Sustained release of PKR inhibitor C16 from mesoporous silica nanoparticles significantly enhances mRNA translation and anti-tumor vaccination
Graphical abstract
Introduction
In vitro transcribed messenger RNA (mRNA) has recently emerged as a promising class of nucleic acid therapeutics with the potential to treat a broad range of diseases. [1], [2], [3] There is a surge of interest in using mRNA for various biomedical applications, such as cancer immunotherapies [4], [5], [6], [7], [8], infectious disease vaccines [9], [10], [11], protein replacement or supplement therapies [12], [13], [14], generation of pluripotent stem cells [15], [16], and genome engineering [17], [18].
One of the major challenges in the field is to achieve high translation of mRNA in target cells, as it significantly determines the magnitude of the desired therapeutic outcome. It has been demonstrated that exogenously transfected mRNA is recognized by cytoplasmic RNA sensors, the key function of which is to distinguish self from non-self and respond to viral RNA by inducing innate immune responses [1], [19]. These cytoplasmic RNA sensors, such as toll-like receptors (TLRs) [20], [21], the RNA-activated protein kinase (PKR) [22], retinoic acid-inducible gene 1 (RIG-I) [23], and melanoma differentiation-associated protein 5 (MDA5) [24], attenuate mRNA translation through direct or indirect PKR upregulation and activation. The main function of PKR is to phosphorylate the eukaryotic translation initiation factor 2α (eIF2α) and ultimately inhibit mRNA translation [22], [25]. Incorporation of modified nucleosides, such as pseudouridine, 5-methyluridine, 5-methylcytidine or N6-methyladenosine during in vitro synthesis of mRNA has been shown to enhance the translation through reduction of these immune-stimulatory effects, which partially attenuate PKR activation [26], [27]. However, double-stranded RNA in nucleoside-modified mRNA, inevitably created during in vitro transcription, still upregulates PKR albeit at a lower level [28]. Taken together, PKR is a desirable target because it plays a crucial role in modulating the translation of mRNA.
We have recently pioneered an immune evasion approach based on the co-delivery of a transgene with mRNA encoding influenza A virus derived non-structural protein 1 (NS1) [29]. We showed that NS1 not only could significantly enhance mRNA translation more effectively than nucleoside-modified mRNA, but NS1 could also further enhance translation of nucleoside-modified mRNA itself. In this study, we further discover that the application of small molecule imidazolo-oxindole PKR inhibitor C16 [30], [31] is an equally effective approach to achieve non-linear mRNA translation enhancement. C16 is a selective inhibitor of the enzyme double-stranded RNA-dependent protein kinase (PKR), which has been shown to effectively inhibit PKR function in vivo [30] and has neuroprotective [32] and nootropic [33] effects in animal studies. In this study, we show that C16 not only can enhance mRNA translation more effectively than nucleoside-modified mRNA, but it can also further enhance translation of nucleoside-modified mRNA itself as well as HPLC purified mRNA. When we apply C16 in vivo with naked mRNA via subcutaneous administration, a mesoporous silica nanoparticles (C16@MSNs) system is introduced as a depot for sustained release of C16, which is necessary as an interface to maintain enhanced in vivo mRNA translation and prolonged expression kinetics. Lastly, we demonstrate therapeutic impact by applying the MSN-mRNA vaccine formulation composed of naked OVA mRNA and C16@MSNs on a xenograft E.G7-OVA prophylactic tumor model, resulting in very potent tumor inhibition. Our study demonstrates that the application of C16 is a novel immune evasion approach for mRNA translation enhancement with potency comparable to that of NS1. It is also a practical approach because it can be orthogonally applied without changes to users’ existing protocols. Our study also demonstrates that a simple MSN-mRNA delivery system can translate the potent effects of C16 in vivo, leading to improved efficacy of subcutaneous mRNA anti-tumor vaccine. Importantly, our study illustrates a simple yet novel approach to enhance efficacy of mRNA therapeutics via nanotechnology.
Section snippets
Cells and reagents
HepG2 and NIH 3 T3 cells were purchased from the American Type Culture Center (ATCC). HepG2 cells and NIH 3T3 cells were cultured in MEM and DMEM, respectively, supplemented with 10% heat-inactivated FBS, 100 units/mL penicillin and 100 mg/mL streptomycin. Mouse bone marrow derived dendritic cells (BMDCs) were generated as previously described. [34] BMDCs were cultured in maintained in RPMI 1640 supplemented with 5% FBS, 5 mM L-glutamine, 1 mM sodium pyruvate, 10 mm Hepes, 1xnon-essential amino
Results
As RNA-activated protein kinase (PKR) plays an inhibitory role in RNA translation, inhibiting its up-regulation during mRNA transfection would enhance the translation efficiency. Imidazolo-oxindole C16 is a selective small molecule inhibitor of PKR, which binds to PKR’s ATP-binding site [30]. We discovered the potency of C16 during our routine cell transfection of HepG2 using unmodified luciferase mRNA (UM Luc) and observed 100 fold enhancement in luciferase expression (Figure S1). This
Conclusion
In this study, we developed a mesoporous silica nanoparticles based mRNA (MSN-mRNA) delivery system composed of naked mRNA and a subcutaneous depot of PKR inhibitor C16 and demonstrated its potent capacity to enhance in vitro and in vivo mRNA translation. In vitro mRNA translation enhancement was correlated to suppressed PKR up-regulation. To apply C16 for in vivo mRNA transfection, C16@MSNs was developed as a subcutaneous depot to sustainably release C16. Finally, we show that the MSN-mRNA
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This study is supported by NMRC/OFYIRG/0028/2016 (R-279-000-492-511). We thank Dr Drew Weissman and Dr Norbert Pardi for providing HPLC purified luciferase mRNA in unmodified and nucleoside-modified formats.
Author contributions
KKLP conceived the idea. WZ performed the experiments. YL and JMC assisted in some of the experiments. KKLP and WZ troubleshooted experiments, analyzed the results and wrote the manuscript.
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