One of the major challenges involved in treating brain diseases is the delivery of therapeutic agents into specific types of brain cells. Cell penetrating peptides (CPPs), a group of small and positively charged peptides, have been shown to be able to deliver a wide variety of cargos into mammalian cells in vitro and in vivo while the cell integrity is maintained. TAT, one of most studied CPPs, has been reported to be capable of delivering β-galactosidase into murine brain tissues in vivo. However, the non-specific delivery into other tissues has hampered TAT as a candidate for therapeutic delivery. We are working towards engineering novel specific cell penetrating peptides (SCPPs) to deliver cargos into specific types of brain cells through an innovative directed evolution approach using a plasmid display system to deliver a fluorescent transgene. The transduction ability of TAT for delivery to the brain has been verified by the efficient delivery of green fluorescent protein (GFP) into PC12 cells (31.4±9.2%) and primary astrocytes (10.8±3.4% ) through the fusion of TAT at the carboxyl-terminal end of GFP. In addition, HA2, a cell membrane-destabilizing peptide, has been shown to further improve the efficiency of TAT-mediated GFP delivery when it is fused to the amino-terminal end of GFP (64.8±7.8% for PC12 cells and 69.2±15.7% for astrocytes). A plasmid display system used for directed evolution of SCPPs has been established. However, the delivery efficiency of the plasmid display complex requires improvements to enable our directed evolution experiments. The pronounced negative charge of the plasmid DNA likely inhibits the transduction efficiency of TAT. In addition, the size dependence of the delivered DNA cargo on TAT-mediated delivery is being explored using linear DNA fragments with lengths ranging from 20 to 1000 base pairs. The insights gained through these efforts are enabling us to redesign our system for the directed evolution of novel SCPPs.

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