187. Modular Cationic Peptides as Gene Delivery Agents

• Published in: Molecular therapy Vol. 13; no. S1; p. S73
• Main Authors: Rajagopalan, Rukkumani, Xavier, Jennifer, Rao, Nalam M., Gopal, Vijaya
• Format: Journal Article
• Language: English
• Published: Milwaukee Elsevier Limited 01.05.2006
• Subjects: Bioengineering; Efficiency; Localization; Microscopy; Peptides; Proteins; Japan
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1016/j.ymthe.2006.08.211

Peptide-based gene delivery is novel and allows a combinatorial approach to suit specific needs.Our strategy uses the recombinant approach where the major advantage would be the ease with which a large number of peptide combinations could be prepared by bacterial fermentation. By combining motifs with the desired properties of DNA condensation and membrane transduction, the efficiency of transfection could be greatly enhanced particularly in cells that are non-dividing.We have recently shown that the recombinant peptides, expressed as N-terminal His-Tag fusion proteins, TAT-Mu, TAT-NLS, Mu and Mu-Mu, are capable of transporting DNA effectively into MCF-7, CHO, COS, HeLa and macrophages. TAT constitutes a 11 amino acid region of the HIV-TAT while Mu, of the adenoviral core protein, has 19 amino acids. The nuclear localization sequences (SV 40 NLS) constitutes 3 copies of the sequence present in tandem. These sequences have been cloned in frame in an expression vector. In the case of TAT-Mu and TAT-NLS, transfection was enhanced ∼3 fold in the presence of the cationic lipid DDAB:DOPE. When transfections were performed in the presence of chloroquine, a lysosomotropic agent, the DNA transfection was further enhanced. These results and colocalization with endosomal markers suggests involvement of endocytic pathways in the uptake of the lipopolyplexes. Immunolocalization of the peptide DNA complexes by confocal microscopy, showed that macrophages, HeLa, and MCF-7 cells internalized the peptide:DNA complexes in a time dependent manner with strong nuclear staining in all the cells.Recombinant methodology to produce a large variety of modular, cationic peptides allows rapid development of cell tropic molecules for gene delivery. Such molecular entities would have great potential in the development and advancement of molecular medicine.