Results

This page shows the results of our project, along with a detailed analysis and discussion of their significance. Future plans and reflections on the impact of our project are also outlined here.

Results

Overview

We present LEGO (Light-induced Expression of GLP-1 and Insulin for Obesity and diabetes treatment), an innovative optogenetic gene therapy system. LEGO is designed for the simultaneous, precise co-expression of insulin and GLP-1 within the body. Through systematic design and validation, we have created a robust toolkit for light-regulated hormone delivery with therapeutic potential.

On this page, we provide a comprehensive description of all experimental results obtained during the design and validation of the LEGO system, along with corresponding analysis and discussion. While we encountered certain challenges and limitations during implementation, we remain committed to continuously improving the LEGO system to unlock its full potential for future in vivo therapeutic applications.

Design and Simulation of LEGO Components

Through literature research, we identified the iLID-sspB system as the optogenetic switch for LEGO (Figure 1A) (Guntas et al, 2015), with NanoLuc serving as a potential endogenous activation trigger (England et al., 2016). The transcriptionally inactive Gal4DBD was employed as the DNA-targeting module, 5×UAS as the binding motif (Figure 1B), and VP64 as the highly efficient transcriptional activation module (Figure 1C).

The light-responsive activator plasmid was designed using the most streamlined configuration (Figure 2).

We subsequently designed the amino acid and DNA sequences of human insulin and GLP-1 optimized for secretion by HEK-293T cells. The processing of native human insulin relies on pancreatic β-cell-specific prohormone convertases PC1/3 and PC2 for post-translational modification and precise C-peptide excision (Figure 3A). To engineer a universally processable insulin sequence compatible with diverse mammalian cell types, we replaced the native PC1/3 and PC2 cleavage sites with a Furin cleavage motif (RRKR) while retaining the N-terminal signal peptide, thereby ensuring efficient secretion (Figure 3B).

To thoroughly validate the design feasibility, we employed the protein disulfide bond prediction tool Disulfide by Design 2.0 to analyze the inter/intra-chain disulfide bonds of the engineered human insulin (Craig et al., 2013). Comparative analysis with native human insulin confirmed complete conservation of all disulfide bond positions (Figure 4).

The active fragment of human GLP-1 (7-37) was engineered with an A8G substitution to enhance resistance to DPP-IV enzymatic hydrolysis, and fused with the secretion signal peptide of Exendin-4 (Parsons et al., 2007) (Figure 5), thereby enabling efficient expression and secretion of bioactive GLP-1 in mammalian cells.

Subsequently, we integrated the two engineered hormone genes using an IRES element to create a dual-hormone expression cassette for mammalian cell expression. A reporter gene encoding the red fluorescent protein mCherry was incorporated to visualize expression dynamics and secretion competence. The dual-hormone expression cassette is driven by a 5×UAS-Hsp70 promoter, where the 5×UAS sequence serves as the targeted binding motif for Gal4DBD, providing a binding scaffold for the transcriptional activator. (Figure 6)

To confirm the feasibility of targeted binding between the Gal4DBD domain of the optogenetic activator protein and the UAS motif, we utilized AlphaFold 3 to predict their interaction interface, validating the reliability of Gal4DBD's specific binding to UAS (Figure 7) (Abramson et al., 2024).

The light-responsive activator and dual-hormone expression vector, as two core components, constitute the LEGO system. Through rigorous design principles and minimalist configuration, we have established a streamlined toolkit for light-controlled engineered hormone expression regulation.

Plasmid Construction

The optogenetic activator plasmid was constructed by assembling the fragments into the pLVX-Hygro vector digested by XhoI/XmaI. We successfully PCR-amplified the insert fragments—NLS-Gal4DBD (538 bp), VP64 (193 bp), miRFPnano3-sspB (839 bp), IRES (634 bp), NLS-Nluc (609 bp), and iLID (470 bp)—from existing plasmids and confirmed their sizes by agarose gel electrophoresis (Figure 8). The vector plasmid pLVX-Hygro was digested with restriction enzymes XhoI and XmaI, and the digestion products were also verified by agarose gel electrophoresis (Figure 8).

Subsequently, we ligated the insert fragments with the linearized vector using homologous recombination. The recombinant products were transformed into DH5α competent cells and plated. After 16 hours of incubation at 37°C, scattered single colonies were observed on the bacterial plates, indicating successful transformation (Figure 9). We selected six colonies for inoculation in 3 mL LB medium and cultured them with shaking at 37°C for 5 hours. Plasmid sequencing was performed using primers CMV-F and PGK-R, and bidirectional sequencing results confirmed that the plasmid sequences were consistent with the expected designs, validating the successful construction of the optogenetic activator plasmid.

The plasmids used for validation and the dual-hormone expression vector plasmid were synthesized and constructed by Tsingke Biotechnology. All plasmids were extracted from E. coli cultures using the alkaline lysis method, followed by purification via silica-column chromatography. DNA concentrations were quantified via spectrophotometry, yielding high-purity plasmid preparations ranging from 1246.1 to 1311 ng/µL.

Validation of the Feasibility and Reversibility of the Light-Responsive System

Before validating the overall functionality of the LEGO system, we systematically characterized the basic parts within the composite modules. This included validation of the optogenetic iLID-sspB system's nuclear localization stability, light responsiveness, reversible dimerization kinetics, and feasibility of NanoLuc-mediated activation.

To validate whether the two core optogenetic components of the LEGO system, iLID and sspB, exhibit stable and consistent nuclear localization under blue-light illumination in HEK-293T cells (Pathak et al., 2017), we designed and constructed plasmids NLS-Nluc-iLID-mCherry and NLS-miRFPnano3-sspB, each fused with a nuclear localization sequence (NLS) and a fluorescent protein tag. After 24 hours of co-transfection of these two validation plasmids into HEK-293T cells, confocal microscopy imaging revealed that both under short-term (200 s) continuous blue-light stimulation and long-term (18 h) pulsed blue-light illumination, the components exhibited clear and sustained nuclear localization. These results confirm the nuclear localization stability and reliability of the light-responsive system (Figure 10).

To visually validate the blue light-responsive interaction between the optogenetic components, we monitored their spatial redistribution in real time. The sspB protein was anchored to the plasma membrane via a membrane localization sequence (NLYN), while iLID remained freely diffusible in the cytoplasm. Both components were tagged with fluorescent proteins and encoded in the plasmids Nluc-iLID-mCherry and NLYN-miRFPnano3-sspB. After 24 hours of co-transfection in HEK-293T cells, live-cell confocal imaging demonstrated that upon brief blue light illumination (100 s), iLID was rapidly recruited to the plasma membrane. Following light withdrawal, iLID quickly reverted to its diffuse cytoplasmic distribution under dark conditions (Figure 11), confirming the rapid light responsiveness and excellent reversibility of this optogenetic module.

Validation of Bioluminescence-Induced Activation of the Light-Responsive System

We next sought to determine whether bioluminescence generated by NanoLuc (Nluc) could activate the iLID-sspB interaction. Upon adding 40μM of the Nluc substrate fluorofurimazine (FFz) to the culture medium, we observed robust recruitment of iLID to the plasma membrane, closely mirroring the localization pattern induced by blue light illumination (Figure 12). These results validate the feasibility of using intracellular bioluminescence to activate the light-responsive components within the LEGO system.

Validation of Light-Activated Co-expression of Insulin and GLP-1

We next proceeded to evaluate the functionality of the LEGO system's composite parts. Using confocal live-cell imaging, we validated both the feasibility and light-responsive characteristics of target gene activation by the LEGO system. Following 24 hours of pulsed blue-light stimulation, a marked enhancement in co-expressed mCherry fluorescence was observed compared to the dark-treated control group (Figure 13A). Statistical analysis further confirmed reliable significance (Figure 13B), thereby validating the successful blue-light-responsive activation of the dual-hormone expression.

We also measured the transcript levels of insulin, GLP-1, and mCherry in HEK-293T cells by RT-qPCR. Consistently, the mRNA levels of insulin (INS), GLP-1, and mCherry were markedly upregulated compared to the dark control group (Figure 14). These results demonstrate that the LEGO system enables robust, blue light-dependent co-expression of Insulin and GLP-1.

Validation of Engineered Human Insulin Activity

Since the engineered insulin was independently designed, it was essential to characterize its bioactivity. Native human insulin initiates a precise signaling cascade upon binding to insulin receptors on target cells, ultimately leading to the activation of key signaling molecule Akt, which translocates to the plasma membrane during this process. To confirm the secretion and bioactivity of insulin produced by LEGO system, we monitored the activation of the PI3K-Akt signaling pathway using live-cell confocal microscopy in response to conditioned medium from HEK-293T cells expressing the LEGO system. Culture medium collected from cells exposed to either blue light stimulation or dark incubation was applied to reporter cells expressing AktPH-EGFP (the PH domain of Akt fused with EGFP). We observed significant translocation of AktPH to the plasma membrane in cells treated with medium from blue light-stimulated conditions, confirming blue light-dependent production, secretion, and bioactivity of the insulin generated by the engineered LEGO gene circuits (Figure 15A and 15B). To exclude potential effects of cellular heterogeneity, the same reporter cells were sequentially treated with conditioned medium from the dark-incubated group followed by that from the blue-light-stimulated group. Results showed that PI3K-Akt pathway activation occurred only after treatment with the blue-light-stimulated conditioned medium (Figure 15C), further validating both the light responsiveness of the LEGO system and the functional activity of the secreted products.

Discussion

The successful establishment of the LEGO system represents a significant advancement in optogenetic gene therapy. Our modular design—integrating the iLID/sspB optogenetic switch, NanoLuc bioluminescence activation, and engineered insulin/GLP-1 expression cassettes—demonstrates robust spatiotemporal control over metabolic hormone production. The system's tight regulation, evidenced by minimal basal leakage and strong light-induced responses, along with the confirmed bioactivity of the engineered insulin through PI3K/Akt pathway activation, positions LEGO as a reliable platform for future translational applications.

Despite these results, several limitations warrant attention. Though basal leakage is low, further optimization of promoter strength and target gene design could enhance the dynamic range. the translational potential depends on efficient and safe delivery methods for both genetic constructs and the FFz substrate in vivo, which presents technical and regulatory hurdles.

Looking forward, the LEGO system holds substantial promise for diabetes treatment and beyond. Its modular architecture allows straightforward adaptation to express other therapeutic proteins, enabling broad applications in hormone replacement therapies. Furthermore, the bioluminescent activation mechanism could be adapted for autonomous feedback-controlled therapy, where hormone secretion is triggered by physiological cues detected by engineered sensors. By addressing these challenges and leveraging its unique capabilities, the LEGO platform may ultimately pave the way for a new generation of smart, precise, and personalized gene therapies for metabolic and other diseases.

References

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Craig, D. B., & Dombkowski, A. A. (2013). Disulfide by Design 2.0: a web-based tool for disulfide engineering in proteins. BMC bioinformatics, 14, 346. https://doi.org/10.1186/1471-2105-14-346

England, C. G., Ehlerding, E. B., & Cai, W. (2016). NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence. Bioconjugate chemistry, 27(5), 1175–1187. https://doi.org/10.1021/acs.bioconjchem.6b00112

Guntas, G., Hallett, R. A., Zimmerman, S. P., Williams, T., Yumerefendi, H., Bear, J. E., & Kuhlman, B. (2015). Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins. Proceedings of the National Academy of Sciences of the United States of America, 112(1), 112–117. https://doi.org/10.1073/pnas.1417910112

Parsons, G. B., Souza, D. W., Wu, H., Yu, D., Wadsworth, S. G., Gregory, R. J., & Armentano, D. (2007). Ectopic expression of glucagon-like peptide 1 for gene therapy of type II diabetes. Gene therapy, 14(1), 38–48. https://doi.org/10.1038/sj.gt.3302842

Pathak, G. P., Spiltoir, J. I., Höglund, C., Polstein, L. R., Heine-Koskinen, S., Gersbach, C. A., Rossi, J., & Tucker, C. L. (2017). Bidirectional approaches for optogenetic regulation of gene expression in mammalian cells using Arabidopsis cryptochrome 2. Nucleic acids research, 45(20), e167. https://doi.org/10.1093/nar/gkx260