A method for generating a molecular glue gate ectopic thymus and an imaging monitoring platform

Abstract

The present application relates to the field of biotechnology, and relates to immune monitoring and imaging, and particularly discloses a molecular glue-gated ectopic thymus model and a preparation and evaluation method thereof, the ectopic thymus model comprising engineered cells expressing NOX2 complex on the surface, T cell surface death receptor FAS, and humanized CRBN protein in the cell; the engineered cells are used for targeted degradation of transcription factors by reaction of a molecular glue drug with the CRBN protein, and for inhibition of type I interferon synthesis and inflammatory storm. The engineered cells constructed in the present application are transplanted into developed organoids, which can be regulated by a molecular glue drug, avoiding problems such as uncontrollable gene silencing and inflammatory storm caused by traditional organoids, and can maintain the effect for a long time. Based on expansion microscopy and methods such as nucleic acid, protein and lipid imaging, the present application can realize quantitative analysis of the drug action mechanism at the molecular level in the cell, which is beneficial to effect monitoring and related immune mechanism analysis and drug development.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to immune monitoring and medical imaging, and more specifically to a molecularly glue-gated ectopic thymus model and its preparation and evaluation methods. Background Technology

[0002] Traditional drug therapies are often accompanied by toxic side effects, and their mechanisms of action are difficult to assess, relying mainly on pathological indicators. Cell therapy, which treats patients with autologous / allogeneic cells, offers advantages such as long-lasting efficacy and the ability to be specifically programmed, but it also suffers from off-target toxicity and difficulty in in vivo regulation. Organoids are multicellular structures that develop into organ-like structures in vivo / in vitro, and studies have shown that they can compensate for the function of damaged organs in the body.

[0003] Lupus is a common autoimmune disease mediated by multiple genetic and environmental factors, often affecting multiple tissue systems and organs, and is currently incurable. Lupus patients exhibit thymic cortical atrophy and medullary disorder. Engineered immunotherapy is an advanced strategy for the clinical management of lupus. Studies have shown that transplanting allogeneic healthy thymic cells or organoids into mice can rebuild their immune function and alleviate their condition. However, organoids present challenging scientific problems, such as triggering bursts of inflammatory cytokines like type I interferon and gene silencing, leading to short-lived efficacy.

[0004] Engineered cells, through genetic and molecular engineering methods, allow for reprogramming and reprogramming, providing a solution for cell transplantation and organoid therapy. For example, thalidomide analogues can form a complex with the E3 ubiquitin ligase substrate CRBN protein, leading to the degradation of the type I interferon synthesis pathway transcription factor IKZF3 protein. This effectively reduces type I interferon release levels and blocks gene silencing, potentially improving the prognosis of complex diseases. However, mouse CRBN protein does not exhibit this reaction with the molecular glue. Monitoring in vivo ubiquitin-mediated transcription factor complexes requires a resolution of less than 100 nanometers in the one-dimensional spatial domain and less than a few minutes in the temporal domain, making it difficult to accurately monitor the dynamic processes of target molecular complex formation and degradation at single-cell spatial resolution. Currently, commonly used molecular glue monitoring methods are based on intermolecular affinity and dose-response relationships, such as IC50 and EC50, monitoring environments outside of tissue cells. These methods are unsuitable for monitoring effective intermolecular interactions at high spatial resolution and elucidating immune regulatory mechanisms, thus delaying the translation of engineered immune cells and organoid therapy strategies. Therefore, the current applications of engineered immune cells to construct organoids for autoimmune diseases such as lupus suffer from problems such as decreased efficacy due to gene silencing and uncontrollable cytotoxicity; engineered cells regulated by molecular glue do not react in mice; and organoid regulation detection cannot achieve single-molecule resolution labeling within tissue cells and suffers from long detection delays. Summary of the Invention

[0005] Based on this, the present invention proposes a molecular glue-gated microscopic imaging monitoring platform for ectopic thymus development and expansion. By using molecular glue-gated engineered cells, the in vivo regulation of thymus organoid failure and toxicity can be addressed, thereby achieving in vivo regulation.

[0006] According to a first aspect of the present invention, a molecular glue-gated ectopic thymus model is provided, comprising engineered cells expressing a NOX2 complex on the surface, a T cell surface death receptor FAS, and an intracellular expression of humanized CRBN protein.

[0007] The engineered cells are used to target and degrade transcription factors by reacting molecular glue drugs with CRBN proteins, thereby inhibiting type I interferon synthesis and cytokine storm.

[0008] According to an embodiment of the present invention, the engineered cells are obtained through ectopic organoids induced by small molecule drug intervention;

[0009] The ectopic organoids were obtained by introducing cells from wild-type B6 mice into the subcutaneous lymph nodes of disease model mice.

[0010] According to an embodiment of the present invention, the disease model mouse is a B6.Fas mouse obtained through crossbreeding. lpr .Ncf1 m1J .Lyz2 Akaluc .Crbn I391V A mouse model of receptor disease.

[0011] According to a second aspect of the present invention, a method for establishing the above-described molecular glue-gated ectopic thymus model is provided, characterized by comprising the following steps:

[0012] A disease model was established using crossbreeding of mice;

[0013] Ectopic organoids were constructed by introducing cells from wild-type B6 mice into subcutaneous lymph nodes of a disease model mouse.

[0014] A molecular glue-gated ectopic thymus model was constructed using small molecule drug intervention.

[0015] According to an embodiment of the present invention, the method of establishing a disease model through mouse crossbreeding includes:

[0016] By hybridizing mice of different strains, a genotype marker named B6.Fas was obtained. lpr .Ncf1 m1J .Lyz2 Akaluc .Crbn I391V A mouse model of receptor disease.

[0017] According to an embodiment of the present invention, the method of constructing ectopic organoids by introducing cells from wild-type B6 mice into subcutaneous lymph nodes of a disease model mouse includes:

[0018] To B6.Fas lpr .Ncf1 m1J .Lyz2 Akaluc .Crbn I391V Wild-type B6 donor mouse thymocytes were transplanted into recipient mice to construct ectopic organoids.

[0019] According to an embodiment of the present invention, the method of constructing a molecularly glue-gated ectopic thymus model using small molecule drug intervention includes:

[0020] A molecular glue substance was injected into the ectopic organoid to construct a molecular glue-gated ectopic thymus model;

[0021] The molecular adhesive is a thalidomide analogue;

[0022] The molecular gel is used to form a complex with the E3 ubiquitin ligase substrate receptor CRBN protein, thereby binding to and targeting the degradation of transcription factors.

[0023] According to a third aspect of the present invention, a method for evaluating the above-described molecular glue-gated ectopic thymus model is provided, characterized by comprising the following steps:

[0024] Recipient mice that have been used to construct the ectopic thymus model according to any one of claims 1-3;

[0025] The organoid thymus of the recipient mouse, the thymus, kidney, heart, lung, aorta, and serum of the host mouse were obtained and prepared into sample sections;

[0026] The sample slices were incubated with the expanded monomer solution in a 0.1 mm thick gel chamber at 4 °C for the first time.

[0027] The sample slices after the first incubation were incubated a second time in homogenization buffer.

[0028] The sample slices after the second incubation were expanded and subjected to spatial multi-omics staining to obtain observation slices;

[0029] Acquire image data of the observed slices, including molecular glue drug, CRBN protein, subcellular location of transcription factors, molecular structure of complexes, and relative positions;

[0030] The organoid function compensation effect was obtained through the analysis of the image data.

[0031] According to an embodiment of the present invention, the expanding monomer solution comprises: dimethacrylamide, sodium acrylate, acrylamide, N,N-methylenebisacrylamide, sodium chloride, dissolved in PBS for storage, and ammonium persulfate, tetramethylethylenediamine, 4-hydroxytamoxifen and methacrolein are added before use;

[0032] The homogenization buffer contains sodium dodecyl sulfate and urea.

[0033] According to an embodiment of the present invention, the step of expanding the sample slice after the second incubation and performing spatial multi-omics staining to obtain the observation slice includes:

[0034] The sample slices after the second incubation were trimmed to a suitable shape and then treated with a blocking solution at room temperature for one hour.

[0035] After washing three times with PBS, the molecular gel was used to stain downstream transcription factor proteins such as IKZF3 antibody and intracellular lipid staining reagents (such as mitochondrial staining), nucleic acid staining reagents and other biomolecules of interest.

[0036] Cell nuclei were stained with Hoechest 33258 dye at room temperature for 30 minutes, followed by washing with PBS.

[0037] The sample slices were soaked in double-distilled water at room temperature under light-protected conditions to allow them to fully expand, thus obtaining the observation slices.

[0038] As can be seen from the above technical solutions, the molecular glue-gated ectopic thymus model and its preparation and evaluation method provided by the present invention have the following beneficial effects:

[0039] This invention utilizes mouse crossbreeding to construct engineered cells that express the NOX2 complex and the T cell death receptor FAS on their surface, and humanized CRBN protein intracellularly. During the in vivo development of these engineered cells into thymus organoids, common phenomena such as inflammatory storms and gene silencing can be addressed by targeting and degrading transcription factors through the interaction of molecular gel drugs with CRBN protein. This inhibits type I interferon synthesis and inflammatory storms, blocks the gene silencing process, and maintains the effect.

[0040] This invention employs dilatational microscopy based on methacrolein, combined with spatial protein imaging, lipid imaging, and transcriptomics techniques, to simultaneously achieve 20-nanometer resolution imaging of proteins, nucleic acids, and lipids. This enables a subcellular resolution spatial multi-omics imaging method, which quantitatively monitors the interaction between molecular gel drugs and CRBN proteins and transcription factors at the intracellular molecular level. It constructs a universal method for monitoring the efficacy of molecular-level drugs and cell transplantation, along with its image processing and analysis methods.

[0041] The molecular glue-gated ectopic thymus development and expansion microscopic imaging monitoring platform provided by this invention amplifies the physical size of interacting molecules using expansion microscopy, achieving nanoscale imaging. It enables real-time dynamic detection of intermolecular interactions within the cellular environment, elucidating the mechanisms of intermediate immune processes, and monitoring the aforementioned cell transplantation and regulatory effects. The method designed in this invention is not limited to any specific molecular glue drug; it has universal applicability for evaluating a class of molecular glue analogs capable of CRBN protein gating and their interactions.

[0042] The engineered cell transplantation-derived organoids constructed in this invention can be regulated by molecular gel-like drugs, avoiding the problems of uncontrollable gene silencing and inflammatory storms caused by traditional organoids, and can maintain the effect for a long time.

[0043] This invention, based on expansion microscopy and nucleic acid, protein and lipid imaging, enables quantitative analysis of drug efficacy and mechanisms of action at the molecular level within cells, which is beneficial for efficacy monitoring, immune mechanism analysis and related drug development. Attached Figure Description

[0044] Figure 1 This is a schematic diagram illustrating the process of establishing an ectopic thymus model according to an embodiment of the present invention;

[0045] Figure 2 This is a schematic diagram of the effect monitoring and dilatational microscopy multi-omics imaging analysis platform used in Embodiment 1 of the present invention. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0047] According to a first aspect of the present invention, a molecular glue-gated ectopic thymus model is provided, comprising engineered cells expressing a NOX2 complex on the surface, a T cell surface death receptor FAS, and an intracellular expression of humanized CRBN protein.

[0048] Engineered cells are used to target and degrade transcription factors by reacting molecular glue drugs with CRBN proteins, thereby inhibiting type I interferon synthesis and cytokine storm.

[0049] According to an embodiment of the present invention, engineered cells are obtained through ectopic organoids induced by small molecule drug intervention;

[0050] Among them, ectopic organoids were obtained by introducing cells from wild-type B6 mice into the subcutaneous lymph nodes of disease model mice.

[0051] Thalidomide analogues, molecular gels, form a complex with the E3 ubiquitin ligase substrate receptor CRBN protein, binding to and targeting the degradation of transcription factors. Mouse CRBN does not respond to thalidomide analogues, but a mutation at position 391 of the Crbn gene, replacing isoleucine with valine, enables mouse CRBN to exhibit the human response to thalidomide analogues, inducing the degradation of downstream transcription factors and thus regulating cells. This invention establishes a monitoring platform using a wild-type C57BL / 6J (B6) mouse model, employing cell transplantation and small molecule intervention strategies, combined with dilatational microscopy imaging.

[0052] According to an embodiment of the present invention, the disease model mouse is B6.Fas obtained through mouse crossbreeding. lpr .Ncf1 m 1J .Lyz2 Akaluc .Crbn I391V A mouse model of receptor disease.

[0053] According to a second aspect of the present invention, a method for establishing the above-described molecular glue-gated ectopic thymus model is provided, characterized in that, as Figure 1 As shown, it includes the following steps:

[0054] S1: Establish a disease model through crossbreeding of mice;

[0055] S2: Ectopic organoids were constructed by introducing cells from wild-type B6 mice into subcutaneous lymph nodes of disease model mice;

[0056] S3: A molecular glue-gated ectopic thymus model was constructed using small molecule drug intervention.

[0057] According to an embodiment of the present invention, in S1, establishing a disease model through mouse crossbreeding includes:

[0058] By hybridizing mice of different strains, a genotype marker named B6.Fas was obtained. lpr .Ncf1 m1J .Lyz2 Akaluc .Crbn I391V A mouse model of receptor disease.

[0059] According to an embodiment of the present invention, S1 specifically involves using B6.Fas lpr strain, B6.Ncf1 m1j strain, B6.Crbn tm1.1Ble (i.e., B6.Crbn) I391V ) strain of mice and B6.Lyz2 Akaluc Establish breeding cages for strain B6.Stat1 mice.fl / fl strain, B6.Cd68 Cre / Cre Breeding cages were established for the strain, crossbreeding was carried out, and the genotype marker B6.Fas was ultimately constructed. lpr .Ncf1 m1J .Lyz2 Akaluc .Crbn I391V Receptor disease model mice and B6.Stat1 fl / fl .Cd68 Cre / Cre (Hereinafter referred to as B6.Stat1) CKO Donor control model mice. Wild-type B6 mice were used as donors.

[0060] According to an embodiment of the present invention, in S2, the method of constructing ectopic organoids by introducing cells from wild-type B6 mice into subcutaneous lymph nodes of disease model mice includes:

[0061] To B6.Fas lpr .Ncf1 m1J .Lyz2 Akaluc .Crbn I391V Wild-type B6 donor mouse thymocytes were transplanted into recipient mice to construct ectopic organoids.

[0062] According to an embodiment of the present invention, S2 specifically involves extracting the thymus from wild-type B6 mice progeny. During the gestation period of B6 mice from 14.5 to 15.5 days, thymus tissue from the progeny is collected, prepared into a single-cell suspension, and cell counting and other operations are performed.

[0063] Cell transplantation and organoid heterotopia. Using a 21G syringe, the prepared single-cell suspension was injected into B6. Fas... lpr .Ncf1 m1J .Lyz2 Akaluc .Crbn I391V Subcutaneous lymph nodes in mice. Anesthesia and pain management can be considered as alternatives during injection, but are normally unnecessary. After cell transplantation, small animal imaging techniques are used for continuous dynamic monitoring.

[0064] According to embodiments of the present invention, a molecularly glue-gated ectopic thymus model is constructed using a small molecule drug intervention method, comprising:

[0065] A molecular glue-gated ectopic thymus model was constructed by injecting molecular glue substances into ectopic organoids.

[0066] Among them, the molecular adhesive is a thalidomide analogue;

[0067] The molecular gel is used to form a complex with the E3 ubiquitin ligase substrate receptor CRBN protein, which binds to and targets the degradation of transcription factors.

[0068] According to an embodiment of the present invention, in the fourth week after cell transplantation, an intracellular effect assessment is performed. The method is to euthanize the model animal with carbon dioxide and extract tissues such as ectopic thymus, orthotopic thymus, spleen, kidney, heart, lungs and aortic arch for effect evaluation.

[0069] According to embodiments of the present invention, intracellular efficacy evaluation includes in vivo monitoring. Further, based on engineered intracellular Lyz2... Akaluc Genetically modified luminescent groups were used to assess disease progression in vivo using a small animal in vivo imaging system; cheek blood was taken every two weeks to assess the level of antinuclear antibody dsDNA, obtain thymus function indicators and blood antinuclear antibody indicators, and evaluate the effect and disease condition.

[0070] According to embodiments of the present invention, intracellular efficacy assessment includes in vitro efficacy assessment. Organs are collected four weeks post-surgery, and the collected organ samples are evaluated using methods such as expansion microscopy. Pathological staining and spatial multi-omics imaging are performed on thymus and lymphoid thymus organoids from each group to detect their function. Furthermore, expansion microscopy is used to detect transcription factors, CRBN protein, and thalidomide analogs in mouse thymus and lymphoid thymus organoids treated with molecular gel, and spatial multi-omics analysis is performed to detect their upstream and downstream pathway responses and subcellular structural localization.

[0071] According to embodiments of the present invention, the intracellular effect assessment includes processing the images obtained from imaging, analyzing the organoid functional compensation effect, the autofluorescence of thalidomide analogs, the relative positions and subcellular structural positions of CRBN protein and transcription factors, and assessing the organoid effect and the organoid gating effect of thalidomide.

[0072] According to a third aspect of the present invention, a method for evaluating the above-described molecular glue-gated ectopic thymus model is provided, characterized by comprising the following steps:

[0073] Recipient mice from which an ectopic thymus model of any of claims 1-3 has been constructed;

[0074] P1: Obtain organoids from recipient mice (thymus), host mice (thymus, kidney, heart, lung, aorta, and serum), and prepare sample sections;

[0075] P2: The sample slices and the expanded monomer solution were incubated for the first time in a 0.1 mm thick gel chamber at 4°C;

[0076] P3: After the first incubation, the sample slices were incubated a second time in homogenization buffer.

[0077] P4: The sample slices after the second incubation were expanded and subjected to spatial multi-omics staining to obtain observation slices;

[0078] P5: Obtain image data of the observed slices, including the molecular glue drug, CRBN protein, subcellular location of transcription factors, molecular structure of complexes, and relative positions;

[0079] P6: The organoid functional compensation effect was obtained through image data analysis.

[0080] Expanded microscopy, based on expandable hydrogel networks, immobilizes target biomolecules. Through the digestion of tissue structures and the expansion of the gel network's physical size, the physical size of biomolecules is increased, achieving nanoscale resolution under conventional optical microscopes. Methacrolein-based expanded microscopy has enabled the immobilization of various biomolecules, including proteins, lipids, and nucleic acids, within hydrogel networks, facilitating super-resolution analysis using imaging methods. By combining expanded microscopy with protein immunofluorescence, spatial transcriptomics, and lipid imaging, it is possible to achieve full-process super-resolution imaging of spatially located genes, RNA, proteins, and lipids. This allows for intracellular molecular-level detection of cell transplantation and molecular gel-mediated regulation methods, enabling organoid monitoring and elucidating mechanisms of action.

[0081] According to an embodiment of the present invention, P1 specifically involves: euthanizing recipient mice four weeks post-surgery and collecting materials such as the thymus, thymus, kidney, heart, lung, aorta, and serum from the developed organoids for efficacy evaluation. Recipient mice were anesthetized with 1% isoflurane, and after thoracotomy, the heart was perfused with PBS. The collected samples were stored in PBS in an icebox, then rapidly transferred to freshly prepared 4% paraformaldehyde for 12 hours, followed by immersion in PBS on a shaker for 1 hour, repeated three times. The tissues were then sectioned into 5-micron sections using a cryostat for later use.

[0082] According to an embodiment of the present invention, the effectiveness of collected organ and other sample sections is evaluated based on dilatational microscopy. Specifically, the proposed dilatational microscopy technique achieves approximately 11-fold dilatation, reaching a resolution of 20 nanometers. Dilatational microscopy was performed on thymus organoids from three groups of recipient mice, focusing on thymus and lymphoid development. Sections requiring immunofluorescence staining were mounted on charged slides, incubated in sodium citrate antigen retrieval solution at 95°C for 20 minutes, and then washed with PBS at room temperature for 15 minutes.

[0083] According to an embodiment of the present invention, in P2, the expanding monomer solution includes: dimethylacrylamide, sodium acrylate, acrylamide, N,N-methylenebisacrylamide, sodium chloride, dissolved in PBS for storage, and ammonium persulfate, tetramethylethylenediamine, 4-hydroxytamoxifen and methacrolein are added before use.

[0084] According to an embodiment of the present invention, P2 specifically involves incubating the sample slices with the expanding monomer solution in a 0.1 mm thick gel chamber at 4°C. In this step, the hydrogel monomer components permeate into the tissue cells, synthesizing a hydrogel network in the biological tissue through polymerization. The anchoring molecule, methacrolein, can link with proteins, nucleic acids, and lipids, immobilizing them within the hydrogel network and ensuring that the molecules remain in situ during subsequent expansion, enabling spatial multi-omics imaging. The incubated slices are then transferred to 37°C and incubated overnight to allow gelation.

[0085] According to an embodiment of the present invention, in P3, the homogenization buffer contains sodium dodecyl sulfate and urea, which can disrupt the structure of proteins without damaging their amino acid sequences, thereby relieving the internal constraints of tissue swelling while preserving the aforementioned fixed biomolecular signals. The digested sample is washed three times with PBS on a shaker at room temperature for 15 minutes each time, and then can be stored at 4°C in PBS containing 0.02% sodium azide.

[0086] According to an embodiment of the present invention, in P4, the sample slices after the second incubation are expanded and subjected to spatial multi-omics staining to obtain observation slices including:

[0087] After the second incubation, the sample slices were trimmed to a suitable shape and then treated with a blocking solution at room temperature for one hour.

[0088] After washing three times with PBS, the target protein molecular targeted staining reagents, such as IKZF3 antibody and lipid and nucleic acid targeted staining reagents, such as mitochondrial staining reagents, were used to stain multiple groups of biological molecules. After incubation overnight at 4°C in the dark, the staining was followed by washing with PBS.

[0089] Cell nuclei were stained with Hoechest 33258 dye at room temperature for 30 minutes, followed by washing with PBS.

[0090] The sample slices were soaked in double-distilled water at room temperature under dark conditions to allow them to fully expand, thus obtaining the observation slices.

[0091] According to an embodiment of the present invention, the sealing fluid comprises 5% FBS and 0.3% Triton-100;

[0092] IKZF3 antibody was diluted 1:1000;

[0093] Mitochondrial staining reagent was diluted 1:200 before staining; Hoechest 33258 was diluted 1:1000.

[0094] According to an embodiment of the present invention, P5 specifically involves: using a Leica DMi8 inverted confocal microscope with 63x magnification to observe the molecular glue drug, CRBN protein, subcellular location of transcription factors, molecular structure of complexes, and relative positions.

[0095] According to an embodiment of the present invention, P6 specifically involves: analyzing the obtained image data using Python. Algorithms such as 3D-UXNet are used to segment three-dimensional fluorescent points along the Z-axis. The locations of thalidomide analogs, transcription factors, CRBN proteins, and intracellular spatial locations are segmented based on channels. The outer contour of the complex is then segmented using these three points, and its area and number of complexes are calculated. Based on the segmentation results, the process of molecular glue regulation is evaluated. A dataset is constructed from the segmented images of different stages of action to assess the time-molecular glue interaction relationship and efficiency, thereby identifying the actual molecular mechanism of action and reaction efficiency of the molecular glue drug.

[0096] The technical solution of the present invention will be described in detail below through preferred embodiments. It should be noted that the specific embodiments in the following text are for illustrative purposes only and are not intended to limit the present invention.

[0097] Example:

[0098] Construct animal models of recipient diseases and animal models of donor diseases.

[0099] During the process, the inventors used B6.Fas from Jackson Laboratory in the United States. lpr strain, B6.Nc f1 m1j strain, B6.Crbn tm1.1Ble (i.e., B6.Crbn) I391V ) strain mice and B6.Lyz2 mice from Shanghai Southern Model Animal Company Akaluc Establish breeding cages for strain B6.Stat1 fl / fl strain, B6.Cd68 Cre / Cre Breeding cages were established for the strain of mice, and crossbreeding was carried out to ultimately construct a genotype marker of B6.Fas. lpr .Ncf1 m1J .Lyz2 Akaluc .Crbn I391V Disease model mice and B6.Stat1 fl / fl .Cd68 Cre / Cre Donor control mouse model.

[0100] Thymus was extracted from B6 mouse progeny.

[0101] During the process, when B6 mice were 14.5 to 15.5 days pregnant, thymus tissue from offspring was collected, a single-cell suspension was prepared, and cell counting and other operations were performed.

[0102] Mice were anesthetized at 14.5-15.5 days of gestation (0.5 days of gestation was confirmed by the detection of vaginal plugs). After removing abdominal hair with depilatory cream, the mice were fixed in an upright position. The thymus of the offspring was extracted and treated with 0.25% trypsin-EDTA. The mixture was then filtered through a 40-micron cell filter with 1 mL of PBS to prepare a cell suspension. 5 μL of the prepared cell suspension was mixed with 5 μL of trypan blue for cell counting.

[0103] Step Sc: Cell transplantation and organoid development.

[0104] Mice were anesthetized with 1% isoflurane, and recipient mice were fixed in a supine position. The abdomens of the recipient mice were shaved to expose the right inguinal lymph nodes. Extracted thymocytes were slowly injected into each mouse using a 1 ml syringe, ensuring that at least 10 cells were injected per mouse. 6 One. Suture the wound continuously with medical sutures, and decide whether to administer 2 mg / kg ketoprofen for pain relief two days after surgery, depending on the situation. Disinfect the wound regularly.

[0105] Construction of an imaging monitoring platform.

[0106] The effects were evaluated in vivo over four weeks. Furthermore, for the effect evaluation, four groups of B6.Fas were used. lpr .Ncf1 m 1J .Lyz2 Akaluc .Crbn I391V Mice were treated according to the above steps, and a control experiment was set up to complete the construction of the ectopic organoid imaging monitoring platform:

[0107] 1.A: To B6.Fas lpr .Ncf1 m1J .Lyz2 Akaluc .Crbn I391V wild-type B6 donor mouse thymocytes were transplanted into recipient mice.

[0108] 2.B: To B6.Fas lpr .Ncf1 m1J .Lyz2 Akaluc .Crbn I391V Recipient mice transplanted with B6.Stat1 CKO Thymocytes from donor mice;

[0109] 3.C: To B6.Fas lpr .Ncf1 m1J .Lyz2 Akaluc .Crbn I391V Recipient mice were transplanted with wild-type B6 donor mouse thymocytes and treated with molecular gel drugs;

[0110] 4.D: To B6.Faslpr .Ncf1 m1J .Lyz2 Akaluc .Crbn I391V The recipient mice underwent sham surgery.

[0111] Furthermore, such as Figure 2 As shown, for the five groups of mice, fluorescence levels in joint and organ regions were detected weekly using an in vivo small animal imaging system (IVIS) based on engineered cell luminescent groups to assess the inflammatory process in vivo. Every two weeks, cheek blood was collected and antinuclear antibody dsDNA was detected using a pre-ordered test kit to assess the level of antinuclear antibody dsDNA in order to evaluate organoid function and disease progression.

[0112] Figure 2 This demonstrates that by applying dilatational microscopy to overcome the optical diffraction limit, the original structure and interaction positions of biomolecules that are blurred at low resolution can be revealed through improved resolution. The relative positions of target biomolecules can be segmented within the cellular environment to quantitatively assess their mechanisms of action; the action stages and quantities of complexes can be evaluated to quantitatively analyze drug efficacy.

[0113] Evaluation of molecular effects within tissue cells.

[0114] Four weeks post-surgery, recipient mice were sacrificed, and the resulting organoids (thymus, thymus, kidney, heart, lung, aorta, serum, etc.) were collected for efficacy evaluation. Recipient mice were anesthetized with 1% isoflurane, and after thoracotomy, the heart was perfused with PBS. The collected samples were stored in PBS in an icebox, then rapidly transferred to freshly prepared 4% paraformaldehyde for 12 hours, followed by immersion in PBS on a shaker for 1 hour, repeated three times. The tissues were then sectioned into 5-micron sections using a cryostat for later use.

[0115] The effectiveness of the collected organ and other sample sections was evaluated using dilatational microscopy. Specifically, the proposed dilatational microscopy technique achieves approximately 11-fold dilatation, reaching a resolution of 20 nanometers. Dilatational microscopy was performed on thymus organoids from the thymus and lymphoid development of three groups of recipient mice. Sections requiring immunofluorescence staining were mounted on charged slides, incubated in sodium citrate antigen retrieval solution at 95°C for 20 minutes, and then washed with PBS at room temperature for 15 minutes.

[0116] The steps of dilatational microscopy imaging are as follows:

[0117] 1. Incubate the sections with a swelling monomer solution (dimethacrylamide, sodium acrylate, acrylamide, N,N-methylenebisacrylamide, sodium chloride, dissolved in PBS and stored; add ammonium persulfate, tetramethylethylenediamine, 4-hydroxytamoxifen, and methacrolein before use) in a 0.1 mm thick gel chamber at 4°C. In this step, the hydrogel monomer components permeate into the tissue cells and synthesize a hydrogel network within the biological tissue through polymerization. The anchoring molecule methacrolein can link with proteins, nucleic acids, and lipids, immobilizing them within the hydrogel network and ensuring that the molecules remain in situ during subsequent swelling. Transfer the incubated sections to 37°C and incubate overnight to allow gelation.

[0118] 2. Samples were incubated in homogenization buffer. The homogenization buffer contained sodium dodecyl sulfate and urea, which can disrupt the structure of proteins without damaging their amino acid sequences, thus relieving the internal constraints of tissue swelling while preserving the aforementioned fixed biomolecular signals. After digestion, the samples were washed three times with PBS on a shaker at room temperature for 15 minutes each time, and then stored at 4°C in PBS containing 0.02% sodium azide.

[0119] 3. After digestion, the sample swells and undergoes spatial multi-omics staining. Trim the gel to a suitable shape, then treat with blocking buffer (5% FBS, 0.3% Triton-100) at room temperature for one hour. Wash with PBS for 5 minutes at room temperature, then stain with IKZF3 antibody (1:1000 dilution), incubate overnight at 4°C in the dark, and wash with PBS three times for 15 minutes each time. Stain cell nuclei with Hoechst 33258 (1:1000) at room temperature for 30 minutes, then wash with PBS three times for 15 minutes each time. Immerse in double-distilled water at room temperature for 1 hour three times, changing the water each time, to ensure sufficient sample swelling, while keeping the sample protected from light.

[0120] 4. Using a Leica DMi8 inverted confocal microscope at 63x magnification, observe the molecular gel drug, CRBN protein, subcellular location of transcription factors, molecular structure of complexes, and relative positions.

[0121] The images obtained from imaging are processed to analyze the organoid functional compensation effect.

[0122] The obtained image data was analyzed using Python. An algorithm was employed to segment three-dimensional fluorescent points along the Z-axis, and the locations of thalidomide analogs, transcription factors, CRBN proteins, and intracellular spatial locations were segmented based on channels. The outer contours of the complexes were then segmented using these three points, and their areas and complex numbers were calculated. Based on the segmentation results, the regulatory process of the molecular glue was evaluated. A dataset was constructed from the segmented images of different stages of action to assess the time-molecular glue interaction relationship and efficiency, thereby identifying the actual molecular mechanism of action and reaction efficiency of the molecular glue drugs.

[0123] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for establishing a molecular glue gated ectopic thymic model, characterized in that, The ectopic thymus model comprises engineered cells expressing NOX2 complex on cell surface, T cell surface death receptor FAS, and humanized CRBN protein in cells; The engineered cells are used to target degradation of transcription factors by reacting with CRBN protein to inhibit type I interferon synthesis and inflammatory storm; The engineered cells are obtained by small molecule drug intervention in ectopic organoids; The ectopic organoids are obtained by inputting cells from wild type B6 mice into subcutaneous lymph nodes of disease model mice; The disease model mice are B6. Fas lpr . Ncf1 m1J . Lyz2 Akaluc . Crbn I391V receptor disease model mice.

2. The method of establishing a molecular glue gated ectopic thymic model according to claim 1, characterized in that, The method comprises the following steps: A disease model is established by cross-breeding mice; An ectopic organoid is constructed by inputting cells from wild type B6 mice into subcutaneous lymph nodes of disease model mice; An ectopic thymus model is constructed by small molecule drug intervention.

3. The method of claim 2, wherein, The method of establishing a disease model by cross-breeding mice comprises: By crossing different strains of mice, a recipient disease model mouse with the genotype marker B6. Fas lpr . Ncf1 m1J . Lyz2 Akaluc . Crbn I 391V was obtained.

4. The method of claim 2, wherein, The method of constructing an ectopic organoid by inputting cells from wild type B6 mice into subcutaneous lymph nodes of disease model mice comprises: B6. Fas lpr . Ncf1 m1J . Lyz2 Akaluc . Crbn I391V Recipient mice were transplanted with wild-type B6 donor mouse thymocytes to construct ectopic organoids.

5. The method of claim 2, wherein, The method of constructing an ectopic thymus model by small molecule drug intervention comprises: A molecular glue drug is injected into the ectopic organoid to construct a molecular glue gated ectopic thymus model; The molecular glue drug is a thalidomide analogue; The molecular glue drug is used to form a complex by reacting with E3 ubiquitin ligase substrate receptor CRBN protein, bind and target degradation of transcription factors.

6. An evaluation method of the molecular glue-gated ectopic thymic model established by the method of establishing a molecular glue-gated ectopic thymic model according to claim 1, characterized in that, The method comprises the following steps: A recipient mouse of the ectopic thymus model of claim 1 is taken; An organoid thymus, host mouse thymus, kidney, heart, lung, aorta, and serum of the recipient mouse are obtained to prepare sample sections; The sample sections are subjected to first incubation with an expanding monomer solution in a 0.1 mm-thick gel chamber at 4°C; The sample sections after the first incubation are subjected to second incubation in a homogenate buffer; The sample sections after the second incubation are expanded and subjected to spatial multi-omics staining to obtain observation sections; Image data of the observation sections containing molecular glue drug, CRBN protein, transcription factor subcellular location, complex molecular structure, sample multi-omics information, and relative position are obtained; The image data analysis obtains organoid functional compensation effect and analyzes immune system reaction mechanism.

7. The evaluation method according to claim 6, characterized in that The expanding monomer solution comprises 4% v / v dimethyl acrylamide, 30% w / v sodium acrylate, 10% w / v acrylamide, 0.01% w / v N,N-methylene bisacrylamide, 1% w / v sodium chloride, and is dissolved in a phosphate buffer for storage, and 0.25% w / v ammonium persulfate, 0.04% v / v tetramethyl ethylenediamine, 0.001% w / v 4-hydroxytamoxifen, and 0.1% v / v methyl propyl aldehyde are added before use, and 200 microliters are used for each sample; The homogenate buffer contains sodium dodecyl sulfate and urea.

8. The evaluation method according to claim 6, characterized in that The sample slice after the second incubation is inflated and subjected to spatial multi-omics staining to obtain an observation slice, which comprises: The sample slice after the second incubation is trimmed to a proper shape and then treated with a blocking solution at room temperature for one hour; After being washed with a phosphate buffer solution for 3 times, the sample slice is stained with a molecular glue downstream transcription factor protein and intracellular lipid staining reagent, and then subjected to dark incubation at 4°C overnight, and then washed with a phosphate buffer solution; The sample slice is stained with a DNA dye Hoechest 33258 at room temperature for 30 minutes, and then washed with a phosphate buffer solution; The sample slice is sufficiently inflated by being soaked in double-distilled water at room temperature in the dark to obtain an observation slice.

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