• CRISPR-Cas9
• gold nanoparticles
• melanoma
• off-target

Melanoma is a cancer resulting from the malignant transformation of melanocytes into cancerous cells. The main cause of melanoma is excessive or uncontrolled exposure to UV radiation. The treatment of malignant melanoma depends on the stage of the tumor. Surgical resection is the first strategy in the case of circumscribed melanomas; while radiotherapy or chemotherapy are used in case of large and irresectable tumor or in presence of metastases. Chemo/radio-therapy are used in combination with immunotherapy to improve the chances of tumor eradication. In addition, targeted therapies have been approved about ten years ago and aim to block pathways involved in proliferation, survival and metastasis formation. Even if these therapies have an impact on tumor dimension, they may fail to completely eradicate the tumor causing the development of resistant cells that might re-form tumor masses. For these reasons, an effective and safe therapy against malignant melanoma is still lacking and the prognosis for all patients diagnosed with advanced metastatic melanoma is poor. The development of new therapies that combines the induction of cell death that cannot be escaped by single mutagenic events, with a safe drug delivery system could be effective in treating malignant melanoma avoiding side effects, metastasis and recurrence. The CRISPR/Cas9 system might provide the right tool to induce high-rate chromosomal damage into melanoma cells, without the possibility to overcome the damage. The optimal treatment for Cas9-based eradication of melanoma cells might have the following characteristics: (i) the activity of Cas9 should be controlled by the operator ; (ii) Cas9 delivery must be improved to increase the bio-distribution of the protein in the tumor mass. These tasks might be obtained by fusing Cas9 proteins to nanoparticles, that might spontaneously penetrate into melanoma cells without transfection reagents. Moreover, several nanoparticle formulations might be activated by the operator, providing the tool to better control where the Cas9 might be active and in which time window. To contribute to the design of these nanoparticles, the present thesis validate a new Cas9-delivery sistem based on gold nanoparticles and screen several guide RNAs for their ability to kill melanoma cells through hundreds of thousands of cuts at the same time in melanoma genomes. The hypothesis behind this approach is based on the assumption that even the more resistant melanoma cell might not survive the complete disruption of its genome.