851 RNA therapy for congenital melanocytic naevi

Congenital melanocytic naevi (CMN) are moles present at birth, with severe cases associated with life-limiting neurological abnormalities and melanoma. There is an urgent need for treatments. In 2013 we established that the commonest cause of CMN is a mosaic heterozygous oncogenic mutation in NRAS, most commonly c.181C>A p.(Q61K). The aim of this work was to design precision genetic therapies for CMN and to test them in appropriate cellular and animal models. We designed and optimized siRNAs to discriminate between mutant NRASc.181C>A and wildtype (WT) human mRNA transcripts, selecting the lead candidate (anti-NRASQ61K) on the basis of allelic discrimination and fewest off-target effects on RNA-seq. Primary naevus cell culture from 9 patients allowed extensive characterization pre- and post-treatment with anti-NRASQ61K. Treatment led to significant knockdown of the mutant allele with preservation of the WT, normalization of MAPK pathway activation, a reduction in dividing cell fraction, and resultant rescue of baseline proliferation. Design and assembly of receptor-targeted lipid nanoparticles (RTNPs) demonstrated protection of siRNA from degradation. Intradermal delivery of anti-NRASQ61K-RTNPs (CMN nanomedicine) into patient skin explants confirmed successful targeting of therapy to naevus cells. Transgenic mice harboring humanized NRASQ61K(Tyr::NRASQ61K) were then treated with intradermal injection of the CMN nanomedicine, and biopsied to confirm intradermal delivery and assess response. Knockdown of the mutant NRAS allele with preservation of the murine endogenous NRAS allele was demonstrated at RNA level, clearly establishing proof of concept of in vivoprecision medicine targeting for this condition. This work paves the way for clinical trials of the first genetic therapy for CMN, and potentially for other NRAS-driven melanocytic diseases.