Development of Therapeutic siRNAs for Pachyonychia Congenita

Abstract

Pachyonychia congenita (PC) is an autosomal-dominant keratin disorder where the most painful, debilitating aspect is plantar keratoderma. PC is caused by mutations in one of four keratin genes; however, most patients carry K6a mutations. Knockout mouse studies suggest that ablation of one of the several K6 genes can be tolerated owing to compensatory expression of the others. Here, we have developed potent RNA interference against K6a as a paradigm for treating a localized dominant skin disorder. Four small interfering RNAs (siRNAs) were designed against unique sequences in the K6a 3'-untranslated region. We demonstrated near-complete ablation of endogenous K6a protein expression in two keratinocyte cell lines, HaCaT and NEB-1, by transient transfection of each of the four K6a siRNAs. The siRNAs were effective at very low, picomolar concentrations. One potent lead K6a inhibitor, which was highly specific for K6a, was tested in a mouse model where reporter gene constructs were injected intradermally into mouse paw and luciferase activity was used as an in vivo readout. Imaging in live mice using the Xenogen IVIS system demonstrated that the K6a-specific siRNA strongly inhibited bicistronic K6a-luciferase gene expression in vivo. These data suggest that siRNAs can specifically and very potently target mutated genes in the skin and support development of these inhibitors as potential therapeutics.

Designed guanidinium-rich amphipathic oligocarbonate molecular transporters complex, deliver and release siRNA in cells

“…Furthermore, the relative td-TOM knockdown observed for the various amphipathic cooligomers mirrored the flow cytometry results; 8b, which had a more moderate level of knockdown, exhibited some observable red fluorescence whereas 8a, 9a, and 9d, all co-oligomers which exhibit >80% tdTOM knockdown by flow cytometry, gave no detectable red fluorescence. Reflecting the potential generality of these findings, the ability of these co-oligomers to deliver a different siRNA for a different target protein [K6a siRNA (34) to target keratin 6a expression] was also confirmed by a reduction in target K6a mRNA levels in HaCaT cells as determined by quantitative reverse transcription PCR (SI Appendix, Fig. S4).…”

Increased interstitial pressure improves nucleic acid delivery to skin enabling a comparative analysis of constitutive promoters

“…administration 8,9,[36][37][38] possibly via a protein-mediated event. 36 We have previously used BLI to quantitatively assess plasmid uptake and expression in skin 37,[39][40][41] following i.d. injection of a plasmidencoding modified firefly luciferase as a reporter.…”

Therapeutic siRNAs for dominant genetic skin disorders including pachyonychia congenita

“…Alternatively, all possible siRNAs targeting the mutation site can be prepared (sequence walk) and tested, assuring that all possible effective siRNAs are identified. Both approaches have been used with success [14][15][16] and a combination of the two may be most effective, eliminating those sequences that are known to be ineffective or non-discriminating. Efficient and effective screening requires an assay that will quickly and accurately identify suitable candidates.…”