Nucleic acid nanoparticles (NANPs) as molecular tools to direct desirable and avoid undesirable immunological effects

Abstract

Nucleic acid nanoparticles (NANPs) represent a highly versatile molecular platform for the targeted delivery of various therapeutics. However, despite their promise, further clinical translation of this innovative technology can be hindered by immunological off-target effects. All human cells are equipped with an arsenal of receptors that recognize molecular patterns specific to foreign nucleic acids and understanding the rules that guide this recognition offer the key rationale for the development of therapeutic NANPs with tunable immune stimulation. Numerous recent studies have provided increasing evidence that in addition to NANPs' physicochemical properties and therapeutic effects, their interactions with cells of the immune system can be regulated through multiple independently programmable architectural parameters. The results further suggest that defined immunomodulation by NANPs can either support their immunoquiescent delivery or be used for conditional stimulation of beneficial immunological responses.

Keywords: Drug delivery; Immune responses; Immunostimulation; Immunotherapy; NANPs; PRR; RNA nanotechnology.

Figure 1:. Examples of mechanisms of TNA action.

For efficient intracellular delivery, some TNAs require a carrier with several of them exemplified in the upper panel. (A) Aptamers, composed of either DNA, RNA or their chemical analogs, function by binding a specific target molecule. As an example, Pegaptanib[14] (shown using RNAComposer[15, 16]) is schematically shown to bind to VEGF (PDB: 1VPF) for its inhibition to prevent downstream angiogenesis. (B) Delivery of mRNA into the cytoplasm is translated via the ribosome (PDB: 6Y0G) to yield a protein of interest. Spike protein from SARS-CoV-2 (PDB: 6VXX) is shown as a protein product example of mRNA vaccines. (C) For RNAi-induced gene silencing, either Dicer Substrate (DS) RNAs may be introduced for processing by Dicer, or siRNAs may be introduced exogenously. siRNAs are incorporated into the RNA-induced silencing complex (RISC) and guide strands direct sequence-specific mRNA cleavage. For illustration purposes, only the Ago2 component of RISC is shown (PDB: 6CBD). (D) ASOs bind to an endogenous mRNA sequence, where they may act as a steric hinderance for further splicing and translation, or may serve as a target for degradation by RNase H (PDB: 2QK9). (E) CRISPR Cas9 (PDB: 5F9R) utilizes a guide RNA sequence as a template to promote the double strand breakage of a gene. Repair mechanisms including homology directed repair (HDR) or non-homologous end joining (NHEJ) can be implemented for gene editing. Created with Biorender.com

Figure 2:. Design strategies and functionalization of NANPs.

(A) For the formation of cubes, intermolecular hydrogen bonds occur between six oligonucleotides. As these are canonical WC bps, the cubes may be composed of any combination of RNA and/or DNA. (B) For the formation of rings, intramolecular hydrogen bonding first occurs within each strand, exposing single-stranded regions (RNA kissing loop motifs) which can then interact intermolecularly. (C) By extending the sequences in their compositions, NANPs can be functionalized with Dicer Substrate (DS) RNAs which can then enter the RNA interference (RNAi) pathway. Due to their hexameric nature, up to six DS RNAs can be added to each ring for simultaneous knockdown of six different target genes. Cryo-EM (from ref. [119]) demonstrates the structure of the functional RNA rings. (D) Functional NANPs must be combined with a delivery carrier for their transfection into cells, where they may then be processed by Dicer to begin RNAi. Created with Biorender.com.

Figure 3:. Non-functional RNA/DNA hybrid NANPs can be used for the coordinated activation of RNAi and NF-κB decoys.

Re-association of hybrid fibers in the cytosol yields two products. First, DS RNAs that are cleaved by Dicer produce functional siRNAs for the silencing of target genes. Second, synthetic dsDNA decoys that readily bind to NF-κB prevent nuclear translocation and activation of NF-κB-induced cytokines. PDBs: 1NFK and 1NFI. Created with Biorender.com

Figure 4:. QSAR modeling employs a set of NANPs to predict pro-inflammatory immune responses.

A panel of representative DNA, RNA, and DNA/RNA NANPs was designed with varying descriptors such as molecular weight, melting temperature, size, and stability. A training set composed of 80% of this batch was used for machine learning, where descriptors were matched against outcomes of experimentally found immunostimulations. A validation set was then used to confirm the predicted trends and validate the model. Created with Biorender.com

Figure 5:. PRRs are localized to specific subcellular compartments to screen for PAMPs and DAMPs.

The endosomal and cytosolic sensors display composition, sequence, length, and structure-dependent recognition of nucleic acid ligands. The same ligand preferences determine binding to NANPs. The figure highlights key features of NANPs that meet the necessary ligand characteristics and have been experimentally confirmed to activate nucleic acid PRRs (A) with trends in relative responses in IFN productions across some representative categories of NANPs (B). Created with Biorender.com