Humanized zinc finger-truncated nuclear receptor fused small molecule response type gene regulation system and application thereof

Abstract

The invention discloses a human zinc finger-truncated nucleus receptor fused small molecule response type gene regulation system and application thereof, and relates to the technical field of gene engineering. The gene regulation and control system contains transcriptional regulation and control protein, and the transcriptional regulation and control protein is formed by fusing a human zinc finger DNA binding module and a truncated nuclear receptor; the human zinc finger DNA binding module can specifically recognize and bind a target DNA regulatory sequence, and the truncated nuclear receptor is combined with a ligand to serve as a transcriptional regulatory module to realize controllable expression of a target gene; the human zinc finger DNA binding module is a human or humanized zinc finger array; according to the truncated nuclear receptor, an inherent DNA binding domain and an N-terminal transcriptional activation region are removed, and a hinge region and a ligand binding region are mainly reserved. The gene regulation system disclosed by the invention has the characteristics of humanization, compact structure, capability of being regulated by small molecules and the like, and in some embodiments, in-vivo or in-vitro controllable expression of a target gene can be realized.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a small molecule responsive gene regulation system based on human zinc finger-truncated nuclear receptor fusion and its applications. Background Technology

[0002] In the fields of gene therapy and cell therapy, the key to balancing therapeutic efficacy and safety lies in safely, controllably, and reversibly regulating the expression of target genes in vivo or in vitro. Existing mammalian gene or genetic regulatory systems, such as Tet-On / Tet-Off, Gal4-UAS, and Cre-ERT2, typically rely on transcriptional regulatory elements derived from non-mammals (e.g., bacteria, yeast, or viruses) or artificially synthesized sensory protein modules obtained through significant structural modifications and non-physiological mutations. Furthermore, antibiotics or hormones are often used as inducers. Long-term or in vivo application of these systems may lead to immunogenicity risks, pharmacological incompatibility, or background leakage, thus limiting their clinical application. Simultaneously, some nuclear receptor-based regulatory systems are susceptible to non-specific activation by endogenous ligands (such as retinoic acid and vitamin D metabolites), resulting in high basal expression and making it difficult to achieve a strictly "off" state.

[0003] To reduce immunogenicity risks and improve drug accessibility, existing technologies have proposed various gene regulation strategies relying on clinically compatible small molecules. For example, engineered zinc finger regulators (such as synZiFTR) utilize the DNA-binding domain of human zinc finger proteins as a localization module and couple it to different types of regulatory elements in a modular manner to achieve drug regulation of gene expression. However, in such systems, the regulatory modules used are mostly derived from plants, viruses, or engineered estrogen receptor ligand-binding domains (ERT2) that respond to tamoxifen. Tamoxifen has certain contraindications in clinical use and has a long drug half-life, which may affect the flexibility of regulation in some application scenarios. The feasibility of orthogonal control within a pharmacologically relevant concentration range has been demonstrated for caffeine-responsive nanobody-zinc finger protein (Nanobody-ZF) fusion switches. However, since the nanobody domains used are not entirely human, additional immunogenicity considerations may be introduced compared to fully human protein domains. Although the zinc finger DNA binding module itself is humanized, the control module used still relies on non-fully human or hormone-responsive elements in terms of structural origin or pharmacological response characteristics, and further optimization is still needed for clinical translation and long-term in vivo application.

[0004] Zinc finger proteins (ZFs) are the most abundant family of transcription factors in the human genome, providing compact, programmable DNA-binding modules. Each zinc finger domain typically consists of about 30 amino acids and can be assembled into tunably specific zinc finger arrays, making them valuable for genome editing and synthetic transcriptional control. Importantly, zinc finger proteins can be configured to recognize synthetic DNA sequences that are present or absent at low frequencies in the human genome, thereby minimizing crosstalk with host transcription. Previous studies have demonstrated the feasibility of human zinc finger proteins in constructing controllable gene regulatory systems and highlighted their potential for applications in human-related transcriptional regulation. However, these systems employ non-human effector domains or ligands with limited drug applicability, restricting their translational applications.

[0005] For example, Beerli et al. reported fusing artificially designed zinc finger proteins with the ligand-binding domains of estrogen receptors, progesterone receptors, or retinoic acid X receptors, and further introducing exogenous transcriptional activation domains (such as VP16 or VP64) to achieve small molecule-induced transcriptional activation. These systems provide the primary transcriptional activation capacity through exogenous effector domains, while the nuclear receptor ligand-binding domains are mainly used to confer responsiveness to specific small molecules. Their engineering goal is to construct artificial transcription factors with strong induction amplitude. In contrast, these systems do not primarily aim to achieve low-background, finely regulated gene switches under highly humanized constraints.

[0006] Transcriptional regulation systems based on nuclear receptors (NRs) are typically influenced by endogenous ligands under physiological conditions. Many natural nuclear receptors (such as retinoic acid receptors and vitamin D receptors) can bind to endogenous metabolites in vivo, and their ligand-binding domains can be activated within physiological concentration ranges, leading to increased background activity in transcriptional regulation. In applications requiring the construction of humanized, small-molecule-controlled gene regulatory systems, these characteristics present significant engineering challenges for natural nuclear receptor systems as strictly controllable gene switches.

[0007] Therefore, there is still an urgent need for a novel gene expression regulation system that is entirely based on human protein domains, has minimal interference with the host genome transcriptional background, and can be precisely regulated through clinically relevant small molecules. Summary of the Invention

[0008] To address the engineering challenges faced by natural nuclear receptors in constructing controllable gene regulation systems, as described above, this invention provides a small-molecule responsive gene regulation system fused with a human zinc finger-truncated nuclear receptor and its applications. By fusing a human or humanized zinc finger DNA localization module with a truncated natural nuclear receptor and systematically engineering around the characteristics of different nuclear receptors, this invention provides multiple pathways for using natural nuclear receptors to construct controllable gene switches, thereby achieving controllable regulation of target gene expression while maintaining the humanization of the system. The specific technical solution is as follows: A small-molecule responsive gene regulation system based on a human zinc finger-truncated nuclear receptor fusion is disclosed. The system comprises a fusion transcriptional regulatory protein, a target DNA regulatory sequence containing a zinc finger DNA binding site, and a target gene operably linked to the target DNA regulatory sequence. The fusion transcriptional regulatory protein is formed by fusing a human or humanized programmable zinc finger DNA binding module (ZF) with a domain of a truncated nuclear receptor (ΔNR). The zinc finger DNA binding module specifically recognizes and binds to the target DNA regulatory sequence containing the zinc finger DNA binding site. The domain of the truncated nuclear receptor binds to a ligand and, under the action of the ligand, regulates the expression of the target gene. It should be noted that the truncated nuclear receptor in the gene regulation system of this invention performs transcriptional regulation, directly regulating the transcription process of the target gene, rather than merely regulating the nuclear localization or stability of the fusion protein.

[0009] The truncated nuclear receptor domain no longer possesses the endogenous DNA binding function of its inherent DNA-binding domain (DBD), and preferably further weakens or eliminates the transcriptional activation function of the N-terminal transcriptional activation region (AF-1), thereby mainly retaining the hinge region and ligand-binding region (LBD) to reduce ligand-independent transcriptional activation activity.

[0010] The zinc finger DNA binding module is a zinc finger array with low crosstalk to the host genome's endogenous DNA sequence, used to specifically recognize the target DNA sequence. In a preferred embodiment, the zinc finger array is ZF10, but it can also be ZF1, ZF3, or other zinc finger arrays with similar DNA recognition capabilities.

[0011] In this invention, the zinc finger DNA binding module is not limited to the complete absence of any potential binding sites in the host genome. In some embodiments, the zinc finger array may also identify DNA sequences that are present at low frequencies in the host genome. As long as such binding does not have a materially adverse effect on the intended function or application purpose of the gene regulatory system described in this invention, it can still be considered to meet the technical requirements of this invention.

[0012] In some embodiments, the truncated nuclear receptor is selected from one or more of the following: retinoic acid receptors (RARα, RARγ), vitamin D receptor (VDR), retinoic acid X receptor (RXRα), and thyroid hormone receptor (THRA1). In other embodiments, the truncated nuclear receptor may also be selected from other nuclear receptor superfamily members, as long as it contains a ligand-binding domain, retains its responsiveness to small molecules after truncation, and participates in the regulation of target gene expression as a transcriptional regulatory module in the gene regulation system, it can be used to construct the gene regulation system described in this invention.

[0013] In one embodiment, a mutation can be introduced into the nuclear receptor ligand-binding region to reduce its responsiveness to physiological concentrations of endogenous ligands, thereby allowing the gene regulatory system to maintain low background expression in the absence of exogenous ligands and exhibit an enhanced inducible response upon administration of pharmacologically relevant concentrations of exogenous ligands. In a preferred embodiment, the truncated nuclear receptor is a mutant of the retinoic acid receptor (RAR), specifically a K207N mutant with reduced sensitivity to all-trans retinoic acid (ATRA) and an increased response threshold; this mutant reduces its responsiveness to physiological concentrations of endogenous ligands.

[0014] The ligand may be an endogenous or exogenous small molecule capable of binding to the ligand-binding domain of a truncated nuclear receptor and activating or inhibiting the activity of the gene regulatory system. In some embodiments, the ligand may be, for example, but not limited to, one or more of endogenous all-trans retinoic acid, exogenous all-trans retinoic acid, retinoic acid receptor antagonists, vitamin D-related metabolites, or analogues thereof.

[0015] The target gene can be either an exogenous gene or an endogenous gene in the host genome.

[0016] In one embodiment, the human zinc finger DNA binding module can recognize and bind to the promoter of the target gene containing a zinc finger DNA binding site.

[0017] In some embodiments, the zinc finger DNA binding module may also be designed to recognize and bind to enhancer regions of target genes, distal regulatory elements, or specific sites in the host genome to achieve controllable regulation of endogenous gene expression.

[0018] The present invention also provides the application of the human zinc finger-truncated nuclear receptor fusion small molecule responsive gene regulation system in regulating the expression of reporter genes, therapeutic proteins, immune receptors (e.g., CARs) or cell death-related effectors (e.g., GSDMD-NT).

[0019] In some embodiments, the gene regulation system may be applied in vitro or in vivo in the form of plasmids, viral vectors, or engineered cells.

[0020] The present invention also provides a gene regulation method, wherein the gene regulation method employs the aforementioned human zinc finger-truncated nuclear receptor fusion small molecule responsive gene regulation system; the method includes introducing the gene regulation system into cells and, under the action of ligands, regulating the expression of target genes.

[0021] Therefore, the gene regulation system of the present invention can achieve a variety of controllable regulatory modes by means of endogenous ligand-driven and combined with antagonist inhibition, exogenous ligand-induced activation, or by mutating truncated nuclear receptors to regulate their response threshold to endogenous ligands.

[0022] Compared with the prior art, the beneficial effects of the present invention are: 1. The gene regulation system of this invention fuses a human or humanized zinc finger DNA binding module (preferably ZF10) with a truncated nuclear receptor hinge and ligand-binding domain responsible for transcriptional regulation, thereby achieving small-molecule responsive regulation of clinically relevant ligands. This invention is based entirely on human or humanized protein domains, which helps reduce the risk of immunogenicity. Simultaneously, the truncated nuclear receptor design helps reduce the system's background expression. This invention provides a well-adapted regulatory framework for precise, small-molecule responsive control of gene expression, and has safe and tunable application potential in gene- and cell-based therapies.

[0023] 2. The truncated nuclear receptor in this invention mainly retains the hinge region and ligand-binding region, which can reduce crosstalk with the host transcription program. In particular, removing the DNA-binding domain from the nuclear receptor can reduce the recognition of native genomic sites, and removing the N-terminal transcriptional activation region can reduce ligand-independent activation, thereby improving the precision and safety of gene regulation at the structural level.

[0024] 3. The gene regulation system described in this invention achieves activation or inhibition regulation of target gene expression through the interaction of small molecules with truncated nuclear receptor ligand binding domains, thereby providing a gene regulation method with good temporal controllability; and in some embodiments, the gene regulation state can be maintained without continuous exogenous small molecule intervention.

[0025] 4. The gene regulation system described in this invention can be used to regulate exogenously introduced target genes, and also has the potential applicability to regulate endogenous genes in the host genome. It can be adapted to different gene delivery or expression forms, thereby improving the versatility and engineering adaptability of the system in different application scenarios. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. The symbols, abbreviations, dosages, treatment times, statistical labels, or experimental results shown in the drawings are only used to exemplify specific experimental conditions and should not be construed as limiting the scope of protection of the present invention.

[0027] Figure 1 This is a schematic diagram of the construction and optimization of the ZF-NR ligand-responsive gene regulation system in Example 1; wherein, (a) is a schematic diagram of the ZF10-ΔRARα structure; (b) is an exemplary performance comparison of the truncated RARα configuration fused with ZF10; (c) is an exemplary evaluation of different zinc finger arrays fused with ΔRARα (approximately 149-457 aa); (dh) is a schematic diagram of the response of the ZF10-ΔNR chimera to ligand stimulation; (i) is a schematic diagram of the comparison between ZF10-ΔRARα and ZF10-ΔVDR and the Tet-On system under the same detection conditions; Figure 2 This is a schematic diagram illustrating the inhibitory and regulatory effect of the selective RARα antagonist YCT529 on the ZF10-ΔRARα system in Example 2; wherein, (a) is a schematic diagram of the ZF10-ΔRARα system; (b) ZF10-ΔRARα in stably carrying P ZF10 (c) Schematic diagram of the response of the SEAP reporter gene to ATRA stimulation in HEK293T cells; (dg) Schematic diagram of the inhibition of ATRA-induced activity by YCT529; (h) Schematic diagram of the regulation of ZF10-ΔRARα-related reporter signaling in C57BL / 6J mice by oral administration of YCT529 after microencapsulated cell transplantation; (i) Schematic diagram of the in vivo regulation of ZF10-ΔRARα-related reporter signaling. Figure 3This is a schematic diagram illustrating the in vitro and in vivo evaluation of the ZF10-ΔVDR system's response to calcipotriol in Example 3; wherein, (a) is a schematic diagram of the ZF10-ΔVDR system; (b) is a schematic diagram of changes in ZF10-ΔVDR-related SEAP expression in HEK293T cells under conditions of increased vitamin D precursor or precursor-related metabolites (e.g., vitamin D3); (c) is a schematic diagram of the ZF10-ΔVDR response to calcipotriol stimulation; (di) is a functional example of ZF10-ΔVDR in different cell types; (j) is a schematic diagram of the local activation of ZF10-ΔVDR in C57BL / 6J mice by calcipotriol ointment; and (k) is a schematic diagram of the in vivo regulation of ZF10-ΔVDR. Figure 4 Example 4 illustrates the ZF10-ΔRARα system based on mutations in the nuclear receptor ligand binding region; wherein, (a) a schematic diagram of the response of ZF10-ΔRARα_K207N to ATRA stimulation; (b, c) schematic diagrams of the control of ZF10-ΔRARα_WT and ZF10-ΔRARα_K207N under in vivo conditions; (d) a schematic diagram of local activation of ZF10-ΔRARα_K207N in vivo; (e) Jurkat cells with CD19 + (f) Schematic diagram of ZF10-ΔRARα_K207N-related CAR expression and CD69 phenotype changes under K562 cell co-culture conditions; (f) Functional example of ZF10-ΔRARα_K207N in Jurkat cells; Figure 5 This is an application example of ZF10-ΔRARα regulating GSDMD-NT expression in Example 5; wherein, (a) is a schematic diagram of the changes in GSDMD-NT expression and cell death-related phenotypes of ZF10-ΔRARα under in vivo environmental conditions related to endogenous retinoic acid-related factors; (b, c) are schematic diagrams of engineered tumor cell death under ATRA-induced conditions; (d) is a schematic diagram of the experimental procedure of the tumor re-excitation model; (e) is a schematic diagram of tumor growth and survival in the re-excitation model; Figure 6 This is an example of the application of the ZF10-ΔRARα system in regulating luciferase reporter gene expression in other cell types, as described in Example 2. The cells shown express ZF10-ΔRARα and P... ZF10 - Luciferase reporter system, and detection of luciferase activity under different ligand or antagonist treatment conditions; Figure 7This is an example of the application of the ZF10–ΔVDR system in mice to the response to calcitriol stimulation, as described in Example 3. (a) is a schematic diagram illustrating the pharmacological regulation of ZF10–ΔVDR in C57BL / 6J mice via microencapsulation; (b) shows the stable delivery of P... ZF10 HEK293T cells expressing the SEAP reporter gene and ZF10–ΔVDR were transplanted into mice via intraperitoneal transplantation, and serum SEAP expression was detected after calcitriol treatment. The in vivo or in vitro experimental results shown in the figures are used to exemplify the feasibility and regulatory characteristics of the gene regulation system described in this invention; the specific experimental parameters and detection methods are as described in the embodiments of the specification. Detailed Implementation

[0028] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise defined, all technical terms used below have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in the present invention are commercially available or can be prepared by existing methods.

[0029] Terms and abbreviations ADSCs (Adipose-Derived Stromal Cells), adipose-derived mesenchymal stromal cells; AF-1 (Activation Function-1), transcriptional activation functional region 1; APA, alginate-poly-L-lysine-alginate microcapsules; ATRA (All-trans Retinoic Acid), all-trans retinoic acid; ATRA-ip, exogenous all-trans retinoic acid administered via intraperitoneal injection; BAT-SVF, stromal vascular component cells derived from brown adipose tissue; BDM (Binding DNA Motif), a zinc finger protein binding DNA binding module or binding motif; BSD (Blasticidin Sdeaminase), blastcinin S deaminase; Calcitriol, an endogenous active ligand for the vitamin D receptor; Calcipotriol, an exogenous vitamin D analog that selectively activates the vitamin D receptor; CAR (Chimeric Antigen Receptor); CD69, a marker molecule for T cell activation; Conv. CAR (Conventional CAR), constitutively expressed chimeric antigen receptor; DBD (DNA-binding domain), DNA-binding domain; Δ, used to indicate a truncated or deleted conformation relative to the full-length protein; ΔNR (Truncated Nuclear Receptor), a truncated nuclear receptor without endogenous DNA binding function; EC50, half-maximal effective concentration; EGFP (Enhanced Green Fluorescent Protein), enhanced green fluorescent protein; Endo-ATRA, in vivo endogenous retinoic acid condition; EV (Empty Vector), empty vector control conformation; GSDMD-NT, N-terminal domain of Gasdermin D, used as an effector to induce pyroptosis-like cell death; HMEC-1, human microvascular endothelial cell line; IC50, half-maximal inhibitory concentration; ip(Intraperitoneal Injection); IRES (Internal Ribosome EntrySite); LBD (Ligand-binding domain); LTR (Long Terminal Repeat); Luc / Luciferase, luciferase used as a reporter protein; MCS (Multiple Cloning Site); MIN6, mouse pancreatic β-cell line; NR (Nuclear Receptor); Null, control condition without added exogenous ligands or inhibitors; Ointment, topical ointment administration; OVA (Ovalbumin); P2A, virus-derived 2A self-cleaving peptide used to achieve bicistronic gene expression in mammalian cells; pA, polyadenylation signal; P. EF1α Human elongation factor 1α (EF1α) promoter; P hCMV Early promoter of human cytomegalovirus; P hPGK Human phosphoglycerate kinase gene promoter; P SFFV The splenic lesion forms the viral promoter; P TRE3GV Doxycycline-responsive promoter; PuroR, puromycin resistance gene; pybTATA, minimal promoter sequence containing TATA elements; Pyroptosis, pyroptosis-like cell death process, used in this specification to describe cell lysis mediated by GSDMD-NT; P ZF An artificial promoter containing multiple zinc finger protein-specific binding sites; P ZF1 Artificial promoters containing ZF1 binding sites; P ZF3 Artificial promoters containing ZF3 binding sites; P ZF10Artificial promoters containing ZF10 binding sites; RARα, retinoic acid receptor α; RARγ, retinoic acid receptor γ; RLU (Relative Light Unit); rtTA3, tetracycline-inverse control transactivator; RXRα, retinoic acid X receptor α; SEAP (Secreted Alkaline Phosphatase), human placental secretory alkaline phosphatase; SV40p, simian virus 40 promoter; Tet-On, tetracycline-inducible gene expression regulation system; Tet-free FBS, tetracycline-free fetal bovine serum; THRA1, thyroid hormone receptor α1; 3T3-L1, mouse preadipocyte cell line; VDR (Vitamin D Receptor); Vitamin D3, vitamin D precursor metabolite; WT (Wild-Type); YCT529, a selective retinoic acid receptor α (RARα) antagonist; ZF (Zinc Zinc finger proteins or zinc finger arrays; ZF1, ZF3, and ZF10 represent zinc finger protein arrays with different DNA binding specificities.

[0030] method plasmid construction Representative plasmids used in this study are listed in Table 1. Plasmid construction employed conventional methods in the field, including seamless cloning (homological recombination) and restriction endonuclease digestion and ligation. After construction, the correctness of the inserted fragment and its reading frame consistency could be verified through restriction enzyme digestion analysis and / or sequencing.

[0031] Reagent and Ligand Preparation Representative small molecules used for system induction or inhibition include: all-trans retinoic acid (ATRA), retinoic acid receptor antagonists (such as YCT529), 9-cis retinoic acid, vitamin D analogs (such as calcipotriol) and active vitamin D ligands (such as calcitriol), vitamin D precursors or precursor-related metabolites (such as vitamin D3), thyroid hormone (T3), and doxycycline. In in vitro experiments, these small molecules are typically prepared into stock solutions using DMSO or other pharmaceutically acceptable solvents, diluted appropriately, and added to cell culture media. The stock solutions can be stored at low temperatures and protected from light to reduce repeated freeze-thaw cycles and degradation.

[0032] In in vivo experiments, small molecules can be dissolved or dispersed in vegetable oils (such as corn oil) or other pharmaceutically acceptable carriers and administered orally via gavage or injection. For local induction, retinoids or vitamin D analogs can be further prepared into topical formulations (such as ointments / gels / creams or ethanol-based solutions) and applied topically to shaved skin areas. The specific solvent-to-matrix ratio, drug concentration, and frequency of administration in topical formulations can be adjusted within the conventional range in this field according to the experimental objectives.

[0033] Cell culture Representative cell types include HEK293T, human keratinocytes HaCaT, hTERT immortalized brown adipose-derived stromal vascular fraction cells (hBAT-SVF), primary human dermal fibroblasts, Jurkat, K562, B16-F10 (with or without stable OVA expression), MIN6, HMEC-1, primary human adipose-derived stromal cells (ADSCs), and 3T3-L1, etc.

[0034] Adherent cells can be cultured in media such as DMEM, DMEM / F-12, or MCDB 131, while suspension cells can be cultured in RPMI 1640. Appropriate proportions of serum, antibiotics, and necessary growth factors should be added according to the cell type. Cell culture is typically performed at 37°C and 5% CO2. Specific culture medium formulations and supplementation factors can be performed according to standard conditions in this field or cell line recommendations.

[0035] Isolation of primary human dermal fibroblasts Skin tissue samples were obtained with informed consent. After removing subcutaneous tissue, a stepwise digestion strategy was used to separate the epidermis and dermis. The dermal fraction was further digested with collagenase and DNase, filtered to obtain a cell suspension, and then cultured and expanded in fibroblast growth medium. The obtained fibroblasts were characterized and confirmed by flow cytometry detection of typical surface markers (e.g., CD90 and CD105 positive, but CD45 negative) and used for subsequent experiments.

[0036] White adipocyte differentiation of 3T3-L1 cells After 3T3-L1 preadipocytes were expanded to confluence in conventional culture medium, they were induced to differentiate using an induction system containing insulin, IBMX, dexamethasone, and T3. Subsequently, they were switched to maintenance medium containing insulin and T3 for continued culture. The differentiation cycle and medium change frequency could be adjusted according to the degree of lipid droplet formation and downstream assay requirements.

[0037] Brown adipocyte differentiation of BAT-SVF cells After BAT-SVF cells have expanded to a high degree of confluence, differentiation is induced using brown adipose tissue induction medium, with the medium being changed at fixed intervals. The induction medium may contain IBMX, dexamethasone, insulin, T3, indomethacin, pantothenic acid, and biotin to promote the formation of the brown adipose tissue phenotype. The induction medium can be freshly prepared or aliquoted and reconstituted, and must be aseptically treated before use.

[0038] Instantaneous transfection HEK293T and other adherent cells can be transiently transfected using polyethyleneimine (PEI) or other commonly used transfection reagents in this field. Generally, cells are seeded to an appropriate density (e.g., 70–80% confluence) before adding the transfection complex, incubating for a certain period, and then replacing with fresh culture medium. For construct screening or mutant screening, the transfection system can be scaled down proportionally in a multi-well plate system and paralleled.

[0039] Preparation of lentiviruses and establishment of stable cell lines Lentiviral particles can be prepared in HEK293T cells using commonly used packaging systems in this field. After a certain period of transfection, the viral supernatant is collected, cell debris is removed, and the viral fluid is filtered to obtain the viral solution, which can be stored at low temperatures for a short period or cryopreserved for a long period.

[0040] For suspension cells such as Jurkat and K562, viral supernatant containing infection-promoting agents can be used for transduction, and centrifugation can be combined to improve efficiency. For adherent cells, viral supernatant can be added directly for infection at an appropriate confluence level. After infection, a stable expression population can be obtained by flow cytometry sorting or antibiotic screening. The type and working concentration of antibiotic can be determined within the conventional range in this field based on cell tolerance.

[0041] Cytotoxicity and cell death detection Cell viability can be assessed using colorimetric or luminescent assay kits based on metabolic activity. Cell death can be assessed using flow cytometry with Annexin V and membrane integrity dyes to evaluate phosphatidylserine eversion and changes in membrane permeability. Samples were stained under dark conditions before being analyzed using standard gating strategies.

[0042] Animal experiments Laboratory animals can be immunocompetent mice (such as C57BL / 6J) or immunodeficient mice (such as BALB / c nude mice), and should be housed under SPF conditions. Mice of appropriate sex can be selected for research purposes.

[0043] Transgenic induction in mice To assess in vivo induction effects, engineered cells can be encapsulated in microcapsules (e.g., alginate-polylysine-alginate microcapsules) or mixed with matrix gel before implantation. Cells can be administered via intraperitoneal injection or subcutaneous implantation in the back. The number of cells implanted per mouse and the volume of the carrier can be adjusted within the conventional range in this field according to the experimental design.

[0044] Following implantation, mice can receive systemic administration (e.g., oral or injectable) or local administration (e.g., topical formulations or ethanol-based solutions) to induce or inhibit target gene expression. Dosage frequency, dosage range, and treatment duration can be optimized based on induction kinetics and safety requirements. Blood samples can be collected at specific time points after implantation to detect serum reporter protein (e.g., SEAP) levels, thereby assessing in vivo induction efficiency.

[0045] mouse model of melanoma In vivo functional validation can be performed using the B16-F10-OVA melanoma model. Engineered tumor cells are subcutaneously seeded to form a primary tumor. After a certain period of tumor growth, the primary tumor is resected, and parental tumor cells are then introduced on the contralateral side to assess tumor growth-related phenotypic changes. Tumor volume can be measured periodically with calipers, and volume changes are calculated using formulas commonly used in the field.

[0046] Statistical analysis In vitro experiments can be repeated and expressed as mean and dispersion indices; in animal experiments, randomization can be used and conventional statistical methods can be employed to compare differences between groups. Statistical methods and significance thresholds should be performed in accordance with prevailing practices in the field.

[0047] Table 1. Exemplary nucleic acid vectors of the present invention and their functional descriptions (Note: The vectors listed in this table are exemplary constructions used to illustrate the implementation of the gene regulation system of the present invention, and should not be construed as limiting the vector backbone, promoter type, or element combination.) Note: In the exemplary embodiments of the present invention, the truncated construction of RARα is based on cloning of a known isotype sequence (e.g., RefSeq NP_001019980.1). For ease of description and comparison, the mutation site numbering described herein refers to the amino acid numbering system of another commonly used RARα isotype (e.g., RefSeq NP_000955.1) to maintain consistency with existing research reports. It should be noted that different RARα isotypes have sequence differences in the N-terminal region, while their DNA-binding domain (DBD), hinge region, and ligand-binding domain (LBD) are structurally and functionally conserved; therefore, the equivalent amino acid sites and their functionally equivalent mutations corresponding to different numbering systems should all be considered to fall within the protection scope of the present invention.

[0048] It should be noted that the exemplary nucleic acid vectors listed in Table 1 are only used to illustrate the construction form and functional implementation path of the gene regulation system of the present invention in different embodiments, and do not constitute a limitation on the vector backbone, promoter type, cistron linkage method, reporter gene, or selection marker. In different embodiments, the transcriptional regulatory protein may be expressed by a constitutive or conditional promoter, and the target gene, reporter gene, or selection marker may be linked by, for example, IRES, 2A self-cleaving peptide, or other polycistron expression methods, and the reporter gene or selection marker used may be selected or replaced according to experimental or application requirements. Equivalent substitutions, deletions, or combinations of the above-mentioned vector components made by those skilled in the art without departing from the technical concept and claims of the present invention should be considered as falling within the protection scope of the present invention.

[0049] Table 2. Exemplary ZF-NR constructs of the present invention and their corresponding nucleic acid sequences. The nucleic acid sequences listed in Table 2 are exemplary embodiments of the present invention, used to illustrate the construction methods of zinc finger-nuclear receptor fusion transcriptional regulatory proteins and their associated regulatory elements. The present invention is not limited to the specific nucleic acid sequences or codon formats listed. Those skilled in the art, without departing from the technical concept and claims of the present invention, may make equivalent substitutions, synonymous mutations, degenerate codon substitutions, domain boundary adjustments, or other changes to the relevant nucleic acid sequences that do not affect their ligand response and transcriptional regulatory functions; all such changes should be considered to fall within the protection scope of the present invention.

[0050] Example 1 Construction and exemplary performance description of the zinc finger-truncated retinoic acid receptor gene regulatory system In this embodiment, following the materials and methods described above, a transcriptional regulatory protein was constructed by fusing a human or humanized zinc finger DNA binding module with a truncated retinoic acid receptor domain. This protein was then combined with an artificial promoter containing multiple zinc finger binding sites and a reporter gene to evaluate the feasibility of this type of structure as a small molecule responsive gene regulatory module.

[0051] When the gene regulatory system was expressed in mammalian cells, it was observed that the reporter gene remained at a low expression level without the addition of a ligand; however, after administration of all-trans retinoic acid, the reporter gene expression level increased, demonstrating that the fusion transcriptional regulatory protein can convert small molecule ligand signals into transcriptional regulatory outputs, thereby achieving ligand-responsive gene regulation. These results exemplify the feasibility of the zinc finger-truncated nuclear receptor structure as a gene regulatory module.

[0052] In one embodiment, a zinc finger-truncated nuclear receptor fusion transcriptional regulator protein is constructed by fusing a human or humanized orthogonal zinc finger array (ZF10) with a truncated domain (preserving the hinge region and ligand-binding domain) of the retinoic acid receptor RARα (see [link to original text]). Figure 1 a). This design utilizes the interaction between the retinoic acid receptor and its ligand to achieve transcriptional regulation, while also reducing non-specific recognition of endogenous DNA sequences in the host genome based on the design characteristics of the selected zinc finger DNA binding module.

[0053] In one exemplary embodiment, a full-length retinoic acid receptor RARα (1-457 amino acid residues) is fused with a zinc finger array ZF10 and coupled with an artificial promoter (P) containing zinc finger binding sites. ZF10 The SEAP reporter gene, driven by a ligand, was co-introduced into mammalian cells (such as HEK293T). Upon administration of all-trans retinoic acid (ATRA), upregulation of the reporter gene expression was observed, exemplifying the ligand-dependent transcriptional activation characteristics of this system (see [link to documentation]). Figure 1 b).

[0054] In a further exemplary embodiment, to reduce basal expression and optimize ligand-inducible performance, various nuclear receptor truncated configurations were constructed and compared. These configurations include, but are not limited to: (i) a configuration in which the zinc finger array ZF10 is inserted between the N-terminal transcriptional activation region (approximately 1–82 amino acid residues) of the retinoic acid receptor and its hinge-ligand binding region (approximately 149–457 amino acid residues); (ii) a configuration in which the zinc finger array ZF10 is directly fused to both the hinge region and the ligand binding region of the retinoic acid receptor, which in one embodiment may be referred to as ZF10-ΔRARα; and (iii) a further truncated configuration starting from approximately 195 amino acid residues, which removes the hinge region and retains only the ligand binding region. The above amino acid ranges are for illustrative purposes only and do not constitute a limitation on the nuclear receptor truncation methods of the present invention. In the tested configurations, the ZF10-ΔRARα configuration maintained low basal expression without ligand addition, while still inducing reporter gene expression under ligand stimulation (see [link to relevant documentation]). Figure 1 (b) In one embodiment, this configuration will be selected as an exemplary configuration for illustration in subsequent embodiments.

[0055] The impact of the zinc finger DNA binding module on system performance was then evaluated. In addition to ZF10, other zinc finger arrays (e.g., ZF1 and ZF3) were further constructed and tested with fusion configurations of the same truncated nuclear receptor domain. Under ligand stimulation, all of these configurations induced reporter gene expression (see [link to relevant documentation]). Figure 1 c), indicating that different zinc finger DNA binding modules are substitutable in this gene regulatory system. Under the tested conditions, ZF10 exhibited a relatively balanced characteristic between basal expression and induced response (see c). Figure 1 (b, c), and therefore used as an exemplary zinc finger DNA binding module in subsequent embodiments. In a transient transfection reporter system, dose-response analysis was performed on the system, and ZF10-ΔRARα-mediated SEAP expression was observed to respond to changes in all-trans retinoic acid concentration, with a half-maximal effective concentration of approximately 22 nM (see [link to relevant documentation]). Figure 1 d), This result exemplifies that the system possesses tunable small molecule response characteristics.

[0056] To evaluate the modularity of the system, the truncated domain of RARα was replaced with corresponding truncated domains of other nuclear receptors, including RARγ, RXRα, THRA1, and VDR, to construct various zinc finger-truncated nuclear receptor chimeras (ZF–ΔNR). Under the administration of the corresponding ligands (e.g., ATRA for RARγ, 9-cis-retinoic acid for RXRα, iodothyronine for THRA1, and calcipotriol for VDR), all tested chimeras exhibited ligand-dependent reporter gene induction (see [link to relevant documentation]). Figure 1(eh), indicating that the fusion of the orthogonal zinc finger module with the truncated nuclear receptor domain can be used to construct gene regulatory systems with ligand-responsive properties. In the tested conformations, the ZF10-ΔVDR conformation, while exhibiting low basal expression, could still be induced by the corresponding ligand ( Figure 1 h), which will be described as one of the exemplary configurations in subsequent embodiments.

[0057] Furthermore, ZF10-ΔRARα and ZF10-ΔVDR were compared with commonly used classical Tet-On gene regulation systems. Under the same detection conditions, ligand-induced transgene expression was observed in all systems, while the zinc finger-truncated nuclear receptor-based gene regulation system showed lower background expression in the uninduced state (see [link to study]). Figure 1 i) illustrates its potential applicability in application scenarios that require strict gene expression regulation.

[0058] In summary, this embodiment exemplifies the characteristics of zinc finger-truncated retinoic acid receptors and their related conformations in terms of low basal activity and ligand-induced response, providing a basis for further functional verification in subsequent embodiments.

[0059] Example 2 Examples of the inhibitory regulation of gene regulatory systems by retinoic acid receptor antagonists Based on the gene regulation system described in Example 1, this example exemplifies the inhibitory regulatory effect of retinoic acid receptor antagonists on the activity of this gene regulation system. Under conditions of all-trans retinoic acid (ATRA)-induced system activation, the introduction of a retinoic acid receptor antagonist resulted in a decrease in reporter gene expression levels, indicating that the system described in this invention, in addition to being activated by ligands, can also achieve inhibitory regulation through antagonists, thereby forming a regulatory mode combining activation and inhibition.

[0060] In one embodiment, a stable SEAP reporter gene (P) driven by an artificial promoter was established. ZF10 The regulatory activity of ZF10-ΔRARα was assessed in mammalian cells (HEK293T polyclonal stable line) with SEAP-mediated retinoid receptor agonism (RTR) or its antagonist YCT529. Reporter gene expression was detected after treatment (see [link to study]). Figure 2 a). In this experimental system, the half-maximal effective concentration induced by all-trans retinoic acid was observed to be approximately 1.6 nM (see [link to experimental system]). Figure 2 (b) This value is lower than the response concentration observed in transient transfection reporter systems (approximately 22 nM, see [reference]). Figure 1d). In one embodiment, this difference may be related to the genomic integration status of the reporter construct. Further, the aforementioned response concentration range is comparable to the reported physiological concentration range of circulating ATRA in vivo. In one embodiment, this observation suggests that, in vivo, this gene regulatory system may be influenced by endogenous retinoic acid-associated ligands.

[0061] Based on the above observations, in one embodiment, it can be inferred that the system can maintain a low background expression state under in vitro conditions, while under in vivo conditions, activation driven by endogenous ligands may occur, thereby maintaining transgene expression without the need for continuous administration of exogenous ligands. However, in some application scenarios, it is still necessary to introduce pharmacological inhibition methods to achieve more precise regulation.

[0062] Based on this, the feasibility of pharmacologically inhibiting the system using an antagonist was further investigated. In one embodiment, the retinoic acid receptor antagonist YCT529 was selected as an example for verification. In cultured cells, the addition of YCT529 showed an inhibitory effect on all-trans retinoic acid-induced reporter gene activity, causing a rightward shift in the system's response curve. In one embodiment, the corresponding half-maximal effective concentration was approximately 436 nM (see [link to relevant documentation]). Figure 2 b). Meanwhile, under all-trans retinoic acid (e.g., 10 nM) induction conditions, the half-maximal inhibitory concentration (IC50) of YCT529 on reporter gene activity is approximately 25 nM (see [link to relevant documentation]). Figure 2 c). Furthermore, ZF10-ΔRARα and corresponding reporter systems were introduced into various mammalian cell models for detection, including but not limited to human brown adipocyte progenitor cells (BAT-SVF). Figure 2 d) and differentiated brown fat-like cells (see Figure 6 a) Human microvascular endothelial cells (HMEC-1, Figure 2 e) Mouse pancreatic β cells (MIN6, Figure 2 f) Primary human adipose-derived stromal cells (ADSCs, Figure 2 g), as well as mouse 3T3-L1 precursor adipocytes and differentiated adipocytes, etc. (see g), and mouse 3T3-L1 precursor adipocytes and differentiated adipocytes, etc. (see g) Figure 6 (b, c) A decrease in reporter signal was observed in both cases when an antagonist was added under ligand-inducible conditions (see [link to relevant documentation]). Figure 2 (d–g), which exemplifies that this inhibitory regulation phenomenon can be achieved in different cell backgrounds and differentiation states.

[0063] To assess whether the ZF10-ΔRARα gene regulatory system can be activated under physiological conditions, in one embodiment, a gene expressing ZF10-ΔRARα and stably carrying P... ZF10HEK293T cells containing the SEAP reporter gene were encapsulated in semi-permeable microcapsules and implanted into immunocompetent mice (e.g., C57 / BL6J). Under control conditions, SEAP signaling was detectable in serum without the administration of exogenous ligands (see [link to control treatment]). Figure 2 i, labeled as the endo ATRA group, endogenous retinoic acid condition. Compared with the control treatment, a decrease in serum SEAP levels was observed after administration of a retinoic acid receptor antagonist (e.g., oral YCT529), suggesting that the detected reporter signal in this embodiment may be related to the activation of the ZF10-ΔRARα system by endogenous retinoic acid-associated ligands. Conversely, an increase in SEAP expression levels was further observed after treatment with exogenous all-trans retinoic acid (e.g., intraperitoneal injection of ATRA), indicating that the system remains responsive to exogenous ligand stimulation in vivo. Similar endogenous retinoic acid-driven ZF10-ΔRARα activation was also observed in another independent experiment (see [link to study]). Figure 4 (b) This provides an example of how to support the reproducibility of the in vivo response.

[0064] The above results exemplify that the ZF10-ΔRARα gene regulatory system can be activated by endogenous retinoic acid-related ligands in one embodiment, and can be enhanced by exogenous retinoic acid, while being inhibited by retinoic acid receptor antagonists (e.g., YCT529). These results also exemplify that this gene regulatory system can achieve a hierarchical regulatory scheme in one embodiment: in vitro OFF, in vivo autonomous ON, and pharmacological OFF when necessary, providing a basis for constructing a regulatory strategy combining activation and inhibition in specific application scenarios.

[0065] The above results indicate that, in the presence of endogenous ligands, the gene regulation system of the present invention can be in an activated state, and this activated state can be inhibited by corresponding antagonists, thereby constructing a gene regulation mode that combines an endogenous ligand-driven default activation state with pharmacologically controllable shutdown.

[0066] Example 3 Construction of a zinc finger-truncated vitamin D receptor gene regulatory system and its responsiveness to specific vitamin D analogs: An exemplary illustration In this embodiment, a truncated domain of the vitamin D receptor was used instead of the retinoic acid receptor domain in Example 1 to construct a zinc finger-truncated vitamin D receptor fusion transcriptional regulatory protein. This protein was then combined with an artificial promoter containing a zinc finger DNA binding site and a reporter gene to exemplarily evaluate the gene regulatory characteristics of this type of structure under vitamin D ligand conditions.

[0067] In one exemplary embodiment, the gene regulation system maintains a low reporter gene expression level in the presence of endogenous vitamin D metabolites within physiologically relevant concentration ranges; however, upon administration of a specific vitamin D analog, an increase in reporter gene expression can be observed. This exemplifies that the zinc finger-truncated nuclear receptor architecture can respond to different small molecules by replacing the nuclear receptor ligand response module.

[0068] Furthermore, it was evaluated whether the system could be selectively activated by exogenous ligands rather than endogenous metabolites. Figure 3 a). Since endogenous vitamin D-related metabolites are ubiquitous in the circulatory system, in one embodiment, their effect on systemic background activation was first examined. Under the test conditions, no significant activation of the ZF10–ΔVDR configuration was observed in vitamin D precursor metabolites (e.g., vitamin D3) within a certain concentration range. Figure 3 b). In contrast, the major endogenous active ligand of the vitamin D receptor, 1,25-dihydroxyvitamin D (calcitriol), induces only limited reporter gene activation under typical serum concentration conditions, below the observed half-maximal effective response concentration (see [link to relevant documentation]). Figure 3 c). The above results illustrate, in one embodiment, that the ZF10–ΔVDR configuration is less responsive to endogenous vitamin D metabolites, while in certain embodiments it can still be selectively activated by exogenous vitamin D analogs (e.g., calcipotriol), thereby maintaining a low basal expression level under physiological conditions.

[0069] To evaluate the applicability of this system in different cellular contexts, a stable reporter cell line was constructed in one exemplary embodiment, wherein the ZF10-ΔVDR construct was coupled with a luciferase reporter gene (e.g., P) regulated by a zinc finger binding site. ZF10 -Luciferase reporter was stably integrated. Subsequent detection was performed in various cell types, including but not limited to precursor cells derived from brown adipose tissue (…). Figure 3 d) Human microvascular endothelial cells ( Figure 3 e) Mouse pancreatic β-cell-like cells ( Figure 3 f) Primary adipose-derived stromal cells ( Figure 3 g), primary dermal fibroblasts ( Figure 3 h) and keratinocytes ( Figure 3i) After treatment with exogenous vitamin D analogs (e.g., calcipotriol), reporter gene expression induction can be observed in various cell types, while endogenous active ligands at physiological concentrations (e.g., calcitriol (100 pM)) show little or no activation. In one embodiment, vitamin D analog (e.g., calcipotriol)-induced changes in reporter gene expression can also be observed in skin-associated cell types (e.g., fibroblasts and keratinocytes), exemplifying that the system also possesses ligand-responsive capabilities in a skin-associated cell environment.

[0070] In a further exemplary in vivo embodiment, a SEAP reporter gene (e.g., P) expressing ZF10-ΔVDR and stably carrying a zinc finger binding site-regulated SEAP reporter gene is used. ZF10 Mammalian cells (e.g., HEK293T) containing the SEAP reporter gene are encapsulated in semi-permeable microcapsules and implanted into immunocompetent mice (e.g., C57BL / 6). In one embodiment, changes in serum SEAP levels can be detected after treatment with an exogenous vitamin D active ligand (e.g., calcitriol). Figure 7 (a, b) exemplify that the system retains its responsiveness to ligand stimulation in an in vivo environment. To assess the feasibility of local induction, in one embodiment, the microcapsule is subcutaneously implanted, and a vitamin D analog (e.g., calcipotriol) is administered locally at the implantation site. Figure 3 j). Under the tested conditions, elevated serum SEAP levels were observed ( Figure 3 In contrast, in the same model, no detectable reporter gene activation was observed with oral vitamin D supplementation (a dietary precursor metabolite). Figure 3 k).

[0071] The above in vivo results illustrate, in one embodiment, that the ZF10–ΔVDR gene regulatory system can be activated by exogenous ligands under in vivo conditions, and can also be spatially induced by local administration, while maintaining a low response to dietary vitamin D-related metabolites under the tested conditions.

[0072] In summary, this embodiment exemplifies that the ZF10–ΔVDR gene regulation system can mediate selective small molecule responsive gene expression in multiple cell types and achieve systemic or spatially restricted activation under in vivo conditions, providing an exemplary basis for its use in different application scenarios.

[0073] Example 4 An exemplary illustration of the regulation of endogenous ligand response thresholds through mutations in the nuclear receptor ligand binding region. Based on the gene regulation systems described in Embodiments 1 and 3 above, this embodiment exemplifies how to reduce the system's responsiveness to endogenous ligands at physiological concentrations by introducing amino acid substitution mutations into the nuclear receptor ligand binding region, thereby further regulating the response threshold of the gene regulation system. In one embodiment, a zinc finger-truncated nuclear receptor fusion transcriptional regulator conformation with altered response threshold is obtained by mutating the nuclear receptor ligand binding region. Under physiologically relevant endogenous ligand conditions, the system corresponding to the mutated conformation can maintain a low basal expression level; however, reporter gene expression can still be induced after stimulation with exogenous pharmacologically relevant concentrations of ligand. These results exemplify that by mutating and engineering the nuclear receptor domain, its sensitivity to endogenous ligands can be regulated while retaining ligand responsiveness.

[0074] In one exemplary embodiment, ZF10–ΔRARα_K207N was selected as a representative mutant for functional characterization. Compared to the wild-type ZF10-ΔRARα configuration without the introduced mutation, the K207N variant exhibited a rightward shift in the dose-response curve in the dose-response analysis, corresponding to a higher half-maximal effective response concentration (approximately 72.7 nM, compared to approximately 1.6 nM for the unmutated configuration; see [link to relevant documentation]). Figure 4 a) This exemplifies that the mutant exhibits reduced sensitivity to retinoic acid signaling under physiological conditions while retaining its responsiveness to higher concentrations of exogenous ligands. To further evaluate the regulatory characteristics of the mutant in vivo, in one embodiment, a SEAP reporter gene (e.g., P) expressing wild-type or K207N mutant ZF10-ΔRARα and stably carrying a zinc finger binding site is used. ZF10 Cells carrying the K207N mutant SEAP were implanted into animals. In the absence of exogenous retinoic acid, animals carrying the wild-type conformation showed higher serum SEAP levels, while animals carrying the K207N mutant conformation showed lower SEAP levels (see [link to original text]). Figure 4 (b) As an example, in one embodiment, the mutation reduces the system's background response to endogenous retinoic acid-related signals. In a further exemplary embodiment, by treating with exogenous retinoic acid, an increase in SEAP expression levels corresponding to the K207N mutant was observed, while no similar change was observed under control conditions (see [link to documentation]). Figure 4(c) This exemplifies how the mutant retains its responsiveness to exogenous ligand stimulation under in vivo conditions. Furthermore, to evaluate the system's usability in local drug delivery scenarios, in one embodiment, engineered cells containing the mutant and the SEAP reporter gene were encapsulated in microcapsules and subcutaneously implanted. Subsequently, a retinoid preparation was administered locally to the implantation site, and serum SEAP levels were observed to change with treatment conditions under the tested conditions (see [link to relevant documentation]). Figure 4 d) This example illustrates that the activation of the mutant gene regulatory system can also be achieved through local administration.

[0075] To extend this mutant gene regulatory system to immune cell-related applications, in one exemplary embodiment, a chimeric antigen receptor (CAR) signaling model based on Jurkat cells is employed. The ZF10–ΔRARα_K207N conformation is used to regulate the expression or function of a CD19-specific CAR in Jurkat cells, and the cells are co-cultured with CD19-positive target cells. Upon stimulation with exogenous retinoic acid, signal changes associated with CAR activation are observed and quantified using T cell activation markers such as CD69 (see [link to relevant documentation]). Figure 4 f). Without ligand, the mutant system maintains low baseline activity, while enhanced CAR-related signals are observed under exogenous ligand stimulation, illustrating the system's suitability for applications requiring strict baseline control and activation when needed.

[0076] In summary, this embodiment exemplifies how introducing amino acid substitution mutations into the nuclear receptor ligand binding region can regulate the response threshold of the zinc finger-truncated nuclear receptor gene regulatory system to endogenous and exogenous ligands, thereby reducing background response under physiological conditions while maintaining ligand induction ability. This provides an exemplary basis for functional verification in different application scenarios in subsequent embodiments.

[0077] Example 5 ZF10-ΔRARα-driven GSDMD-NT regulation example In this embodiment, cell death-related effector genes are selected as the regulatory targets to illustrate the controllability of the ZF10–ΔRARα-based gene regulation system in the scenario of regulating the expression of genes with high cytotoxic effects.

[0078] In one exemplary embodiment, the N-terminal domain of Gasdermin D (GSDMD-NT) is selected as an example of an effector gene. GSDMD-NT can disrupt cell membrane structure and induce lytic cell death; such effector genes typically require strict expression control in in vitro and in vivo applications. This embodiment exemplarily utilizes a zinc finger-truncated nuclear receptor gene regulation system to regulate the expression of GSDMD-NT. In one embodiment, an engineered B16-F10-OVA tumor cell line was constructed for in vitro and in vivo experiments. The constructed B16-F10-OVA... ZF10–ΔRARα / GSDMD-NT In cells, GSDMD-NT expression is controlled by an artificial promoter containing a zinc finger binding site and regulated by the ZF10–ΔRARα gene regulatory system. As a control, B16-F10-OVA cells that do not express ZF10–ΔRARα were constructed. EV / GSDMD-NT Cells, as well as B16-F10-OVA cells carrying only the empty vector, were used for comparative analysis.

[0079] Under in vitro culture conditions, the proliferation status of different engineered cell lines was comparable without exogenous retinoic acid treatment, demonstrating that basal expression of GSDMD-NT did not significantly affect cell growth under uninduced conditions. Since physiological levels of retinoic acid signaling are lacking in conventional in vitro culture conditions, low nanomolar concentrations of all-trans retinoic acid were used as an exemplary stimulus in in vitro experiments to simulate endogenous activation conditions.

[0080] Following treatment with all-trans retinoic acid, decreased cell viability and an increased proportion of cell death were observed in B16-F10-OVA ZF10–ΔRARα / GSDMD-NT cells (see [link to original text]). Figure 5 b, 5c), while no significant changes were observed in control cells. Furthermore, SYTOX Green staining of cell membrane permeability allowed for the observation of lysis-related cell death signals (see [link to SYTOX Green staining]). Figure 5 (b, 5c) This phenomenon is consistent with the characteristics of GSDMD-NT mediated lytic cell death.

[0081] To assess the feasibility of in vivo application, a contralateral re-stimulation tumor model was used in one exemplary embodiment. B16-F10-OVA was applied... ZF10–ΔRARα / GSDMD-NT ZF10–ΔRARα / GSDMD-NT cells were implanted into the left side of syngeneic mice to form an initial tumor, which was surgically removed after a certain period of time. Under the tested conditions, the initial tumor derived from ZF10–ΔRARα / GSDMD-NT cells showed a different volume change compared to the control group (see [link to test]). Figure 5 e).

[0082] Subsequently, parental B16-F10-OVA cells were implanted into the contralateral flank of mice. In some animals, differences in tumor formation and growth behavior compared to the control group were observed (see...). Figure 5 f), and during the follow-up process, different trends in survival status were recorded (see f). Figure 5 g). The above in vivo observations exemplify that, in one embodiment, the ZF10–ΔRARα gene regulatory system may be affected by endogenous retinoic acid-related factors in the in vivo environment, thereby driving the expression of GSDMD-NT and triggering corresponding cell death-related biological effects.

[0083] In summary, this embodiment exemplifies that the gene regulation system based on ZF10–ΔRARα can maintain low background expression of GSDMD-NT under in vitro conditions and be activated under specific stimuli in vivo, thereby achieving controllable expression of cell death effector genes and providing an exemplary basis for its application in scenarios requiring strict regulation of effector gene expression.

[0084] The above embodiments illustrate, from different perspectives, various ways to achieve controllable regulation of gene expression based on nuclear receptor engineering design.

[0085] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A small-molecule responsive gene regulatory system based on human zinc finger-truncated nuclear receptor fusion, characterized in that, The gene regulation system includes a fusion transcription regulatory protein, a target DNA regulatory sequence containing a zinc finger DNA binding site, and a target gene operatively linked to the target DNA regulatory sequence; wherein the fusion transcription regulatory protein is formed by fusing a human or humanized programmable zinc finger DNA binding module with a truncated nuclear receptor domain; the zinc finger DNA binding module is capable of specifically recognizing and binding the target DNA regulatory sequence containing the zinc finger DNA binding site; the truncated nuclear receptor domain binds to a ligand and, under the action of the ligand, regulates the expression of the target gene; The truncated nuclear receptor's structural domain no longer possesses the endogenous DNA-binding function of its inherent DNA-binding domain; after binding with the ligand, the truncated nuclear receptor mediates the regulation of the target gene's expression level through its transcriptional regulatory function.

2. The small molecule responsive gene regulation system based on human zinc finger-truncated nuclear receptor fusion according to claim 1, characterized in that, The function of the N-terminal transcriptional activation region (AF-1) of the truncated nuclear receptor is weakened or eliminated, thereby mainly preserving the hinge region and ligand-binding region.

3. The small molecule responsive gene regulation system based on human zinc finger-truncated nuclear receptor fusion according to claim 1, characterized in that, The truncated nuclear receptor is one or more of the following: retinoic acid receptor (RAR), vitamin D receptor (VDR), retinoic acid X receptor (RXR), thyroid hormone receptor (THR), or other nuclear receptors with ligand-binding domains and mutants thereof that maintain transcriptional regulatory function. The nuclear receptor is used in the gene regulation system as a transcriptional regulatory module to regulate the transcription process of target genes, rather than merely as a ligand-dependent nuclear localization or protein stability regulatory module.

4. The small molecule responsive gene regulation system based on human zinc finger-truncated nuclear receptor fusion according to claim 3, characterized in that, The truncated nuclear receptor is a mutant of the retinoic acid receptor (RAR), which reduces its responsiveness to physiological concentrations of endogenous ligands.

5. The gene regulation system according to claim 4, characterized in that, The mutant is the K207N mutant located in the α-ligand binding domain of the retinoic acid receptor.

6. The gene regulation system according to claim 1, characterized in that, The zinc finger DNA binding module is a zinc finger array with low crosstalk to the host genome endogenous DNA sequence, preferably a ZF10, ZF1, or ZF3 zinc finger array.

7. The small molecule responsive gene regulation system based on human zinc finger-truncated nuclear receptor fusion according to claim 1, characterized in that, The ligand is an endogenous or exogenous small molecule capable of binding to the ligand-binding domain of the truncated nuclear receptor and activating or inhibiting the activity of the gene regulatory system.

8. The small molecule responsive gene regulation system based on human zinc finger-truncated nuclear receptor fusion according to claim 1, characterized in that, The target gene can be an endogenous gene or an exogenous gene.

9. A small molecule responsive gene regulation system based on a human zinc finger-truncated nuclear receptor fusion according to claim 1, characterized in that, The zinc finger DNA binding module specifically recognizes and binds to zinc finger DNA binding sites located in the promoter region, enhancer region, or other regulatory elements of the target gene.

10. The gene regulation system according to claim 1, characterized in that, When endogenous ligands reach a level sufficient for activation, the gene regulatory system is in an activated state, and this activation state can be inhibited by an antagonist corresponding to the truncated nuclear receptor, thereby achieving a gene regulatory mode that combines an endogenous ligand-driven default activation state with pharmacologically controllable shutdown.

11. The gene regulation system according to claim 1, characterized in that, When the truncated nuclear receptor is a truncated version of the vitamin D receptor, the gene regulatory system maintains a low basal expression level under physiologically relevant conditions of endogenous ligands, and can induce the expression of target genes through exogenously applied vitamin D analogs.

12. The gene regulation system according to claim 1, characterized in that, The gene regulatory system is controlled to be regulated by at least one of the following methods: (i) by antagonist inhibition in an activation state driven by endogenous ligands; (ii) by activation induced by exogenous ligands; and (iii) by mutating the truncated nuclear receptor to modulate the response threshold to endogenous ligands.

13. The use of a small molecule responsive gene regulatory system based on a human zinc finger-truncated nuclear receptor fusion as described in any one of claims 1-12 in regulating the expression of reporter genes, therapeutic proteins, immune receptors, or cell death-related effector factors.

14. The application according to claim 13, characterized in that, The gene regulation system is applied in vitro or in vivo in the form of plasmids, viral vectors, or engineered cells.

15. A gene regulation method, characterized in that, The gene regulation method employs a small molecule responsive gene regulation system based on the human zinc finger-truncated nuclear receptor fusion as described in any one of claims 1-12; the method includes introducing the gene regulation system into cells and regulating the expression of target genes under the action of ligands.

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