posted on 2023-11-03, 00:20authored byJuliana Bolsoni, Danny Liu, Fatemeh Mohabatpour, Ronja Ebner, Gaurav Sadhnani, Belal Tafech, Jerry Leung, Selina Shanta, Kevin An, Tessa Morin, Yihang Chen, Alfonso Arguello, Keith Choate, Eric Jan, Colin J.D. Ross, Davide Brambilla, Dominik Witzigmann, Jayesh Kulkarni, Pieter R. Cullis, Sarah Hedtrich
Despite exciting
advances in gene editing, the efficient
delivery
of genetic tools to extrahepatic tissues remains challenging. This
holds particularly true for the skin, which poses a highly restrictive
delivery barrier. In this study, we ran a head-to-head comparison
between Cas9 mRNA or ribonucleoprotein (RNP)-loaded lipid nanoparticles
(LNPs) to deliver gene editing tools into epidermal layers of human
skin, aiming for in situ gene editing. We observed
distinct LNP composition and cell-specific effects such as an extended
presence of RNP in slow-cycling epithelial cells for up to 72 h. While
obtaining similar gene editing rates using Cas9 RNP and mRNA with
MC3-based LNPs (10–16%), mRNA-loaded LNPs proved to be more
cytotoxic. Interestingly, ionizable lipids with a pKa ∼ 7.1 yielded superior gene editing rates (55%–72%)
in two-dimensional (2D) epithelial cells while no single guide RNA-dependent
off-target effects were detectable. Unexpectedly, these high 2D editing
efficacies did not translate to actual skin tissue where overall gene
editing rates between 5%–12% were achieved after a single application
and irrespective of the LNP composition. Finally, we successfully
base-corrected a disease-causing mutation with an efficacy of ∼5%
in autosomal recessive congenital ichthyosis patient cells, showcasing
the potential of this strategy for the treatment of monogenic skin
diseases. Taken together, this study demonstrates the feasibility
of an in situ correction of disease-causing mutations
in the skin that could provide effective treatment and potentially
even a cure for rare, monogenic, and common skin diseases.