The immunorecognition, subcellular compartmentalization, and physicochemical properties of nucleic acid nanoparticles can be controlled by composition modification

Abstract

Nucleic acid nanoparticles (NANPs) have become powerful new platforms as therapeutic and diagnostic tools due to the innate biological ability of nucleic acids to identify target molecules or silence genes involved in disease pathways. However, the clinical application of NANPs has been limited by factors such as chemical instability, inefficient intracellular delivery, and the triggering of detrimental inflammatory responses following innate immune recognition of nucleic acids. Here, we have studied the effects of altering the chemical composition of a circumscribed panel of NANPs that share the same connectivity, shape, size, charge and sequences. We show that replacing RNA strands with either DNA or chemical analogs increases the enzymatic and thermodynamic stability of NANPs. Furthermore, we have found that such composition changes affect delivery efficiency and determine subcellular localization, effects that could permit the targeted delivery of NANP-based therapeutics and diagnostics. Importantly, we have determined that altering NANP composition can dictate the degree and mechanisms by which cell immune responses are initiated. While RNA NANPs trigger both TLR7 and RIG-I mediated cytokine and interferon production, DNA NANPs stimulate minimal immune activation. Importantly, incorporation of 2′F modifications abrogates RNA NANP activation of TLR7 but permits RIG-I dependent immune responses. Furthermore, 2′F modifications of DNA NANPs significantly enhances RIG-I mediated production of both proinflammatory cytokines and interferons. Collectively this indicates that off-target effects may be reduced and/or desirable immune responses evoked based upon NANPs modifications. Together, our studies show that NANP composition provides a simple way of controlling the immunostimulatory potential, and physicochemical and delivery characteristics, of such platforms.

Induction of Cytokines by Nucleic Acid Nanoparticles (NANPs) Depends on the Type of Delivery Carrier

“…The field of RNA and DNA nanotechnology is rapidly growing. In the past decade, researchers have established various approaches to synthesize RNA and DNA nanoassemblies of different sizes, shapes, and compositions and generated proof-of-concept data intended for the use of these materials in biology and medicine [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 ]. A growing library of nucleic acid nanoparticles (NANPs), the design of which takes advantage of natural RNA (and DNA) motifs and canonical Watson–Crick base pairings, have been demonstrated to assemble into precise nanoscaffolds exemplified by hexagonal rings [ 12 ], various polygons [ 13 ], and fibrous structures [ 14 ], to name a few [ 15 ].…”

In Vivo Production of RNA Aptamers and Nanoparticles: Problems and Prospects

“…The integration of aptamers with RNA nanoparticles allows the targeting of a number of molecules, particularly cellular receptors, and aids in the active penetration of nanoparticles into cells through receptor-mediated endocytosis, or the passive penetration through the plasma membrane [ 140 , 141 , 142 ]. Lin et al have extensively reviewed various types of RNA nanoparticles used for the treatment of cancer and immunomodulation [ 143 ].…”

The Recognition of and Reactions to Nucleic Acid Nanoparticles by Human Immune Cells

“…The modular functionalization of NANPs with aptamers, antibodies, or small molecules for their targeted delivery allows NANPs to integrate and deliver various TNAs into cells for synergistic therapeutic effects. However, despite these developments, NANPs have yet to advance to clinical translation due to concerns that need to be investigated and resolved including their specific delivery to target cells, their enzymatic degradation, and their ability to induce an immune response upon cellular uptake [ 4 , 12 , 14 , 15 ]. While targeting and stability are not immediate life-threatening issues, the excessive immune recognition of NANPs and overreaction by immune cells can have potentially deleterious effects.…”

“…While targeting and stability are not immediate life-threatening issues, the excessive immune recognition of NANPs and overreaction by immune cells can have potentially deleterious effects. Thereby, the immunostimulatory properties of NANPs are being extensively investigated [ 14 , 16 , 17 ].…”

“…Several physicochemical properties of NANPs determine their recognition by the immune cells; the most notable properties are 3D structure, composition (RNA to DNA ratio), molecular size, and the NANP’s sequence. In addition, the immune response could be modulated by the type of delivery carriers used [ 6 , 14 , 18 , 19 , 20 , 21 , 22 ]. The proper design of NANPs with respect to immunostimulatory properties has the potential to activate innate and adaptive immune responses by activating nucleic acid immune sensors, thus having high potential as vaccine adjuvants and pan-antivirals [ 2 , 14 , 20 , 21 , 23 , 24 , 25 ].…”