Targeted Gene Delivery to Human Airway Epithelial Cells with Synthetic Vectors Incorporating Novel Targeting Peptides Selected by Phage Display

Abstract

Human airway epithelial cell targeting peptides were identified by biopanning on 1HAEo-cells, a well characterised epithelial cell line. Bound phage were recovered after three rounds of binding, high stringency washing and elution, leading to the production of an enriched phage peptide population. DNA sequencing of 56 clones revealed 14 unique sequences. Subsequent binding analysis revealed that 13 of these peptides bound 1HAEo-cells with high affinity. Three peptides, SERSMNF, YGLPHKF and PSGAARA were represented at high frequency. Three clearly defined families of peptide were identified on the basis of sequence motifs including (R/K)SM, L(P/Q)HK and PSG(A/T)ARA. Two peptides, LPHKSMP and LQHKSMP contained two motifs. Further detailed sequence analysis by comparison of peptide sequences with the SWISSPROT protein database revealed that some of the peptides closely resembled the cell binding proteins of viral and bacterial pathogens including Herpes Simplex Virus, rotavirus, Mycoplasma pneumoniae and rhinovirus, the latter two being respiratory pathogens, as well as peptide YGLPHKF having similarity to a protein of unknown function from the respiratory pathogen Legionella pneumophila. Peptides were incorporated into gene delivery formulations with the cationic lipid Lipofectin and plasmid DNA and shown to confer a high degree of transfection efficiency and specificity in 1HAEo-cells. Improved transfection efficiency and specificity was also observed in human endothelial cells, fibroblasts and keratinocytes. Therefore, on the basis of clone frequency after biopanning, cell binding affinity, peptide sequence conservation and pathogenic similarity, we have identified 3 novel peptide families and 5 specific peptides that have the potential for gene transfer to respiratory epithelium in vivo as well as providing useful in vitro transfection reagents for primary human cell types of scientific and commercial interest.

Multifunctional nanocomplexes for gene transfer and gene therapy

“…The addition of a cell-targeting function, such as a peptide, enhances transfection efficiency of nanoparticles by a fivefold to tenfold, depending on the cell and ligand (Writer et al 2004;Hart et al 1998). In many cases, nanoparticles with targeting properties that comprise cationic components will also retain a net positive surface charge, which, if not shielded, will provide further cell-binding functions.…”

“…The first formulation of this system (LID vector) transfected bronchial epithelium in vivo with high efficiency in rats (Jenkins et al 2000), mice (Jenkins et al 2003) and pigs (Cunningham et al 2002). Recently, we have optimised the peptide sequence for transfection of human airway epithelial cells (Writer et al 2004). The selected peptide has the sequence K 16 GACSERSMNFCG, in which K 16 mediates DNA binding, and SERSMNF is a receptorbinding ligand.…”

“…A new RTN formulation, LYD, comprising a peptide with the targeting domain, YGLPHKF ("peptide Y"; Writer et al 2004), and a modified cationic diether lipid with a 14-carbon alkyl tail (CH315) instead of the C18 alkyl tail of DOTMA (Table 2) used in the LID formulation, was found to transfect both rabbit aortic smooth muscle cells and primary cultures of human saphenous vein smooth muscle cells with greater efficiency than the original integrin-targeted formulation. The YGLPHKF ligand was identified in phage display, biopanning studies on epithelial cells, and its receptor is unknown (Writer et al 2004).…”

“…Phage peptide display systems offer a convenient technology for identifying candidate ligands from libraries of random peptide sequences by panning on the desired protein receptor target (Koivunen et al 1993). Alternatively, phage libraries can be panned directly on the target cell type, and this approach has been used to identify target ligands for endothelial cells (Nicklin et al 2000) and airway epithelial cells (Writer et al 2004) where the selected peptides were subsequently used to target nanocomplexes to airway epithelium in vivo (Tagalakis et al 2008).…”

Airway Deposition of Nebulized Gene Delivery Nanocomplexes Monitored by Radioimaging Agents

“…The RTN formulation comprises cationic liposomes, a receptor-targeting peptide and plasmid DNA that self-assembles electrostatically to form cationic nanoparticles of approximately 150 nm (5). We previously tested this vector system for the transfection of airway epithelial cells in vitro and in vivo in murine lungs and tracheas via instillation and nebulization (5)(6)(7). Pigs are used widely in preclinical studies, because their lung physiology and anatomy are closer to those of humans than are those of small rodents, and their size allows for regional studies of nebulized deposition within the lung after inhalation.…”