Background: Chronic rejection is now the major cause of allograft failure. A prominent characteristic of the histopathology is extensive intimal proliferation of vascular smooth muscle cells. Targeting vascular smooth muscle cells by gene therapy techniques offers a possible avenue for arresting or reversing chronic rejection. Defining suitable non-viral DNA vectors for this application is the objective of this study.
Methods: A 31 amino acid synthetic peptide has been evaluated as a DNA vector for primary cultures of vascular smooth muscle cells of man, rabbit, and rat. The vector comprises a 15 amino acid integrin-binding domain and a chain of 16 lysines for electrostatic binding of DNA. Three agents known to promote exit of vector/DNA complexes from endocytic vesicles were studied systematically to define optimal, non-toxic conditions for gene delivery.
Results: Initial binding studies on frozen sections showed that the integrin-binding domain binds strongly to vascular smooth muscle cells in all three species, thereby establishing vascular smooth muscle cells as a potential target for this receptor-targeted DNA vector system. Primary cultures of vascular smooth muscle were therefore studied. The use of chloroquine to assist endocytic exit, which works well on immortalized cell lines, was of little value because of toxicity to the primary vascular smooth muscle cells. The addition of cationic lipids to polylysine-molossin/DNA conjugates gave excellent reporter gene expression, but required mildly toxic doses of cationic lipid, and resulted in some loss of integrin specificity of the vector system. The optimal system involved the use of the amino terminal 20 amino acids of the hemagglutinin of the influenza virus. This peptide, when added to polylysine-molossin/DNA complexes at an optimal w/w ratio of 5:1:2 (polylysine-molossin/DNA/fusogenic peptide) resulted in 25-30% transfection of vascular smooth muscle cells with good levels of gene expression and no toxicity.
Conclusion: This represents an effective and safe DNA vector, comprised entirely of small synthetic peptides, and therefore readily standardized for clinical and experimental application.