Progress in microRNA delivery

Abstract

MicroRNAs (miRNAs) are non-coding endogenous RNAs that direct post-transcriptional regulation of gene expression by several mechanisms. Activity is primarily through binding to the 3' untranslated regions (UTRs) of messenger RNAs (mRNA) resulting in degradation and translation repression. Unlike other small-RNAs, miRNAs do not require perfect base pairing, and thus, can regulate a network of broad, yet specific, genes. Although we have only just begun to gain insights into the full range of biologic functions of miRNA, their involvement in the onset and progression of disease has generated significant interest for therapeutic development. Mounting evidence suggests that miRNA-based therapies, either restoring or repressing miRNAs expression and activity, hold great promise. However, despite the early promise and exciting potential, critical hurdles often involving delivery of miRNA-targeting agents remain to be overcome before transition to clinical applications. Limitations that may be overcome by delivery include, but are not limited to, poor in vivo stability, inappropriate biodistribution, disruption and saturation of endogenous RNA machinery, and untoward side effects. Both viral vectors and nonviral delivery systems can be developed to circumvent these challenges. Viral vectors are efficient delivery agents but toxicity and immunogenicity limit their clinical usage. Herein, we review the recent advances in the mechanisms and strategies of nonviral miRNA delivery systems and provide a perspective on the future of miRNA-based therapeutics.

Keywords: 1,2-Di-O-octadecenyl-3-trimethylammonium propane; 2′-MOE; 2′-Me; 2′-methoxyethyl; 2′-methyl; AMO; APP; BBB; CNS; CSC; DDAB; DGCR8; DNA; DOTMA; DiGeorge syndrome critical region gene; FANA; GC4; HCV; Human disease; IR; LAC; LNA; LPH; MicroRNA; NLE; NSCLC; Nonviral delivery; ODN; PEI; PNA; PU; RISC; RNA; RNA induced silencing complex: scFv, single-chain variable fragment; Small RNA delivery; TEPA-PCL; TPGS; UTR; VEGF; amyloid precursor protein; anti-miRNA oligonucleotides; base pairs; blood–brain barrier; bp; cancer stem cell; central nervous system; d-alpha-tocopheryl polyethylene glycol 1000 succinate; deoxyribonucleic acid; dicetyl phosphate-tetraethylenepentamine-based polycation liposomes; dimethyldioctadecylammonium bromide; fluorine derivatives nucleic acid; hepatitis C virus; iNOP; ionizing radiation; liposome-hyaluronic acid; locked nucleic acid; lung adenocarcinoma; miRNA; miRNA therapeutics; microRNA; nanotransporter interfering nanoparticle-7; neutral lipid emulsion; non-small cell lung cancer; nt; nucleotides; oligodeoxynucleotides; peptide nucleic acids; phage identified internalizing scFvs that target tumor sphere cells; polyethyleneimine; polyurethane; ribonucleic acid; siRNA; small interfering RNA; untranslated regions; vascular endothelial growth factor.

Figure 1. Schematic representation of the biogenesis of miRNA

From genomic DNA, RNA is transcribed either as (1) an independent miRNA sequence or (2) an intron that is removed from mRNA. Following loop formation, the pri-miRNA is processed by DROSHA into pre-miRNA. The pre-miRNA is exported from the nucleus by Exportin5 before maturation by Dicer. Upon loading into the RISC complex, the miRNA duplex is unwound and the mature strand (red strand) retained within the miRISC complex. The passenger strand (green strand) is degraded.

Figure 2. Mechanisms of natural miRNA-mRNA action

The roles of miRNA in protein production have been proposed to be through many routes. The majority of the actions inhibit protein production, but several modes of promotion of protein production have also been proposed. Binding of miRNA to DNA may promote transcription directly or through the recruitment of other factors. In addition, translation can be promoted by 5’UTR binding. Inhibitory action is likely through a combination of the mechanisms shown which include (1) direct mRNA degradation, (2) deadenylation, (3) initiation repression, (4) ribosomal stalling, (5) ribosomal drop-off, (6) co-translational degradation, and (7) translation repression following DNA binding.

Figure 3. Chemistry and structure of miRNA therapeutic molecules

RNA molecules (RNA) has several bonds that have been modified for stability including the alpha oxygen of phosphate (red), 4’ hydrogen (green), 2’ hydroxide (orange), 4’ hydrogen (purple), and the 1’ amide linkage to the base (blue). Peptide nucleic acids (PNAs) substitute a peptide bond at the 1’ amide linkage to the base. Phosphorothioates, methylphosphonates, and boranophosphates substitute a sulfur, methyl, and a borano group, respectively, for the alpha-oxygen of the phosphate. Locked nucleic acids (LNAs) add a secondary linkage between the 4’ carbon and the 2’ hydroxide while 2’-O-(2-methoxyethyl)-(2’-O-MOE), 2'-O-methyl-(2’-O-Me), and 2’-fluoro-(2’-F) RNAs substitutes less reactive groups for the 2’ hydroxyl group.

Figure 4. Therapeutic Strategies for miRNA activity

Delivered DNA or RNA (green) can act to augment, mimic miRNA activity, or prevent activity of miRNA. MicroRNA can be produced by plasmid DNA (miRNA gene therapy) that is introduced into the cell, or by introducing agents to increase by translation promoters or cofactors, or delivered directly (in the forms of pri−, pre−, or mature with or without passenger), i.e. miRNA mimic. To prevent the activity of expressed miRNAs, masks that compete with the binding site on mRNA inhibit miRNA binding with mRNA. In addition, binding of miRNA with the RISC complex may be inhibited with a sponge or a single miRNA binding anti-miRNA. Finally, translation may be directly repressed at the DNA level.