936. Phage-Selected Motifs Internalize Via Multiple Endocytic Pathways and Facilitate Gene Delivery

Abstract

Top of pageAbstract The ability to direct internalization of macromolecules into cells or across endothelial barriers efficiently has tremendous therapeutic implications. Several internalization motifs (IM) that exist in nature have been defined and exploited, including the third -helix of the Drosophila Antennapedia homeodomain and the HIV TAT protein. To investigate the general properties required for protein-based macromolecule internalization we constructed and performed peptide phage display (PPD) with T7 bacteriophage random 7-mer peptide libraries. Functional biopanning with the T7 libraries was performed on microvascular endothelial cells (EC) to select peptide motifs that could internalize phage. The titer of internalized phage increased through sequential rounds of biopanning and resulted in selection of phage expressing IM with a finite shared sequence relationship between charged, polar, and hydrophobic amino acids. Selected IM were synthesized as rhodamine-labeled free peptides and their internalization properties were investigated in EC. Internalization of IM was inhibited at 4 degrees, suggesting an energy dependent mechanism of internalization. Confocal data showed that the IM was internalized via at least two different endocytic pathways: clathrin-coated pits and lipid rafts. Treatment with inhibitors of clathrin-mediated and lipid raft endocytosis inhibited uptake of IM, consistent with the result from confocal study. Further, in vitro and in vivo studies indicated that precomplexing of adenovirus with the IM facilitated the degree of viral transduction into endothelial cells and across endothelial barriers. These data suggest that relatively short peptide motifs are capable of directing macromolecules to diverse internalization pathways. It may be possible to exploit these characteristics for targeted delivery of therapeutic macromolecules to cells and tissues, and potentially to develop more efficient methods of gene transfer.