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

Ex vivo 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 ex vivo 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 Trpv3 gene. Specifically, PVLP-delivered SaCas9 and shRNA effectively disrupted the Trpv3 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 ex vivo skin gene therapy.