Cello-biose-responsive mammalian transgenic expression regulation system and use thereof

Abstract

The present application provides a cellobiose-responsive mammalian transgene expression regulation system, which comprises CebR-O CebR a cellobiose-sensing module, which is adapted to a mammalian cell; the CebR-O CebR The cellobiose-sensing module comprises: (a) a first fusion protein, which is fused from a CebR transcriptional regulator and a transcriptional regulatory domain; and (b) an artificial promoter, which comprises at least one O CebR operator sequence and a core promoter sequence; the O CebR operator sequence can be specifically bound by the CebR transcriptional regulator; the CebR-O CebR The cellobiose-sensing module comprises at least one of the following two regulation modes: Cb OFF mode; and Cb ON mode. The present application also provides corresponding applications.

Description

Technical Field

[0001] This invention relates to the fields of synthetic biology, gene therapy, cell therapy, and biopharmaceutical manufacturing, specifically to a control system for regulating transgenic expression by cellobiose, its construction method, and its application. Background Technology

[0002] Trigger-induced gene switches are important tools in biological research and synthetic biology applications, including gene- and cell-based therapies. Trigger-induced transcriptional control systems reversibly fine-tune transgene expression in response to molecular cues, significantly advancing the rational reprogramming of mammalian cells. Gene therapy applications may be risky due to adverse side effects or off-target effects of transgene products. Therefore, turning transgene expression on and off, or modulating its expression levels, can significantly improve the safety of gene therapy. Bacterial transcription factors (TFs) with helix-turn-helix (HTH) DNA-binding domains have been extensively explored and used to construct orthogonal transcriptional regulatory systems in mammalian cells.

[0003] In recent years, the range of synthetic gene regulatory systems that respond to small molecules (such as caffeine, spearmint, and menthol) or physical stimuli (such as heat, light, or electrical signals) has greatly expanded, providing a variety of options for specific in vivo gene regulation. Furthermore, substantial progress has been made in the development of gene switches using natural products, offering new strategies for synthetic biology applications, including remote control of gene expression, complex protein logic computation, and the treatment of neuropathic pain and metabolic diseases. The therapeutic capabilities of these devices demonstrate that natural molecular activation switches can precisely regulate the dosage of therapeutic proteins, and these switches hold immense potential for future basic research and clinical applications.

[0004] Most currently available mammalian transgenic control devices share common design principles that ensure basic transcriptional controller compatibility and allow for integration into higher-order control networks. The basic control device consists of synthetic transcription factors and prokaryotic response regulators fused with transactivation or silencing domains, which bind to and induce or inhibit chimeric promoters containing specific operand sites. Transcription can be fine-tuned by specific triggering compounds that modulate the promoter affinity of the chimeric transcription factors in a dose-dependent manner.

[0005] However, several challenges need to be considered before these natural molecular control devices can be successfully applied in clinical practice. First, the natural molecules should be completely non-toxic, highly safe, and readily enter specific cells and tissues. Second, these safety-triggered molecules must have appropriate half-lives to ensure good bioactivity of a single injection or oral dose over an appropriate period without leading to excessive production of therapeutic proteins over time. Therefore, future research needs to screen for more natural molecules that meet these requirements. Third, although recent studies have successfully induced the production of therapeutic proteins, high concentrations of induction are still required. Moreover, dose control of transgenic encoded proteins is crucial in clinical treatment, and unlike optogenetic tools, many natural molecules are difficult to remove immediately once the system is activated. Therefore, a feedback strategy that can control the smart switch of gene expression should be considered.

[0006] Furthermore, to improve the stability of gene expression control devices, a simpler programming procedure can be achieved by reducing the number of circuit elements. Besides optimal regulatory performance, the properties of trigger molecules are also crucial when designing control circuits for future gene and cell therapies. Therefore, the latest generation of transgenic control systems considers inducer molecules with minimal potential side effects, such as inducer molecules derived from endogenous metabolites or physiologically inert molecules, such as licensed cosmetic compounds.

[0007] CebR repressor proteins are typical transcriptional regulators widely found in prokaryotes. They specifically bind to the promoter regions of target genes, thereby inhibiting gene transcription. CebR protein activity is typically regulated by small molecule inducers, and this regulatory mechanism provides a theoretical basis for designing novel gene expression systems. Cellobioose, a natural disaccharide molecule, has been shown to effectively bind to and regulate CebR protein activity, making it a potential gene inducer. Summary of the Invention

[0008] The purpose of this invention is to overcome at least one of the shortcomings of the prior art and to provide a cellobiose-responsive mammalian transgenic expression regulation system and its application.

[0009] To address the aforementioned technical problems, a first aspect of the present invention provides a cellobiose-responsive mammalian transgenic expression regulatory system, characterized in that the system comprises CebR–O CebR A cellobiose sensing module, wherein the sensing module is adapted to mammalian cells; The CebR–O CebR The cellobiose sensing module includes: (a) The first fusion protein, formed by the fusion of the CebR transcription factor and a transcriptional regulatory domain; and (b) An artificial promoter containing at least one O CebR The manipulation subsequence and the core starter subsequence, the O CebR The operon sequence can be specifically bound by the CebR transcriptional regulatory factor; The CebR–O CebR The cellobiose sensing module includes at least one of the following two regulation modes: Cb OFF The pattern, in the aforementioned Cb OFF In this model, the first fusion protein is aCebR, the transcriptional regulatory domain is the transcriptional activation domain, and the artificial promoter is P. aCebR Promoter; in the absence of cellobiose, aCebR binds to P aCebR The promoter activates the expression of downstream target genes; in the presence of cellobiose, cellobiose binds to aCebR, causing it to exit P... aCebR Dissociation at the promoter reduces or shuts down the expression of downstream target genes; Cb ON The pattern, in the aforementioned Cb ON In this model, the first fusion protein is iCebR, the transcriptional regulatory domain is a transcriptional repression domain, and the artificial promoter is P. iCebR Promoter; in the absence of cellobiose, iCebR binds to P iCebR The promoter inhibits and suppresses the expression of downstream target genes; in the presence of cellobiose, cellobiose binds to iCebR, causing it to exit P... iCebR Dissociation at the promoter releases transcriptional repression and enables the expression of downstream target genes.

[0010] Preferably, in the aCebR, the transcriptional activation domain is selected from VP64 or VPR, and the core promoter is selected from miniCMV, miniTK, TATA, or YB_TATA; in the P aCebR The promoter contains 1 to 5 copies of O, arranged in tandem, located upstream of the core promoter. CebR Manipulated subsequences.

[0011] Preferably, the transcriptional activation domain is VPR, the core promoter is miniCMV, and O CebR The number of copies of the operon sequence is 3.

[0012] Preferably, in the iCebR, the transcriptional repressor domain is KRAB, and the core promoter is selected from SV40, CMV, EF-1α, or PGK; in the P iCebR The promoter contains the first O CebROperant subclusters and second O CebR Manipulating subclusters, the first O CebR The operon cluster is located upstream of the core promoter, the second O CebR The operator cluster is located downstream of the core promoter.

[0013] Preferably, the first O CebR The manipulator subcluster contains n copies of O CebR The manipulator sequence, n is an integer from 1 to 10, preferably an integer from 1 to 3; the second O CebR The sub-cluster contains m copies of O CebR The manipulator sequence, m is an integer from 1 to 10, preferably an integer from 1 to 5.

[0014] Ideally, n is 3 and m is 2.

[0015] Preferably, the CebR transcriptional regulatory factor is selected from CebR homologs, CebR functional mutants, or CebR coding sequences codon-optimized to adapt to mammalian cell expression.

[0016] Preferably, the downstream target gene encodes a therapeutic protein, which is insulin or a functional fragment or variant thereof; or, the downstream target gene encodes VZV gE protein, IL-17A protein, S-HBsAg protein, or α-Syn protein.

[0017] Preferably, the mammalian cells are selected from HEK293 cells, CHO cells, HeLa cells, NIH3T3 cells, HepG2 cells, Jurkat cells, or primary cells or stem cells derived from mice, rats, monkeys or humans.

[0018] Preferably, the CebR–O CebR The cellobiose sensing module is stably coupled with the protocatechuic acid response system to form a dual-input logic gate, which is selected from AND logic gate, NAND logic gate, NOR logic gate, Cb NIMPLYPCA or PCA NIMPLY Cb.

[0019] A second aspect of the invention provides the use of the system in the preparation of a medicament for treating diabetes.

[0020] Preferably, the diabetes mellitus is type 1 diabetes mellitus.

[0021] Preferably, the drug is a gene therapy drug or a cell therapy drug. Attached Figure Description

[0022] Figure 1 For Cb OFF Schematic diagram of the switch design.

[0023] Figure 2 To evaluate the effects of different chimeric transcription activators and core promoter combinations on Cb OFF Optimization and screening of switching performance. Among them, Figure 2 A is a P constructed using aCebR1 (VP64-CebR) combined with different core promoters. aCebR Report carrier, thereby affecting Cb OFF A diagram illustrating the system's optimization and filtering strategies; Figure 2 B represents HEK293T cells (1×10⁻⁶). 5 Co-transfect plasmid pRF1(P) hCMV -aCebR1-P2A-CDT1-pA) and report carrier pRF3 (P aCebR1 -eGFP-pA), pRF4 (P aCebR2 -eGFP-pA), pRF5 (P aCebR3 -eGFP-pA) or pRF6 (P aCebR4 Representative fluorescence images following (-eGFP-pA). Among them, P... aCebR1–4 In a single O CebR The binding site connects downstream of the core promoter P miniCMV P miniTK P TATA or P YB_TATA Cells were cultured for 48 h with or without 0.6 mmol / L cellobiose before imaging. Figure 2 C is constructed using aCebR2 (VPR-CebR) combined with different core promoters to build P. aCebR Report carrier, for Cb OFF A diagram illustrating the system's optimization and filtering strategies; Figure 2 D represents HEK293T cells (1×10⁻⁶). 5 Co-transfect plasmid pRF2 (P) hCMV Representative fluorescence images of cells after inoculation with the reporter vector pRF3–6 (-aCebR2-P2A-CDT1-pA). Imaging was performed after cells were cultured for 48 h with or without 0.6 mmol / L cellobiose. Scale bar: 100 μm. Figure 2 B and Figure 2 All images in D.

[0024] Figure 3 For Cb OFF The switch's starter has been optimized. Among other things, Figure 3 A is P aCebR Schematic diagram of promoter optimization strategy. By adjusting the series O... CebRBy replicating the number of copies of the operating elements and combining them with different core promoters, a series of P... aCebR Variants; Figure 3 B– Figure 3 E is P aCebR Screening of variants for their inhibitory effect on cellobiose-mediated glucose expression. HEK293T cells (1×10⁻⁶) 5 ) Co-transfected 125 ng pRF2 (P hCMV -VPR-CebR-pA) and 125 ng reporter plasmid pRF7–26, different reporter plasmids contain different P aCebR A variant promoter drives Gluc expression. The normalized Gluc luminescence signal is represented by log2(Fold change).

[0025] Figure 4 For Cb OFF Regulatory performance analysis of the switch in different mammalian cell lines. HEK-293F, HeLa, CHO-K1, and HA1800 cells were cultured under conditions of no cellobiose (0 mmol / L) or with 1.5 mmol / L cellobiose, and the luminescent signal of the reporter gene was detected. The ordinate represents relative luminescent units (RLU, ×10⁻¹⁰). 5 The values ​​in the figure represent the inhibition fold of the 1.5 mmol / L cellobiose treatment relative to the untreated group.

[0026] Figure 5 The effect of cellobiose treatment on the viability of HEK293T cells.

[0027] Figure 6 For Cb OFF Construction and screening of stable cell clones. HEK293T cells were co-transfected with pRF27 (IR / DR-P). aCebR6 -Gluc-IRES-mCherry-pA-IR / DR; P aCebR6 , (O CebR )3-P miniCMV pRF28 (IR / DR-P) hCMV -VPR-CebR-IRES-eGFP-pA-P mPGK After being processed with CDT1-pA-IR / DR and pCMV(CAT)T7-SB100, cells were sorted by flow cytometry and expanded for 2 weeks. Subsequently, eight single clones were randomly selected and cultured for 48 h with or without 1.5 mmol / L cellobiose, and their ability to regulate Gluc expression in response to cellobiose was examined. Data are presented as mean ± SEM value. n = 3 independent experiments).

[0028] Figure 7 For Cb OFF Dynamic response and reversible control of a switch-regulated monoclonal transistor. Among them, Figure 7 A is Cb OFF Temporal analysis of Gluc expression in the switch. In the stable cell line Cb OFF In C2, after treatment with 1.5 mmol / L cellobiose for different durations, the expression level of Gluc was detected at the corresponding time points; Figure 7 B is Cb OFF The reversible switching characteristics of the system under alternating ON / OFF conditions were investigated. Cells were cultured alternately under conditions of no cellobiose (ON) and containing 1.5 mmol / L cellobiose (OFF), with the culture medium changed every 2 days and Gluc expression levels measured every 12 h. Vertical dashed lines indicate the switching time points between the ON and OFF states. Data are expressed as mean ± SEM (mean ± scalar image size). n = 3 independent experiments).

[0029] Figure 8 For Cb ON Schematic diagram of the switch design.

[0030] Figure 9 To adjust the position of the KRAB structural domain to affect Cb ON The switch has been optimized. Among other things, Figure 9 A is a schematic diagram of the iCebR variant, in which the KRAB structural domain is fused to the N-terminus or C-terminus of CebR respectively; Figure 9 B represents HEK293T cells (1×10⁻⁶). 5 pRF29 (P) was co-transfected separately. hCMV -iCebR1-P2A-CDT1-pA; iCebR1, KRAB-CebR) or pRF30 (P hCMV -iCebR2-P2A-CDT1-pA; iCebR2, CebR-KRAB) and transfected reporter plasmid pRF31 (P iCebR1 -eGFP-pA). Among them, P iCebR1 By (O) CebR )1-P SV40 -(O CebR )2. Cells were cultured for 48 h with or without 0.6 mmol / L cellobiose before fluorescence imaging. Scale bar, 100 μm, applicable to all images in (B).

[0031] Figure 10 For Cb ON P in the switch iCebR Optimized filtering of variants. Among them, Figure 10 A is PiCebR Schematic diagram of promoter variant optimization strategy. By adjusting P... SV40 O on both sides CebR The number of copies and their arrangement of operating elements are used to construct a series of P... iCebR Variants; the rest are different P iCebR Screening results of variants against cellobiose-induced Glucose expression. HEK293T cells (1×10⁻⁶) 5 ) 125 ng pRF29 (P hCMV Glucose activity was assessed after culturing with 125 ng of the reporter plasmid pRF32–51 (-KRAB-CebR-pA) and cellobiose at the indicated concentrations. Data are presented as mean ± SEM value. n = 3 independent experiments).

[0032] Figure 11 For different constitutive promoter pairs of Cb ON P in the switch iCebR Performance optimization screening. Among them, Figure 11 A is P iCebR Schematic diagram of promoter variants. (Through O) CebR By inserting different compositional promoters between operating elements, a series of P... iCebR Variants; Figure 11 B–11E contains promoters P with different configurations. SV40 P CMV P EF1α or P PGK P iCebR Assessment of the effect of variants on cellobiose-induced Glucose expression levels. HEK293T cells (1×10⁻⁶) 5 ) A total of 125 ng pRF29 (P hCMV Glucose activity was assessed after culturing with 125 ng reporter plasmid pRF52–55 containing the indicated concentration of cellobiose. Data are presented as mean ± SEM value. n = 3 independent experiments).

[0033] Figure 12 For Cb ON The dose-response characteristics and cross-cell line applicability of the switch. Among them, Figure 12 A represents the detection of Cb in HEK293T cells co-transfected with pRF29 and pRF48 under different concentrations of cellobiose treatment. ON Switch-mediated dose-dependent expression of glucose; Figure 12 B is Cb ON Switching cellobiose-dependent Glucose expression in different mammalian cell lines. Data are presented as mean ± SEM.n = 3 independent experiments).

[0034] Figure 13 For Cb ON Validation of heterologous protein output from the switch. Among them, Figure 13 A shows the protein expression levels of VZV gE, IL-17A, S-HBsAg, and α-Syn under different concentrations of cellobiose treatment. VZV gE and IL-17A were detected from cell culture supernatant; S-HBsAg and α-Syn were detected from cell lysates. Apparent molecular weights in the figure are expressed as ranges to reflect migration heterogeneity caused by protein processing and post-translational modifications. Figure 13 B represents the quantitative analysis of protein expression levels. The signal intensities of VZV gE and IL-17A were directly quantified based on the immunoblot bands; the signal intensities of S-HBsAg and α-Syn were normalized to their corresponding GAPDH values. Data are expressed as mean ± SEM. n = 3 independent experiments).

[0035] Figure 14 For Cb ON Diagram showing the construction and screening of stable cell clones.

[0036] Figure 15 For Cb ON Dynamic reversibility analysis of switch-regulated monoclonal electron microscopy. Among them, Figure 15 A is Cb ON Temporal analysis of Glucum expression in the switch. In Cb ON -C5 cells were treated with 1.5 mmol / L cellobiose for different durations, and the expression level of Gluc was detected at the corresponding time points. Figure 15 B is Cb ON Analysis of the reversibility of the switch. Cb ON -C5 cells were cultured alternately in media containing 1.5 mmol / L cellobiose (ON) or without cellobiose (OFF), with the medium changed every 2 days, and Gluc expression levels were measured every 12 h. Data are presented as mean ± SEM value. n = 3 independent experiments).

[0037] Figure 16 A schematic diagram of the experimental design for evaluating cellobiose-regulated transgene expression in mice.

[0038] Figure 17 This study validates the regulatory capacity of the cellobiose-responsive regulatory system in mice. Specifically, Figure 17 A is Cb OFFFunctional validation of the switch in mice. Wild-type mice were hydrodynamically injected via the tail vein with pRF27, pRF28, and pCMV(CAT)T7-SB100 to deliver Cb. OFF The system was subsequently treated with different doses of cellobiose, and the level of glucose in the blood circulation was measured on days 2, 4, 8 and 15 post-injection. Figure 17 B is Cb ON Functional validation of the switch in mice. Wild-type mice were hydrodynamically injected via the tail vein with pRF55, pRF56, and pCMV(CAT)T7-SB100 to deliver Cb. ON The system was subsequently treated with different doses of cellobiose, and circulating glucose levels were measured on days 2, 4, 8, and 15 post-injection. Data are presented as mean ± SEM. n = 3 mice / group). An asterisk indicates a statistically significant difference compared to the 0 g / kg group. p <0.01, p <0.0001.

[0039] Figure 18 This is the AND transcriptional regulatory logic gate jointly controlled by Cb and PCA in HEK293T cells. Figure 18 A is a schematic diagram of the AND logic gate design. This system is built upon a Cb response module and a PCA response module, and outputs high-level eGFP expression only when both input signals are present simultaneously. Figure 18 B is a heatmap showing the output response of the AND logic gate under different input conditions. HEK293T cells were co-transfected with the AND logic gate plasmid combination pRF29 / pRF61 / pRF62 as shown, cultured for 48 h in a solution containing the indicated concentrations of Cb and PCA, and the eGFP fluorescence intensity was detected. The data shown in the heatmap are the average fluorescence intensity obtained from three independent experiments, and the color scale represents the fluorescence signal intensity in any unit (AU). Figure 18 C represents representative eGFP fluorescence images of the AND logic gate under different input conditions. Scale bar, 100 μm, applicable to all images.

[0040] Figure 19 This is a NAND transcriptional regulatory logic gate jointly controlled by Cb and PCA in HEK293T cells. Figure 19 A is a schematic diagram of a NAND logic gate. The system is designed to suppress eGFP expression only when both input signals are present simultaneously, while maintaining a high level of output under other input conditions. Figure 19 B shows the output response heatmap of the NAND logic gate under different input conditions. HEK293T cells were co-transfected with the indicated NAND logic gate plasmid combination pRF2 / pRF63 / pRF64 and cultured for 48 h with the indicated concentrations of Cb and PCA. eGFP fluorescence intensity was then measured. The heatmap data represents the average fluorescence intensity obtained from three independent experiments; the color scale indicates fluorescence signal intensity in arbitrary units (AU). Figure 19 C represents representative eGFP fluorescence images of NAND logic gates under different input conditions. Scale bar, 100 μm, applicable to all images.

[0041] Figure 20 This is a NOR transcriptional regulatory logic gate jointly controlled by Cb and PCA in HEK293T cells. Figure 20 A is a schematic diagram of a NOR logic gate. The system is designed to generate high-level eGFP expression only when neither of the two input signals is present, while suppressing the output under either single-input or dual-input conditions. Figure 20 B shows the output response heatmap of the NOR logic gate under different input conditions. HEK293T cells were co-transfected with the indicated NOR logic gate plasmid combination pRF2 / pRF65 / pRF66 and cultured for 48 h with the indicated concentrations of Cb and PCA. The eGFP fluorescence intensity was then detected. The heatmap data are the average fluorescence intensity obtained from three independent experiments, and the color scale represents the fluorescence signal intensity in any unit (AU). Figure 20 C represents representative eGFP fluorescence images of NOR logic gates under different input conditions. Scale bar, 100 μm, applicable to all images.

[0042] Figure 21 This is the Cb NIMPLY PCA transcriptional regulatory logic gate in HEK293T cells, jointly controlled by Cb and PCA. Figure 21 A is a schematic diagram of the Cb NIMPLY PCA logic gate design. The system is designed to produce a high-level eGFP output only when Cb is present but PCA is absent. Figure 21 B shows the output response heatmap of the Cb NIMPLY PCA logic gate under different input conditions. HEK293T cells were co-transfected with the indicated Cb NIMPLY PCA logic gate plasmid combination pRF29 / pRF67 / pRF64 and cultured for 48 h with the indicated concentrations of Cb and PCA. eGFP fluorescence intensity was then measured. The heatmap data represents the average fluorescence intensity obtained from three independent experiments; the color scale indicates fluorescence signal intensity in arbitrary units (AU). Figure 21C represents representative eGFP fluorescence images of the Cb NIMPLY PCA logic gate under different input conditions. Scale bar, 100 μm, applicable to all images.

[0043] Figure 22 This is the PCA NIMPLY Cb transcriptional regulation logic gate in HEK293T cells, jointly controlled by Cb and PCA. Figure 22 A is a schematic diagram of the PCA NIMPLY Cb logic gate design. The system is designed to generate a high-level eGFP output only when PCA is present and Cb is absent. Figure 22 B shows the output response heatmap of the PCA NIMPLY Cb logic gate under different input conditions. HEK293T cells were co-transfected with the indicated PCA NIMPLY Cb logic gate plasmid combination pRF2 / pRF68 / pRF62 and cultured for 48 h in a medium containing the indicated concentrations of Cb and PCA. The eGFP fluorescence intensity was then measured. The heatmap data represents the average fluorescence intensity obtained from three independent experiments; the color scale indicates fluorescence signal intensity in any unit (AU). Figure 22 C represents representative eGFP fluorescence images of the PCA NIMPLY Cb logic gate under different input conditions. Scale bar, 100 μm, applicable to all images.

[0044] Figure 23 The construction and in vitro validation of a Cb / PCA logic gate-driven insulin expression system were carried out. Figure 23 A is a schematic diagram of an insulin expression system controlled by a Cb / PCA dual-input logic gate; Figure 23 B represents the in vitro characterization of the Cb and PCA dual-input controlled insulin switch. HEK293T cells were co-transfected with pRF69 and pRF70 and cultured for 48 h with the indicated concentrations of Cb and PCA. Nluc output is shown as a heatmap.

[0045] Figure 24 The therapeutic effect of a Cb / PCA-regulated insulin-lowering device on T1D mice. Figure 24 A is a schematic diagram of the T1D treatment circuit. The treatment plasmid system pRF69 (IR / DR-P) is delivered via tail vein hydrodynamics. Ca1 -mINS-T2A-Nluc-pA-IR / DR; P Ca1 , (O CebR )3-P SV40 -(O CebR )2-(O PcaV )2) pRF70 (IR / DR-P CMVAfter delivering KRAB-CebR-T2A-PcaV-KRAB-pA-IR / DR) and pCMV(CAT)T7-SB100 into T1D mice, treatment with cellobiose and PCA can activate insulin expression in transfected host cells. Figure 24 B– Figure 24 C represents the detection of fasting blood glucose and serum insulin levels in mice 2 days after treatment. T1D mice with an insulin switch received no treatment or the indicated input combination. Treatment group mice were given PCA (500 mg / (kg·day)) and / or cellobiose (2 g / (kg·day)) twice daily. Blood glucose levels were measured 6 hours after fasting. Figure 24 B, then blood was drawn to measure serum insulin levels, such as Figure 24 C. Data are expressed as mean ± SEM (mean ± SEM value). n = 3 mice / group). p <0.05, p <0.01, p <0.001, p <0.0001.

[0046] Figure 25 This relates to the regulatory role of the Cb / PCA insulin switch in glucose tolerance and long-term insulin secretion. Among other things, Figure 25 A shows the glucose tolerance test results of fasting mice in each group; Figure 25 B represents weekly fasting serum insulin levels in T1D mice with an insulin switch, under conditions of treatment with or without cellobiose and PCA. Data are expressed as mean ± SEM. n = 3 mice / group). p <0.05, p <0.01, p <0.001, p <0.0001. Detailed Implementation

[0047] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments.

[0048] This invention aims to develop a CebR repressor protein system based on cellobiose metabolic regulation for precise control of exogenous gene expression. This invention is expected to have wide applications in synthetic biology, biopharmaceuticals, and gene therapy. By overcoming the limitations of existing induction systems, this invention aims to provide a highly sensitive, metabolically compatible, and reversible mammalian cell-specific induction and regulation strategy, while also providing a theoretical basis for the research and application of cellobiose as a novel metabolic signaling molecule in eukaryotes.

[0049] This invention specifically provides a CebR-O based CebR A cellobiose-responsive mammalian transgenic expression regulatory system for sensing axes, its construction method, and its application. This invention utilizes microbial-derived CebR–O... CebR The cellobiose sensing module was engineered and adapted to mammalian cells, and combined with mammalian transcriptional activation / repression domains, an artificial promoter architecture, and a cellobiose uptake module to construct a Cb OFF and Cb ON Two opposing regulatory modes.

[0050] Specifically, Cb OFF In the system, CebR fuses with the transcriptional activation domain to form aCebR, which binds to P in the absence of cellobiose. aCebR The promoter activates the expression of downstream target genes, while the presence of cellobiose causes aCebR to be released from P. aCebR The upper dissociation reduces or shuts down the expression of the target gene. Cb ON In the system, CebR fuses with the transcriptional repressor domain to form iCebR, which binds to P in the absence of cellobiose. iCebR The promoter inhibits the expression of downstream target genes, while the presence of cellobiose causes iCebR to dissociate and relieve transcriptional repression, thereby enabling the expression of the target gene.

[0051] This invention solves the problem of microbial source CebR–O CebR This invention addresses the expression, nuclear localization, promoter adaptation, low-background regulation, intracellular accessibility of cellobiose, and in vivo availability of the sensory axis in mammalian cells. It utilizes mammalian promoters, transcriptional activation / repression domains, and P... aCebR / P iCebR Artificial promoters and cellobiose transport modules such as CDT1 were used to construct a cellobiose-responsive transgenic expression control system that can operate in mammalian cells and animals.

[0052] This invention provides a Cb that can operate independently.OFF and Cb ON A dual-mode transcription switch is provided. A PCA module is also included as part of subsequent orthogonal logic extension applications.

[0053] Microbial source CebR–O CebR The module retains its cellobiose responsiveness in mammalian cells; the same cellobiose input signal can achieve two opposite modes of expression—expression off and expression on—through different effector domains and promoter conformations; via O CebR Optimization of the number, location, and promoter core of the operating elements significantly improves background leakage, maximum output, and dynamic range. The system is dose-dependent, reversible, has long-term availability in stable cell lines, and is controllable in mice. It is also adaptable to a variety of reporter proteins, functional proteins, and therapeutic proteins.

[0054] All vectors involved in this invention were constructed using the Gibson seamless cloning strategy. The brief process is as follows: PCR amplification of the target fragment was performed according to the design scheme; the DNA fragment containing homologous ends was seamlessly cloned and assembled with the linearized vector according to the seamless cloning kit instructions; after transformation into competent cells, single clones were picked, and the correct expression plasmids were obtained after verification by bacterial PCR, enzyme digestion, and sequencing. Detailed configurations, nomenclature, and amino acid / DNA sequences of related tool molecules for the vector plasmids are shown in Tables 1 and 2 below.

[0055] Note: Table 2 only lists P. aCebR6 P iCebR17 The specific DNA sequence information can be obtained by combining the vector information and sequence information in Tables 1 and 2. The DNA sequence information of other related promoters can be obtained by combining the vector information and sequence information in Tables 1 and 2.

[0056] Cb based on cellobiose OFF Construction and optimization of transcriptional repression switches Cb OFF System design principles To establish a transcriptional repressor gene expression system regulated by cellobiose in mammalian cells, this study used microbial-derived CebR–O CebR The sensing module is designed with a cellobiose-responsive OFF switch (Cb). OFF CebR can recognize specific DNA manipulation elements, and its binding ability to target DNA is weakened in the presence of cellobiose.

[0057] Based on this characteristic, this study fused CebR with the mammalian transcription activation domain to construct an artificial transcription activator, which binds to the target promoter and activates the expression of downstream reporter genes under cellobiose-free conditions; however, after the addition of cellobiose, the binding of CebR to the promoter is weakened, thereby weakening the transcriptional activation effect and inhibiting the expression of reporter genes.

[0058] like Figure 1 As shown, a cellobiose-responsive artificial transcription activator aCebR was constructed by fusing CebR with a transcription activation domain and constitutively expressed in cells. The target gene (GOI) is expressed by the chimeric promoter P. aCebR ((O CebR ) n -P mini Driven by ) under cellobiose-free (-Cb) conditions, aCebR binds to P aCebR It also activates GOI expression; in the presence of cellobiose (+Cb), aCebR from P aCebR The dissociation of GOI inhibits its expression.

[0059] Based on the above design concept, this study further constructed a chimeric promoter composed of a CebR-responsive element and a core promoter, and placed it upstream of the reporter gene to form a complete CebR promoter. OFF Transcriptional regulatory system. The core logic of this system is: in the absence of cellobiose, the downstream reporter gene is in an "on" state; while in the presence of cellobiose, the system output is suppressed, thereby achieving reversible OFF regulation mediated by exogenous small molecules, specifically as follows: Figure 1 As shown in the figure. This design lays the foundation for subsequent system optimization and functional expansion of the cellobiose-responsive switch.

[0060] Cb OFF Screening of transcription activators and core promoters in the system To obtain Cb with low background leakage, strong output signal and good response characteristics to cellobiose. OFF In this study, we first systematically screened transcriptional activators and core promoters. Given that different transcriptional activation domains can significantly affect the transcriptional output level and dynamic range of the system, we fused CebR with two commonly used mammalian transcriptional activation domains, VP64 and VPR, to construct two classes of artificial transcriptional activators. Figure 2 A and Figure 2 As shown in C, in order to evaluate the impact of core promoters on system performance, core promoter elements such as miniCMV, miniTK, TATA, and YB_TATA were selected and combined with CebR operators to construct a series of report carriers.

[0061] Preliminary screening results in HEK293T cells indicate that different combinations of transcriptional activation domains and core promoters can support cellobiose-responsive Cb. OFF The systems are constructed differently, but there are significant differences in output level and response amplitude. For example... Figure 2 B and Figure 2 As shown in D, overall, the VPR fusion-type transcriptional activator exhibited higher reporter gene expression levels and a more significant cellobiose repressive effect in most conformations, while the overall output of the VP64 fusion vector was relatively low. This result indicates that in Cb... OFF In the system, a strong transcriptional activation domain is beneficial for increasing the maximum system output and expanding the expression difference before and after induction, thereby improving the regulatory dynamic range of the system.

[0062] Besides the transcriptional activation domain, different core promoters also significantly impact system performance. Screening results showed differences among core promoters in basal expression levels, maximum output intensity, and responsiveness to cellobiose. Some promoters, while providing higher expression output, also exhibited higher background leakage (P<0.05). miniCMV , P YB_TATA While some promoters have lower basic expression, their overall output signal capability is limited (P). miniTK , P TATA Therefore, for Cb OFF The evaluation of system performance should not rely on a single indicator, but should comprehensively consider multiple parameters such as the basic expression level, the degree of induced inhibition, and the overall dynamic range.

[0063] In summary, cellobiose-responsive Cb OFF The performance of the switch depends not only on the sensing protein itself, but also on the synergistic influence of the strength of the transcriptional activation domain and the characteristics of the core promoter. Systematic screening of these key components is a crucial foundation for constructing high-performance mammalian inducible transcriptional regulatory systems.

[0064] Cb OFF Optimization of the system's response promoter configuration After determining VPR-CebR as the optimal activation configuration, this study further optimized the system structure around the CebR-responsive promoter. To improve the system's response amplitude to cellobiose and reduce leakage expression in the basal state, this study modified the CebR operating element (O... CebR The copy number of ) was calculated and combined with different core promoters to construct a series of P aCebR Variants were used to systematically compare the transcriptional output characteristics of different responding promoter conformations in HEK293T cells; see details in [link to relevant documentation]. Figure 3 A. For example Figure 3 B to Figure 3 As shown in D, the results indicate that O CebR The copy number and its combination with the core promoter can significantly affect system performance. Overall, with the increase in the number of manipulators, the recruitment capacity of artificial transcription activators is enhanced, and the expression output of downstream reporter genes is correspondingly increased; simultaneously, the basal expression level of the system and its response characteristics to cellobiose also change significantly. This indicates that O CebR The copy number is not necessarily better the higher it is; rather, it needs to be matched with the strength of the core promoter and the overall system configuration to achieve better control effects.

[0065] Through systematic comparison of multiple promoter variants, the Cb with the best performance was finally selected. OFF Response promoter configuration (aCebR2–P) aCebR6 This configuration exhibits a large dynamic range before and after the addition of cellobiose, indicating that it achieves a good balance between output intensity and background leakage. Furthermore, this configuration achieves optimal response at a cellobiose concentration of 1.5 mmol / L.

[0066] Cb OFF Evaluation of the system's cross-cell line applicability and cellobiose cell compatibility After obtaining the optimal promoter configuration, this study further evaluated the Cb OFF The applicability of the system in different mammalian cell lines. Because different cell lines vary in transfection efficiency, promoter activity, endogenous transcriptional background, and cellular physiological state, the expression level and response amplitude of the same regulatory system may differ in different hosts. Therefore, this study further validated the system in HEK293T cells using various mammalian cell lines, including HEK293F, CHO-K1, HeLa, and HA1800.

[0067] like Figure 4 As shown, the results indicate that Cb OFF The system not only demonstrated good regulatory ability in HEK293T cells, but also achieved cellobiose-dependent reporter gene expression inhibition in HEK293F, CHO-K1, HeLa, and HA1800 cells. Although there were some differences in the absolute output level and inhibition magnitude among different cell lines, which may be related to cell transfection efficiency, exogenous regulatory protein expression level, promoter basal activity, and inducer uptake capacity, the overall response trend remained consistent. These results indicate that Cb... OFF The system exhibits good cross-cell line applicability, which also suggests that CebR–O CebR The sensing module can perform relatively stable transcriptional regulation functions in different mammalian cell backgrounds.

[0068] Considering the biocompatibility of exogenous inducers in subsequent applications, this study further evaluated the effect of cellobiose on cell viability within the experimental concentration range. Figure 5 As shown, HEK293T cells (1×10⁻⁶) 4 Cell viability was assessed using the CCK-8 assay after culturing in a medium containing the indicated concentration of cellobiose for 72 h. Data are presented as mean ± SEM value, and scatter plots represent independent replicates. n = 4). Student's t-test was used to compare each treatment group with the 0 mmol / L control group, and the results showed no statistically significant differences among the groups. ns The results showed that, at the tested concentrations, cellobiose did not significantly adversely affect the viability of HEK293T cells, indicating that it had good cell compatibility under the conditions used in this study. This result provides experimental evidence for the subsequent application of cellobiose as a regulatory input signal in the design of more complex cell circuits and potential in vivo regulatory systems.

[0069] In summary, through systematic optimization of the response promoter structure, this study established a Cb PCR model that is stable, has a large dynamic range, and exhibits good cross-cell line applicability. OFF Transcription switch. The acquisition of this system not only laid the foundation for the subsequent construction of stable cell lines, but also provided support for its further application in complex gene circuit design and functional expansion.

[0070] Cb OFF Construction of stable cell lines and dynamic reversibility analysis To further evaluate Cb OFF To assess the system's long-term stability and its dynamic regulation under continuous culture conditions, this study constructed a Cb system using the Sleeping Beauty transposon subsystem. OFF Stable transfection of cell lines. Specifically, cell lines containing Cb... OFF The pRF27 vector of the response element, the pRF28 reporter gene expression vector, and the Sleeping Beauty transposase were introduced into HEK293T cells. After obtaining a double-positive cell population by flow cytometry, single-cell cloning culture was further carried out using the limiting dilution method, and finally multiple candidate stable monoclonal cell lines were obtained.

[0071] Subsequently, functional identification was performed on eight randomly selected monoclonal antibodies. Figure 6 As shown, the results indicated that all monoclonal clones exhibited varying degrees of downregulation of reporter gene expression levels upon cellobiose stimulation, but their basal expression levels, fold reduction, and overall dynamic range showed significant differences. This interclonal variation may be related to factors such as the integration site of the exogenous gene, the number of integrated copies, and the local chromatin environment. After comprehensively comparing various performance indicators, this study selected the monoclonal clone (Cb) with the best response performance.OFF -C2) is used for subsequent research.

[0072] Based on this, this study further analyzed the time response characteristics of this stable cell line to cellobiose treatment. For example... Figure 7 As shown in Figure A, the results indicate that the expression level of Gluc gradually decreased with the extension of cellobiose treatment time, suggesting that Cb... OFF The system retains good time-dependent regulatory characteristics even under stable conditions. This result indicates that the system can still effectively respond to exogenous input signals under long-term stable expression conditions. Figure 7 As shown in Figure B, further reversibility experiments demonstrated that when cells were alternately cultured under conditions containing and without cellobiose, the expression level of Gluc could be repeatedly switched between "off" and "on" states, and the system maintained good response consistency and reproducibility even after multiple rounds of switching. This result indicates that Cb... OFF The switch can not only respond to a single exogenous stimulus, but also has the ability to be reversible and repeatedly modulated, showing good dynamic regulation characteristics.

[0073] The establishment of a stable cell model further proves that Cb OFF The system is not only suitable for short-term expression regulation under transient transfection conditions, but also possesses the potential for long-term, stable, and reversible gene regulation. Combining the aforementioned response promoter optimization and cross-cell line validation results, this study successfully constructed a mammalian transcriptional repressor switch with high dynamic range, good biocompatibility, and strong application potential, using cellobiose as an input signal.

[0074] Cb based on cellobiose ON Construction and optimization of transcription activation switches Cb ON System design principles The aforementioned study has successfully constructed a cellobiose-responsive transcriptional repression switch Cb based on the CebR–cellobiose sensing module. OFF To expand the application of this sensing module in mammalian transcriptional regulation, this study designed and constructed a cellobiose-inducible transcriptional activation switch, Cb. ON With Cb OFF The system's regulatory logic differs from Cb, which weakens artificial transcriptional activation through cellobiose and thereby inhibits downstream gene expression. ON The system aims to utilize cellobiose to relieve transcriptional repression, thereby inducing the expression of the target gene under saccharification conditions. In other words, Cb... OFF It belongs to the "sugar-off" system, while Cb ONThis belongs to the "sugar-activated" type of system. This design helps to verify the feasibility of the same cellobiose sensing core achieving opposite output behaviors under different regulatory architectures, and further expands its programmability in mammalian synthetic biology.

[0075] Based on the above approach, this study fused CebR with the classic mammalian transcriptional repressor domain KRAB to construct the artificial transcriptional repressor iCebR, and further designed it to contain O CebR Operating elements and compositional promoters of responsive promoters P iCebR To establish a complete Cb ON Regulatory loop. This system achieves the induction and activation of the target gene by maintaining transcriptional repression in the absence of cellobiose and releasing the repression in the presence of cellobiose. For details, please refer to... Figure 8 .

[0076] By fusing CebR with the transcriptional repressor domain KRAB, a cellobiose-responsive transcriptional repressor iCebR (KRAB-CebR) was constructed and constitutively expressed in cells. The reporter gene is generated by the chimeric promoter P. iCebR ((O CebR ) n -P const. -(O CebR ) n Driven by [the mechanism of action]. Under conditions without cellobiose (-Cb), iCebR binds to P [a specific component]. iCebR It also inhibits GOI expression; in the presence of cellobiose (+Cb), iCebR from P iCebR The ion dissociates, thereby relieving the inhibition and restoring GOI expression levels.

[0077] KRAB fusion method and optimization of response promoter configuration To improve Cb ON To improve the system's induction efficiency and reduce background leakage under cellobiose-free conditions, this study first compared the effects of different KRAB-CebR fusion methods on system performance. Specifically, two artificial transcriptional repressor variants of KRAB located at the N-terminus and C-terminus of CebR were constructed, and their response characteristics to cellobiose were evaluated using eGFP as a reporter gene. (See [link to details]). Figure 9 A. For example Figure 9 As shown in Figure B, the results indicate that the KRAB-CebR conformation exhibits stronger repression of downstream genes under cellobiose-free conditions, while showing more significant expression recovery and a more ideal inducible ON response characteristic after the addition of cellobiose. In contrast, the CebR-KRAB conformation shows lower eGFP expression levels and a relatively limited response to cellobiose stimulation. Therefore, KRAB-CebR was selected as the primary transcriptional repressor conformation for subsequent experiments.

[0078] After determining the optimal repressor configuration, this study further investigated the response promoter P. iCebR System optimization was carried out. First, by adjusting O... CebR The copy number and arrangement of the operating elements were used to construct a series of P... iCebR Promoter variants were used to compare the effects of different conformations on the system's inhibitory and inducible expression capabilities; see [link to relevant documentation] for details. Figure 10 Simultaneously, the system performance of different promoter compositions as core promoters was further compared, aiming to achieve the optimal balance between low background leakage and high induction amplitude. For details, please refer to [link to relevant documentation]. Figure 11 The results showed that O CebR The number, arrangement, and type of core promoters can all significantly affect system performance. Overall, moderately increasing the number of O... CebR The copy number helps enhance transcriptional repression under cellobiose-free conditions, while a reasonable element arrangement facilitates post-induction expression recovery, thereby improving the system's dynamic range. After comprehensively comparing the background expression, post-induction output, and fold induction of various promoter variants, the optimal combination iCebR1–P was finally selected. iCebR17 Its induction fold is the highest, approximately 27 times; see details for more information. Figure 10 The above results indicate that P iCebR The regulatory performance depends on the synergistic optimization of the number of operating elements, spatial organization, and core promoter selection, further illustrating that synthetic promoter engineering is crucial for improving Cb. ON A key aspect of system response performance.

[0079] Cb ON Validation of the system's dose response characteristics, cross-cell line applicability, and heterologous protein export. After determining the optimal repressor configuration and the responsive promoter, this study further evaluated Cb. ON System dose-response characteristics. First, using Gluc as a reporter gene, the expression changes of cellobiose after treatment with different concentrations were analyzed in HEK293T cells. For details, please refer to [link to relevant documentation]. Figure 12 A. The results showed that, under cellobiose-free conditions, the system maintained a low basal expression level; with increasing cellobiose concentration, Gluc expression levels gradually increased, exhibiting a clear dose-dependent effect, reaching a peak at 1.5 mmol / L. Further increases in inducer concentration did not further enhance expression output; instead, they slightly decreased. This indicates that Cb... ON The system has certain continuously adjustable characteristics, and 1.5 mmol / L can be used as a suitable inducing agent concentration for setting subsequent experimental conditions.

[0080] Based on this, this study further verified Cb ONThe system's applicability in different mammalian cells. For example... Figure 12 As shown in Figure A, HEK293T cells co-transfected with pRF29 and pRF48 were analyzed for Cb under different concentrations of cellobiose treatment. ON Switch-mediated dose-dependent expression of glucose. Figure 12 As shown in B, this system can achieve cellobiose-dependent gene expression activation in various cell types, including HEK293F, CHO-K1, HeLa, and HA1800. This result is consistent with the aforementioned Cb. OFF The system validation results were consistent, further demonstrating that the cellobiose / CebR sensing module has good universality in mammalian cells.

[0081] To evaluate Cb ON To assess the system's adaptability to different types of heterologous protein output, this study selected VZV gE, IL-17A, S-HBsAg, and α-Syn as representative validation targets. These proteins represent different categories, including membrane-associated virus glycoproteins, secreted cytokines, viral surface antigens, and intracellular functional proteins. Specifically, VZV gE depends on proper glycosylation and is suitable for evaluating the system's ability to regulate the expression of complex membrane glycoproteins; IL-17A is a typical secreted cytokine and can be used to verify the system's suitability for secreted protein output; S-HBsAg has secretory and particle assembly characteristics, reflecting the system's compatibility with viral-derived antigen protein expression; and α-Syn is a non-secretory intracellular protein that can be used to further evaluate the system's ability to regulate the expression of intracellular functional proteins. Therefore, selecting these four protein classes helps to comprehensively validate Cb. ON The system's output expansion capabilities and its applicability to different protein types were examined. Furthermore, in the functional protein expression validation, to obtain stronger protein output and a more stable detection signal, this study selected the CMV promoter to drive heterologous protein expression.

[0082] like Figure 13 A and Figure 13 As shown in Figure B, Western blot results revealed that the expression levels of proteins such as VZV gE, IL-17A, S-HBsAg, and α-Syn were significantly increased after cellobiose treatment. The different proteins showed a consistent upregulation trend before and after induction, indicating that Cb... ON The system is applicable not only to the regulation of reporter gene expression but also to the controlled expression of various heterologous proteins. These results demonstrate that the cellobiose-responsive ON switch exhibits good output compatibility and shows potential for application in both basic research and biomedicine.

[0083] In summary, the optimized Cb ONThe system exhibits low basal leakage, clear dose dependence, good cross-cell line applicability, and strong heterologous protein export capability, indicating that it is a mammalian inducible transcriptional activation tool with high engineering applicability and also expands the application of cellobiose response modules in mammalian cell gene regulation.

[0084] Cb ON Construction of stable cell lines and dynamic reversibility analysis To further evaluate Cb ON This study investigated the stability, time-response characteristics, and reversible controllability of the system under long-term culture conditions using the Sleeping Beauty transposition system to construct a Cb... ON Stable transfected cell lines were established. After obtaining several candidate single clones, their cellobiose response performance was screened.

[0085] like Figure 14 As shown, HEK293T cells were co-transfected with pRF55 (IR / DR-P). iCebR17 -Gluc-IRES-mCherry-pA-IR / DR; P iCebR17 , (O CebR )3-P SV40 -(O CebR 2) pRF56 (IR / DR-P) hCMV -KRAB-CebR-IRES-eGFP-pA-P mPGK After being processed with CDT1-pA-IR / DR and pCMV(CAT)T7-SB100, cells were sorted by flow cytometry and amplified for 2 weeks. Eight single clones were randomly selected and cultured for 48 h with or without 1.5 mmol / L cellobiose. Their ability to regulate Glucose expression in response to cellobiose was then assessed. The fold changes shown in the bars above the graphs are calculated based on the 0 mmol / L cellobiose group. Data are expressed as mean ± SEM value. n = 3 independent experiments).

[0086] The results showed that all monoclonal clones exhibited varying degrees of upregulation of Gluc expression upon cellobiose stimulation, but there were significant differences in their basal expression levels, maximum output intensity, and fold induction. This interclonal variation is consistent with the aforementioned Cb... OFF The results were consistent with those of the stable gene transfer system, which may be related to factors such as the integration site of the exogenous gene, the number of integrated copies, and the local chromatin environment. After comprehensively comparing various performance indicators, the monoclonal antibody (Cb) with the highest induction fold, lowest background leakage, and best overall performance was finally selected. ON -C5) is used for subsequent functional verification.

[0087] Based on this, this study further analyzed the time response characteristics of this stable cell line to cellobiose treatment. For example... Figure 15 As shown in A, after the addition of cellobiose, Cb ON The expression level of Gluc in C5 cells gradually increased with prolonged treatment time, indicating that Cb ON The system retains good time-dependent activation characteristics even under stable conditions. This result indicates that the system can still effectively sense exogenous input signals and achieve continuous expression regulation under long-term integrated expression conditions. Further reversibility experiments show that, as Figure 15 As shown in B, when cells are alternately cultured between cellobiose-containing and cellobiose-free conditions, Cb ON -Gluc expression levels in C5 cells exhibit reversible fluctuations with the addition and withdrawal of the inducer: they gradually increase in the presence of cellobiose, and gradually decrease to lower levels after cellobiose removal. Even after multiple rounds of alternating treatments, the system maintains a stable induction and decline trend, indicating that Gluc expression in C5 cells... ON The switch has good reversible activation capability and dynamic response stability.

[0088] In summary, stable cell experiments indicate that cellobiose-responsive Cb ON Even under long-term integrated expression conditions, the switch maintains stable induction activation ability and good dynamic response characteristics. This indicates that the system is not only suitable for short-term expression regulation under transient transfection conditions, but also has the potential to be used as a long-term ON-type transcriptional regulation tool, providing a foundation for subsequent construction of complex gene circuits and in vivo functional verification.

[0089] Functional validation of cellobiose-responsive switches in mice In vivo validation experimental design In the aforementioned study, this project has successfully constructed and optimized cellobiose-responsive Cb in mammalian cells. OFF and Cb ON A transcriptional regulatory switch was developed, demonstrating good dose-dependency, reversibility, and cross-cell line applicability. However, the relatively simplified in vitro experimental conditions cannot fully reflect the distribution characteristics of exogenous small molecules, plasmid expression persistence, and the influence of tissue microenvironment on transcriptional regulation efficiency in the complex in vivo environment. Especially at the whole-animal level, the absorption, distribution, and clearance of exogenous inducers, as well as the delivery efficiency of exogenous expression vectors in specific tissues, can significantly alter the actual performance of the system. Therefore, relying solely on in vitro cell experiments is insufficient to fully assess the in vivo operational capability and application potential of the cellobiose-responsive switch; further functional validation at the whole-animal level is necessary.

[0090] Based on the above considerations, this study used in vivo experiments in mice to investigate the effects of Cb. OFFand Cb ON The control performance of the switch is evaluated. For example... Figure 16 As shown, the corresponding plasmid was delivered via tail vein hydrodynamic injection, and mice were treated with different doses of cellobiose after plasmid injection. Blood samples were collected at multiple time points, and the dynamic response characteristics of the glucose activity analysis system in vivo were detected by detecting the activity in the blood circulation.

[0091] This part of the experiment mainly focuses on two questions: First, can cellobiose, as an exogenous input signal, effectively regulate transgene expression in mice? Secondly, the Cb established in vitro in the early stage OFF and Cb ON Can the two types of regulatory logics maintain a consistent response trend in the complex physiological environment in vivo as in vitro?

[0092] If the two systems still exhibit the expected inhibitory and activating effects in vivo, it indicates that the cellobiose / CebR module is not only suitable for gene regulation at the in vitro cellular level, but also has the potential to be further applied to disease models and therapeutic regulation research.

[0093] like Figure 16 As shown, the corresponding plasmids were delivered into the liver of mice via tail vein hydrodynamic injection, and the regulatory performance of the cellobiose-responsive transcription switch in vivo was evaluated by administering different doses of cellobiose.

[0094] Cb OFF and Cb ON System regulatory capacity test in mice To verify the regulatory capacity of cellobiose-responsive transcriptional switches in vivo, this study investigated the effects of Cb... OFF and Cb ON The system was functionally evaluated in vivo in mice. Specifically, the corresponding plasmid was delivered to wild-type mice via tail vein hydrodynamic injection, followed by treatment with different doses of cellobiose. Blood samples were collected on days 2, 4, 8, and 15 post-injection, and the system's response to exogenous input signals was assessed by detecting circulating glucose activity. This experimental design allows for comparison of the effects of different doses of cellobiose on the system's output, and also enables dynamic observation of the sustained response capabilities of the two types of switches in the in vivo environment, thus providing a more comprehensive evaluation of their regulatory performance at the whole animal level.

[0095] like Figure 17 As shown in A, for Cb OFFThe results showed that, compared with the control group not treated with cellobiose, the overall level of Glucose in the blood of mice treated with cellobiose decreased, exhibiting the expected "glucose-off" characteristic. Especially in the early stages after plasmid delivery, such as day 2 after injection, the Glucose level in the cellobiose-treated group was significantly lower than that in the untreated group, indicating that cellobiose can effectively inhibit Cb in vivo. OFF System-driven reporter gene expression. Further comparison of different treatment groups showed that the reporter signal generally decreased with increasing cellobiose dosage, with the high-dose group exhibiting a more significant inhibitory effect, suggesting that Cb... OFF The system also exhibits a certain dose-dependency in vivo.

[0096] like Figure 17 As shown in B, in contrast, Cb ON The system exhibits similarities to Cb in vivo. OFF The opposite regulatory trend was observed. Results showed that cellobiose treatment increased overall Gluc levels in the blood of mice, consistent with the expected "glucose-activated" characteristic. Particularly in the early stages after plasmid delivery, the report signal in the cellobiose-treated group was significantly higher than in the untreated group, indicating that cellobiose can effectively activate Cb in vivo. ON System-driven gene expression. Comparison of different dosage groups further indicates that cellobiose input can enhance Cb expression within a certain range. ON The system's output intensity suggests that the system also retains a good induced response capability in vivo.

[0097] Further analysis of the results at different time points revealed that, with the extension of time after injection, regardless of Cb OFF Or Cb ON The overall glucose levels in all experimental groups decreased, which may be related to the transient expression characteristics of plasmids mediated by hydrodynamic injection. Nevertheless, significant differences remained between the cellobiose-treated and untreated groups on days 4, 8, and 15, indicating that both systems do not only have transient response capabilities in vivo, but can continuously sense exogenous input signals within a certain time range and maintain inhibitory or activating effects on downstream outputs, respectively.

[0098] In summary, Cb OFF and Cb ON In mice, the system consistently demonstrated predictable regulatory responses to cellobiose input, exhibiting complementary transcriptional control logics of "glucose-on" and "glucose-off." This indicates that the cellobiose / CebR module not only achieves stable regulation in in vitro mammalian cells but also maintains good functional activity in the complex physiological environment of vivo, providing a foundation for its application in disease models and therapeutic gene regulation research.

[0099] Construction and Validation of a Two-Input Transcriptional Regulation Logic Gate Based on a Cellobiose (Cb) and Protocatechuic Acid (PCA) Response Module Cb / PCA dual-input logic gate design strategy Logic gates are fundamental functional units in synthetic biology for information integration and decision control. By integrating multiple external input signals into a specific transcriptional output, cells can perform combinational logic operations similar to those in electronic logic circuits, thereby enabling more precise control over gene expression at a higher level. Based on this idea, this study couples a cellobiose response module with an established protocatechuic acid response module to construct a two-input transcriptional logic gate, and evaluates the scalability of the cellobiose module in complex gene circuits and its orthogonality with the PCA module.

[0100] In the specific design, cellobiose and PCA are used as two independent small molecule input signals. Based on their respective transcriptional regulatory proteins and operon elements, five two-input logic gates—AND, NAND, NOR, Cb NIMPLY PCA, and PCA NIMPLY Cb—are constructed in the same cell. Each logic gate is based on the aforementioned Cb... OFF / Cb ON System and PCA OFF / PCA ON The response system achieves specific eGFP output modes under different input conditions by recombining operating elements and adjusting activation and inhibition relationships. Compared to single-input transcription switches, dual-input logic gates better demonstrate the modularity, orthogonality, and programmability of artificial gene circuits. If the cellobiose module can be stably coupled with the protocatechuic acid module and produce output results that conform to the expected truth table under different input combinations, it indicates that the system can not only be used as an independent inducible transcription switch but also as a basic module to participate in the construction of higher-level biological computing circuits. This will provide an important foundation for the subsequent development of multi-input, programmable mammalian gene regulation systems.

[0101] Construction and Validation of AND, NAND, and NOR Transcriptional Regulation Logic Gates To verify the applicability of the cellobiose response module in multi-input logic operations, this study first constructed three typical dual-input logic gates: AND, NAND, and NOR, and then detected their eGFP expression output levels under different input conditions in HEK293T cells.

[0102] In the design of AND logic gates, such as Figure 18 As shown in Figure A, this study constructed a system containing O. CebR and O PcaV The chimeric promoter of the response element ensures that the system should output a high level of eGFP only when both Cb and PCA input signals are present simultaneously. Experimental results show that, Figure 18 B and Figure 18 As shown in Figure C, the eGFP output remained at a low level when either Cb or PCA was added alone; however, the system exhibited the strongest fluorescence signal when both input signals were present simultaneously, consistent with the theoretical truth table of the AND logic gate. This result indicates that the Cb module can synergistically work with the PCA module to achieve dual-input-dependent transcriptional activation.

[0103] In the construction of NAND logic gates, such as Figure 19 As shown in Figure A, this study introduces an inverse control relationship based on the AND gate design, enabling the system to maintain a high output under most input conditions, while switching to an inhibited state only when both Cb and PCA are present. Experimental results show that, as Figure 19 B and Figure 19 As shown in Figure C, the NAND logic gates maintain high eGFP output when there is no input or only a single input signal; however, when both input signals are present simultaneously, the fluorescence signal decreases significantly, consistent with the expected output pattern of the NAND logic gates. This demonstrates that the cellobiose response module can not only participate in positive activation logic operations but also achieve a reverse inhibition loop response by combining with other regulatory modules.

[0104] like Figure 20 As shown in Figure A, in a NOR logic gate, the system is designed to output a high-level signal only when neither of the two input signals is present, and to shut down downstream gene expression when either input is present. Figure 20 B and Figure 20 As shown in Figure C, the experimental results indicate that significant eGFP expression can be observed in cells under conditions without Cb and PCA; however, fluorescence output is significantly reduced under conditions with the addition of any single input or the simultaneous presence of both inputs. This result is consistent with the theoretical truth table of NOR logic gates, further demonstrating that the Cb module possesses good reconfigurability and logical adaptation capabilities in multi-level transcriptional regulatory networks.

[0105] Overall, the AND, NAND, and NOR dual-input logic gates all achieved the expected output patterns in HEK293T cells, indicating that the Cb response module can be stably coupled with the PCA system and perform various basic logical operations. This not only verifies the functional orthogonality of the cellobiose / CebR module but also demonstrates that it can serve as an important basic component for constructing complex mammalian transcriptional regulatory circuits.

[0106] Construction and validation of NIMPLY transcriptional regulatory logic gates After validating three typical two-input logic gates—AND, NAND, and NOR—this study further constructed and analyzed two types of NIMPLY logic gates: Cb NIMPLY PCA and PCA NIMPLY Cb. NIMPLY logic gates produce high-level outputs only when one input is present and the other is absent, thus enabling more precise identification of specific input combinations. This has potential application value in constructing highly selective, low-false-trigger therapeutic gene circuits. Compared to logic gates that only distinguish between "present / absent input" or "coexistence of two inputs," NIMPLY circuits have a stricter dependence on input relationships, enabling targeted identification of unilateral conditions. Therefore, they are more suitable for engineered regulatory scenarios with high requirements for expression boundaries.

[0107] like Figure 21 As shown in Figure A, in the Cb NIMPLY PCA loop, the system is designed to output a high level of eGFP only in the presence of Cb and the absence of PCA. Figure 21 B and Figure 21 As shown in Figure C, the experimental results indicate that the system produces the strongest fluorescence signal when only cellobiose is added; however, the eGFP output is significantly reduced when PCA is present alone or when both inputs are present simultaneously. This result demonstrates that the circuit can effectively distinguish between the two input states of "Cb only" and "Cb and PCA present simultaneously," conforming to the theoretical truth table of the CbNIMPLY PCA logic gate.

[0108] Accordingly, such as Figure 22 As shown in Figure A, in the PCA NIMPLY Cb loop, the system is designed to produce a high-level output only when PCA is present and Cb is absent. Figure 22 B and Figure 22 As shown in Figure C, the experimental results indicate that eGFP expression is significantly enhanced when PCA is present alone; however, fluorescence output remains at a low level when Cb is present alone, both inputs are present simultaneously, or both inputs are absent. These results further demonstrate that by adjusting the arrangement of response elements in the promoter and the combination relationship between transcriptional activation and repression modules, the Cb module can flexibly participate in the construction of different types of single-input-specific logic gates.

[0109] The successful construction of two types of NIMPLY logic gates further demonstrates the versatility and adaptability of the cellobiose response module in two-input transcriptional logic operations. Compared with basic logic gates such as AND, NAND, and NOR, NIMPLY logic gates have a stronger ability to distinguish input combinations and can achieve higher-precision conditional discrimination, thus possessing greater potential application value in intelligent therapeutic circuit design. Especially in disease intervention scenarios, the expression of therapeutic genes often needs to be activated only when multiple external signals satisfy specific combinations to reduce the risk of false activation. The Cb and PCA modules can stably realize such unilateral conditional responses, providing an experimental foundation for the subsequent development of higher-order, multi-level logic control networks. Simultaneously, this also demonstrates that the cellobiose response module can maintain good functional compatibility in more complex combinatorial logic, providing more possibilities for its extension to therapeutic protein expression control and intelligent cell circuit design.

[0110] In summary, all five dual-input logic gates constructed in this study can produce output patterns consistent with the theoretical truth table in mammalian cells, indicating that the cellobiose response module is not only suitable for the design of a single transcription switch, but can also participate in the construction of more complex transcriptional biological computing systems as a standardized regulatory element.

[0111] An Investigation into Type 1 Diabetes Treatment Based on Cb / PCA Logic Gates Insulin is the most critical effector molecule in the treatment of type 1 diabetes, and its supplementation can directly correct the glucose metabolism disorder caused by absolute insulin deficiency. However, continuous or uncontrolled insulin expression often leads to safety risks such as hypoglycemia, thus limiting its application in gene therapy and cell therapy. Therefore, compared with a single continuous expression strategy, a hypoglycemic device with conditional restriction and precise regulation capabilities has greater application advantages. An ideal therapeutic circuit should be able to activate insulin expression only when specific input conditions are met, thereby ensuring therapeutic efficacy while minimizing the adverse consequences of false activation. Based on this, this chapter couples cellobiose with a protocatechuic acid response module to construct a dual-input AND gate-controlled insulin expression system, and applies this system to a diabetic mouse model to evaluate its feasibility and potential therapeutic value in precise in vivo hypoglycemia control.

[0112] Design and in vitro validation of a Cb / PCA-gated insulin expression system In the aforementioned studies, our project successfully constructed a transcriptional logic gate system based on Cb and PCA dual inputs and demonstrated its good logical operation capabilities in HEK293T cells. To further evaluate the application potential of this system in disease treatment, this study used type 1 diabetes as a model and placed therapeutic insulin expression under the control of a Cb / PCA dual-input AND logic gate, constructing an insulin expression circuit that can be co-regulated by exogenous small molecules, such as... Figure 23 As shown in Figure A. To verify the in vitro functional characteristics of this circuit, this study co-transfected HEK293T cells with the insulin regulatory system vectors pRF69 and pRF70, and treated them with different concentrations of Cb and PCA, as shown in Figure A. Figure 23 As shown in Figure B, since this system uses the mINS-T2A-Nluc expression conformation, the insulin expression level can be indirectly reflected by detecting the Nluc signal. The results showed that the Nluc output had a significant dual-input dependence: under conditions with no input or only a single input, the signal remained at a low level; however, when Cb and PCA were present simultaneously, the system output was significantly enhanced and gradually increased with the increase of the concentration of the two inducers, reaching the strongest signal under the combined treatment of high concentrations of Cb (3 mmol / L) and PCA (1 mmol / L).

[0113] The results demonstrate that coupling the Cb / PCA dual-input logic gate with the therapeutic insulin expression module maintains a clear input-output correspondence and exhibits an expression pattern consistent with the AND truth table. Compared to single-input induction systems, this dual-input design helps improve the selectivity and safety of therapeutic gene expression and reduces the risk of false activation. Therefore, it is more suitable for therapeutic scenarios requiring high expression precision, such as insulin, an effector molecule that requires strict control of dosage and timing.

[0114] The therapeutic effect of Cb / PCA logic gate-regulated insulin expression system on T1D mice To evaluate the therapeutic effect of the Cb / PCA dual-input insulin expression circuit in vivo, this study used an STZ-induced T1D mouse model and delivered a therapeutic plasmid system consisting of pRF69, pRF70, and pCMV(CAT)T7-SB100 to mice via tail vein hydrodynamic injection. After plasmid delivery, mice were treated with Cb, PCA, or a combination of both according to the experimental design, and fasting blood glucose and serum insulin levels were further measured to assess the functional output of the therapeutic circuit in vivo. For details, please refer to [link to relevant documentation]. Figure 24 A. For example Figure 24As shown in Figure B, the results indicate that T1D mice that did not receive induction treatment maintained high fasting blood glucose levels, exhibiting a typical diabetic phenotype. For mice given only a single input signal, although blood glucose levels fluctuated somewhat, the overall improvement was limited, remaining above 20 mmol / L. In contrast, in the dual-input treatment group (simultaneous administration of cellobiose and protocatechuic acid), fasting blood glucose levels significantly decreased to approximately 12 mmol / L, significantly lower than the uninduced group and the single-input treatment group. This result demonstrates that dual-input combined stimulation can effectively activate the insulin expression circuit in vivo, thereby improving the hyperglycemic state in T1D mice. More importantly, no significant hypoglycemic effect was observed under single-input conditions, indicating that the system is highly dependent on the coexistence of dual signals, which helps reduce non-specific activation induced by a single inducer.

[0115] like Figure 24 As shown in Figure C, consistent with the trend of blood glucose changes, serum insulin levels in the dual-input treatment group mice were significantly increased and returned to near-normal levels, while insulin levels in the uninduced group and the single-input treatment group were below 0.5 μg / L. This indicates that the logic gate system can effectively drive therapeutic insulin expression in vivo, and its output has a clear dual-input dependence. Combined with the aforementioned in vitro cell experiments, it can be seen that the Cb / PCA dual-input logic gate can not only achieve the expected AND-type input integration at the cellular level, but also maintain the corresponding therapeutic output characteristics at the whole animal level. This demonstrates that after coupling the dual-input logic gate with the therapeutic gene expression module, the system still has good functional fidelity and can produce physiologically significant therapeutic effects in disease models.

[0116] Monitoring the long-term hypoglycemic effect of the Cb / PCA logic gate-regulated insulin expression system in T1D mice Building upon the aforementioned short-term treatment trials, this study further evaluated the ability of the Cb / PCA dual-input insulin expression circuit to improve the diabetic phenotype through a glucose tolerance test (GTT) and long-term monitoring. Figure 25 As shown in Figure A, the GTT results revealed that uninduced T1D mice maintained a persistently high blood glucose level after glucose loading, with a slow rate of decline, exhibiting significant glucose metabolism disorders. In contrast, T1D mice treated with a combined Cb / PCA dual-input therapy showed a significantly lower increase in blood glucose and a faster decline at subsequent time points, with an overall trend closer to that of normal mice. These results indicate that the Cb / PCA dual-input treatment circuit not only reduces basal blood glucose levels but also improves the clearance capacity of diabetic mice from glucose loading to some extent, suggesting a positive impact on overall glucose homeostasis.

[0117] In the long-term observation of treatment effects, this study monitored changes in insulin expression levels in mice over several consecutive weeks after treatment. Figure 25 As shown in Figure B, the results indicate that, compared to the uninduced group, the dual-input treatment group maintained higher circulating insulin levels throughout the observation period, demonstrating the sustained function of the therapeutic circuit in vivo. Meanwhile, the overall signal gradually decreased with prolonged observation, but significantly higher expression levels than the control group were still detected at the end of the observation period.

[0118] Given that this study used the Sleeping Beauty transposon system for in vivo delivery, this signal attenuation phenomenon may be related to limited integration efficiency and the gradual loss of unintegrated plasmids. In other words, the high expression levels detected early on may have originated from both transient expression of unintegrated plasmids and sustained expression after partial successful integration. As time progresses, unintegrated plasmids are gradually cleared or inactivated, leading to a decrease in overall signal intensity. The remaining expression in later stages is more likely to originate primarily from expression units that have already been integrated. Furthermore, factors such as hepatocyte renewal, changes in the local chromatin environment, and epigenetic silencing may also contribute to the long-term expression intensity.

[0119] The combined results of fasting blood glucose, circulating insulin levels, and glucose tolerance tests indicate that the Cb / PCA dual-input logic gate-controlled insulin expression circuit can achieve physiologically significant therapeutic output in T1D mice and significantly improve abnormal glucose metabolism phenotypes. Although this system is currently based on hydrodynamic injection-mediated in vivo plasmid expression, its long-term stability and translational application potential still require further optimization. However, the validation results in this disease model fully demonstrate that the cellobiose response module has the potential to be developed into a therapeutic gene regulation platform.

[0120] In this specification, the invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and variations can be made without departing from the spirit and scope of the invention. Therefore, this specification should be considered illustrative rather than restrictive.

Claims

1. A cellobiose-responsive mammalian transgenic expression regulatory system, characterized in that, The system described includes CebR-O CebR A cellobiose sensing module, wherein the sensing module is adapted to mammalian cells; The CebR–O CebR The cellobiose sensing module includes: (a) The first fusion protein, formed by the fusion of the CebR transcription factor and a transcriptional regulatory domain; and (b) An artificial promoter containing at least one O CebR The manipulation subsequence and the core starter subsequence, the O CebR The operon sequence can be specifically bound by the CebR transcriptional regulatory factor; The CebR–O CebR The cellobiose sensing module includes at least one of the following two regulation modes: Cb OFF The pattern, in the aforementioned Cb OFF In this model, the first fusion protein is aCebR, the transcriptional regulatory domain is the transcriptional activation domain, and the artificial promoter is P. aCebR Promoter; in the absence of cellobiose, aCebR binds to P aCebR The promoter activates the expression of downstream target genes; in the presence of cellobiose, cellobiose binds to aCebR, causing it to exit P... aCebR Dissociation at the promoter reduces or shuts down the expression of downstream target genes; Cb ON The pattern, in the aforementioned Cb ON In this model, the first fusion protein is iCebR, the transcriptional regulatory domain is a transcriptional repression domain, and the artificial promoter is P. iCebR Promoter; in the absence of cellobiose, iCebR binds to P iCebR The promoter inhibits and suppresses the expression of downstream target genes; in the presence of cellobiose, cellobiose binds to iCebR, causing it to exit P... iCebR Dissociation at the promoter releases transcriptional repression and enables the expression of downstream target genes.

2. The system according to claim 1, characterized in that, In the aforementioned aCebR, the transcriptional activation domain is selected from VP64 or VPR, and the core promoter is selected from miniCMV, miniTK, TATA, or YB_TATA; in the aforementioned P aCebR The promoter contains 1 to 5 copies of O, arranged in tandem, located upstream of the core promoter. CebR Manipulated subsequences.

3. The system according to claim 2, characterized in that, The transcriptional activation domain is VPR, the core promoter is miniCMV, and O CebR The number of copies of the operon sequence is 3.

4. The system according to claim 1, characterized in that, In the iCebR, the transcriptional repressor domain is KRAB, and the core promoter is selected from SV40, CMV, EF-1α, or PGK; in the P iCebR The promoter contains the first O CebR Operant subclusters and second O CebR Manipulating subclusters, the first O CebR The operon cluster is located upstream of the core promoter, the second O CebR The operator cluster is located downstream of the core promoter.

5. The system according to claim 4, characterized in that, The first O CebR The manipulator subcluster contains n copies of O CebR Manipulated subsequence, where n is an integer from 1 to 10; the second O CebR The manipulator sub-cluster contains m copies of O CebR Manipulate the subsequence, where m is an integer from 1 to 10.

6. The system according to claim 4, characterized in that, n is 3 and m is 2.

7. The system according to claim 1, characterized in that, The CebR transcriptional regulatory factors are selected from CebR homologs, CebR functional mutants, or CebR coding sequences that have been codon-optimized to adapt to mammalian cell expression.

8. The system according to claim 1, characterized in that, The downstream target gene encodes a therapeutic protein, which is insulin or a functional fragment or variant thereof; or, the downstream target gene encodes VZV gE protein, IL-17A protein, S-HBsAg protein, or α-Syn protein.

9. The system according to claim 1, characterized in that, The mammalian cells mentioned are selected from HEK293 cells, CHO cells, HeLa cells, NIH3T3 cells, HepG2 cells, Jurkat cells, or primary cells or stem cells derived from mice, rats, monkeys or humans.

10. The system according to claim 1, characterized in that, The CebR–O CebR The cellobiose sensing module is stably coupled with the protocatechuic acid response system to form a dual-input logic gate, which is selected from AND logic gate, NAND logic gate, NOR logic gate, Cb NIMPLY PCA or PCA NIMPLY Cb.

11. Use of the system according to any one of claims 1 to 10 in the preparation of a medicament for treating diabetes.

12. The application according to claim 11, characterized in that, The diabetes mentioned is type 1 diabetes.

13. The application according to claim 11, characterized in that, The drug in question is a gene therapy drug or a cell therapy drug.

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