Immunostimulating capacities of stabilized RNA molecules

Abstract

Since direct injection of naked mRNA induces an immune response, we tested the capacity of RNA to signal danger. We show here that mRNA molecules that are protected from immediate degradation either through interaction with cationic proteins (trans protection) or through chemical modification of the phosphodiester backbone (phosphorothioate RNA; cis protection) act as sequence-independent danger signals on mouse DC. As opposed to CpG DNA, the cis-stabilized RNA is degraded in a few minutes, does not activate B cells and, in contrast to double-stranded RNA, requires MyD88 for activation of the DC. We postulate that phosphorothioate RNA, which mimics trans-stabilized RNA, is a new PAMP.

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

“…For this reason, our studies concentrated on protamine-stabilized RNA. Based on our previously published reports [18,19] and reports from others [15,16], we propose that the capacity of naked and protamine-RNA complexes to stimulate immune cells through TLR, and in particular through mouse TLR7 (also for protamine-RNA: S. Bauer, personal communication), is the basic mechanism that accounts for the observed anti-tumor activity.…”

“…6) and reported by others [12,29], the repeated injection of very stable phosphorothioate CpG DNA oligonucleotides leads to uncontrolled immune stimulation (evidenced by splenomegaly and potentially leading to autoimmunity). On the contrary, stabilized RNA that do not induce the proliferation of B cells [18] and that have a half-life of approximately 110 h in serum [19] are naturally cleared with reliable and reproducible kinetics from the site of injection, thereby offering a controllable therapeutic window. Thus, although they are similar in their mode of action, RNA may be preferred over ODN for the safe and transient immunostimulation that is required for immunotherapies and vaccinations in human patients.…”

“…We have shown that synthetic ssORN stabilized by a phosphorothioate backbone have an immunostimulatory effect in mice [18]. 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].…”

“…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).…”

“…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.…”