Papillomavirus as a novel viral vector 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 the 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 that can deliver up to 8 kilobases of double stranded DNA, 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 shRNA to keratinocytes, offering advantages over other viral vectors such as AAV and lentivirus. PVLPs can be produced in two ways: through cell-based assembly, like other viral vectors, or in a cell-free reaction environment. We developed a workflow that combines the strengths of both approaches. First, we used the cell-free assembly method to quickly screen a library of plasmids on cell lines. After identifying the most effective candidate, we transitioned to the cell-assembly method, which, although more time-consuming, achieves better transduction efficiency in primary keratinocytes. We showed that PVLPs can be used for gene therapy for Olmsted syndrome, a monogenic skin disorder 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.