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. 2012 May 7;9(5):1481-8.
doi: 10.1021/mp300081s. Epub 2012 Apr 18.

Polymer nanoparticle-mediated delivery of microRNA inhibition and alternative splicing

Christopher J Cheng  1 , W Mark Saltzman
Affiliations

Polymer nanoparticle-mediated delivery of microRNA inhibition and alternative splicing

Christopher J Cheng et al. Mol Pharm. .

Abstract

The crux of current RNA-based therapeutics relies on association of synthetic nucleic acids with cellular RNA targets. Antisense oligonucleotide binding to mature microRNA and splicing junctions on pre-mRNA represent methods of gene therapy that respectively inhibit microRNA-mediated gene regulation and induce alternative splicing. We have developed biodegradable polymer nanoparticles, which are coated with cell-penetrating peptides, that can effectively deliver chemically modified oligonucleotide analogues to achieve these forms of gene regulation. We found that this nanoparticle system could block the activity of the oncogenic microRNA, miR-155, as well as modulate splicing to attenuate the expression of the proto-oncogene, Mcl-1. Regulation of these genes in human cancer cells reduced cell viability and produced pro-apoptotic effects. These findings establish polymer nanoparticles as delivery vectors for nonconventional forms of gene therapy activated by cellular delivery of RNA-targeted molecules, which have strong therapeutic implications.

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Conflict of interest statement

Conflict of Interest

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Workflow schematic of alternative splicing and miRNA inhibition induced by nona-arginine-coated nanoparticles (ARG-NPs) that deliver charge-neutral oligonucleotide analogs. ARG-NPs comprise a spherical PLGA core coated with PEGylated ARG. Dehydrated ARG-NPs were visualized by scanning electron microscopy (SEM), intracellular uptake of osmium tetroxide-loaded ARG-NPs was visualized by transmission electron microscopy (TEM); scale bars for SEM and TEM micrographs represent 1 µm.
Figure 2
Figure 2
Comparison of Coumarin 6-labeled NPs coated with ANTP, ARG, and TAT; unmodified NPs and PEGylated NPs were used as controls. CPP density (~3, 8, and 15 ug of peptide per mg of NP) on the surface of NPs increases from left-to-right for individual groups. Cellular association and internalization was monitored using flow cytometry and Trypan Blue quenching of extracellular fluorescence. Shown is the mean channel (FL-1) fluorescence, n=3; statistical analysis was performed with respect to unmodified nanoparticles for either the associated or internalized groups, ***p < 0.001; **p < 0.01; *p < 0.05.
Figure 3
Figure 3
(A) Controlled release profiles of nucleic acids from uncoated NPs. The inset depicts initial burst release; axes are the same as the parental figure. Cation refers to the positively-charged counter-ion, spermidine, which was used to enhance loading of anionic ssDNA. (B) Antisense translation-based inhibition of in vitro expression of luciferase. Each group comprised the total amount of PMOs released from NPs over the indicated time interval. ***p < 0.0005; *p < 0.05.
Figure 4
Figure 4
(A) miR-155 dual luciferase sensor demonstrating the inhibition of miR-155 activity in KB cells by anti-miRs (PMOs or PNAs as indicated) that were delivered by ARG-NPs. Renilla luciferase was normalized to Firefly luciferase signal. ***p < 0.0001. (B) Relative levels of miR-155 in KB cells treated with PMO anti-miRs loaded in NPs. ***p < 0.0001.
Figure 5
Figure 5
(A) Confocal microscopy of cellular localization of ARG-NPs delivering FITC-labeled PMOs. Green = PMO; red = actin; blue = nucleus; scale bar represents 25 µm (B) Electrophoretic fractionation of Mcl-1 splicing isoforms, Mcl-1L and Mcl-1S. Both isoforms were amplified from the same set of primers; Mcl-S (757 bp) is a truncation of Mcl-1L (1005 bp). β-actin was used as a loading control.
Figure 6
Figure 6
(A) Dose response of KB cells to treatment with anti-mir-155 or Mcl-1 splice-shifting PMOs. Data presented as percent survival relative to untreated cells. 2-way ANOVA was used for statistical analysis relative to ARG-NP anti-CRL group. ***p < 0.001; **p < 0.01; *p < 0.05. (B) Extent of apoptotic DNA fragmentation measured by quantifying cytosolic mono- and oligonucleosomes in cells treated with anti-mir-155 or Mcl-1 splice-shifting PMOs. Data presented as fold-change relative to untreated cells. **p < 0.005.
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