Nucleic Acid Armor: Fortifying RNA Therapeutics through Delivery and Targeting Innovations for Immunotherapy

Abstract

RNA is a promising nucleic acid-based biomolecule for various treatments because of its high efficacy, low toxicity, and the tremendous availability of targeting sequences. Nevertheless, RNA shows instability and has a short half-life in physiological environments such as the bloodstream in the presence of RNAase. Therefore, developing reliable delivery strategies is important for targeting disease sites and maximizing the therapeutic effect of RNA drugs, particularly in the field of immunotherapy. In this mini-review, we highlight two major approaches: (1) delivery vehicles and (2) chemical modifications. Recent advances in delivery vehicles employ nanotechnologies such as lipid-based nanoparticles, viral vectors, and inorganic nanocarriers to precisely target specific cell types to facilitate RNA cellular entry. On the other hand, chemical modification utilizes the alteration of RNA structures via the addition of covalent bonds such as N-acetylgalactosamine or antibodies (antibody-oligonucleotide conjugates) to target specific receptors of cells. The pros and cons of these technologies are enlisted in this review. We aim to review nucleic acid drugs, their delivery systems, targeting strategies, and related chemical modifications. Finally, we express our perspective on the potential combination of RNA-based click chemistry with adoptive cell therapy (e.g., B cells or T cells) to address the issues of short duration and short half-life associated with antibody-oligonucleotide conjugate drugs.

Keywords: RNA drugs; antibody–oligonucleotide conjugates; immunotherapy; nanotechnology; nucleic acid delivery.

Figure 6

Non-viral and lipid nanomaterials for RNA delivery. (A) Polyethylenimine-modified Porous Silica Nanoparticle (L-PPSN) for the delivery of engineered IL-2 for antitumor immunity. Figure is adapted from [63]. (B) A biodegradable polymeric nanocarrier that can deliver in vitro transcribed mRNA encoding disease-specific chimeric antigen receptors (CARs) or T cell receptors (TCRs) directly to circulating T cells in vivo. Figure is adapted from [66].

Figure 7

The preparation and intracellular mechanism of action of ADC drugs and antibody–oligonucleotide conjugate (AOC) drugs.

Figure 8

Recent clinical studies of antibody–oligonucleotide conjugates for immunotherapy. (A) Evaluation of the activity of GalNAc–siRNA conjugates in pre-clinical animal models with reduced asialoglycoprotein receptor expression. Figure is adapted from [76]. (B) Evaluation of mannose–siRNA and GalNAc–siRNA conjugates for hepatocyte infection. Figure is adapted from [78]. (C) Development of antibody–oligonucleotide conjugates for targeted delivery of RNA therapeutics to skeletal and cardiac muscle. Figure is adapted from [82].

Figure 1

The timeline for RNA-based therapeutic development for various diseases and immunotherapy.

Figure 2

Preparation method of delivery vehicles. (A) Workflow of conventional and novel approaches for LNP preparation. Figure is adapted from [22]. (B) Overview of adeno-associated virus (AAV) production/purification. Figure is adapted from [23]. (C) Schematic diagram showing the preparation of mesoporous silica nanoparticles (MSNs). Figure is adapted from [24].

Figure 3

Lipid and liposome-based vehicles for immunotherapy. (A) Lipid nanoparticles (LNPs) for promoting immunogenic cancer cell death (ICD), delivery of RNA to stimulate danger sensors in transfected cells, and delivery of RNA-encoded interleukin (IL)-12 fusion protein production in cancer cells for boosting immune cells’ antitumor activities. Figure is adapted from [45]. (B) A nanobooster targeting programmed cell death protein 1 (PD-L1) and inhibiting heme oxygenase-1 (HO1) (siRNA) for cancer chemo-immunotherapy. Figure is adapted from [46]. Scale bars indicate a 100 nm scale.

Figure 4

Schematic illustration of various RNA delivery approaches with their cellular entry pathway mechanisms.

Figure 5

Virus-based RNA vectors for immunotherapy. (A) High-throughput in vivo selection to engineer new adeno-associated virus vectors specifically designed for local neuronal transduction at the site of focused ultrasound blood–brain barrier opening (FUS-BBBO); (B) visualization of the copy number in each hemisphere. Yellow dots represent 5 clones (AAV.FUS.1−5) selected for low-throughput testing.; (C) all AAV.FUS candidates had higher neuronal tropism. Black dots represent data from 3 male and 3 female mice for corresponding serotypes. Image AAV9 represents the transduce both neurons (blue, NeuN staining, example neurons designated by an arrow) and non-neuronal cells (example non-neuronal cells designated by an arrowhead). Image AAV.FUS.3 represents more of the cells transduced with AAV.FUS (green) are neurons (example neurons designated by an arrow), rather than non-neuronal cells (example cell designated by an arrowhead). Figures are adapted from [55]. The scale bars are 50 μm. (**** p < 0.0001, ANOVA) (D) Heterologous prime-boost vaccination, using the chimpanzee adenovirus ChAd68 followed by self-amplifying RNA (samRNA) to induce broad and long-lasting CD8⁺ T cell responses in non-human primates. Figure is adapted from [56].