Abstract 3812: Identification of a Cardiac Specific Protein Transduction Domains by in vivo Biopanning using a M13 Phage Peptide Display Library
To identify cardiac-specific protein transduction domains (PTD), able to deliver peptides/ proteins of therapeutic potential using a combination of in vitro and in vivo phage display. H9C2 cells were incubated with a random 12 amino acid M13 phage display library. The internalized phage were recovered, amplified, injected in BalbC mice, phage from heart and kidneys recovered, amplified and reinjected for a subsequent round of biopanning. After 4th round of biopanning, 6 out of 10 sequenced plaques were identical. The peptide, termed cardiac targeting peptide (CTP), was synthesized labeled with 6-carboxyfluorosene (6CF), biotinylated or fused to the NEMO-binding peptide (NBD), an inhibitor of the inducible NF-kappa B Kinase. Multiple different cell lines were incubated with CTP-6CF and confocal microscopy performed. H9C2 cells were transfected with a reporter plasmid containing an NF-kappa B dependent promoter-driven luciferase expression, followed by treatment with varying concentrations of CTP-NBD and TNF-α, and luciferase activity measured. Biotinylated CTP, a random peptide and 8-Lysine (8K), a known PTD, were conjugated with fluorescently labeled streptavidin (SA488), injected intravenously into mice, euthanized 30 minutes later and fluorescent microscopy performed on multiple organs. Confocal microscopy showed Increasing intracellular localization of the CTP in a concentration dependent manner in cardiomyocytes. CTP-NBD also inhibited luciferase activation significantly in a dose-dependent and cell type-dependent manner. The CTP+SA488 conjugate transduced heart tissue efficiently in vivo with very little uptake by lung and kidney capillaries, and none in liver, skeletal muscle, spleen or brain. This level of heart uptake was not seen with 8K, which had uptake primarily in liver and kidney glomeruli. No uptake was seen with the random peptide. Biopanning using a peptide phage display library identified a peptide able to transduce cardiomyoblasts in vitro, heart tissue in vivo, deliver a biologically active peptide and a marker protein conjugate in a cell type specific manner. This peptide could be used to deliver peptides/proteins of therapeutic potential to cardiac cells in vivo.