Nucleic Acid Therapeutics

Disease detection and management might benefit from external imaging of disease gene mRNAs. Previously we designed molecular imaging nanoparticles (MINs) based on peptide nucleic acids complementary to cancer gene mRNAs. The MINs included contrast agents and analogs of insulin-like growth factor 1 (IGF-1). Analysis of MIN tumor uptake data showed stronger binding in tumors than in surrounding tissues. We hypothesized that MINs with an IGF-1 analog stay in circulation by binding to IGF-binding proteins. To test that hypothesis, we fit the tissue distribution results of several MINs in xenograft-bearing mice to a physiological pharmacokinetics model. Fitting experimental tissue distribution data to model-predicted mass transfer of MINs from blood into organs and tumors converged only when the parameter for MINs bound to circulating IGF-binding proteins was set to 10%–20% of the injected MIN dose. This result suggests that previous mouse imaging trials used more MINs than necessary. This prediction can be tested by a ramp of decreasing doses.

The objective of this study was to investigate the transfection efficiency of chitosan hydroxybenzotriazole (CS-HOBT) for in vitro nucleic acid delivery. The results revealed that CS-HOBT was able to condense with DNA/small interfering double-stranded RNA molecules (siRNA). Illustrated by agarose gel electrophoresis, complete complexes of CS-HOBT/DNA were formed at a weight ratio of above 3, whereas those of CS-HOBT/siRNA were formed at a weight ratio of above 4 (CS molecular weights [MWs] 20 and 45 kDa) and above 2 (CS MWs 200 and 460 kDa). Gel electrophoresis results indicated that binding of CS-HOBT and DNA or siRNA depended on the MW and weight ratio. The particle sizes of CS-HOBT/nucleic acid complexes were in nanosize range. The highest transfection efficiency of CS-HOBT/DNA complex was found at a weight ratio of 2, with the lowest CS MW of 20 kDa. The CS-HOBT-mediated siRNA silencing of the enhanced green fluorescent protein gene occurred maximally with 60% efficiency. The CS-HOBT/siRNA complex with the lowest CS MW of 20 kDa at a weight ratio of 80 showed the strongest inhibition of gene expression. For cytotoxicity studies, over 80% the average cell viabilities of the complexes were observed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. This study suggests that CS-HOBT is straightforward to prepare, is safe, and exhibits significantly improved nucleic acid delivery potential in vitro.

Signaling pathways for caspase-2-mediated apoptosis are poorly defined. This is partially due to a lack of a reproducible stimulus to trigger caspase-2 activation. We present the oligonucleotide Dz13, a DNA enzyme that cleaves c-Jun mRNA and is capable of inhibiting various model tumors in mice, which potently induces caspase-2 resulting in apoptosis in a panel of tumor cell lines. Dz13-mediated cell death occurred even in the absence of known caspase-2 molecular partners in p53-induced protein with a death domain, RIP-associated Ich-1/CED homologous protein with death domain, or DNA-dependent protein kinase catalytic subunit, or other caspases in cell lines of breast cancer, prostate cancer, osteosarcoma, and liposarcoma. z-VDVAD-fmk, caspase-2^−/− mouse embryonic fibroblasts and siRNA silencing of caspase-2 in tumor cells abrogated Dz13-mediated cell death. In an orthotopic tumor model, expression of caspase-2 increased as the tumor metastasized and caspase-2 expression was sporadic in patient tumor specimens. These findings provide hope that Dz13, and other agents that evoke activation of caspase-2, may be therapeutic clinically.

Skin delivery of antisense oligonucleotides (AsODNs) has exciting potential in the treatment of skin diseases. However, the therapeutic applications of oligonucleotide-based therapies are limited by the instability of these molecules toward nucleases, short half-life in vivo, and insufficient cellular uptake. The purpose of this study was to investigate in vivo antisense effect of AsODN-loaded chitosan nanoparticles after topical application. AsODN-loaded chitosan nanoparticles were topically applied to Sprague Dawley rats (adult and baby). At 1, 3, 6, 9, and 12 days posttransfection, animals' skin samples were taken for measurement of β-galactosidase (β-Gal) expression and histological control. After topical application of AsODN-loaded chitosan nanoparticles in different doses, β-Gal expression reduced significantly. Highest inhibition was observed after 6 days of transfection of nanoparticles. Free AsODNs exhibited 35% of β-Gal inhibition on the first day. β-Gal expression was inhibited in approximately 82–85% with transfection of nanoparticles containing 30 μg AsODNs at 6 days. The antisense effect of AsODN-loaded chitosan nanoparticle in baby skin was evaluated at 6 days: 77–86% of β-Gal suppression was measured and differences between the doses were not significant. Thus, chitosan nanoparticles are useful carrier for delivery of AsODNs into skin cells of rats and may be used for topical application on human skin.

Mutations in the KRAS gene are required for early occurrence and maintenance of tumorigenesis and are the most frequently found in many types of human malignant diseases. Therefore, approaches targeting RAS function have been proposed for cancer therapy. However, no selective and specific inhibitors of KRAS have yet been developed as anticancer agents. In this study, by employing counter-systematic evolution of ligands by exponential enrichment technique, we identified and characterized an RNA aptamer that specifically bound to mutant KRAS protein with a point mutation in codon 12 of the KRAS gene. Real-time polymerase chain reaction analysis, surface plasmon resonance measurements, and competitive precipitation experiments showed that the selected aptamer contained activities of specific and high-affinity binding to the mutant KRAS (K_D ∼4.04 nM) but much less binding to the wild type (K_D ∼227 nM). This RNA aptamer could be useful as a ligand for specific therapeutics and diagnostics against mutant KRAS-mediated cancers.

Despite high specificity and potency, small interfering RNA (siRNA)-based therapeutics have been limited by their poor biostability and intracellular penetration. Thus, effective nanocarriers that can protect and efficiently deliver siRNA to target cells in vivo are needed. Here we report on the efficiency of imidazole-modified chitosan (chitosan-imidazole-4-acetic acid [IAA])-siRNA nanoparticles to mediate gene silencing after administration via either intravenous (i.v.) or intranasal (i.n.) routes. Poly(ethylene glycol) (PEG)ylated nanoparticles for i.v. delivery demonstrated significant knockdown of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) enzyme in both lung and liver at as low as 1 mg/kg siRNA dose. In addition, the efficient, dose-dependent silencing of apolipoprotein B in the liver was also shown. For i.n. delivery, significant silencing of GAPDH protein expression was seen in the lungs with only 0.5 mg/kg/day siRNA delivered over 3 consecutive days. In summary, imidazole-modified chitosan-IAA nanoparticles are potentially effective carriers for siRNA delivery.