Lipid-Mediated Enhancement of Transfection by a Nonviral Integrin-Targeting Vector

Nonviral vectors consisting of integrin-targeting peptide/DNA (ID) complexes have the potential for widespread application in gene therapy. The transfection efficiency of this vector, however, has been limited by endosomal degradation. We now report that lipofectin (L) incorporated into the ID complexes enhances integrin-mediated transfection, increasing Iuciferase expression by more than 100-fold. The transfection efficiency of Lipofectin/Integrin-binding peptide/DNA (LID) complexes, assessed by β-galactosidase reporter gene expression and X-gal staining, was improved from 1% to 10% to over 50% for three different cell lines, and from 0% to approximately 25% in corneal endothelium in vitro. Transfection complexes have been optimized with respect to their transfection efficiency and we have investigated their structure, function, and mode of transfection. Both ID and LID complexes formed particles, unlike the fibrous network formed by lipofectin/DNA complexes (LD). Integrin-mediated transfection by LID complexes was demonstrated by the substantially lower transfection efficiency of LKD complexes in which the integrin-biding peptide was substituted for K16 (K). Furthermore, the transfection efficiency of complexes was shown to be dependent on the amount of integrin-targeting ligand in the complex. Finally, a 34% reduction in integrin-mediated transfection efficiency by LID complexes was achieved with a competing monoclonal antibody. The role of lipofectin in LID complexes appears, therefore, to be that of a co-factor, enhancing the efficiency of integrin-mediated transfection. The mechanism of enhancement is likely to involve a reduction in the extent of endosomal degradation of DNA. Nonviral vectors, consisting of a targeting ligand and a DNA-binding moiety, have great potential for gene therapy due to their safety, simplicity, and capacity for packaging very large DNA molecules. The major limiting factor in the development and application of these vectors, however, has been poor transfection efficiency due, primarily, to endosomal degradation. We have developed an integrin-targeted vector, consisting of a peptide containing a 16-lysine DNA-binding domain and one of a number of possible integrin-binding domains, of approximately 10 amino acid residues. Complexes of peptide and plasmid DNA transfect a number of different cells. The efficiency of this vector has now been enhanced substantially by incorporating small amounts of lipofectin into the complexes. Transfection is largely integrin mediated and the enhancement effect may be explained by lipofectin destabilizing endosomal membranes. Lipofectin-mediated enhancement may be a general approach to improving the efficiency of receptor-mediated vectors.