Final Activity Report Summary - TRANSFECTAZINE (Low-toxicity azine-based cationic lipids for gene transfection) This work focused on the development of a conceptually new class of reagents for gene delivery, consisting in cationic lipids having a central scaffold supporting three side chains bearing: 1) a lipophilic chain, 2) a polar head, 3) a dimerisable function. The dimeric disulfides have also been evaluated in terms of transfection efficiency and toxicity. First of all, the molecules were chemically and physically characterised. In particular, one cationic lipid (named compound 4) was extremely efficient in transfecting different cell lines in vitro, with a combined toxicity / efficiency profile which is nearly always much superior to commercially available and popular transfection reagents. The easy synthesis in multigram amount from inexpensive and commercially available starting materials, combined with a user-friendly (no formulation or liposome preparation is required) and effective protocol for cell transfection render these triazine-based compounds very attractive reagents for gene delivery applications. Secondly, the project focused my attention on TNF-a gene cloning. TNF-a is a pivotal cytokine with broad ranging effects. TNF-a is a trans membrane protein that is then cleaved in the extracellular domain through the action of an enzyme to shed a mature soluble protein. The project produced two TNF-a cDNA mutant forms: a gene coding for a directly soluble protein named sTNF-a lacking the trans membrane domain and another coding for a non-cleavable, only trans membrane form, named mTNF-a. These genes were introduced into an expression plasmid and are now useful for gene therapy in combination with the safe gene delivery system described above.
