Comb-structured mRNA vaccine tethered with short double-stranded RNA adjuvants maximizes cellular immunity for cancer treatment

Abstract

Integrating antigen-encoding mRNA (Messenger RNA) and immunostimulatory adjuvant into a single formulation is a promising approach to potentiating the efficacy of mRNA vaccines. Here, we developed a scheme based on RNA engineering to integrate adjuvancy directly into antigen-encoding mRNA strands without hampering the ability to express antigen proteins. Short double-stranded RNA (dsRNA) was designed to target retinoic acid-inducible gene-I (RIG-I), an innate immune receptor, for effective cancer vaccination and then tethered onto the mRNA strand via hybridization. Tuning the dsRNA structure and microenvironment by changing its length and sequence enabled the determination of the structure of dsRNA-tethered mRNA efficiently stimulating RIG-I. Eventually, the formulation loaded with dsRNA-tethered mRNA of the optimal structure effectively activated mouse and human dendritic cells and drove them to secrete a broad spectrum of proinflammatory cytokines without increasing the secretion of anti-inflammatory cytokines. Notably, the immunostimulating intensity was tunable by modulating the number of dsRNA along the mRNA strand, which prevents excessive immunostimulation. Versatility in the applicable formulation is a practical advantage of the dsRNA-tethered mRNA. Its formulation with three existing systems, i.e., anionic lipoplex, ionizable lipid-based lipid nanoparticles, and polyplex micelles, induced appreciable cellular immunity in the mice model. Of particular interest, dsRNA-tethered mRNA encoding ovalbumin (OVA) formulated in anionic lipoplex used in clinical trials exerted a significant therapeutic effect in the mouse lymphoma (E.G7-OVA) model. In conclusion, the system developed here provides a simple and robust platform to supply the desired intensity of immunostimulation in various formulations of mRNA cancer vaccines.

Keywords: RNA engineering; cancer vaccine; mRNA delivery; mRNA vaccine; vaccine adjuvant.

Fig. 1.

Comb-structured mRNA with immunostimulatory dsRNA teeth. This formulation is prepared from in vitro transcribed RNA with 5′ triphosphate (5′ppp-RNA), mRNA, and a chemically synthesized counter-RNA strand complementary to the 5′ppp-RNA and particular sequences in mRNA (cRNA). dsRNA teeth from 5′ppp-RNA and cRNA possess blunt-ended 5′ triphosphate, a substrate of RIG-I. The length of the cRNA sequence complementary to mRNA is fixed to 17 nt to maintain mRNA translational activity. In addition, design parameters of comb-structured mRNA, including the lengths and sequences of dsRNA teeth, and lengths of gap sequence in cRNA between two regions complementary to 5′ppp-RNA and mRNA, were optimized.

Fig. 2.

Optimization of tooth design for efficient immunostimulation. (A and B) Effect of dsRNA lengths and sequences. (C and D) Effect of gap RNA lengths in cRNA between two regions complementary to 5′ppp-RNA and mRNA. (E and F) Effect of the dsRNA tooth and mRNA codelivery method. Introduction of a 24-nt GU-repeat tooth alone and separate introduction of mRNA and a 24-nt GU-repeat tooth were performed. (G and H) Effect of tooth numbers. The teeth used possessed dsRNA with 24-bp GU-repeat and a 2-nt gap (dsRNA24-GUrepeat/2-gap). Transcript levels of interferon (IFN)-β (A, C, E, and G) and interleukin (IL)-6 (B, D, F, and H) were measured using quantitative PCR 4 h after mRNA introduction to DC2.4 cells. n = 5 in (A–D) and n = 6 in (E–H).

Fig. 3.

Immunological profiling of BMDCs after mRNA treatment. Protein expression levels of 25 types of cytokines, interferons, and chemokines were quantified 24 h after mRNA introduction to mouse BMDCs. Colors in the heatmap represent protein levels relative to untreated control. Among 25 types of molecules, nine types (IFN-γ, IL-2, 4, 5, 7, 10, 12p40, 12p70, and 15) were below detection limits, and data from the other 16 are shown. n = 6. LF, Lipofectamine; PPP, 5′ triphosphate; G-CSF, granulocyte colony-stimulating factor. In Separate, mRNA and tooth with tooth amount equal to that in 1 tooth were separately formulated with LF for addition to BMDCs.

Fig. 4.

Cancer vaccines using lipoplex in mice. (A) Expression of fLuc in the spleen 24 h after i.v. injection of lipoplex. n = 4. (B) Activation of DC in the spleen 24 h after i.v. injection of lipoplex. n = 4. The expression level of CD86 in CD11c positive splenocytes. (C) CTL immunity against OVA 7 d after mRNA vaccination, evaluated by the in vivo CTL assay. n = 4. (D) Serum levels of IL-6 were measured using ELISA 6 h and 24 h after i.v. injection of lipoplexes. n = 4. n.d.: not detected. (E) Prophylactic model of subcutaneously inoculated lymphoma expressing OVA, treated using OVA mRNA. n = 6 for untreated, n = 4 for the other two groups. (F) Therapeutic model of subcutaneously inoculated lymphoma expressing OVA, treated using OVA mRNA. n = 4. (G) Therapeutic model of subcutaneously inoculated melanoma, treated using Trp2 mRNA. †Average tumor volume was not shown at later time points because of the death of one or more mice. n = 8. *P < 0.05 versus 0 tooth. (E−G) The first chart from the left shows the average tumor volumes in each group. The second to fourth charts from the left shows the tumor volume of individual mice (Right). (F) The right figure shows the mouse survival. (B−F) OVA mRNA dose for each injection was 5 μg, and tooth dose was 0.23 μg for 1 tooth, 0.70 μg for 3 teeth, and 1.2 μg for 5 teeth, respectively. (G) The dose was 10 μg for Trp2 mRNA and 0.34 μg for a tooth.

Fig. 5.

Contribution of innate immune receptors for immunostimulation by mRNA. (A) Comb-structured mRNA was added to RAW cells without knockout of immune receptors (WT) and with MDA-5 or RIG-I knockout (MDA-5-KO, RIG-I-KO). The cells were genetically modified to express Lucia luciferase (lLuc) after proinflammatory stimulation for quantifying immunostimulation intensity based on lLuc expression. n = 6. (B) Comb-structured mRNA was added to HEK 293 cells, genetically modified to express TLR3 or TLR7, or without transformation to express TLRs (Null). The cells were transformed to express SEAP reporter after nuclear factor-κB (NF-κB) stimulation. n = 6. PPP, 5′ triphosphate. **P < 0.01; ***P < 0.001, n.s., nonsignificant versus untreated, respectively.

Fig. 6.

Activation of dendritic cells by comb-structured mRNA. mRNA was added to mouse BMDCs (A–K) and human BMDCs (L and M). (A–H and J) Expression of surface markers, CD40 (A–C), CD86 (D–F and L), MHC I (G), and MHC II (H) was quantified using immunocytochemistry 24 h after mRNA addition. n = 4 (I and M) Expression of gLuc was measured using the cultured medium. n = 6. PPP, 5′ triphosphate. (J and K) The kinetics of innate immune activation and antigen presentation. (J) For evaluating the kinetics of antigen presentation, OVA epitope (SIINFEKL)/MHC class I H-2Kb complexes on the surface of DC2.4 cells were quantified using flow cytometry after the introduction of OVA mRNA with 0 and 1 tooth. n = 4. (K) For evaluating the kinetics of innate immune activation, OVA mRNA with 0 and 1 tooth was added to reporter RAW-Lucia cell lines with and without RIG-I knockout. Lucia luciferase (lLuc) expression was measured as a marker of innate immune activation. n = 6.

Fig. 7.

Vaccines using iLNPs and PMs. (A and B) The in vivo CTL assay was performed at indicated schedules. iLNPs loading OVA mRNA with 0 or 1 tooth were injected into mice via i.m. route. n = 4. (B) PMs loading OVA mRNA with 0, 1, or 5 teeth were injected into mice via i.m. and i.d. routes. Tooth dose was 0.0047 μg for 0.01 μg mRNA, 0.0014 μg for 0.03 μg mRNA, 0.047 μg for 0.1 μg mRNA, and 0.014 μg for 0.3 μg mRNA, respectively. n = 4. (C and D) Luciferase expression was evaluated after i.m. injection of iLNP (C) and after i.m. and i.d. injection of PMs (D). Only expression at the muscle was quantified in (C). Representative images at 4 h are shown. n = 5 in (C) and n = 3 in (D). In i.m. injection, OVA mRNA dose for each injection was 20 μg, and tooth dose was 0.93 μg for 1 tooth and 4.7 μg for 5 teeth, respectively. In i.d. injection, OVA mRNA dose for each injection was 12 μg, and tooth dose was 0.56 μg for 1 tooth and 2.8 μg for 5 teeth, respectively.