In order to construct a gene switch that responds to ultraviolet light for better gene therapy, we have carried out much design work and developed a new type of therapy that uses optogenetic technology as the control end and gene therapy as the foundation. This includes the ultraviolet-responsive interaction between UVR8 and COP1, the binding of GAL4 to 5*UAS, the potent transcriptional activator VP64, the verification of the opening of the ultraviolet switch, the biosafety module, and the construction of precise regulation of gene expression levels.

In Arabidopsis, COP1 is an E3 ubiquitin ligase that targets HY5 for degradation. In absence of UV-B signals, UVR8, a photoreceptor protein, exists in the form of homodimers outside the nucleus. Upon UV-B exposure, UVR8 monomerizes and translocates into the nucleus, where it interacts with COP1 to regulate plant photomorphogenesis.

To make our gene express in response to ultraviolet light, we introduced the interaction between UVR8 and COP1 into the gene expression induction system, allowing UV-B signaling to promote the expression of the target gene through the binding of UVR8 and COP1.

Through literature review, we found that the native sequences of Arabidopsis UVR8 and COP1 can still be used in mammalian cells without the need for sequence optimization. Additionally, we added a nuclear localization signal (NLS) sequence to COP1 to stabilize its location in the cell nucleus, which would be more conducive to initiating gene expression.

GAL4 is a transcriptional activator derived from yeast, capable of recognizing and binding to the upstream activating sequence (UAS), thereby activating the transcription of downstream genes. The term "5xUAS" refers to the repetition of the UAS sequence, which can enhance the binding capacity of the GAL4 protein.

GAL4 typically consists of two domains: the DNA-binding domain (BD) and the activation domain (AD). Considering that our system utilizes the binding of GAL4 to 5xUAS for localization rather than direct initiation of gene expression, we have chosen the BD region of GAL4. By leveraging the localization effect of the tight binding between GAL4 and 5xUAS, we facilitate the next step of the reaction.

VP64 is a potent transcriptional activator derived from the transactivation domain of the VP16 protein of the herpes simplex virus. It can bind to the promoter to achieve transcriptional activation of genes. Compared to VP16, VP64 has stronger activation capabilities and higher stability, which is why our project has chosen VP64 as the transcriptional activator.

In order to construct a gene switch that responds to ultraviolet light, after selecting the photoreceptor protein that responds to ultraviolet light, we need to design the pathway to initiate gene expression.

We attempted to link UVR8 to VP64 and the nuclear-localized COP1 to GAL4, placing the target gene behind the 5xUAS sequence and the CMV promoter. When UV-B signals are present, UVR8 monomerizes and enters the nucleus to interact with COP1. Since COP1 is already present in the nucleus, GAL4 will bind to the 5xUAS. Therefore, when the interaction between UVR8 and COP1 occurs, it brings VP64 closer to the CMV promoter, thereby efficiently initiating the expression of downstream target gene and achieving the construction of a gene switch that responds to ultraviolet light.

To verify that our designed UV gene switch can be activated—specifically, to initiate the expression of genes downstream of the CMV promoter under UV light exposure through the interaction of photoreceptor proteins and transcriptional enhancers—we first performed a qualitative validation.

We attempted to fuse UVR8 with VP64 and link COP1 with a nuclear localization signal to GAL4, placing the GFP gene downstream of the 5*UAS sequence and the CMV promoter. When UV-B signals are present, UVR8 monomerizes, enters the nucleus, and interacts with COP1, bringing VP64 closer to the CMV promoter, thus initiating the expression of the downstream GFP gene. The appearance of green fluorescence in the cell nucleus indicates successful activation. However, if any protein component of the system is missing, the expression of the GFP gene cannot be initiated.

RUP2 is a core protein in plants that regulates the response to UV-B radiation, acting as a negative regulator in the UV-B signaling pathway. It can dissociate the interaction between UVR8 and COP1, promoting the re-dimerization and inactivation of UVR8, thus limiting the intensity and duration of this response.

Our project aims to achieve gene therapy by introducing a normal XPC gene. To avoid the potential overexpression hazards that might be caused by the delivery of non-integrative exogenous genes into mammalian cells, we have inserted the RUP2 gene behind the XPC gene. This allows RUP2 to competitively bind with UVR8 after a certain duration of UV-B exposure, forming a negative feedback regulation of the system, controlling the expression level of the target gene within a safe range. Moreover, when the UV-B signal is removed, RUP2 can completely dissociate the interaction between UVR8 and COP1, thereby turning off the gene switch.

P2A is a self-cleaving peptide derived from Porcine teschovirus, which can translate a polycistronic mRNA into two or more independent proteins. When the ribosome translates to the P2A sequence, it causes the ribosome to skip at a specific location, thereby achieving separate translation of sequences before and after the P2A.

To more precisely regulate the expression level of the XPC gene, so that the expression level of XPC stabilizes at the level corresponding to that of epidermal cells in normal organisms upon UV-B exposure, we introduced the P2A sequence between the XPC and RUP2 genes, enabling the independent translation of XPC and RUP2 proteins.

Firstly, we used the luciferase gene for quantitative validation. The luciferase gene was placed behind the CMV promoter, and plasmids were constructed with or without the P2A sequence and the RUP2 gene following the luciferase gene. By detecting the luminescence intensity of luciferase protein reacting with the substrate in cell lysates after transferring different plasmids and turning on the UV-B signal, we quantitatively analyzed the expression level of the target gene, thereby verifying the negative feedback effect of RUP2 on the UV-induced gene switch.

Then, we replaced the target gene with the XPC gene and used Western blotting to further determine the effect of the UV gene switch on the expression of the XPC gene and the function of RUP2 in stabilizing the concentration of XPC with the help of imaging analysis.

In addition, we also attempted a series of sequence optimization tests, replacing the codons in the RUP2 sequence with synonymous codons that encode rare codons. By adjusting the number of replacements, we ultimately made the expression level of XPC close to that in normal organism cells.