(512g) Directed Evolution of Novel Cell Penetrating Peptides for Delivery to the Brain

Cell penetrating peptides (CPPs) can traverse cellular membranes and deliver cargos including proteins and DNA into most cell types, including the brain, but new methods are needed to engineer them for enhanced delivery and specificity. We have developed an innovative directed evolution (DE) strategy based on a plasmid display (PD) platform to discover new specific CPPs. Successful penetration of cells by members of a randomized peptide library expressed using PD were indicated by the expression of a fluorescent transgene included in the plasmid DNA cargo. We have extensively characterized the ability of the well-known TAT CPP to deliver fluorescent proteins to PC-12 cells and we have also used TAT to deliver DNA oligonucleotides of varying lengths to PC-12 cells using the genotype/phenotype linkage method employed in the PD platform. We have also performed DE experiments using the PD platform and a novel CPP, SG3, has been successfully selected from the library. This newly identified CPP, SG3, is capable of delivering the green fluorescent protein, a model therapeutic protein cargo, to PC12 cells. The delivery efficiency, cell specificity and likely delivery mechanism of SG3 into differentiated PC12 cells, astrocytes and other brain cells will also be presented. Peptides developed using this new DE approach may represent a new strategy for the targeted delivery of therapeutics to the brain.