Toll‐like receptor‐dependent activation of several human blood cell types by protamine‐condensed mRNA

Abstract

We reported that RNA condensed on protamine is protected from RNase-mediated degradation and can be used for vaccination. Here, we show that such complexes are also danger signals that activate mouse cells through a MyD88-dependent pathway. Moreover, mRNA-protamine complexes stimulate human blood cells. They strongly activate DC and monocytes, leading to TNF-a and IFN-a secretion. In addition, protamine-RNA complexes directly activate B cells, NK cells and granulocytes. The detailed analysis of the activated cell types, the study of the cytokines released from PBMC cultured with protamine-RNA complexes and recently published results suggest that TLR-7 and TLR-8 may be involved in the recognition of protamine-stabilized RNA. Our data indicate that protamine-stabilized RNA, which may be similar to RNA condensed in the nucleocapsids of RNA viruses, is a strong danger signal. Thus, similarly to plasmid DNA, protamine-RNA combines antigen production and non-specific immunostimulation. The studies presented here explain the capacity of protamine-RNA to act as a vaccine, and pave the way towards the development of safe and efficient mRNA-based immunotherapies.

Therapeutic anti‐tumor immunity triggered by injections of immunostimulating single‐stranded RNA

“…1C). We have already demonstrated that several cytokines are produced by human immune cells stimulated by protamine-RNA [19]. Here we show that IL-12 was produced by mouse splenocytes incubated with protamine-RNA (Fig.…”

“…Condensation of in vitro transcribed mRNA to the cationic peptide protamine is another method for generating stabilized ssRNA capable of stimulating mouse DC [18] and several human cell types [19]. To determine whether protamine-mRNA stimulates cell types in mice other than DC, we incubated splenocytes for 16 h with protamine-condensed mRNA at a concentration of 20 lg/mL (10 lg RNA plus 10 lg protamine).…”

“…1) and their relatively high stability [19,26], which makes them ideal candidates for use in the clinic, we focused our studies on these compounds rather than on naked RNA. At day 21 after tumor implantation, the treated mice that were tumor free (in the presented experiment: two in the CpGtreated group, three in the intra-tumor protaminemRNA injected group and one in the protamine-mRNA contralateral injected group) were re-challenged with 1 Â 10 6 SMA-560 tumor cells injected into the opposite flank.…”

“…This receptor is expressed by myeloid DC (mDC), NK cells and macrophages [14]. In contrast to DNA and dsRNA, ssRNA are very sensitive to degradation by ubiquitous RNases, and must be protected in vitro by encapsulation in liposomes [15][16][17], condensation to a cationic peptide [18,19] or chemical modifications (phosphorothioate oligoribonucleotides) [16,18] to reveal their immunostimulatory potency. Immunostimulatory ssRNA are recognized by TLR7 and TLR8 [15,16], which are both localized in endosomal and lysosomal vesicles within the cell.…”

Evading innate immunity in nonviral mRNA delivery: don’t shoot the messenger

“…Although this naturally occurring protein is demonstrated to protect the mRNA from degradation, mRNA:protamine complexes were shown to strongly induce innate immune response [73]. Scheel et al indicate that protamine condensed mRNA stimulates the immune system through a MyD88-dependent pathway, suggesting that TLR7 and TLR8 are probably the receptors involved [74]. This immune activating capacity of protamine can be exploited for vaccination strategies, but seemed to inhibit the primary goal of mRNA delivery, i.e.…”