Part:BBa_K5073030
L7Ae-CD63
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 620
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
To develop an exosome-based targeting and RNA packaging and delivery system for GPC3 mRNA delivery, we investigated a method for efficiently encapsulating specific RNA molecules into exosomes and deliver them to target cells. After reviewing the literature [1] [2] [3], we focused on the archaeal ribosomal protein L7Ae, which can specifically binds to the C/D box RNA structures.
In our system design, we fused the L7Ae protein with the C-terminus of CD63 to form the L7Ae-CD63 fusion protein, where CD63 is a well-known exosome marker protein that allowed its presence on the exosome membrane. To package the therapeutic RNA, we modified the 3'untranslated region (3'UTR) of GPC3 mRNA by inserting a C/D box sequence. The interaction between L7Ae on the surface of the exosome and the C/D box in GPC3 mRNA enables effective encapsulation of GPC3 mRNA into the exosomes.
In addition, by integrating hypoxia-responsive elements, we can further enhance the targeted delivery of these exosomes to liver cancer cells, which are typically situated in a hypoxic tumor microenvironment. This ensured that exosomes packaged with GPC3 mRNA can selectively deliver to liver cancer cells, thereby improving the safety and targeting of the therapy.
We experimentally verified the effectiveness of the L7Ae-C/D box system, demonstrating the interaction between CD63 and GPC3 mRNA, and successfully loaded GPC3 mRNA into exosomes.
To validate the ability of EXOtic to carry GPC3 mRNA in exosomes, we employed qPCR to measure the GPC3 mRNA content within the exosomes (Fig1.A). Meanwhile, to assess the role of L7Ae-C/D box in the exosome loading process, we conducted an RIP experiment combined with a CD63 antibody to analyze the binding of the exosome-specific protein CD63 to GPC3 mRNA The results showed that CD63 antibody significantly enriched GPC3 mRNA, and the binding of GPC3 mRNA was significantly increased compared to IgG-negative control (P<0.01), indicating that LA7e-C/D box plays a key role in the binding process between GPC3 mRNA and CD63 protein (Fig1.B-C).
A. qPCR results of exosome samples (****, P<0.0001) ;
B. Schematic diagram of the L7Ae-C/D box system;
C. RIP results;
References
[1] Kojima, R., Bojar, D., Rizzi, G., Hamri, G. C., El-Baba, M. D., Saxena, P., Ausländer, S., Tan, K. R., & Fussenegger, M. (2018). Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson's disease treatment. Nature communications, 9(1), 1305.
[2] Zhang, M., Hu, S., Liu, L., Dang, P., Liu, Y., Sun, Z., Qiao, B., & Wang, C. (2023). Engineered exosomes from different sources for cancer-targeted therapy. Signal transduction and targeted therapy, 8(1), 124.
[3] Zhang, X., Zhang, H., Gu, J., Zhang, J., Shi, H., Qian, H., Wang, D., Xu, W., Pan, J., & Santos, H. A. (2021). Engineered Extracellular Vesicles for Cancer Therapy. Advanced materials (Deerfield Beach, Fla.), 33(14), e2005709.
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