scut-china-b
1. To propose a new therapeutic option for the treatment of tumors.
2. Designed and successfully constructed a tumor-specific plasmid that effectively promotes T-cell activation.
3. Designed the iCLAN nanodrug delivery system.
4. Successfully synthesized a novel tumor-specific expression gene nanomedicine, which can significantly promote T cell
In contrast to traditional cancer therapies, immunotherapy is recognized as one of the most effective therapies with the longest patient survival and is one of the hottest areas of research, attracting the attention of many experts and scholars. Our focus on immunotherapy cleverly avoids the challenge of finding immune targets. Due to the difficulty in finding immune targets, as well as possible off-targeting and immune resistance, immunotherapy is often costly and ineffective in the long term. Inspired by the widespread example of CD28, CD3 dual signaling to activate T cells in vitro, our team designed a tumor-specific gene-based nanomedicine with the aim of expressing CD3, CD28 signaling molecules on the surface of tumor cells and activating T cell immune responses in vivo.
We narrowed down the problem to a short-term experimental goal of treating melanoma.
(1)We selected the Tyr promoter, which is regulated by melanocyte-inducing factor, a bioactive substance that plays an important role in the skin and other tissues, and whose content is particularly high in cancerous melanocytes, and whose binding to the Tyr promoter significantly enhances the transcriptional strength of downstream genes. This ensures that the gene nanomedicine is highly expressed only in melanoma cells.
(2)CD3 is an important signaling molecule on the surface of T cells, and forms TCR-CD3 complex with T cell receptor (TCR), which is involved in T cell activation. In immunotherapy, by mimicking the natural activation process of T cells, it can promote the recognition and killing of tumor cells by T cells.CD86 is a co-stimulatory molecule on the surface of T cells, which provides a second signal to enhance the activation of T cells. Activation of CD86 enhances the proliferation, survival and cytokine production of T cells, which is essential to maintain the immune response of T cells.1) CD3 is an important signaling molecule on the surface of T cells and forms a TCR-CD3 complex with the TCR, which is involved in T cell activation.
(3)Gene Synthesis: Synthesize genes encoding anti-CD3 scFv, anti-CD86 scFv and mut IL-2. The target genes are cloned into expression vectors using restriction enzymes and DNA ligases, and tandem expression of multiple genes is achieved by linking the genes with appropriate signal peptides and post-transcriptional regulatory sequences (e.g. T2A, P2A, etc.) using ligases.
(4)Plasmid construction verification: Verify the correct insertion of the target gene into the vector by PCR and restriction enzyme analysis, and confirm the correctness of the insertion sequence and reading frame by DNA sequencing.
Common attributes of nucleic acid drugs, such as high molecular weight, negatively charged nature, and susceptibility to degradation by nucleases, exhibit limited bioavailability in the naked form, severely limiting their delivery. Traditional gene delivery systems involve recombinant viruses such as lentiviruses and adenoviruses. Although these viral vectors have demonstrated very effective results in several clinical trials and marketed products (e.g., Gendicine, Neovasculgen, and Glybera), the inherent weaknesses of potential tumorigenicity, immunogenicity, limited payload capacity, and inapplicability to all nucleic acid types have limited further development. In contrast, the inherent properties of conventional cationic materials, including associated cytotoxicity, large and heterogeneous complex sizes, and rapid clearance by the reticuloendothelial system, limit their clinical application.
To overcome the challenges of nucleic acid delivery, we chose a cationic lipid-assisted PEGb-PLA nanoparticle (CLAN) that has a high encapsulation rate and is able to protect nucleic acids by encapsulating them within an aqueous core and deliver them to the target cell while maintaining or improving nucleic acid function. Our team utilizes a one-step encapsulation plasmid with a 95% encapsulation rate.
We successfully constructed we constructed four tumor-specific gene nanodrugs, iCLANTyr-mut IL-2, iCLANTyr-αCD3-CD86, iCLANTyr-αCD3-CD86+Tyr-mut IL-2, iCLANTyr-αCD3-CD86+Tyr-mut IL-2, and successfully sequenced and examined protein expression.
Xu, C. F., Iqbal, S., Shen, S., Luo, Y. L., Yang, X., & Wang, J. (2019). Development of "CLAN" Nanomedicine for Nucleic Acid Therapeutics. Small (Weinheim an der Bergstrasse, Germany), 15(16), e1900055. https://doi.org/10.1002/smll.201900055.