This year, our project is primarily dedicated to constructing a UV-responsive gene switch to address the issues faced by patients with Xeroderma Pigmentosum Type C. Due to mutations in the XPC gene, these patients' genomes are impaired in their ability to repair damage when exposed to ultraviolet radiation. We have not only added a new basic part, RUP2, to the Registry Part Pages, providing a negative feedback mechanism for the regulation of UVR8 and COP1, but have also successfully verified the interaction between UVR8, COP1, and RUP2 in mammalian cells.

Creatively, we have designed a UV-responsive gene switch for the gene therapy of XP, using RUP2 for negative feedback to stabilize the expression level of the target gene. This also provides a tool for other iGEM teams to use UV-sensitive proteins to regulate gene expression. In terms of modeling, we have constructed a scalable RNA large language model for protein expression prediction. At the same time, we are exploring a microneedle-based AAV transdermal delivery system for potential future clinical applications.

We sincerely hope that our work this year will inspire and assist more iGEMers and synthetic biologists, enhance the public's understanding of gene research and its applications in medical treatment, and improve societal respect, understanding, and care for XP patients. Ultimately, we aim to provide humanitarian care for XP patients, both in terms of disease treatment and social awareness.

Although there have been reports on the interaction between UVR8 and COP1 in mammalian cells, the interaction between these two proteins is irreversible, meaning that UVR8 and COP1 cannot effectively dissociate after UV irradiation. We have introduced the RUP2 element and incorporated it into the UVR8 and COP1 system. RUP2 is a core protein that regulates the response to UV-B in plants, acting as a negative regulator in the UV-B signaling pathway. It can dissociate the interaction between UVR8 and COP1, promote the re-dimerization and inactivation of UVR8, and limit the intensity and duration of this response. When UV irradiation induces the interaction between UVR8 and COP1, the presence of RUP2 can dissociate their interaction, and regulating the expression time and quantity of RUP2 can effectively control the duration of the interaction between UVR8 and COP1.

Furthermore, the introduction of RUP2 (BBa_K5182003) into the iGEM parts library has enriched the available gene engineering toolkit, and the interaction of these three proteins can create a UV switch for designing a series of tools that respond to ultraviolet light, providing a reference for subsequent iGEM teams.

After introducing the new synthetic biology component RUP2, we have, for the first time, verified the interaction between UVR8, COP1, and RUP2 in 293T cells. We fused UVR8 with GFP and COP1 with mCherry, which has a nuclear localization sequence, and placed RUP2 under the control of the tetracycline-inducible TRE promoter. In the absence of UV-B signals, a large amount of UVR8 dimers exist in the cytoplasm, resulting in green fluorescence in the cytoplasm, while the presence of COP1 in the nucleus produces red fluorescence in the nucleus.

When UV-B signals are present, UVR8 monomerizes and enters the nucleus to interact with COP1, leading to the co-localization of green and red fluorescence in the nucleus. At this point, the addition of tetracycline to induce the expression of RUP2 results in the gradual disappearance of co-localization and a decrease in green fluorescence in the nucleus, successfully verifying the interaction between UVR8, COP1, and RUP2 in mammalian cells.

We have constructed a UV-responsive gene switch that can be adapted for other gene therapies by simply changing the target gene. It can also be combined with gene-editing systems, such as CRISPR/Cas, to achieve better spatiotemporal control of gene manipulation.

We fused UVR8 with VP64 and linked COP1, which contains a nuclear localization signal, to the GAL4 sequence. The target gene was placed under the control of the 5UAS sequence and the CMV promoter, enabling UVR8 to monomerize and enter the nucleus to interact with COP1 in the presence of UV-B signals. Due to the localization effect of GAL4 binding to 5UAS, the distance between VP64 and the CMV promoter is shortened, thereby initiating downstream gene expression. Additionally, we linked RUP2 to the target gene via a P2A sequence to achieve controllable expression of the target gene.

Building on a pre-trained mRNA large language model, we developed a deep neural network called CodonBERTER, capable of end-to-end prediction of protein expression levels from mRNA sequences. CodonBERTER, a state-of-the-art model, offers extensibility for input sequence length, ease of fine-tuning, and significantly outperforms existing competitor models designed for similar tasks. Additionally, we propose a next-generation mRNA coding sequence design solution that leverages RNA large language models, optimizing both speed and accuracy in mRNA sequence design. This approach may lead to a more promising future for mRNA coding sequence design in synthetic biology, enabling more efficient and precise control over protein expression and driving innovative applications in the field.

After conducting a detailed analysis of the gene circuits in the project, we mathematically abstracted the circuits and established a series of ordinary differential equations based on the Michaelis-Menten equation and first-order kinetic equations to dynamically represent each step of the metabolic process. By incorporating early wet-lab data, this model helps calculate feasible ranges for several critical experimental parameters, such as plasmid dosage for cell transfection, UV exposure duration, and disease treatment cycles. This provides theoretical guidance for optimizing experimental efficiency.

After deciding to use microneedles as the delivery tool for the project, we referred to drug diffusion and metabolic kinetics models in the bloodstream. Based on Fick's second law of diffusion, we simulated the diffusion of AAV in the dermis and its clearance into the bloodstream following microneedle injection into the epidermis. This simulation provided calculation methods for key clinical parameters such as dosage timing, frequency, and amount, offering valuable insights for the clinical use of microneedle-based drug delivery systems.

One of the main contributions our project makes to synthetic biology is raising awareness about the importance of genetic research and its applications in medical treatments, while also fostering appreciation and recognition of the field within society. We have engaged with various stakeholders, including patients, healthcare professionals, and regulatory authorities, to examine the technological, ethical, safety, and legal aspects of XPCures. This collaborative approach has been instrumental in understanding diverse perspectives and incorporating them into our project design.

Recognizing the societal benefits and potential future impact of our project, we have created a patient journey timeline to illustrate the experiences of XP patients, providing insight into their daily challenges. We have also mapped our stakeholders into distinct categories and conducted a value-sensitive analysis to derive the necessary criteria for our design. This analysis ensures that our project aligns with the key values of our stakeholders and is managed strategically through a power-interest map.

Safety is of utmost importance in our project, and we have ensured that all aspects of our work meet the highest safety and ethical standards. We are committed to protecting the rights and safety of patients, interviewees, and the broader society.

Our proposed implementation strategy involves a phased approach, starting with research and development, followed by clinical trials, licensing, and eventual marketization. This strategy has been designed in consultation with key stakeholders to ensure the successful rollout of XPCures.

The Human Practices component of our project has been crucial in developing a comprehensive business plan for XPCures. This plan outlines our target customer segments, financial projections, market potential, and the overall strategy for our future venture. By integrating stakeholder feedback and ensuring that safety and ethical considerations are at the forefront, we aim to create a socially responsible and desirable design that benefits patients.

For more detailed information and analysis, please refer to our Human Practices page on the iGEM wiki.