNA library used (Scishare, #SSLP044S) targets 903 genes and contains 9391 unique guide sequences (10 sgRNAs per gene). Lentiviral viruses targeting ubiquitin-related proteins were used to infect HeLa cells at a multiplicity of infection (MOI) of 0.3. Forty-eight hours after transduction, cells were treated with 1.5 μg / ml⁻¹ puromycin for two days for selection. After puromycin selection, Pex3-mCherry-GBP IRES-EGFP was transfected into puromycin-resistant cells and cultured for 48 hours. Cells with the top 5% GFP positivity were sorted using a BD FACSAria Fusion flow cytometer, and flow cytometry data were collected using BD FACSDiva software. The collected cells were centrifuged, genomic DNA was extracted, and the sgRNA coding regions were amplified using a one-step CRISPR NGS library construction kit (Yomebio, #PK201) according to the kit instructions. PCR products were sequenced on an Illumina NovaSeq X Plus sequencer, and data analysis was performed using MaGeCK software. Genes enriched in the sorted cells were ranked according to the positive selection-log (PosScore).

[0069] 14. siRNA knockdown HeLa cells were seeded in 24-well plates and cultured to 80% confluence. Following the kit instructions, 20 pmol of the SYVN1 siRNA complex (SYVN1 siRNA1: 5'-CAGGCUUCAUCAAGGUUCUTT-3' (SEQ ID NO.3); SYVN1 siRNA2: 5'-AGAACCUUGAUGAAGCCUGTT-3' (SEQ ID NO.4)) was co-transfected into the cells with Lipofectamine 3000 transfection reagent. Cells were cultured at 37°C in a 5% CO2 incubator for 48-72 hours, and the knockdown effect of the siRNA was verified by Western blotting.

[0070] 15. Western blot (WB) Primary Antibodies: Discard the blocking buffer and dilute the primary antibodies with the prepared 5% skim milk. The primary antibodies used include: GFP-tagged rabbit monoclonal antibody (Zenbio, #R24437, 1:1000 dilution), β-actin rabbit monoclonal antibody (ABclonal, #AC026, 1:50000 dilution), glyceraldehyde-3-phosphate dehydrogenase polyclonal antibody (Bioworld, #AP0063, 1:5000 dilution), glutathione peroxidase 4 rabbit monoclonal antibody (Zenbio, #R381958, 1:1000 dilution), glucose-regulated protein 78 rabbit polyclonal antibody (Zenbio, #350216, 1:1000 dilution), ROR1 mouse monoclonal antibody (Zenbio, #222703, 1:1000 dilution), and RhoA rabbit polyclonal antibody (Zenbio, #346086, 1:1000 dilution), activated caspase-3 p17 rabbit polyclonal antibody (Zenbio, #341034, 1:1000 dilution), K48 ubiquitinated rabbit polyclonal antibody (Zenbio, #381517, 1:1000 dilution), K63 ubiquitinated rabbit monoclonal antibody (Zenbio, #R381564, 1:1000 dilution), SYVN1 mouse monoclonal antibody (Zenbio, #660171, 1:1000 dilution), AKT1 / 2 / 3 rabbit monoclonal antibody (Zenbio, #R23412, 1:1000 dilution), phosphorylated AKT1 (Ser473) rabbit monoclonal antibody (Zenbio, #R381555, 1:1000 dilution), ERK1 / 2 rabbit polyclonal antibody (Zenbio, #R343830, 1:1000 dilution) and phosphorylated ERK1 / 2 (Thr202 / Tyr204) / (Thr185 / Tyr187) rabbit monoclonal antibody (Zenbio, #R381555, 1:1000 dilution).

[0071] 16. EdU proliferation experiment (1) Select cells in good growth state and in the logarithmic growth phase. In a 96-well plate suitable for confocal microscope, seed 15,000 cells per well and culture the cells to the normal growth stage. Depending on the concentration of the added drug, a total of 6 wells of cells need to be seeded. This experiment was repeated twice.

[0072] (2) After adding nanobody drugs of different concentration gradients, the EdU solution was diluted with whole culture medium hDMEM to a final concentration of 50 μM, and 100 μL was added to each well to incubate the cells for 2 h. After 2 h, the culture medium was removed.

[0073] (3) Wash the cells gently with PBS solution and let them stand for 5 min. The washing process needs to be repeated twice.

[0074] (4) Remove the PBS solution, then add 50 μL of PBS solution containing 4% paraformaldehyde to each of the 6 wells, incubate the cells for about 30 min, and then remove the fixative from the wells.

[0075] (5) Add 50 μL of 2 mg / mL glycine solution to each well. This solution needs to be prepared in advance before the experiment. Then place the 96-well plate on a shaker and shake it for 5 min. After that, remove the 96-well plate from the shaker and remove the glycine solution from the well.

[0076] (6) Add 100 mL of PBS solution to each well, incubate on a shaker for 5 min, and remove the PBS solution from the well.

[0077] (7) Add 100 μL of pre-prepared PBS solution containing 0.5% Triton X-100 to each of the 6 wells, incubate on a shaker for about 10 min, and remove the permeabilizer from the wells.

[0078] (8) Add 100 mL of PBS solution to each well, incubate on a shaker for 5 min, and remove the PBS solution from the well.

[0079] (9) Prepare 1 mL of 1×Apollo staining solution required for the experiment. Mix 938 μL of sterile water, 50 μL of reaction buffer, 10 μL of catalyst, 9 mg of buffer additive and 3 μL of fluorescent dye solution together and add them to a 1.5 mL EP tube.

[0080] (10) Add 100 μL of 1×Apollo staining reaction solution to each well, wrap the 96-well plate with tin foil, protect it from light, place it on a shaker, incubate for 30 min, and then remove the staining reaction solution from the well.

[0081] (11) Add 100 μL of pre-prepared PBS solution containing 0.5% Triton X-100 to each well and incubate it on a shaker for about 10 min. Repeat this process 3 times.

[0082] (12) Dilute Hoechst33342 with sterile water to make a 1×Hoechst33342 reaction solution, wrap the 96-well plate with tin foil, and store it in the dark.

[0083] (13) Add 100 μL of the pre-diluted 1×Hoechst33342 reaction solution to each well. Wrap the 96-well plate with tin foil at room temperature, protect it from light, and place it on a shaker. Incubate for 30 min and then remove the reaction solution from the well.

[0084] (14) Add 100 mL of PBS solution to each well, incubate on a shaker for 5 min, and remove the PBS solution from the well. Repeat 3 times.

[0085] (15) Add 100 mL of PBS solution to each well and observe under a microscope. 17. Quantitative proteomics analysis Cells were lysed using commercial RIPA lysis buffer containing a 1× protease inhibitor (Beyotime Biotechnology Co., Ltd.) and incubated at 4°C for 10 minutes. Cells were scraped, further disrupted by probe sonication, and the supernatant was collected by centrifugation. Protein concentration was determined using the BCA method, and 100 μg of total protein from each sample was used for in-solution enzymatic digestion. Specific steps: 100 μl of lysis buffer was taken, reduced with 10 mM dithiothreitol (DTT), alkylated with 20 mM indole-3-acetic acid (IAA), and then 1 μg / μl trypsin was added. The digestion was incubated overnight at 37°C. The digested peptides were collected by centrifugation, and the digestion was terminated by adding trifluoroacetic acid. The acidified peptides were desalted using a C18 pipette tip, eluted with 50% acetonitrile, vacuum dried, and then reconstituted with 25 μl of 0.1% trifluoroacetic acid.

[0086] Desalted peptides were separated by online nano-level liquid chromatography (NLC) and mass spectra were obtained using an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) in data-independent acquisition (DIA) mode. All raw data files were aligned with the UniProt human protein sequence database in Proteomics Discovery (PD) software (version 2.5). The peptide precursor mass tolerance was 10 ppm, the fragment ion mass tolerance was 0.02 Da, two missed cleavages at the trypsin restriction site were allowed, and cysteine ​​carbamoyl methylation was set as a fixed modification. Each group contained three biological replicates. 18. Hematoxylin-eosin (H&E) staining Tumor tissues and major organs were fixed in 4% paraformaldehyde at 4°C for 12 hours, followed by dehydration, clearing, embedding, and sectioning. After dewaxing and antigen retrieval, the sections were stained with hematoxylin and eosin, then dehydrated and mounted. The stained sections were observed and photographed under an optical microscope. Ki67 immunohistochemical (IHC) staining was performed by Nanjing Friss Biotechnology Co., Ltd.

[0087] 19. Image Analysis Microscopic images were analyzed and processed using ImageJ / Fiji software, and images were created using Microsoft Office PowerPoint for presentation. Image processing included only brightness adjustment (linear stretching), background subtraction, cropping, rotation, scaling, and pseudo-color encoding using lookup tables (LUTs). Colocalization analysis was performed using the Pearson correlation coefficient (PCC) plugin from the "Manders_Coefficients.class" plugin for ImageJ / Fiji, which is provided as supplementary software. Typically, only one cell is present per imaging field; if multiple cells are present, a single cell is selected using the polygon selection tool, and redundant cells are removed (edit / clean the exterior) before PCC analysis. Images were converted to 8-bit depth (image / type / 8-bit) before analysis, and at least 10 cells were analyzed per PCC analysis.

[0088] The procedure for determining the peroxisome / cytoplasmic fluorescence intensity ratio is as follows: First, in ImageJ / Fiji software, perform automatic threshold analysis on the EGFP channel (Image / Adjustment / Threshold / Auto) to identify the mitochondrial region, select and save it using the magic wand tool (Analysis / Tools / ROI Manager / Add); then, measure the average mCherry fluorescence intensity (Iper) and area (Sper) of the peroxisome region (Analysis / Measurement); simultaneously, measure the total cell area (Scell) and average mCherry fluorescence intensity (Icell) of each cell; calculate the average cytoplasmic area (Scytosol=Scell–Sper) and average cytoplasmic fluorescence intensity [Icytosol=(Icell×Scell–Iper×Sper) / Scytosol]; finally, calculate the mitochondrial / cytoplasmic fluorescence intensity ratio (ratio=Iper / Icytosol).

[0089] 20. Image processing and analysis of mitochondrial morphology Mitochondria morphology was analyzed using Fiji software (ImageJ, National Institutes of Health) equipped with the Mitochondria Analyzer plugin, which was installed via the Fiji update site "IJPB-Plugins". First, the images were converted to 8-bit (Image › Type › 8-bit) and the spatial scale was calibrated to micrometers (Image › Attributes). To obtain an accurate binarized mask, the "2D Threshold Optimize" tool was used to visually compare the generated mask with the original image to determine the optimal threshold parameters (e.g., block size = 25, C value = 3). Using the optimized parameters, the final binarized image was generated using the "2D Threshold" module. Quantitative analysis of mitochondrial morphology was performed using the "2D Analysis" module, outputting parameters including mitochondrial coverage area, morphological factor, aspect ratio, and particle number. All measurement results were exported for statistical analysis.

[0090] 21. Statistical Analysis and Experimental Replication Unless otherwise specified, all microscopic imaging experiments were repeated at least three times to ensure representativeness; representative laser confocal microscopy images were derived from at least 10 independent cells, ensuring consistency. This study did not employ a randomized or blinded experimental design. Graphpad and Microsoft Excel were used for plotting, data fitting, and statistical analysis. Student's t-test was used to compare differences between experimental conditions; unless otherwise specified, one-sided unpaired t-tests were used (e.g., comparison between drug-treated and untreated groups). Asterisks were used to indicate p-values ​​for statistical tests when necessary. : P < 0.05; P < 0.01； : P<0.001; ****: P<0.0001), key experiments are labeled with precise P values.

[0091] PexTAC construction method 1. Gene Fragment Synthesis: Genes encoding peroxisome targeting modules (such as PEX3N), nanobodies (such as NbROR1, NbHSPA5, NbGPX4), linkers (GGSGGS), and fluorescent reporter proteins (mCherry) are obtained through gene synthesis. (1) Single-specific PexTAC: GFP-PexTAC (i.e. PEX3N-mCherry-GBP): (SEQ ID NO.5) MLRSVWNFLKRHKKKCIFLGTVLGGVYILGKYGQKKIREIQES( PEX3N ）GRPVATMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKSGGGSGGSVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS*; ROR1-PexTAC (i.e., PEX3N-mCherry-NbROR1): (SEQ ID NO.6) MLRSVWNFLKRHKKKCIFLGTVLGGVYILGKYGQKKIREIQES GRPVATMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKSGGGSGGSRCAAAESGGGLVQPGGSLTLSCAASGRISGFLAMSWYRQPPGKQREFVARISSRSNTAYSDSVRGRFTISRDNAKNTVYLRMNNLKSEDTAVYYCGAGTNWDPGYWGQGTQVTVSS* (2) Bispecific PexTAC: HSPA5 / GPX4-BsPexTAC (i.e., PEX3N-mCherry-NbHSPA5-NbGPX4, abbreviated as PEX3N-NbH5 / G4): (SEQ ID NO.7) MLRSVWNFLKRHKKKCIFLGTVLGGVYILGKYGQKKIREIQESGRPVATMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKSGGSEVQLQQSGGGSVQAGGSLRLSCVASGYTRIHNHVGWFRQDSGKEREGVAAIYTGDGTQTYAASVKGRFTISHDNANNTVYLQMNSLKPEGTGMYYCAQGAYSDIPLLIYDYDTWGQGTLVTVSSLYKSGGGSGGSEVQLQESGGGSVQAGGSLRLSCAASGDTSSRYYMGWFRQAPGKEREVVAGFTSMDGSTSYADSVKGRFTMSQDNAKNTVYLQMDSLKVEDTAMYYCATGRAWAAGRPAYGPFDLRRYNYWGQGTQVTV* Other single-target PexTACs (3)PEX2-PexTAC (i.e., PEX2-mCherry-GBP): (SEQ ID NO.8) MASRKENAKSANRVLRISQLDALELNKALEQLVWSQFTQCFHGFKPGLLARFEPEVKACLWVFLWRFT IYSKNATVGQSVLNIKYKNDFSPNLRYQPPSKNQKIWYAVCTIGGRWLEERCYDLFRNHHLASFGKVKQCVNFVIG LLKLGGLINFLIFLQRGKFATLTERLLGIHSVFCKPQNICEVGFEYMNRELLWHGFAEFLIFLLPLINVQKLKAKL [[ID= ​ PEX2 ）GRPVATMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKSGGGSGGSVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS* (4) PEX5-PexTAC (i.e., GBP-mCherry-PEX5): (SEQ ID NO.9) MVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSSGRPVATMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKSGGGSGGS ​ ​ ​ ​ ​ ​ ​ KELFLAAVRLDPTSIDPDVQCGLGVLFNLSGEYDKAVDCFTAALSVRPNDYLLWNKLGATLANGNQSEEAVAAYRR ALELQPGYIRSRYNLGISCINLGAHREAVEHFLEALNMQRKSRGPRGEGGAMSENIWSTLRLALSMLGQSDAYGAA DARDLSTLLTMFGLPQ( PEX5 ） * (5) PEX6-PexTAC (i.e., PEX6-mCherry-GBP): (SEQ ID NO.10) MALAVLRVLEPFPTETPPLAVLLPPGGPWPAAELGLVLALRPAGESPAGPALLVAALEGPDAGTEEQG PGPPQLLVSRALLRLLALGSGAWVRARAVRRPPALGWALLGTSLGPGLGPRVGPLLVRRGETLPVPGPRVLETRPA LQGLLGPGTRLAVTELRGRARLCPESGDSSRPPPPPVVSSFAVSGTVRRLQGVLGGTGDSLGVSRSCLRGLGLFQG EWVWVAQARESSNTSQPHLARVQVLEPRWDLSDRLGPGSGPLGEPLADGLALVPATLAFNLGCDPLEMGELRIQRY LEGSIAPEDKGSCSLLPGPPFARELHIEIVSSPHYSTNGNYDGVLYRHFQIPRVVQEGDVLCVPTIGQVEILEGSP EKLPRWREMFFKVKKTVGEAPDGPASAYLADTTHTSLYMVGSTLSPVPWLPSEESTLWSSLSPPGLEALVSELCAV LKPRLQPGGALLTGTSSVLLRGPPGCGKTTVVAAACSHLGLHLLKVPCSSLCAESSGAVETKLQAIFSRARRCRPA VLLLTAVDLLGRDRDGLGEDARVMAVLRHLLLNEDPLNSCPPLMVVATTSRAQDLPADVQTAFPHELEVPALSEGQ RLSILRALTAHLPLGQEVNLAQLARRCAGFVVGDLYALLTHSSRAACTRIKNSGLAGGLTEEDEGELCAAGFPLLA EDFGQALEQLQTAHSQAVGAPKIPSVSWHDVGGLQEVKKEILETIQLPLEHPELLSLGLRRSGLLLHGPPGTGKTL LAKAVATECSLTFLSVKGPELINMYVGQSEENVREVFARARAAAPCIIFFDELDSLAPSRGRSGDSGGVMDRVVSQ LLAELDGLHSTQDVFVIGATNRPDLLDPALLRPGRFDKLVFVGANEDRASQLRVLSAITRKFKLEPSVSLVNVLDC CPPQLTGADLYSLCSDAMTAALKRRVHDLEEGLEPGSSALMLTMEDLLQAAARLQPSVSEQELLRYKRIQRKFAAC （ PEX6 ） GRPVATMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKSGGGSGGSVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS* (6) PEX7-PexTAC (i.e., GBP-mCherry-PEX7): (SEQ ID NO.11) MVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSSGRPVATMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKSGGGSGGS MSAVCGGAARMLRTPGRHGYAAEFSPYLPGRLACATAQHYGIAGCGTLLILDPDEAGLRLFRSFDWND GLFDVTWSENNEHVLITCSGDGSLQLWDTAKAAGPLQVYKEHAQEVYSVDWSQTRGEQLVVSGSWDQTVKLWDPTV GKSLCTFRGHESIIYSTIWSPHIPGCFASASGDQTLRIWDVKAAGVRIVIPAHQAEILSCDWCKYNENLLVTGAVD CSLRGWDLRNVRQPVFELLGHTYAIRRVKFSPFHASVLASCSYDFTVRFWNFSKPDSLLETVEHHTEFTCGLDFSL QSPTQVADCSWDETIKIYDPACLTIPA( PEX7 ） * (7) PEX10 - PexTAC (i.e., PEX10 - mCherry - GBP): (SEQ ID NO.12) MAPAAASPPEVIRAAQKDEYYRGGLRSAAGGALHSLAGARKWLEWRKEVELLSDVAYFGLTTLAGYQT LGEEYVSIIQVDPSRIHVPSSLRRGVLVTLHAVLPYLLDKALLPLEQELQADPDSGRPLQGSLGPGGRGCSGARRW MRHHTATLTEQQRRALLRAVFVLRQGLACLQRLHVAWFYIHGVFYHLAKRLTGITYQALRPDPLRVLMSVAPSALQ LRVRSLPGEDLRARVSYRLLGVISLLHLVLSMGLQLYGFRQRQRARKEWRLHRGLSHRRASLEERAVSRNPLCTLC LEERRHPTATPCGHLFCWECITAWCSSKAECPLCREKFPPQKLIYLRHYR( PEX10 ） GRPVATMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKSGGGSGGSVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS* (8) PEX11-PexTAC (i.e., PEX11-mCherry-GBP): (SEQ ID NO.13) MDAFTRFTNQTQGRDRLFRATQYTCMLLRYLLEPKAGKEKVVMKLKKLESSVSTGRKWFRLGNVVHAI QATEQSIHATDLVPRLCLTLANLNRVIYFICDTILWVRSVGLTSGINKEKWRTRAAHHYYYSLLLSLVRDLYEISL QMKRVTCDRAKKEKSASQDPLWFSVAEEETEWLQSFLLLLFRSLKQHPPLLLDTVKNLCDILNPLDQLGIYKSNPG IIGLGGLVSSIAGMITVAYPQMKLKTR( PEX11 ） GRPVATMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKSGGGSGGSVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS* (9) PEX12-PexTAC (i.e., PEX12-mCherry-GBP): (SEQ ID NO.14) MAEHGAHFTAASVADDQPSIFEVVAQDSLMTAVRPALQHVVKVLAESNPTHYGFLWRWFDEIFTLLDL LLQQHYLSRTSASFSENFYGLKRIVMGDTHKSQRLASAGLPKQQLWKSIMFLVLLPYLKVKLEKLVSSLREEDEYS IHPPSSRWKRFYRAFLAAYPFVNMAWEGWFLVQQLRYILGKAQHHSPLLRLAGVQLGRLTVQDIQALEHKPAKASM MQQPARSVSEKINSALKKAVGGVALSLSTGLSVGVFFLQFLDWWYSSENQETIKSLTALPTPPPPVHLDYNSDSPL LPKMKTVCPLCRKTRVNDTVLATSGYVFCYRCVFHYVRSHQACPITGYPTEVQHLIKLYSPEN( PEX12 ）GRPVATMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKSGGGSGGSVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS* (10) PEX13 - PexTAC (i.e., PEX13 - mCherry - GBP): (SEQ ID NO.15) MASQPPPPPKPWETRRIPGAGPGPGPGPTFQSADLGPTLMTRPGQPALTRVPPPILPRPSQQTGSSSV NTFRPAYSSFSSGYGAYGNSFYGGYSPYSYGYNGLGYNRLRVDDLPPSRFVQQAEESSRGAFQSIESIVHAFASVS MMMDATFSAVYNSFRAVLDVANHFSRLKIHFTKVFSAFALVRTIRYLYRRLQRMLGLRRGSENEDLWAESEGTVAC LGAEDRAATSAKSWPIFLFFAVILGGPYLIWKLLSTHSDEVTDSINWASGEDDHVVARAEYDFAAVSEEEISFRAG DMLNLALKEQQPKVRGWLLASLDGQTTGLIPANYVKILGKRKGRKTVESSKVSKQQQSFTNPTLTKGATVADSLDE QEAAFESVFVETNKVPVAPDSIGKDGEKQDL( PEX13 ） GRPVATMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKSGGGSGGSVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS* (11) PEX16 - PexTAC (i.e., PEX16 - mCherry - GBP): (SEQ ID NO.16) MEKLRLLGLRYQEYVTRHPAATAQLETAVRGFSYLLAGRFADSHELSELVYSASNLLVLLNDGILRKELRKKLPVSLSQQKLLTWLSVLECVEVFMEMGAAKVWGELGRWLVIALIQLAKAVLRILLLLWFKAGLQTSPPIVPL DRETQAQPPDGDHSPGNHEQSYVGKRSNRVVRTLQNTPSLHSRHWGAPQQREGRQQQHHEELSATPTPLGLQETIA EFLYIARPLLHLLSLGLWGQRSWKPWLLAGVVDVTSLSLLSDRKGLTRRERRELRRRTILLLYYLLRSPFYDRFSE ARILFLLQLLADHVPGVGLVTTSQRAASPCLPARPHTQPWSPPAFLPGHP( PEX16 ） GRPVATMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKSGGGSGGSVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS* (12) PEX19 - PexTAC (i.e., GBP - mCherry - PEX19): (SEQ ID NO.17) MVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSSGRPVATMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKSGGGSGGS MAAAEEG CSVGAEADRELEELLESALDDFDKAKPSPAPPSTTTAPDASGPQKRSPGDTAKDALFASQEKFFQELFDSELASQA TAEFEKAMKELAEEEPHLVEQFQKLSEAAGRVGSDMTSQQEFTSCLKETLSGLAKNATDLQNSSMSEEELTKAMEG LGMDEGDGEGNILPIMQSIMQNLLSKDVLYPSLKEITEKYPEWLQSHRESLPPEQFEKYQEQHSVMCKICEQFEAE TPTDSETTQKARFEMVLDLMQQLQDLGHPPKELAGEMPPGLNFDLDALNLSGPPGASGEQCLIM( PEX19 ） * (13) PEX26-PexTAC (i.e., PEX26-mCherry-GBP): (SEQ ID NO.18) MKSDSSTSAAPLRGLGGPLRSSEPVRAVPARAPAVDLLEEAADLLVVHLDFRAALETCERAWQSLANH AVAEEPAGTSLEVKCSLCVVGIQALAEMDRWQEVLSWVLQYYQVPEKLPPKVLELCILLYSKMQEPGAVLDVVGAW LQDPANQNLPEYGALAEFHVQRVLLPLGCLSEAEELVVGSAAFGEERRLDVLQAIHTARQQQKQEHSGSEEAQKPN LEGSVSHKFLSLPMLVRQLWDSAVSHFFSLPFKKSLLAALILCLLVVRFDPASPSSLHFLYKLAQLFRWIRKAAFS RLYQLRIRD( PEX26 ） GRPVATMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKSGGGSGGSVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS* (14) PMP70-PexTAC (i.e., PMP70-mCherry-GBP): (SEQ ID NO.19) MAAFSKYLTARNSSLAGAAFLLLCLLHKRRRALGLHGKKSGKPPLQNNEKEGKKERAVVDKVFFSRLI QILKIMVPRTFCKETGYLVLIAVMLVSRTYCDVWMIQNGTLIESGIIGRSRKDFKRYLLNFIAAMPLISLVNNFLK YGLNELKLCFRVRLTKYLYEEYLQAFTYYKMGNLDNRIANPDQLLTQDVEKFCNSVVDLYSNLSKPFLDIVLYIFK LTSAIGAQVLGKILWH ( PMP�ŀ ）GRPVATMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKSGGGSGGSVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS* (15) Fis1-PexTAC (i.e., Fis1-mCherry-GBP): (SEQ ID NO.20) MEAVLNELVSVEDLLKFEKKFQSEKAAGSVSKSTQFEYAWCLVRSKYNDDIRKGIVLLEELLPKGSKE EQRDYVFYLAVGNYRLKEYEKALKYVRGLLQTEPQNNQAKELERLIDKAMKKDGLVGMAIVGGMALGVAGLAGLIG LAVSKSKS ( Fis1 ） GRPVATMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKSGGGSGGSVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS* (16) PTS1-PexTAC (i.e., GBP-mCherry-PTS1): (SEQ ID NO.21) MVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSSGRPVATMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPF AWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKSGGGSGGS PRYHLKP LHSKL ( PTS1 ） * 2. Vector construction: All vectors were constructed using Gibson cloning or subcloning techniques. The above-mentioned nanobody gene fragments were inserted into the expression vector (such as mCherry-C1) by PCR to construct the PexTAC expression plasmid. The peroxisome targeting module can be fused to the N-terminus or C-terminus of the nanobody (selected according to the peroxisome targeting requirements). 3. Construction of inducible expression vector: The PexTAC encoding gene was inserted into the tetracycline-induced expression vector (Tet-on) by PCR to construct a DOX-induced PexTAC expression plasmid, thereby achieving DOX-regulated conditional expression.

[0092] II. In vitro activity verification of PexTAC 1. Cell transfection: PexTAC expression plasmid is transfected into target cell lines (such as HeLa, A549) via liposome transfection (e.g., Lipomaster 3000); 2. Degradation activity detection: Fluorescence detection: Changes in fluorescence intensity of target proteins (such as EGFP) are observed using laser confocal microscopy to quantitatively analyze degradation efficiency; Western blotting (WB): Detects changes in the protein level of target proteins and verifies the degradation effect; 3. Mechanism verification: Inhibitor experiments: The degradation pathway dependence was verified by adding proteasome inhibitor MG132, lysosomal inhibitor BafA1, or autophagy inhibitor chloroquine (CQ), respectively. Ubiquitination detection: The K48-linked ubiquitination level of the target protein was detected by pull-down assay; 4. Cell function testing: Proliferation assay: The inhibitory effect on tumor cell proliferation was detected by the EdU assay; Apoptosis detection: The expression of activated caspase-3 was detected by Western blotting, or the proportion of Annexin V positive cells was detected by flow cytometry; Ferrocyte detection: Lipid peroxidation levels are detected using a lipid peroxidation probe (BDPY581 / 591 C11) or mitochondrial membrane potential probe (TMRE) is used to detect changes in mitochondrial function.

[0093] III. In vivo activity verification of PexTAC 1. Virus preparation: The PexTAC encoding gene was inserted into an AAV vector (such as AAV6), and recombinant AAV was prepared by packaging in AAV293T cells. After purification, the viral titer was measured. 2. Construction of xenograft tumor model: Tumor cells (such as A549) were subcutaneously injected into the axilla of BALB / c nude mice to construct a xenograft tumor model; 3. In vivo administration: After tumor formation (1-2 weeks), AAV-PexTAC (1×10¹² vg・μl⁻¹, 20 μl) is administered via intratumoral injection, once every 4 days, for a total of 4 injections; 4. Evaluation of therapeutic effect: Tumor growth monitoring: The length and width of the tumor are measured using calipers, and the tumor volume is calculated (V = (length × width²) / 2). Histological examination: After tumor tissue dissection, Western blot was used to verify the degradation effect of target proteins, H&E staining was used to observe tumor tissue apoptosis, and Ki67 immunohistochemistry was used to detect the inhibitory effect on tumor cell proliferation. Safety evaluation: Monitor mouse weight changes, perform H&E staining on major organs (heart, liver, spleen, lung, and kidney) to assess systemic toxicity.

[0094] PEX3N is a truncated peptide (amino acids 1-45) of the peroxisome-targeting protein PEX3. In this study, PEX3N was used as a peroxisome-targeting module and fused with the N-terminus of a nanobody to construct peroxisome-targeting chimeric structures (PexTACs), enabling the degradation of the target protein recognized by the nanobody (Figure 1a). To validate the design concept of PexTACs, we constructed a PEX3N-mCherry-GBP chimeric structure containing the PEX3N sequence, green fluorescent protein-binding protein (GBP, dissociation constant Kd = 1.2 nM), and mCherry fluorescent reporter protein.

[0095] For transient transfection, dissolve 0.25 μg of DNA in 12.5 μl of Gibco Opti-MEM medium using Lipomaster 3000 transfection reagent, add 0.5 μl of T3000 enhancer, and gently pipette to mix. Dissolve 0.5 μl of Lipomaster 3000 transfection reagent in 12.5 μl of Gibco Opti-MEM medium and mix with the DNA mixture. Add the mixture to a 2.5 × 10⁻⁶ m³ / h layer of medium. 4 In an 8-well plate containing HeLa cells, each well contained 250 μl of complete DMEM medium. After culturing the cells in a 37°C, 5% CO2 incubator for about 2 hours, the medium was replaced with preheated complete DMEM medium, and the cells were cultured in a 37°C, 5% CO2 incubator for more than 20 hours (when co-transfecting multiple plasmids, the amount of DNA used above refers to the total amount of plasmids used). After that, the cells were observed under a confocal microscope. The cells were divided into three groups: (1) EGFP expression alone; (2) EGFP co-expression with PEX3N-mCherry; (3) EGFP co-expression with PEX3N-mCherry-GBP. Compared with EGFP expression alone or co-expression of EGFP and PEX3N-mCherry, co-expression of EGFP and PEX3N-mCherry-GBP resulted in a significant decrease in the fluorescence intensity of EGFP (Figure 1b, left and upper right).

[0096] In 24-well plates, EGFP was transfected alone, co-transfected with EGFP and PEX3N-mCherry, and co-transfected with EGFP and PEX3N-mCherry-GBP. After 48 h of transfection, cell samples were collected for Western blot analysis. Cells were incubated with a GFP-tagged rabbit monoclonal antibody. The results further confirmed that PEX3N-mCherry-GBP can induce EGFP degradation (Figure 1b, bottom right), verifying the effectiveness of the PexTAC strategy.

[0097] To achieve precise temporal regulation of PexTAC expression, we constructed a tetracycline-induced expression system (Tet-on). Figure 2 a) The PexTAC encoding gene was inserted into a tetracycline-induced expression vector (Tet-on). Different concentrations (0, 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2 μg / ml) of doxycycline (DOX) were added to HeLa cells. After 48 h of induction, cell samples were collected and subjected to Western blotting (WB). Incubation with a GFP-tagged rabbit monoclonal antibody showed that PexTAC expression induced EGFP degradation in a dose-dependent manner (Figure 2b). Further analysis showed that PexTAC expression induced EGFP degradation in a time-dependent manner by adding 0.5 μg / ml of doxycycline (DOX) to HeLa cells and collecting cell samples at different time points (0, 3, 6, 12, 24, 36, 48 h). After all cell samples were collected, WB was performed, and incubation with a GFP-tagged rabbit monoclonal antibody showed that PexTAC expression induced EGFP degradation in a time-dependent manner (Figure 2c).

[0098] By adding 0.5 μg / ml doxycycline (DOX) to HepG2, A549, Capan-1, MDA-MB-231, MCF-7, OVCAR3, and SK-BR-3 cells, cell samples were collected after 48 h. Western blot analysis was performed on all collected cell samples, followed by incubation with a GFP-tagged rabbit monoclonal antibody. The results showed that DOX-regulated PexTAC-induced efficient EGFP degradation was also observed in several other cell lines, confirming the broad applicability of this strategy. Figure 3 ).

[0099] Based on the localization characteristics of different peroxisome proteins, a series of novel PexTACs were constructed by fusing multiple peroxisome proteins (such as PEX2, PEX3, PEX10, PEX12, and PMP70) to the N-terminus or C-terminus of PexTAC. All vector constructions employed Gibson cloning technology. The EGFP nanobody gene fragment was inserted into the expression vector (pmCherry-C1) via PCR to construct the PexTAC expression plasmid. The peroxisome targeting module could be fused to the N-terminus or C-terminus of the nanobody according to its localization. Figure 4a) In the experiment, 0.5 μg of DNA was dissolved in 25 μl of Gibco Opti-MEM medium, 1 μl of T3000 enhancer was added, and the mixture was gently pipetted to mix. 1 μl of Lipomaster 3000 transfection reagent was dissolved in 25 μl of Gibco Opti-MEM medium and mixed with the DNA mixture. The mixture was then added to a medium bed of 6 × 10⁻⁶ cells / mL. 4 In 24-well plates containing adherent HeLa cells, each well contained 500 μl of complete DMEM medium. (When co-transfecting multiple plasmids, the above DNA amount refers to the total amount of plasmids used.) Cell samples were collected after 48 h and then subjected to Western blotting. Incubation with a GFP-tagged rabbit monoclonal antibody showed that, compared with mCherry-GBP, co-expression of EGFP and PexTACs led to varying degrees of EGFP degradation. Figure 4 b).

[0100] Based on the localization characteristics of different peroxisome proteins, a series of novel PexTACs were constructed by fusing multiple peroxisome proteins (such as PEX2, PEX3, PEX10, PEX12, and PMP70) to the N-terminus or C-terminus of PexTAC. All vector constructions employed Gibson cloning technology. The EGFP nanobody gene fragment was inserted into the expression vector (pmCherry-C1) via PCR to construct the PexTAC expression plasmid. The peroxisome targeting module could be fused to either the N-terminus or C-terminus of the nanobody according to its localization. For transfection, 0.5 μg of DNA was dissolved in 25 μl of GibcoOpti-MEM medium, and 1 μl of T3000 enhancer was added and gently mixed. 1 μl of Lipomaster 3000 transfection reagent was dissolved in 25 μl of GibcoOpti-MEM medium and mixed with the DNA mixture. The mixture was then added to a 6×10⁶ layer of medium. 4 In 24-well plates containing adherent A549 and HepG2 cells, each well contained 500 μl of complete DMEM medium. (When co-transfecting multiple plasmids, the above DNA amount refers to the total amount of plasmids used.) Cell samples were collected after 48 h and then subjected to Western blotting. Incubation with a GFP-tagged rabbit monoclonal antibody showed that, compared with mCherry-GBP, co-expression of EGFP and PexTACs led to varying degrees of EGFP degradation. Figure 5Therefore, the PexTAC strategy, which achieves target protein degradation by fusing peroxisome-anchored proteins with nanobodies, is a widely applicable platform for targeted protein degradation.

[0101] EGFP, PEX3N-mCherry-GBP, and Lamp1-TagBFP, TagBFP-LC3B, and TagBFP-p62 were co-expressed in HeLa cells in 8-well dishes. After 24 hours, confocal microscopy was used to observe whether the green fluorescent spots of EGFP overlapped with lysosomal, autophagosome, and autophagy receptor spots. The results showed that EGFP did not have significant colocalization with the lysosomal marker LAMP1, or the autophagy markers LC3B and p62, indicating that the degradation process was independent of the lysosomal and autophagy pathways (Figures 6a-c).

[0102] Secondly, in 24-well plates, cells were expressed with EGFP only, co-expressed with EGFP and PEX3N-mCherry, and co-expressed with EGFP and PEX3N-mCherry-GBP. Then, 500 nM MG132 and 400 nM BafA1 were added. After 48 hours, cell samples were collected, lysed, and the supernatant was obtained. Western blot analysis showed that the proteasome inhibitor MG132 completely inhibited EGFP degradation, while the autophagy / lysosome inhibitor BafA1 had no such effect, indicating that the degradation process depends on the ubiquitin-proteasome system (UPS). Figure 6 d).

[0103] In 24-well plates, EGFP was expressed only, co-expressed with EGFP and PEX3N-mCherry, and co-expressed with EGFP and PEX3N-mCherry-GBP. Then, 500 nM MG132 and 400 nM BafA1 were added. After 48 h, cell samples were collected, lysed, and the supernatant was incubated overnight with magnetic beads bound to rabbit monoclonal antibodies with a GFP tag. The pulled-down samples were then subjected to Western blotting and incubated with K48 ubiquitination antibodies and K63 ubiquitination antibodies. The pull-down results showed that EGFP underwent K48 ubiquitination modification mediated by the ubiquitin-proteasome system, rather than autophagy-related K63 ubiquitination modification (Figure 6e).

[0104] Secondly, in 24-well plates, EGFP was expressed alone, co-expressed with PEX2-mCherry, and co-expressed with PEX2-mCherry-GBP; EGFP was expressed alone, co-expressed with PEX10-mCherry, and co-expressed with PEX10-mCherry-GBP; EGFP was expressed alone, co-expressed with PEX12-mCherry, and co-expressed with PEX12-mCherry-GBP; EGFP was expressed alone, co-expressed with PMP70-mCherry, and co-expressed with PMP70-mCherry-GBP. Then, HeLa cells were treated with 500 nM MG132, 400 nM BafA1, and 20 μM CQ. After 48 hours, cell samples were collected, lysed, and the supernatant was obtained. Western blotting analysis showed that a series of peroxisome-mediated PexTAC targeting pathways were all mediated through ubiquitin- The proteasome system mediates target protein degradation, independent of lysosomal and autophagy pathways. Figure 7 Therefore, the PexTAC platform performs degradation functions through the ubiquitin-proteasome system.

[0105] A CRISPR knockout gRNA library targeting ubiquitin-related proteins was used for research via CRISPR screening (Figure 8a). Next-generation sequencing (NGS) identified several high-scoring proteins, with SYVN1 being the only E3 ubiquitin ligase (Figure 8b). HeLa cells were seeded in 24-well plates and cultured to 80% confluence. Following the kit instructions, 20 pmol of the SYVN1 siRNA complex (SYVN1 siRNA1: 5'-CAGGCUUCAUCAAGGUUCUTT-3') and SYVN1 siRNA2: 5'-AGAACCUUGAUGAAGCCUGTT-3' were co-transfected into the cells with Lipofectamine 3000 transfection reagent. Cells were cultured at 37°C in a 5% CO2 incubator for 48–72 hours, and the knockdown effect of the siRNA was verified by Western blotting. The results showed that siRNA-mediated SYVN1 knockdown significantly reduced the degradation activity of PEX3N-mCherry-GBP (Figure 8c), suggesting that SYVN1 may be an E3 ubiquitin ligase mediating the degradation process of PexTAC.

[0106] To further verify this conclusion, we constructed a SYVN1 knockout (KO) HeLa cell line and used an electroporator to electrotransfect the Cas9-sgRNA ribonucleoprotein (RNP) complex into HeLa cells to construct the SYVN1 CRISPR-Cas9 knockout cell line. Four sgRNAs targeting the SYVN1 gene (SYVN1 sgRNA1: CAATTCATAGGAGAAAGTGG (SEQ ID NO.22); SYVN1 sgRNA2: TGGTCAGGTACACCACAGTG (SEQ ID NO.23); SYVN1 sgRNA3: CCGCACGGCAGTGATGATGG (SEQ ID NO.24); SYVN1 sgRNA4: ACCAGTCCAGCCCCAGCA (SEQ ID NO.25)) were mixed and electroporated. Successful knockout single-cell clones were then screened. Western blot analysis showed that PEX3N-mCherry-GBP completely lost its ability to induce EGFP degradation in the knockout cell line (Figure 8d).

[0107] In knockout cell lines, wild-type SYVN1 was overexpressed to restore its original function. Then, EGFP, EGFP and PEX3N-mCherry, and EGFP and PEX3N-mCherry-GBP were overexpressed in these cells. After 48 hours of expression, cell samples were collected, and Western blotting showed that reintroducing SYVN1 restored the degradation activity of PEX3N-mCherry-GBP against EGFP (Figure 8e). In knockout cell lines, the mutant SYVN1 (C329S) lost its E3 ubiquitin ligase activity. Similarly, EGFP, EGFP and PEX3N-mCherry, and EGFP and PEX3N-mCherry-GBP were overexpressed in HeLa cells. After 48 hours of expression, cell samples were collected, and Western blotting showed that the expression of the inactive SYVN1 (C329S) mutant could not restore its degradation activity (Figure 8f).

[0108] In KO cells, EGFP was expressed alone, co-expressed with EGFP and PEX3N-mCherry, and co-expressed with EGFP and PEX3N-mCherry-GBP. Then, 500 nM MG132 was added, and cell samples were collected after 48 h. The supernatant was lysed and incubated overnight with protein A / G magnetic beads bound to SYVN1 mouse monoclonal antibody. Western blot analysis was performed on the pulled-down samples. Incubation with GFP-tagged rabbit monoclonal antibody confirmed that K48 ubiquitination modification was detected only in knockout cells expressing SYVN1, and not in cells expressing the inactive SYVN1 (C329S) mutant (Fig. 8g). In the presence of PEX3N-mCherry-GBP, SYVN1 ligase interacted with EGFP (Fig. 8h).

[0109] To verify whether PexTACs fused with other peroxisome proteins also depend on SYVN1 for their function, we performed EGFP degradation experiments using these PexTACs in SYVN1 knockout cell lines. A SYVN1 knockout (KO) HeLa cell line was constructed, and the Cas9-sgRNA ribonucleoprotein (RNP) complex was electroporated into HeLa cells to construct a SYVN1 CRISPR-Cas9 knockout cell line. Four sgRNAs targeting the SYVN1 gene (SYVN1 sgRNA1: CAATTCATAGGAGAAAGTGG; SYVN1 sgRNA2: TGGTCAGGTACACCACAGTG; SYVN1 sgRNA3: CCGCACGGCAGTGATGATGG; SYVN1 sgRNA4: ACCAGTCCAGCCCCAGCA) were mixed and electroporated. In the knockout cell line, for transfection, 0.5 μg of DNA was dissolved in 25 μl of Gibco Opti-MEM medium, 1 μl of T3000 enhancer was added, and the mixture was gently pipetted to mix. 1 μl of Lipomaster 3000 transfection reagent was dissolved in 25 μl of Gibco Opti-MEM medium and mixed with the DNA mixture. The mixture was then added to a medium bed of 6 × 10⁻⁶ cells / mL. 4In 24-well plates containing adherent A549 and HepG2 cells, each well contained 500 μl of complete DMEM medium. (When co-transfecting multiple plasmids, the above DNA amount refers to the total amount of plasmids used.) Cell samples were collected after 48 h and then subjected to Western blotting. Incubation with a GFP-tagged rabbit monoclonal antibody showed that, compared to mCherry-GBP, all PexTACs fused with peroxisome proteins failed to induce EGFP degradation. Figure 9 (a, b). Therefore, the PexTAC platform, which achieves degradation by directing target proteins to the peroxisome membrane, relies on the SYVN1-mediated ubiquitin-proteasome system pathway.

[0110] We further explored the therapeutic potential of PexTAC. Orphan receptor 1 (ROR1), a type I transmembrane pseudokinase receptor, is silenced in most normal adult tissues but is abnormally overexpressed in various hematologic malignancies and solid tumors, promoting tumor cell survival, proliferation, and metastasis. Therefore, ROR1 is considered a potential effective target for cancer therapy, such as non-small cell lung cancer. To achieve precise temporal regulation of PexTAC-ROR1 expression, we constructed a tetracycline-inducible expression system (Tet-on): the PexTAC-ROR1 encoding gene was inserted into a tetracycline-inducible expression vector (Tet-on). By adding different concentrations (0, 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2 μg / ml) of doxycycline (DOX) to A549 cells and inducing cell expression for 48 h, cell samples were collected and subjected to Western blotting (WB) with ROR1 mouse monoclonal antibody. The results showed that PexTAC-ROR1 expression induced ROR1 degradation in a dose-dependent manner (Figure 10a). By adding 0.5 μg / ml of doxycycline (DOX) to A549 cells, cell samples were collected at different time points (0, 12, 24, 36, 48, 60, 72 h). After all cell samples were collected, WB was performed with ROR1 mouse monoclonal antibody incubation. The results showed that PexTAC-ROR1 expression induced ROR1 degradation in a time-dependent manner (Figure 10b). Mechanistic studies confirmed that the proteasome inhibitor MG132 completely inhibited this degradation process, while BafA1 had no such effect, suggesting that the degradation process is based on the ubiquitin-proteasome system and is independent of the lysosomal pathway (Figure 10c). Quantitative proteomics analysis verified the specificity of PexTAC for ROR1 degradation (Figure 10d).

[0111] High-efficiency expression of PEX3N-mCherry-NbROR1 was induced by adding 0.5 μg / ml doxycycline (DOX) to HeLa, HepG2, A549, Capan-1, MDA-MB-231, and OVCAR3 cells. Cell samples were collected after 48 h. After all cell samples were collected, Western blot analysis was performed. The cells were incubated with ROR1 mouse monoclonal antibody. The results showed that DOX-regulated PexTAC induced efficient degradation of ROR1 in a variety of other cell lines, confirming the broad applicability of this strategy (Figure 11).

[0112] We further investigated the biological effects of ROR1 degradation on cancer cells. The Pex3N-mCherry and PexTAC-ROR1 encoding genes were inserted into a tetracycline-induced expression vector (Tet-on). A549 cells were induced with 0.5 μg / ml doxycycline (DOX), and cell samples were collected after 48 h of induction. Western blot analysis was performed using a mouse monoclonal antibody against ROR1. The results showed that ROR1 degradation significantly upregulated activated caspase-3, indicating apoptosis (Figure 12a). Furthermore, flow cytometry was used to detect apoptosis using Annexin V / PI double staining. Cells were collected (suspension cells were directly centrifuged, and adherent cells were gently digested with trypsin without EDTA), washed with pre-chilled PBS, and resuspended in 1×Annexin V binding buffer to a density of approximately 1×10⁻⁶. 6 Cells / mL: Take 100 μL of cell suspension, add Annexin V-FITC and PI staining solution, incubate at room temperature in the dark for 15–20 minutes, then add binding buffer and filter. Analyze within 30 minutes, detecting Annexin V through the FITC channel and PI through the PE / PI channel. Collect at least 1 × 10⁻⁶ cells / mL per sample. 4 The cells were gating according to Annexin V / PI double negative (live cells), single positive (early apoptosis), and double positive (late apoptosis / necrosis). The results further confirmed that apoptosis induction was detected only in cells treated with PexTAC (Figure 12b).

[0113] We investigated the effects of ROR1 degradation on the ROR1-RhoA-YAP signaling axis, which is involved in cancer cell proliferation. Figure 13a) The Pex3N-mCherry and PexTAC-ROR1 encoding genes were inserted into a tetracycline-induced expression vector (Tet-on). A549 cells were induced with 0.5 μg / ml doxycycline (DOX), and cell samples were collected after 48 h of induction. Western blot analysis was performed on all collected cells, and incubation with a mouse monoclonal antibody against ROR1 showed that ROR1 degradation significantly downregulated RhoA (Figure 13b). Laser confocal microscopy analysis showed that the transcription factor YAP translocated from the nucleus to the cytoplasm (Figure 13c), indicating inhibited cell proliferation. Finally, after incubation with EdU, fixation, and permeabilization, EdU was labeled using a click reaction, and nuclei were stained with DAPI or Hoechst. Multiple fields of view were then captured under a confocal microscope, and the proportion of EdU-positive nuclei was counted. EdU cell proliferation experiments further confirmed that ROR1 degradation inhibited the proliferation of A549 lung cancer cells (Figure 13d). Meanwhile, Western blot analysis showed that pAKT and pERK expression was downregulated in the AKT-ERK signaling axis involved in cell proliferation. Figure 13 e).

[0114] Based on the localization characteristics of different peroxisome proteins, a series of novel PexTACs were constructed by fusing multiple peroxisome proteins (such as PEX2, PEX11, PEX19, and PMP70) to the N-terminus or C-terminus of PexTAC. All vector constructions employed Gibson cloning technology. The ROR1 nanobody gene fragment was inserted into the expression vector (pmCherry-C1) via PCR to construct the PexTAC expression plasmid. The peroxisome targeting module can be fused to the N-terminus or C-terminus of the nanobody according to its localization. To further demonstrate the broad applicability of the PexTAC platform, the experiment involved transfection. 0.5 μg of DNA was dissolved in 25 μl of Gibco Opti-MEM medium, and 1 μl of T3000 enhancer was added and gently mixed. 1 μl of Lipomaster 3000 transfection reagent was dissolved in 25 μl of Gibco Opti-MEM medium and mixed with the DNA mixture. The mixture was then added to a 6×10⁻⁶ layer of medium. 4In 24-well plates containing adherent A549 cells, each well contained 500 μl of complete DMEM medium. (When co-transfecting multiple plasmids, the above DNA amount refers to the total amount of plasmids used.) Cell samples were collected after 48 h and then subjected to Western blotting. Incubation with ROR1 mouse monoclonal antibody showed that, compared to PEX3N-mCherry, several PexTAC-ROR1 methods induced high degradation of ROR1, highlighting the high versatility of this platform. Figure 14 ad).

[0115] AAV6-PEX3N-NbROR1: The PexTAC-ROR1 gene sequence was amplified by PCR and inserted into the pAAV-CMV vector, as shown in the above embodiments.

[0116] We further constructed a PexTAC-ROR1 delivery system (AAV6-PEX3N-NbROR1) for adeno-associated virus (AAV) delivery, enabling its in vivo application. Figure 15 a). AAV6-PEX3N (1×10¹² vg·ml⁻¹, 20 μl), AAV6-PEX3N-NbROR1 (1×10¹² vg·ml⁻¹, 20 μl), or PBS was injected intratumorally into mice every 4 days for a total of 4 injections. Tumor size was measured daily using calipers, and tumor volume was calculated using the formula: V = (length × width²) / 2. Tumor growth curves showed that tumor growth was inhibited only in the AAV6-PEX3N-NbROR1 treatment group, while tumor growth was unaffected in the AAV6-PEX3N (control) group and the PBS (blank) group (Figures 15b-c). No significant changes in mouse body weight were observed during the experiment, indicating that PexTAC has extremely low systemic toxicity. Figure 15 d). Tumor weight analysis further confirmed that tumor growth was inhibited in the PexTAC treatment group ( Figure 15 After tumor dissection, Western blot analysis confirmed that ROR1 degradation was detected in tumors only in mice treated with PexTAC (Figure 15g). Finally, hematoxylin-eosin (H&E) staining and Ki67 immunohistochemical (IHC) staining confirmed that PexTAC can induce tumor cell apoptosis and inhibit their proliferation in vivo. Figure 15 The above results indicate that AAV-delivered PexTAC has good in vivo delivery safety and potential therapeutic value.

[0117] A tetracycline-induced expression system (Tet-on) was constructed, and the PexTAC-HSPA5 encoding gene was inserted into the tetracycline-induced expression vector (Tet-on). PEX3N-mCherry-NbHSPA5 expression was induced in HeLa cells by adding 0.5 μg / ml doxycycline (DOX). After 48 h of cell expression, cell samples were collected and analyzed by Western blotting. Incubation with a rabbit monoclonal antibody against HSPA5 showed that PEX3N-mCherry-NbHSPA5 can efficiently induce the degradation of HSPA5. Mechanistic studies confirmed that in 24-well plates, only PEX3N-mCherry and PEX3N-mCherry-NbHSPA5 were expressed. After the addition of 500 nM MG132 and 400 nM BafA1, cell samples were collected after 48 h, and the supernatant was obtained by lysis. The protein degradation mediated by PEX3N-mCherry-NbHSPA5 could be completely blocked by MG132, while BafA1 had no such effect. This verified that its degradation process depends on the ubiquitin-proteasome system and is not related to the lysosomal pathway (Figure 16).

[0118] A tetracycline-inducible expression system (Tet-on) was constructed, and the PexTAC-GPX4 encoding gene was inserted into the tetracycline-inducible expression vector (Tet-on). PEX3N-mCherry-NbGPX4 expression was induced in HeLa cells by adding 0.5 μg / ml doxycycline (DOX). After 48 h of cell expression, cell samples were collected and analyzed by Western blotting. Incubation with a rabbit monoclonal antibody against GPX4 showed that PEX3N-mCherry-NbGPX4 can efficiently induce GPX4 degradation. Mechanistic studies confirmed that in 24-well plates, only PEX3N-mCherry and PEX3N-mCherry-NbGPX4 were expressed. After the addition of 500 nM MG132 and 400 nM BafA1, cell samples were collected after 48 h, and the supernatant was obtained by lysis. The protein degradation mediated by PEX3N-mCherry-NbHSPA5 could be completely blocked by MG132, while BafA1 had no such effect. This verified that its degradation process depends on the ubiquitin-proteasome system and is not related to the lysosomal pathway (Figure 17).

[0119] To further expand the applications of the PexTAC platform, we explored the feasibility of simultaneously degrading two proteins along the same signaling axis to achieve a stronger synergistic effect than single-target degradation. Based on this, we designed a bispecific PexTAC (BsPexTAC) – PEX3N-mCherry-NbHSPA5-NbGPX4 (PEX3N-NbH5 / G4 for short). This chimera contains a bispecific nanobody that can recruit HSPA5 upstream and GPX4 downstream of the ferroptosis pathway, respectively (Figure 18a). The genes encoding PEX3N-mCherry, PEX3N-mCherry-NbHSPA5, PEX3N-mCherry-NbGPX4, and PEX3N-mCherry-NbHSPA5-NbGPX4 were inserted into a tetracycline-induced expression vector (Tet-on). HeLa cells were induced with 0.5 μg / ml doxycycline (DOX), and cell samples were collected after 48 h of induction. Western blot analysis was performed using rabbit monoclonal antibodies against GPX4 and HSPA5. The results showed that in HeLa cells, PEX3N-mCherry-NbGPX4 degraded GPX4, PEX3N-mCherry-NbHSPA5 degraded HSPA5 and partially reduced GPX4 expression, while PEX3N-NbH5 / G4 efficiently degraded both HSPA5 and GPX4 simultaneously. Figure 18 b).

[0120] Mechanistic studies confirmed that in 24-well plates expressing only PEX3N-mCherry, PEX3N-mCherry-NbHSPA5, PEX3N-mCherry-NbGPX4, and PEX3N-mCherry-NbHSPA5-NbGPX4, followed by the addition of 500 nM MG132 and 400 nM BafA1, cell samples were collected after 48 h, and the supernatant was obtained after lysis. The protein degradation mediated by PEX3N-mCherry-NbHSPA5, PEX3N-mCherry-NbGPX4, and the bispecific PEX3N-mCherry-NbH5 / G4 could all be completely blocked by MG132, while BafA1 had no such effect, verifying that its degradation process depends on the ubiquitin-proteasome system and is not related to the lysosomal pathway (Figure 19).

[0121] Cells were seeded at an appropriate density in 96-well plates and treated with PEX3N-mCherry, PEX3N-mCherry-NbHSPA5, PEX3N-mCherry-NbGPX4, and PEX3N-mCherry-NbHSPA5-NbGPX4. 10 μL of CCK-8 reagent was added to each well, and the plates were incubated in the dark for 1 hour (ideally until the OD value of the control wells reached 1.0–1.5). The absorbance (OD value) at 450 nm was measured using a microplate reader. Cell-free blank wells and solvent control wells were included. Cell viability (%) was calculated as (OD of experimental wells – OD of blank wells) / (OD of control wells – OD of blank wells) × 100%. Each group had three replicates. Interference from air bubbles and reducing agents should be avoided during the assay. The CCK-8 assay showed that, compared with the monospecific PexTAC targeting HSPA5 and GPX4, the bispecific PexTAC exhibited significantly higher cytotoxicity, suggesting a synergistic pharmacological effect. Furthermore, the ferroptosis inhibitor Fer-1 inhibited bispecific PexTAC-induced cytotoxicity, while the apoptosis inhibitor Q-VD-OPh had no such effect, indicating that the cell death mechanism was ferroptosis rather than apoptosis, consistent with the roles of HSPA5 and GPX4 in regulating ferroptosis (Figure 20).

[0122] Based on the simultaneous degradation of HSPA5 and GPX4-induced ferroptosis, we analyzed changes in mitochondrial phenotype, including mean branch length and mean perimeter of mitochondria. Cells were seeded at an appropriate density in 8-well dishes. After inducing the expression of PEX3N-mCherry, PEX3N-mCherry-NbHSPA5, PEX3N-mCherry-NbGPX4, and PEX3N-mCherry-NbHSPA5-NbGPX4 by adding doxycycline (DOX) at a concentration of 0.5 μg / ml for 24 h, confocal microscopy revealed, as expected, the most significant mitochondrial shrinkage was observed in HeLa cells expressing PEX3N-mCherry-NbHSPA5-NbGPX4, followed by cells expressing PEX3N-mCherry-NbGPX4, and lastly in cells expressing PEX3N-mCherry-NbHSPA5 (Figure 21).

[0123] We examined lipid peroxidation levels. After seeding and treating cells, we added 2 μM MBDPY581 / 591 C11 medium (Beyotime Biotechnology Co., Ltd., #S0043S) and incubated at 37°C for 20 minutes. After washing twice with PBS, we observed the cells using a Nikon A1 ECLIPSE Ti2 inverted laser confocal microscope within 1 hour. Lipid peroxidation levels were assessed by the decrease in the red / green fluorescence intensity ratio. The results showed that HeLa cells expressing bispecific PexTAC had the highest lipid peroxidation levels (Figure 22).

[0124] Next, we detected the mitochondrial membrane potential. Tetramethylrhodamine ethyl ester (TMRE) accumulated in highly polarized mitochondria, and its fluorescence intensity was positively correlated with the mitochondrial membrane potential (MMP). The mitochondrial membrane potential detection kit (containing TMRE) was purchased from Beyotime Biotechnology Co., Ltd. (#C2001S). After treatment, cells were incubated with 1× TMRE at 37°C in the dark for 30 minutes, washed twice with PBS, and resuspended in culture medium. Cells were observed using a Nikon A1ECLIPSE Ti2 inverted laser confocal microscope within 1 hour, and fluorescence intensity was analyzed using ImageJ software. The results showed that the bispecific PexTAC treatment group exhibited the most significant loss of mitochondrial membrane potential, followed by the GPX4-targeted PexTAC treatment group and the HSPA5-targeted PexTAC treatment group (Figure 23). In summary, the above results confirm that bispecific PexTAC exerts a synergistic effect in inducing ferroptosis in cancer cells by simultaneously degrading HSPA5 and GPX4 on the same signaling axis.

[0125] Organoids were dissociated into small clumps at 37°C for 5-10 minutes. The resulting cell clumps were resuspended in a transduction mixture containing organoid culture medium supplemented with an enhancer, 8 μg / mL polybrene, and concentrated lentivirus. Transduction was performed by centrifugation at room temperature. After centrifugation, the organoids were incubated at 37°C and 5% CO2 for 10 hours, then embedded in a matrix gel and re-inoculated into new 24-well plates. Figure 24 a). Two days after infection, transgene expression was induced by doxycycline treatment, which was continued for 2 days. Western blot (WB) analysis was then performed, showing that the bispecific PexTAC effectively degraded HSPA5 and GPX4. The bar chart represents the statistical results of the WB analysis. Figure 24b). 100 μL of CellTiter-Glo 2.0 reagent was added to each well of a 96-well plate, and the matrix gel was mechanically disrupted. After vigorous mixing on a plate shaker for 5 minutes and incubation at room temperature for 25 minutes, the luminescence value was measured. The results showed that bispecific PexTAC effectively inhibited the growth of cervical cancer organoids. Figure 24 c).

[0126] This study uncovered a novel organelle-targeted protein degradation mechanism and identified the previously little-studied E3 ubiquitin ligase SYVN1. Based on these findings, we constructed a modular PexTAC platform that achieves efficient and specific degradation of various disease-related target proteins by targeting 16 different peroxisome-associated proteins, and validated the anti-tumor efficacy of targeting ROR1 in vivo. This research not only provides new E3 ligases and design paradigms for the field of targeted protein degradation but also deepens our understanding of the regulation of peroxisome protein homeostasis.

[0127] Currently, the field of targeted protein degradation utilizes only a few cytoplasmic or nuclear-localized E3 ubiquitin ligases (such as VHL and CRBN). SYVN1 has not previously been used in PROTAC design, and its endogenous function remains unclear. We hypothesize that SYVN1 may be involved in the quality control of peroxisome proteins, responsible for clearing mislocalized or misfolded peroxisome-associated proteins. The PexTAC platform achieves the degradation of therapeutic target proteins by hijacking this endogenous quality control mechanism.

[0128] Unlike small-molecule degraders that rely on specific E3 ligands (such as PROTACs), PexTAC recruits target proteins to the peroxisome surface physically, without requiring E3 ligands. Importantly, studies targeting 16 peroxisome-associated proteins have demonstrated that PexTAC's function is independent of specific anchoring proteins, but rather driven by the spatial event of peroxisome targeting. This characteristic makes PexTAC highly modular: any ligand capable of recognizing peroxisome surface proteins (such as nanobodies, affinity molecules, or small molecules) can serve as a localization module, and any ligand capable of recognizing the target protein can serve as a recognition module, enabling plug-and-play programmable degradation.

[0129] ROR1 is a clinically validated cancer target, highly expressed in various hematologic malignancies and solid tumors, but lowly expressed in normal tissues, thus possessing a broad therapeutic window. Using AAV-delivered PexTAC-ROR1, we achieved efficient degradation of intratumoral ROR1 and significant inhibition of tumor growth in a xenograft tumor model, with promising preliminary safety, thus validating for the first time the feasibility of a peroxisome-targeted degradation strategy to intervene in ROR1. Furthermore, dual-target degradation (HSPA5+GPX4) further expands the application scope of PexTAC. HSPA5 is a key molecular chaperone in endoplasmic reticulum stress, and its upregulation is closely related to chemotherapy resistance; GPX4 is a core regulator of ferroptosis, and both can mediate ferroptosis resistance. Simultaneous targeting of these two proteins can exert a synergistic anti-tumor effect, suggesting that PexTAC can also be applied to synthetic lethality-based multi-target therapeutic strategies to overcome tumor heterogeneity and drug resistance.

[0130] Compared to existing targeted protein degradation technologies, PexTAC offers unique advantages and complementarity. While PROTACs are convenient to administer and allow for reversible regulation, they are highly dependent on E3 ligands and often exhibit poor pharmacokinetic properties due to their large molecular weight. As a gene-encoding tool, PexTAC can achieve long-term, stable regulation of target proteins via vectors such as AAVs, making it particularly suitable for the treatment of chronic diseases and cancer. Unlike lysosome-targeted LYTACs and autophagy-dependent AUTACs, PexTACs function through the ubiquitin-proteasome pathway, resulting in faster degradation and higher specificity. Furthermore, PexTACs do not rely on the target protein's natural degradation signals or endocytosis mechanisms, theoretically enabling the degradation of any protein that can be recognized by nanobodies, making it an important supplement to the existing degradation tool library.

[0131] This study has certain limitations. Currently, PexTAC relies on AAV delivery, and its immunogenicity and tissue tropism may limit its clinical translation. These issues can be addressed by optimizing AAV serotypes or developing non-viral delivery systems such as lipid nanoparticles. Although nanobodies have extremely low immunogenicity, for some nanobodies with immunogenic risks, the risk can be reduced through humanization or screening for fully human nanobodies. Furthermore, the long-term safety of PexTAC, especially its impact on peroxisome function, still needs to be evaluated in long-term animal models. Future research should focus on these directions to promote the clinical translation of PexTAC.

[0132] In summary, this study discovered a novel peroxisome-mediated protein degradation mechanism, identified a novel E3 ligase SYVN1, and constructed a modular PexTAC degradation platform, providing a new therapeutic strategy for refractory cancers. PexTAC expands the scope of protein degradation research from the cytoplasm to the peroxisome, offering new opportunities for intervention on previously undrugable organelle target proteins, and is expected to become an important component of next-generation targeted protein degradation technologies.

Claims

1. A peroxisome-targeting chimera (PexTAC) comprising, Chimeras that achieve targeted degradation of recruited proteins by recruiting them to peroxisomes.

2. A peroxisome-targeting chimera, PexTAC, characterized in that, The PexTAC consists of a peroxisome targeting module A, a target protein recognition module B, and a linker L, with L connecting A and B, and its general formula is ALB.

3. The peroxisome-targeting chimeric PexTAC according to claim 2, characterized in that, The peroxisome targeting module is a protein or polypeptide that targets the peroxisome; the target protein recognition module is a protein or polypeptide that can bind to the target protein; and the linker is a polypeptide linker or protein.

4. The PexTAC according to claim 2, characterized in that, The peroxisome targeting module is a membrane protein of the peroxisome or the membrane localization sequence of the corresponding membrane protein.

5. The PexTAC according to claim 4, characterized in that, The peroxisome targeting module consists of the peroxisome membrane proteins PEX2, PEX3, PEX5, PEX6, PEX7, PEX10, PEX11, PEX12, PEX13, PEX14, PEX16, PEX19, PEX26, PMP70, Fis1, PTS1, and PEX3N.

6. The peroxisome-targeting chimeric PexTAC according to claim 2, characterized in that, The target protein recognition module is a nanobody of the target protein.

7. The peroxisome-targeting chimeric PexTAC according to claim 2, characterized in that, Nanobodies for the target protein recognition module include green fluorescent protein nanobodies GBP, ROR1 nanobodies NbROR1, GPX4 nanobodies NbGPX4, HSPA5 nanobodies NbHSPA, or bispecific nanobodies combining GPX4 and HSPA5 NbHSPA5-NbGPX4.

8. A nucleic acid molecule, characterized in that, Its encoding is PexTAC as described in any one of claims 1-7.

9. A recombinant plasmid or recombinant host cell containing the nucleic acid molecule of claim 8.

10. The use of PexTAC according to any one of claims 1-7, the nucleic acid molecule according to claim 8, or a recombinant plasmid or recombinant host cell containing the nucleic acid molecule according to claim 9 in the preparation of a targeted protein degradation drug.

11. The application according to claim 10, characterized in that, This is achieved through viral delivery of PexTAC or plasmid transfection expressing PexTAC.

12. The application according to claim 10, characterized in that, This refers to the application of PexTAC, delivered via a viral vector, that degrades ROR1, GPX4, or HSPA5 individually or simultaneously, in the preparation of drugs for treating diseases.

13. The use of PexTAC according to any one of claims 1-7, the nucleic acid molecule according to claim 8, or a recombinant plasmid or recombinant host cell containing the nucleic acid molecule according to claim 9 in the preparation of drugs that inhibit cancer cell proliferation or induce programmed cell death.

14. The use of the PexTAC of any one of claims 1-7, the nucleic acid molecule of claim 8, or a recombinant plasmid or recombinant host cell containing the nucleic acid molecule of claim 9, in the preparation of a medicament for treating lung cancer, breast cancer, pancreatic cancer, hematologic malignancies, cervical cancer, ovarian cancer, liver cancer, and colorectal cancer.

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