Papillomavirus-like particles as vectors for ex vivo gene therapy of the skin

<i>Ex vivo</i> gene delivery to the skin utilizing retroviral vectors has been demonstrated to be a viable clinical option for replacement of defective genes. However, because these vectors integrate their cargo into the genome, safety issues arise when utilizing them to deliver gene-editing nucleases. Here, we explored the use of Papillomavirus, a non-integrating viral vector, for <i>ex vivo</i> skin gene editing, exploiting its natural tropism for basal keratinocytes. We demonstrated that Papillomavirus-like particles (PVLPs) can deliver a variety of DNA constructs encoding fluorophores, Cre recombinase, calcium indicators, Cas9, and short hairpin RNA (shRNA) to keratinocytes, offering advantages over other viral vectors such as adeno-associated virus (AAV) and Lentivirus. We further showed that PVLPs can be used for gene therapy for Olmsted syndrome, a genetic skin disease caused by a gain-of-function mutation in the <i>Trpv3</i> gene. Specifically, PVLP-delivered SaCas9 and shRNA effectively disrupted the <i>Trpv3</i> gene or reduced its expression, leading to decreased TRPV3 activity and mitigating the hyperactivity associated with Olmsted syndrome. Skin equivalents generated from PVLP-treated keratinocytes exhibited complete transduction, and PVLP-shRNA treatment significantly reduced hyperkeratosis in skin equivalents from mice bearing the Olmsted syndrome mutation. These findings highlight PVLP as a promising tool for <i>ex vivo</i> skin gene therapy.