Uptake of synthetic naked RNA by skin-resident dendritic cells via macropinocytosis allows antigen expression and induction of T-cell responses in mice

Abstract

Intradermal administration of antigen-encoding RNA has entered clinical testing for cancer vaccination. However, insight into the underlying mechanism of RNA uptake, translation and antigen presentation is still limited. Utilizing pharmacologically optimized naked RNA, the dose-response kinetics revealed a rise in reporter signal with increasing RNA amounts and a prolonged RNA translation of reporter protein up to 30 days after intradermal injection. Dendritic cells (DCs) in the dermis were shown to engulf RNA, and the signal arising from the reporter RNA was significantly diminished after DC depletion. Macropinocytosis was relevant for intradermal RNA uptake and translation in vitro and in vivo. By combining intradermal RNA vaccination and inhibition of macropinocytosis, we show that effective priming of antigen-specific CD8(+) T-cells also relies on this uptake mechanism. This report demonstrates that direct antigen translation by dermal DCs after intradermal naked RNA vaccination is relevant for efficient priming of antigen-specific T-cells.

mRNA vaccines — a new era in vaccinology

“…Although DCs have been shown to internalize naked mRNA through a variety of endocytic pathways 61–63 , ex vivo transfection efficiency is commonly increased using electroporation; in this case, mRNA molecules pass through membrane pores formed by a high-voltage pulse and directly enter the cytoplasm (reviewed in REF. 64).…”

“…Naked mRNA has been used successfully for in vivo immunizations, particularly in formats that preferentially target antigen-presenting cells , as in intradermal 61,65 and intranodal injections 66–68 . Notably, a recent report showed that repeated intranodal immunizations with naked, unmodified mRNA encoding tumour-associated neoantigens generated robust T cell responses and increased progression-free survival 68 (discussed further in BOX 2).…”

Opportunities and Challenges in the Delivery of mRNA-Based Vaccines

“…Hence, it circumvents the need to employ carriers, eventually contributing to reduced cost and potential risk. Another advantage of administering mRNA-based vaccines via the s.c. route is that both cellular and humoral immune responses are induced [94] because the mRNA is expressed by both skin-resident DCs [95] and non-immune cells [96]. However, the outermost stratum corneum layer of the epidermis serves as a tight barrier to the absorption of topically administered drugs [97].…”

Antigen‐Inorganic Hybrid Flowers‐Based Vaccines with Enhanced Room Temperature Stability and Effective Anticancer Immunity

“…To investigate the cellular uptake mechanism of OVA‐Cu‐HVs, different chemical blockers, including cytochalasin D, chlorpromazine, filipin III, and rottlerin, were used to examine the endocytosis pathways for the cell uptake of OVA‐Cu‐HVs . As show in Figure B,C, the uptake of OVA‐Cu‐HVs was significantly suppressed after treatment with rottlerin, indicating that macropinocytosis‐mediated pathway was a major process involved in their cellular uptake …”