Stable RNA nanoparticles as potential new generation drugs for cancer therapy

Abstract

Human genome sequencing revealed that only ~1.5% of the DNA sequence coded for proteins. More and more evidence has uncovered that a substantial part of the 98.5% so-called "junk" DNAs actually code for noncoding RNAs. Two milestones, chemical drugs and protein drugs, have already appeared in the history of drug development, and it is expected that the third milestone in drug development will be RNA drugs or drugs that target RNA. This review focuses on the development of RNA therapeutics for potential cancer treatment by applying RNA nanotechnology. A therapeutic RNA nanoparticle is unique in that its scaffold, ligand, and therapeutic component can all be composed of RNA. The special physicochemical properties lend to the delivery of siRNA, miRNA, ribozymes, or riboswitches; imaging using fluogenenic RNA; and targeting using RNA aptamers. With recent advances in solving the chemical, enzymatic, and thermodynamic stability issues, RNA nanoparticles have been found to be advantageous for in vivo applications due to their uniform nano-scale size, precise stoichiometry, polyvalent nature, low immunogenicity, low toxicity, and target specificity. In vivo animal studies have revealed that RNA nanoparticles can specifically target tumors with favorable pharmacokinetic and pharmacodynamic parameters without unwanted accumulation in normal organs. This review summarizes the key studies that have led to the detailed understanding of RNA nanoparticle formation as well as chemical and thermodynamic stability issue. The methods for RNA nanoparticle construction, and the current challenges in the clinical application of RNA nanotechnology, such as endosome trapping and production costs, are also discussed.

Keywords: Bacteriophage phi29; Biodistribution of nanoparticles; Cancer targeting; Nanobiotechnology; Pharmacokinetics; RNA nanotechnology; RNA therapeutics; Three-way junction; pRNA.

Fig. 1

Approaches to RNA nanotechnology. The construction of RNA nanoparticles starts from a conception design to define the desired properties of the nanoparticles. The RNA structure and folding of building blocks are then computed. After monomeric RNA building block synthesis, the RNA nanoparticles can be assembled following the designed conception. The resulting RNA nanoparticles can be characterized by gel electrophoresis, atomic force microscopy and electron microscopy. After thorough evaluation, the RNA nanoparticles can be used for various applications in vitro and in vivo [29]. This figure was adapted and modified from ref. [29] with permission.

Fig. 2

The bacteriophage phi29 DNA packaging RNA and three “toolkits” for construction of pRNA nanoparticles based on pRNA structural features. (A) pRNA hexamer ring showing the proof-of-concept of RNA nanotechnology in 1998 [36,212]. (B) The primary sequence and secondary structure of pRNA. The 3WJ scaffold domain connects the helical foot domain and central R- and L-hand domain [55]. (C) Three “toolkits” for pRNA nanoparticle construction. Left panel: Toolkit I: extended pRNA hand-in-hand interaction. Middle panel [39,55]: Toolkit II: foot-to-foot interaction. Right panel: Toolkit III: branch extension [31,32,55]. In this figure, (A) was adapted from ref. [36,212], (B) was adapted from ref. [55], (C) was adapted from ref. [31,32,39,55] with permission.

Fig. 3

Comparing RNA nanoparticles with other nano-delivery systems. (A) RNA nanoparticles [70]. (B) Lipid-based nanoparticles [105]. (C) Polymer-based nanoparticles, using dendrimer as an example [213]. (D) Viral nanoparticles [214]. (E) Inorganic nanoparticles [128]. (F) DNA nanoparticles (DNA polygon) [215]. In this figure, (D) was adapted and modified from ref. [214] with permission.

Fig. 4

Methods of labeling oligonucleotides. (A) The architecture of a label, which consists of three components: a small molecule moiety, a spacer and a reactive group. (B1) Reaction of a free amino group on an oligonucleotide with isothiocyanate. (B2) Reaction of a free amino group on an oligonucleotide with an N-hydroxysuccinimide ester. (B3) Click reaction of an azide-modified or alkyne-modified oligonucleotide with an alkyne-modified label and azide-modified label, respectively. (B4) Reaction of a thiol-modified oligonucleotide with maleimide. (B5) Reaction of a phosphoramidite derivative with oligonucleotides.

Fig. 5

Enhanced gene knock-down effects of pRNA-X nanoparticle carrying multiple copies of firefly luciferase siRNAs [32]. RLU: relative luciferase unit. This figure was adapted from ref. [32] with permission.

Fig. 6

Catalytic activity of pRNA nanoparticles harboring HBV ribozyme. (A) Illustration of assembled pRNA-3WJ nanoparticle harboring HBV ribozyme. (B) Assessing the catalytic activity of the HBV ribozyme incorporated into the pRNA-3WJ by denaturing PAGE [31]. This figure was adapted from ref. [31] with permission.

Fig. 7

Specific cell targeting effect of pRNA nanoparticles harboring anti-HIV gp120 aptamer. (A) Illustration of assembled pRNA nanoparticle harboring anti-HIV gp120 aptamer. (B) Cell binding assay of the RNA nanoparticles harboring gp120 aptamer in gp120 positive CHO cells [144]. Red: fluorescent pRNA nanoparticles. Blue represents nuclei. This figure was adapted from ref. [144] with permission. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

pRNA nanoparticle harboring fluorogenic aptamer can be used to monitor RNA folding and degradation profile in cells. (A) Illustration of assembled pRNA nanoparticle harboring MG binding aptamer and spinach aptamer. (B) MG binding aptamer and spinach aptamer retained their function independently after fusion into the pRNA-3WJ complex as shown in gel shift assay and fluorescence assay [75]. MG is the dye binding to MG aptamer, DFHBI is the dye binding to spinach aptamer. (C) The emission spectra of cell lysate acquired at various times for monitoring the pRNA nanoparticle harboring MG binding aptamer [76]. In this figure, (A), (B) were adapted from ref. [75], (C) was adapted from ref. [76] with permission.

Fig. 9

In vitro and in vivo tumor targeting of pRNA nanoparticles. (A) Confocal images showed targeting of folate receptor positive (FR+) KB cancer cells by the co-localization (overlap, 4) of cytoplasm (green, 1) and fluorescent pRNA-3WJ nanoparticles (red, 2) (Magnified, right panel). Blue represents nuclei. The pRNA-X (harboring Folate and Alexa-647) nanoparticles specifically targeted FR+ tumor xenografts upon systemic administration in nude mice, as revealed by whole body imaging (B) and internal organ imaging (C). Control: PBS treated mice. Scale bar: fluorescent intensity [32]. This figure was adapted from ref. [32] with permission. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 10

The packaging RNA (pRNA) nanoparticle did not induce an interferon response in vitro. (A) Interferon (IFN) responsive genes expressions after pRNA nanoparticle injection. (B) TLR-3, 7, and 9 gene expression analysis after pRNA nanoparticle injection. (C) Production of tumor necrosis factor-α (TNF-α) after incubation with different concentrations of pRNA nanoparticles. (D) Activation of Toll-like receptor (TLR)-3 pathway by pRNA nanoparticles differing in helical length (29 nt or 22 nt) and extent of modifications (FF, 2′-F modified helical region; NN, unmodified helical region; all pRNA constructs used had a modified intermolecular interaction domain). Poly I:C was used in all these assays as positive control [54]. This figure and legend were adapted from ref. [54] with permission.

Fig. 11

Ocular delivery of pRNA nanoparticles to cornea and retina following subconjunctival injection. (A) Whole body image and (B) confocal image showing internalization of RNA nanoparticles into retina cells after 4 h [190]. Red: fluorescent pRNA nanoparticles. Blue represents nuclei. This figure was adapted from ref. [190] with permission. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)