A ligand-based system for receptor-specific delivery of proteins.

Mariano Maffei,Balint Doleschall,Luc Reymond,Ivan R Corrêa,Paul A Heppenstall,Jamie A Hackett,Chiara Morelli,Ellie Graham,Fernanda De Castro Reis,Kai Johnsson,Laura Donzelli,Stefano Patriarca,Linda Nocchi,Cora H Chadick

doi:10.1038/s41598-019-55797-1

Abstract

Gene delivery using vector or viral-based methods is often limited by technical and safety barriers. A promising alternative that circumvents these shortcomings is the direct delivery of proteins into cells. Here we introduce a non-viral, ligand-mediated protein delivery system capable of selectively targeting primary skin cells in-vivo. Using orthologous self-labelling tags and chemical cross-linkers, we conjugate large proteins to ligands that bind their natural receptors on the surface of keratinocytes. Targeted CRE-mediated recombination was achieved by delivery of ligand cross-linked CRE protein to the skin of transgenic reporter mice, but was absent in mice lacking the ligand’s cell surface receptor. We further show that ligands mediate the intracellular delivery of Cas9 allowing for CRISPR-mediated gene editing in the skin more efficiently than adeno-associated viral gene delivery. Thus, a ligand-based system enables the effective and receptor-specific delivery of large proteins and may be applied to the treatment of skin-related genetic diseases.

Highlights

Gene delivery using vector or viral-based methods is often limited by technical and safety barriers
We previously described a protein based tool for the delivery of a small molecule photosensitizer to the skin, along with a light-mediated control of itch and inflammatory skin disease[14]. This approach was based upon a SNAP-tagged engineered version of the cytokine interleukin-31 (IL-31K138ASNAP), that binds to its receptors (IL31RA and OSMR) on keratinocytes, but does not provoke cellular signaling
We found that IL-31K138ASNAP is translocated to the nucleus of primary murine keratinocytes upon internalization

Summary

Introduction

Gene delivery using vector or viral-based methods is often limited by technical and safety barriers. Direct access to the interior of a cell enables, among others, gene editing[1,2], modulation of gene expression[3] and ex-vivo cell therapies[4] Realization of these applications is commonly achieved by delivery of exogenous nucleic acids or virus-based methods. This in turn limits the development of strategies for effective therapies of skin-related diseases In this light, a ligand-based system could represent a key technology to gain access to keratinocytes, enabling novel therapeutic applications in the skin. We previously described a protein based tool for the delivery of a small molecule photosensitizer to the skin, along with a light-mediated control of itch and inflammatory skin disease[14] This approach was based upon a SNAP-tagged engineered version of the cytokine interleukin-31 (IL-31K138ASNAP), that binds to its receptors (IL31RA and OSMR) on keratinocytes, but does not provoke cellular signaling. We demonstrate that cross-linked complexes are selectively delivered into primary keratinocytes both in-vitro and in-vivo and can achieve cell-type specific gene editing including homology-directed repair in-vivo

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Conclusion