Graduate student Cornell University, United States
Introduction:: Although RNA-based therapies have shown remarkable success in treating and preventing COVID-19, their potential applications extend far beyond this disease, including gene editing and protein replacement therapy. However, mRNA therapeutics in these applications require much higher standards in the delivery strategy: targeting and efficacy. In this work, we investigated the systemic delivery of mRNA in the absence of monocytes/macrophages. We discovered that depletion of these phagocytes allows an enhanced delivery of the mRNA in the liver characterized by protein expression regardless of LNP formulations. We then applied this mechanism to in vivo mRNA-mediated gene editing and achieved higher efficiency without the need to increase the dosage.
Materials and Methods:: Male C57BL/6J mice at the age of 6-8 weeks were pre-treated with small molecule drugs or antibodies to deplete both circulating and tissue-resident macrophages. LNPs encapsulating mRNA encoding fireflies luciferase (Fluc) were administered to the C57BL/6 mice via intravenous injections. For the evaluation of in vivo protein expression, in vivo bioluminescence imaging of the whole body and isolated organs was conducted on the IVIS platform. For gene editing, LNPs encapsulating mRNA encoding Cre or Cas9 (codelivery with sgRNA) were administered to the Ai14 mice via intravenous injections, respectively. The expression of tdTomato was evaluated via confocal images or flow cytometric analysis to indicate the efficacy of the delivery strategy.
Results, Conclusions, and Discussions:: Compared to untreated mice, LNPs performed more efficient protein expression in mice treated with small molecule drugs to deplete macrophages. (Figure 1A) We also utilized other reagents for macrophage depletion and also achieved enhanced protein expression of mRNA in the liver. Confocal microscopy analysis on tissue sections of the liver demonstrated that our depletion method greatly improves the efficiency of mRNA-mediated gene editing indicated by a higher level of tdTomato expression (Figure 1B). In conclusion, we presented a new approach to improve the delivery efficiency of mRNA, independent of formulations. Building on our previous findings, we discovered that certain LNPs are taken up and eliminated by circulating macrophages and Kupffer cells, leading to reduced efficacy. We have shown that LNPs are targeted and delivered to the hepatocytes after circulating macrophages Kupffer cells are temporarily depleted. Our work provides new insight for designing methods for liver-targeted mRNA delivery.
Acknowledgements (Optional): : We acknowledge start-up support from Cornell University, including Robert S. Langer Professorship and Cornell NEXT Nano Initiative. Imaging data were acquired through the Cornell Institute of Biotechnology's Imaging Facility, with NIH S10OD025049 for the IVIS Spectrum.
