Cell penetrating peptides (CPPs) are a promising tool to deliver therapeutic molecules across the cell membrane into the cytoplasm of target cells. Unfortunately, most traditional CPPs and their linked cargo are trapped to a high degree in the cells’ endosomes and are therefore not efficiently transported into the cytoplasm, where they exert their functional effect.
We have screened ‘Phylomer’ peptide libraries derived from biodiverse bacterial and viral genomes for CPPs with better endosomal escape. A genetic screen called the ‘endosome escape trap’ was used to isolate Phylomer peptides, which were able to deliver T7 phage into the cytoplasm of mammalian cells. These new CPPs, also known as functional penetrating peptides (FPPs), show low toxicity and can be targeted to particular cell types using receptor binding domains.
A variety of assays was used to determine how efficient Phylomer FPPs were in delivering peptide and protein cargoes of different sizes into the cell. One such assay based on split GFP complementation has shown Phylomer FPPs to be 37-160 times more efficient at cytoplasmic delivery than conventional CPPs such as TAT or R9.
Phylomer FPPs have been tested in combination with a variety of cargoes: they were capable of delivering protein conjugates of up to 50 kDa into several types of mammalian cells. In a mouse model of breast cancer, a Phylomer FPP was used to effectively deliver a 90 amino acid MYC inhibitor into cells. In another model, a Phylomer FPP was linked to nucleic acids and gained access into cells causing measurable exon skipping.