Efficacy of a Novel Class of RNA Interference Therapeutic Agents

<div><p>RNA interference (RNAi) is being widely used in functional gene research and is an important tool for drug discovery. However, canonical double-stranded short interfering RNAs are unstable and induce undesirable adverse effects, and thus there is no currently RNAi-based therapy in the clinic. We have developed a novel class of RNAi agents, and evaluated their effectiveness <em>in vitro</em> and in mouse models of acute lung injury (ALI) and pulmonary fibrosis. The novel class of RNAi agents (nkRNA®, PnkRNA™) were synthesized on solid phase as single-stranded RNAs that, following synthesis, self-anneal into a unique helical structure containing a central stem and two loops. They are resistant to degradation and suppress their target genes. nkRNA and PnkRNA directed against TGF-β1mRNA ameliorate outcomes and induce no off-target effects in three animal models of lung disease. The results of this study support the pathological relevance of TGF-β1 in lung diseases, and suggest the potential usefulness of these novel RNAi agents for therapeutic application.</p> </div

Stability of nkRNA, PnkRNA and siRNA directed to mouse TGF-β1.

<p>For evaluating <i>in vitro</i> stability, each RNA was incubated at 37°C in the presence of S7 nuclease (A, B) and then separated on polyacrylamide gel electrophoresis (PAGE). Both nkRNA and PnkRNA were more resistant to degradation than siRNA. Stability of the therapeutic agents in bronchoalveolar lavage fluid (BALF) from mice was also evaluated; each RNA was added to 50 µl of BALF, incubated at 37°C and then separated on PAGE (C). siRNA was completely degraded but nkRNA and PnkRNA remained intact after 10 min (C). -; indicates lane loaded with RNA that was not added to BALF.</p

TGF-β1 nkRNA decreases target expression but had no effect on interferon (IFN) expression in acute lung injury.

<p>The mRNA (A, B) and protein (C) expression of TGF-β1 were significantly decreased in LPS-instilled mice treated with TGF-β1 nkRNA compared to those treated with vehicle or control RNA. The concentration of IFN-α (D) and IFN-β (E) in BALF was not affected by the treatment. Statistical analysis by ANOVA. Data are expressed as the mean ± s.e.m. *p<0.05 vs controls. ‡p<0.05 vs untreated group.</p

Position of deleted nucleotides affects the inhibitory activity of nkRNA.

<p>Numbering of nkRNA is shown in (A). Inhibition of human GAPDH mRNA expression in HCT 116 cells by GAPDH nkRNAs (10 nM) with deleted nucleotides at different positions; nkRNAs with deleted nucleotides near the right or left loop showed increased inhibitory activity (B). The <i>Tm</i> values of the GAPDH nkRNAs with deleted nucleotides at different positions were measured (C); comparison between (B) and (C) shows that the inhibitory activity of GAPDH nkRNAs is proportionally correlated with their <i>Tm</i> values. Comparative <i>Tm</i> values of siRNA, nkRNA and PnkRNA (D). *p<0.05 vs. control groups. Closed squares indicate samples diluted in phosphate-buffered saline; open circles indicate samples diluted in phosphate buffer.</p

Inhibition of human GAPDH expression by novel RNAi agents.

<p>siRNA, nkRNA and PnkRNA directed against human (h)GAPDH significantly inhibited GAPDH gene expression in a dose-dependent fashion in A549 (A), HCT116 (B) and HEK 293 (C) cell lines. Statistical analysis by ANOVA. Data are expressed as the mean ± s.e.m. *p<0.0001 vs. control groups.</p

Sequence of PnkRNA, nkRNA and siRNA directed against human GAPDH mRNA.

<p>Sequence of PnkRNA, nkRNA and siRNA directed against human GAPDH mRNA.</p

hTGF-β1 nkRNA and PnkRNA inhibit acute lung injury in human TGF-β1 TG mice.

<p>Inflammatory cells were conspicuous in BALF from vehicle/LPS and control RNA/LPS groups compared to vehicle/SAL group and mice treated with each RNAi therapeutic agent (A). Histological findings of the lung showed increased infiltration of inflammatory cells (arrows), alveolar thickening (arrow heads) and edema in vehicle/LPS and control RNA/LPS groups compared to the vehicle/SAL group and mice treated with each RNAi agent (B). The BALF total cell count was significantly decreased by nkRNA and PnkRNA and the number of neutrophils by siRNA, nkRNA and PnkRNA compared to vehicle/LPS mice (C, D). The BALF concentration of human TGF-β1 was significantly reduced by each nucleic acid agent compared to untreated mice (E). The scale bars indicate 50 µm. Statistical analysis by ANOVA. Data are expressed as the mean ± s.e.m. *p<0.05 vs controls. ‡p<0.05 vs untreated group.</p

Inhibitory activity of siRNA, nkRNA dn -2 and PnkRNA dn -2 directed against human TGF-β1.

<p>The in vitro inhibitory activity of siRNA, nkRNA dn -2 and PnkRNA dn -2 against TGF-β1 was significant compared to controls in A549 cells. Statistical analysis by ANOVA. Data are expressed as the mean ± s.e.m. *p<0.05 vs controls.</p

Structure of novel RNAi therapeutic agents.

<p>Both nkRNA (A) and PnkRNA (B) were prepared as single-stranded RNA oligomers that then self-anneal as shown. Nucleotides in red indicate sense strand of the target (GAPDH), nucleotides in violet are the antisense strand and nucleotides in blue are the loop cassettes; P indicates a proline derivative.</p

Synthesis of proline diamide amidite.

<p>Compounds 1, 2, 3 and 4 were sequentially prepared to synthesize proline diamide amidite (compound 5). (i) 4-Aminobutanol/N,N′-dicyclohexylcarbodiimide/1-hydroxybenzotriazole; (ii) 4,4′-dimethoxytrityl chloride/pyridine; (iii) piperidine/N,N-dimethylformamide; (iv) 6-Hydroxyhexanoic acid/1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/1-hydroxybenzotriazole/triethylamine; (v) 2-cyanoethyl N,N,N′,N″0tetraisopropylphosphordiamidite/diisopropylammonium tetrazolide.</p