Discovery of a new class of cell-penetrating peptides by novel phage display platform

The primary hurdles for small interference RNA (siRNA) in clinical use are targeted and cytosolic delivery. To overcome both challenges, we have established a novel platform based on phage display, called NNJA. In this approach, a lysosomal cathepsin substrate is engineered within the flexible loops of PIII, that is displaying a unique random sequence at its N-terminus. NNJA library selection targeting cell-expressed targets should yield specific peptides localized in the cytoplasm. That is because phage internalization and subsequent localization to lysosome, upon peptide binding to the cell expressed target, will result in cleavage of PIII, rendering phage non-infective. Such phage will be eliminated from the selected pool and only peptide-phage that escapes lysosomes will advance to the next round. Proof of concept studies with the NNJA library demonstrated cytosolic localization of selected peptide-phage and peptide-siRNA, confirmed through confocal microscopy. More importantly, conjugation of siHPRT to monomeric or multimeric NNJA peptides resulted in significant reduction in HPRT mRNA in various cell types without significant cytotoxicity. Sequence similarity and clustering analysis from NGS dataset provide insights into sequence composition facilitating cell penetration. NNJA platform offers a highly efficient peptide discovery engine for targeted delivery of oligonucleotides to cytosol.