Title: Novel therapies for naevi and treatment-resistant melanoma
Funder: Department of Health and Social Care

Abstract: 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.

Lay abstract: Summary This research proposes to develop new treatments for a rare untreatable birthmark disease of children, and a common cancer of adults, as both have the same underlying causes. These treatments are based on exquisite targeting of the causes, made possible by recent advances in genetics. This approach will bring therapies for the first time to children with the rare disease, and will add a novel attack on the common cancer. Furthermore, the methods used will be applicable and adaptable to other rare skin diseases and other common skin cancers, making this programme of research not only a true paradigm shift in treatment of two diseases, but a valuable investment across Dermatology. The two diseases needing treatment - CMN syndrome and melanoma Congenital melanocytic naevus (CMN) syndrome is a rare disease where children are born with very large, very visible moles covering much of the body. A fifth of patients also have moles in the brain/spine which lead to increased pressure in the head, developmental delay and seizures, and all have a predisposition to the fatal skin cancer melanoma. There are no current treatments for CMN. Our group identified previously that CMN syndrome is caused by one of two faulty genes in 75% of cases, arising by chance in the womb, and these are the same faulty genes that drive most of adult melanoma. Studying CMN is therefore also a very valuable way to study treatments for melanoma. Melanoma is the 5th most common cancer in the UK, and although there have been recent improvements in treatments, death rates from late stage melanoma are still very high. Novel treatment approaches to CMN and melanoma are therefore urgently required. Background clinical observations, and preliminary researchThis research proposal was triggered by the striking clinical observation that some areas of CMN in some children were getting better spontaneously (Figure 1).Skin samples demonstrated that the body was spontaneously silencing the faulty gene in the improving areas. We have therefore copied this concept using cells grown in the laboratory, and have managed to demonstrate that it works. This forms the basis of the potential novel targeted therapies for CMN and melanoma. Research planThis new proposal builds on this by using two different approaches to target the faulty genes. These will be refined using our cell models until we are confident they are as robust, effective and targeted as possible. Then we will extend the testing to small whole CMN skin samples (sometimes small parts are removed surgically), and to normal skin which would otherwise be discarded from other operations.A broad spectrum of tests will check whether the treatments are effective and safe in the lab, leading to refinement and retesting as necessary.In parallel, we will develop two systems to deliver these treatments straight onto the skin (topical). This will be done by two different methods in collaboration with the leaders in these fields, to maximise the chances of success. Simultaneously, via a different grant, safety data for the treatments will be collected from an existing animal model of CMN, which will be necessary for preparing for clinical trials. Patient group involvementI look after the largest group of children with CMN worldwide, who have advised on these research priorities, helped design patient information leaflets, and will participate in our studies, coordinated by the dynamic UK support group Caring Matters Now.This close partnership between patients and clinicians will also allow direct translation of findings from this research back into clinical trials. Translational outcomes Clinical trials of at least one treatment are planned for the end of the five-year programme.

Research activity codes: 6.2 CELLULAR AND GENE THERAPIES
Health categories: CANCER AND NEOPLASMS