(M1130-07-37) Spleen-Targeting Delivery of Small Circular RNA Vaccines

Purpose: Our aim is to synthesize a potent circular RNA vaccine and compare it with current gold standard linear mRNA vaccines in terms of biostability, storage, and efficacy. Since the spleen is the largest immune organ, we try to achieve spleen-targeting delivery of the circular RNA vaccine by developing novel lipid nanoparticle delivery systems ^[1,2].

Methods: Circular RNA will be synthesized using a new splint-dependent RNA ligase method to increase the synthesis efficiency and the efficacy of the circular RNA vaccine will be compared with its linear counterpart in vitro (translatability) and in vivo (immune cell activation). The distribution of circRNA-LNP will be monitored by the in vivo imaging system (IVIS). The blood will be collected in the immunized mice and go through Luminex analysis while the body weight of the mice will also be monitored.

Results: Circular RNA can be successfully synthesized by the new splint-dependent method (figure 1), the 84 mer band in the last lane is the final product circRNA which is resistant to RNase R. By tuning the components of LNPs, we can achieve spleen-targeting delivery (figure2, 3). The in vitro tissue imaging showed that the spleen has higher radiant efficiency than the liver which is the main organ for current LNP accumulation. The blood Luminex analysis will be included later and the T-cell and B-cell activation data will be included after the second dose of this spleen targeting delivery LNP-circRNA vaccine (figure not shown).

Conclusion: The new splint-dependent circular RNA synthesis method has a better efficiency compared to the conventional one. Our LNP system can achieve spleen-targeting delivery which benefits the circRNA vaccine. We expect the Luminex results to bring us a good safety result. According to our previous result, the circular RNA can activate more antigen-specific T cell and B cell responses through s.c. delivery, we expect this i.v. injected spleen-targeting circRNA-NP vaccine can also activate great antigen-specific T cell and B cell responses.

References: 1. Kranz, L., Diken, M., Haas, H. et al. Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy. Nature 534, 396–401 (2016). https://doi.org/10.1038/nature18300
2. Q. Cheng et al., Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing. Nat. Nanotechnol. 15, 313–320 (2020).

Acknowledgments: NIH (R01AI168684, R35GM143014, R21NS114455), DoD CDMRP Breast Cancer Research Program (Breakthrough Award Level II (BC210931/P1), METAvivor Early Career Investigator Award, NIH-NCATS KL2 scholarship (KL2TR002648) via VCU C. Kenneth and Dianne Wright Center for Clinical and Translational Research (UL1TR002649), and pilot grants from VCU Massey Cancer Center Supported by NIH (P30 CA016059)

Figure1. The gel imaging shows the high efficiency of splint-dependent circRNA synthesis



Figure3. The new LNP system can achieve spleen-targeting delivery



Figure2. The new LNP system can accumulate in mice for at least 24 hrs and achieve spleen delivery