Innate immune responses triggered by nucleic acids inspire the design of immunomodulatory nucleic acid nanoparticles (NANPs)

Abstract

The unknown immune stimulation by nucleic acid nanoparticles (NANPs) has become one of the major impediments to a broad spectrum of clinical developments of this novel technology. Having evolved to defend against bacterial and viral nucleic acids, mammalian cells have established patterns of recognition that are also the pathways through which NANPs can be processed. Explorations into the immune stimulation brought about by a vast diversity of known NANPs have shown that variations in design correlate with variations in immune response. Therefore, as the mechanisms of stimulation are further elucidated, these trends are now being taken into account in the design phase to allow for development of NANPs that are tailored for controlled immune activation or quiescence.

Figure 1.

The flow of NANP design and characterization. Structural and long-range interacting motifs that can be either mined from natural NAs, selected via systematic evolution of ligands by exponential enrichment (SELEX), or designed computationally are combined for the rational design of programmable NANPs. All new NANPs are then extensively characterized and their immunostimulation is assessed. Machine learning approaches such as quantitative structure-activity relationship (QSAR) modeling which relates the physicochemical parameters to relative immune response can be utilized to predict and optimize future NANP designs suitable for specific biomedical tasks. Some parts of lower right panel are adapted with permission from Nano Letters 2018, 18 (7), 4309–4321. Copyright 2018 American Chemical Society.

Figure 2.

Possible ways of NANP processing in the cellular environment. NANPs complexed with a polycationic carrier enter the cell via scavenger receptor-mediated endocytosis and get recognized by TLRs (e.g., TLR7 for RNA cubes and TLR9 for both RNA cubes and rings). In the cytoplasm, non-functional RNA/DNA hybrid NANPs can dynamically interact with each other to activate pre-programmed functionalities such as the release of Dicer Substrate (DS) RNAs, later processed into siRNAs, and NF-κB decoy containing dsDNAs which prevent NF-κB translocation into the nucleus and the subsequent production of inflammatory cytokines. The use of longer byproduct dsDNAs helps to activate the cGAS-STING pathway leading to the expression of inflammatory genes.

Figure 3.

Trends in immune stimulation by NANPs. (A) The dimensionality, composition, functionalization, orientation, and sequence of NANPs have been evaluated relative to contributions to immunostimulation. Globular NANPs are more immunostimulatory than planar NANPs, which are in turn more immunostimulatory than fiborous. For composition, an increasing number of RNA strands in an assembly over DNA strands yields a greater subsequent immune response. Increased functionalization of NANPs with DS RNAs increases relative IFN production, while the orientation of DS RNAs within a single fibrous structure can decrease the effect. Finally, the sequences between variations of the structure have no effect on immune stimulation, while the structure itself is what dictates the response. (B) Neither free NANPs without a carrier nor electroporated free NANPs induce any IFN response. Instead, transfection using a polycationic carrier is necessary to trigger the IFN production. Across multiple immune cell types, pDCs show the greatest production of types I and III IFNs in response to various NANPs. (C) A library of RNA, RNA/DNA, and DNA NANP polygons composed of the same set of sequences but varying in relative blood stability, melting temperature, molecular weight, GC content, K_d, and size revealed that those descriptors had the respective impact on NANP-induced immune stimulation. Some parts of (b) are adapted with permission from Nano Letters 2018, 18 (7), 4309–4321. Copyright 2018 American Chemical Society. Some parts of (c) are used with permission from © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.