Evaluation of new gold-based nanoformulations for gene editing: in vitro and in vivo approaches for melanoma immunotherapy

The CRISPR/Cas9 gene-editing system holds immense potential for treating cancer and various diseases, as demonstrated in vitro and in animal models. However, its clinical application remains limited due to two major challenges: the inability to precisely control Cas9 activity in terms of location and duration. This study proposes two innovative gene-editing approaches to overcome these limitations. The first utilizes gold nanoparticles (AuNPs) conjugated to Cas9, enabling spontaneous cellular entry without the need for transfecting agents. The second approach leverages the plasmonic properties of the AuNRs to achieve laser-activated DNA cleavage, providing spatiotemporal control over genome editing. To validate these strategies, we targeted the programmed death-ligand 1 (PD-L1) gene, which is overexpressed in melanoma cells. This gene editing could induce a bystander effect, potentially activating immune cells against both edited and non-edited tumor cells. The nanoformulation was synthesized and tested for cytotoxicity in a cellular monolayer, followed by evaluation of nanoparticle internalization and nuclear localization. Gene-editing efficacy was assessed in A375 melanoma cells. Additionally, nanoparticle penetration and safety were examined using both an in vitro human skin model and a murine model, with accompanying histological and inflammation assessments. Finally, as proof of concept, primary melanoma cells from an in vivo explant were subjected to PD-L1 gene knockout. Our results demonstrate successful cellular uptake of the nanoparticles without external transfection aids, and their ability to penetrate complex structures such as skin. In vitro gene editing was achieved, while in vivo studies are ongoing. These findings highlight the potential of this AuNP-based CRISPR/Cas9 delivery system for precise and controlled gene editing in cancer therapy.