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

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.

New Insights into the Therapeutic Applications of CRISPR/Cas9 Genome Editing in Breast Cancer

“…Additionally, the creation of a selective organ targeting (SORT) system that has been shown to be compatible with both the CRISPR/Cas sgRNA as well as RNP complexes is another particularly impressive approach. This technique involves changing the core charge of lipid nanoparticle transporters by changing the composition of a supplementary ‘SORT’ molecule [ 170 ]. Researchers have used this technique to demonstrate lung-, liver- and spleen-specific targeting in mice, as well as effective gene editing of PTEN, which is a known therapeutic target for cancer [ 170 ].…”

Epigenetic editing: Dissecting chromatin function in context

“…[168] AAV can be further engineered to direct their tropism to the cell type of interest through a ligandreceptor interaction. [169,170] These considerations make therapeutic delivery an important area of focus when it comes to clinical applications of epigenetic editing.…”

Tissue-Specific Delivery of CRISPR Therapeutics: Strategies and Mechanisms of Non-Viral Vectors

“…Remarkable work was seen in the development of a selective organ targeting (SORT) system which is compatible with CRISPR-Cas mRNA/gRNA as well as RNP complexes [ 104 ]. This strategy is based on altering the internal charge of lipid nanoparticle carriers by modifying their molar compositions of an additional “SORT” molecule to change their cell-fate [ 104 ].…”

“…Remarkable work was seen in the development of a selective organ targeting (SORT) system which is compatible with CRISPR-Cas mRNA/gRNA as well as RNP complexes [ 104 ]. This strategy is based on altering the internal charge of lipid nanoparticle carriers by modifying their molar compositions of an additional “SORT” molecule to change their cell-fate [ 104 ]. Using this technique, the research team has shown evidence for lung, liver and spleen specific targeting in mice, and efficient gene editing of PTEN and PCSK9 , therapeutic targets for cancer and hypercholesterolaemia, respectively [ 104 ].…”

“…This strategy is based on altering the internal charge of lipid nanoparticle carriers by modifying their molar compositions of an additional “SORT” molecule to change their cell-fate [ 104 ]. Using this technique, the research team has shown evidence for lung, liver and spleen specific targeting in mice, and efficient gene editing of PTEN and PCSK9 , therapeutic targets for cancer and hypercholesterolaemia, respectively [ 104 ]. Similarly, Wei and colleagues achieved liver and lung-specific delivery of lipid nanoparticle-encapsulated RNPs in mice [ 102 ].…”