Novel therapies for naevi and treatment-resistant melanoma

Summary This research proposes to develop new therapies for both a rare untreatable birthmark disease CMN syndrome, and a common cancer of adults, sporadic melanoma, as both have the same driver mutations. These treatments are based on exquisite allele-specific and cellular targeting.

This novel approach will bring therapies for the first time to children with CMN syndrome, and be a potentially radical addition to melanoma management.

Furthermore, the methods used will be adaptable to other genetic skin diseases and cancers, making this research not only a true paradigm shift in treatment of two diseases, but a valuable investment across Dermatology.

Two disease targets - CMN syndrome and sporadic melanoma Congenital melanocytic naevus (CMN) syndrome is an untreatable rare disease of very large moles, associated neurological abnormalities, and predisposition to melanoma.

We identified previously that 75% of CMN syndrome is caused by the same NRAS and BRAF mutations as drive most sporadic melanoma, but arising in utero.

CMN syndrome therefore also constitutes a pure genetic model of melanoma driver mutations, without the confounding influence of ultraviolet radiation and other 'passenger' variants.

Melanoma is the 5th most common cancer in the UK, with an increasing incidence and despite recent therapeutic advances, a high mortality from metastatic disease. Novel therapeutic approaches to CMN syndrome and melanoma are therefore urgently required.

Preliminary data I made a clinical observation that some children with CMN syndrome have spontaneous partial phenotypic correction. Skin biopsies demonstrated striking silencing of mutation expression in the revertant patches.

Modelling of this phenomenon in melanoma cell lines using allele-specific targeting by CRISPR/Cas9 and silencing by siRNA has recapitulated this natural gene therapy, with downstream correction of MAPK pathway overactivation. These exciting data have prompted this programme of research with a view to genetic therapy for CMN and melanoma.

Research plan Efficiency and allele-specificity will be optimised firstly in cancer cell lines, using allele-specific CRISPR-Cas9 with homology-directed repair from a donor template, and allele-specific siRNA, with multiple contingency plans in place.

Success will be measured by direct sequencing, downstream effector pathway signalling correction, cell morphology, and proliferation/cell cycle assays.

All results will be validated in immortalised CMN patient cell lines, in which on- and off-target effects will be fully characterised using whole genome DNA and RNA sequencing. Two topical genetic therapy delivery systems will be developed in collaboration with leaders in this field. The first is iExosomes (for siRNA), currently in clinical trials for pancreatic cancer.

The second is self-assembling receptor-targeted nanoparticles (for CRISPR or siRNA), highly attractive as they add another layer of targeting at cellular level. Stability of therapy and delivery systems in existing topical vehicles will be tested systematically.

Targeted therapies packaged into delivery systems will then be tested on human skin explants, considered the gold standard for topical therapies.

Readouts will be as for cell models, plus single cell RNA sequencing pre- and post- treatment for on- and off-target effects.

Under a parallel grant, safety data will be collected from existing murine models of CMN, necessary for clinical trials.