In 2018, an American film titled "Midnight Sun" was released. The film tells the story of Katie Price, a young girl who suffers from Xeroderma Pigmentosum (XP), a rare genetic disorder that makes her extremely sensitive to ultraviolet (UV) light. The film follows her love story with Charlie Reed, whom she meets and falls in love with over their shared passion for music. However, Katie has to lead an inverted schedule, dating Charlie only at night because she cannot be exposed to sunlight. Tragically, during one of their dates, they lost track of time and Katie is exposed to daylight, leading to a rapid deterioration of her condition and ultimately her death due to the lack of curative treatment. The lovers, brought together by their love for music, are tragically separated by the progression of XP. Patients like Katie, who cannot live under the sun, suffer immensely from the disease and face significant challenges in seeking medical care. They experience intense conflicts between the need for self-protection and the desire for social interaction, highlighting the demand for greater humanistic care for individuals with XP.

Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder that is found in all races across continents (DiGiovanna et al., 2012). Based on the different gene mutations, XP is classified into several subtypes (e.g., XPA, XPB, XPC, etc.), each with its specific genetic defects (Sancar et al., 1994). XP-C, which accounts for 25% of all cases, is one of the most common subtypes. The condition arises from mutations in the XPC gene, which causes a dificiency in the XPC DNA repair protein. This deficiency hinders the repair of DNA damage caused by ultraviolet (UV) radiation, resulting in a blockage of global genome nucleotide excision repair (NER) (Marteijn et al.,2014).

Patients with XP are highly sensitive to UV light, typically showing symptoms between 6 months and 3 years of age. Sun-exposed areas may develop blisters, freckles, epidermal hyperplasia, scar formation, and tumors. Two-thirds of the patients die before the age of 20 due to multiple tumors (Leung et al.,2022). Traditional treatment methods rely mainly on prevention and management, without directly addressing the genetic defects in XP patients. Strategies for patient care primarily include sun protection, regular skin examinations, and surgical removal of cancerous tissues (Kraemer et al., 2007). However, while these methods can alleviate symptoms, they do not reverse or halt the progression of the disease. The core goal of gene therapy is to restore the normal function of human body by correcting or replacing the defective genes in XP patients. Currently, gene therapy for XP is mainly focused on preclinical research stages. Studies have shown that after gene editing, cells exhibit significantly enhanced tolerance to UV light, resulting in a marked reduction in cancer risk (Rass et al., 2007;Dupuy et al., 2013).

Our project aims to develop a gene switch that responds to ultraviolet light and use this gene switch for the treatment of Xeroderma Pigmentosum Type C. Our project is mainly divided into three modules: First, we have verified the interaction of UVR8, COP1, and RUP2 in mammalian cells and their response to ultraviolet light. Second, we have successfully constructed a UV-responsive gene switch that activates the expression of target genes upon UV irradiation and demonstrated that RUP2 can dissociate the interaction between UVR8 and COP1, thereby stabilizing the expression level of the target gene to a certain extent. Third, through modeling, we have obtained results predicting the expression of the target protein after sequence optimization of RUP2 and successfully constructed plasmids with different numbers of rare codons added to the RUP2 gene, which will be validated through wet lab experiments.

XPCures now takes the stage! We innovatively introduce the plant UV receptor system into the gene therapy of Xeroderma Pigmentosum Type C, using UV-B as an external regulatory factor to induce the expression of the XPC gene, and develop a new type of therapy that uses optogenetic technology as the control end and gene therapy as the foundation. According to the pathogenesis of XPC patients, we have created a gene expression system regulated by UV-B, achieving controllable expression of the XPC gene in patients, turning the pathogenic UV into therapeutic UV.

We firmly believe that the gene switch responding to ultraviolet light is a key focus of future research on genetic skin diseases. In the future, this system may alleviate and even cure an increasing number of skin diseases caused by genetic issues.

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In Arabidopsis thaliana, COP1 is an E3 ubiquitin ligase that targets HY5 for degradation (Lin et al., 2007). UVR8 is a photoreceptive protein that forms a homodimer outside the cell nucleus in the absence of UV-B signals. When UV-B signals are present, UVR8 monomerizes and enters the nucleus, where it interacts with COP1, playing a role in plant photomorphogenesis ( Rizzini et al., 2007). Therefore, our project primarily utilizes the interaction between UVR8 and COP1 in response to UV-B signals to initiate the expression of the target XPC gene.

RUP2 is a core protein in plants that regulates the response to UV-B radiation, serving as a negative regulator in the UV-B signaling pathway (Tilbrook et al., 2013). It can dissociate the interaction between UVR8 and COP1, promote the re-dimerization and inactivation of UVR8, and terminate a series of downstream signal responses triggered by the combination of UVR8 and COP1 induced by UV irradiation.

Existing systems in mammals that use the interaction of UVR8 and COP1 as a switch have a significant drawback: they cannot turn off the signal response triggered by the combination of UVR8 and COP1 when UV-B irradiation ceases. We have addressed this shortcoming by utilizing the characteristics of RUP2's function. We have introduced the RUP2 UV control system such that when the target XPC gene is expressed, it triggers the expression of RUP2 to a certain extent. This leads to the dissociation of the UV-B signal-induced interaction between UVR8 and COP1 and thereby prevents the overexpression of the target XPC gene.

GAL4 is a transcriptional activator derived from yeast which typically composed of two domains: the DNA-binding domain (BD) and the transcriptional activation domain (AD) (Struhl et al., 1995). It can recognize and bind to the upstream activation sequence (UAS), thereby activating the transcription of downstream genes. Our project utilizes the BD domain of GAL4 and the 5UAS sequence to facilitate protein localization through the binding of GAL4 to 5UAS. VP64 is a potent transcriptional activator that, when bound to the promoter, can activate gene transcription. The P2A peptide is a self-cleaving peptide that enables the independent translation of two genes located before and after the P2A sequence (Szymczak & Vignali, 1995).

We have fused UVR8 with VP64 and COP1 with the BD domain of Gal4 and added a nuclear localization sequence (NLS) behind COP1 to stabilize its location in the cell nucleus, which helps to initiate gene expression. The target XPC gene is connected behind the 5UAS sequence and the CMV promoter, and the RUP2 is connected behind the XPC gene through the P2A sequence. Gal4 tightly binds to 5UAS within the nucleus, and when UV-B signal is present, UVR8 monomerizes and enters the nucleus to interact with COP1. Due to the localization effect of Gal4, the distance between VP64 and the CMV promoter is reduced, thereby activating the expression of the downstream XPC and RUP2 genes. The presence of P2A allows for the independent translation of XPC and RUP2. To ensure that the XPC protein reaches a certain concentration before RUP2 inhibits the interaction between UVR8 and COP1, we have optimized the RUP2 sequence by replacing the codons in the RUP2 sequence with synonymous codons that are rare. By adjusting the number of replacements, the expression level of XPC is brought close to that found in normal somatic cells. Due to the repeated interaction and disengagement of UVR8 and COP1, XPC protein is stably maintained at an appropriate concentration. When the UV-B signal is removed, the UVR8 dimer cannot monomerize, ultimately achieving complete dissociation of RUP2 from UVR8 and COP1, constructing a UV-responsive XPC gene switch.

Our project innovatively designed a gene therapy that responds to ultraviolet light, offering possibilities for the treatment of xeroderma pigmentosum. Meanwhile, this UV-responsive control system can be replaced with any target gene behind the CMV promoter, and it can also be combined with gene-editing systems such as CRISPR/Cas, providing solutions for various puzzles of skin genetic diseases, a more sensitive response, and a high degree of spatial and temporal control.

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