O-023. Engineering of a Plasmid Display System for the Directed Evolution of Targeted Cell Penetrating Peptides

S. Gao, M. J. Simon, B. Morrison III, S. Banta;
Columbia Univ., New York, NY.

Considerable effort has been aimed at the development of efficient delivery strategies for introduction of molecules into specific types of cells. Cell penetrating peptides (CPPs) are small and positively charged peptides that are able to deliver a wide variety of cargos into various cell types both in vitro and in vivo while the cell integrity is maintained. However, the non-specific targeting has hampered the use of CPPs in applications such as selective differentiation, imaging, and non-viral gene therapy. We are developing a method to enable the engineering of novel specific cell penetrating peptides (SCPPs) to deliver cargos into specific types of cells using an innovative directed evolution approach using a plasmid display (PD) system. The PD complex uses the DNA-binding domain of the p50 transcription factor to link the plasmid to its fusion protein product during expression in E. coli. The TAT peptide, which is a well-studied CPP, was fused to the p50 gene. A yellow fluorescent transgene was also introduced into the PD vector to indicate the successful delivery of the protein/DNA complex. However, the delivery efficiency of the PD complex into mammalian cells requires improvements to enable our directed evolution experiments. The pronounced negative charge of the plasmid DNA likely inhibits the transduction efficiency of TAT peptide at the molar ratio of protein to plasmid of 2:1, found in the original PD system. To further quantify this effect, the size dependence of the delivered DNA cargo on TAT-mediated delivery is being explored using DNA fragments containing the p50 binding site with lengths ranging from 30 to 4000 base pairs. The results showed that no DNA was delivered by GFP-TAT or p50-GFP controls, which indicates that both binding domain (p50) and penetrating domain (TAT) are necessary for successful DNA delivery. For delivering DNA with p50-GFP-TAT, the optimal molar ratio of protein to DNA is 28 and 280 for linear DNA with 30bp and 294bp. The insights gained through these efforts explain the low delivery efficiency of the PD complex and are enabling us to redesign our PD system for the directed evolution of novel SCPPs.