One of the main problems facing gene therapy is the ability to target the delivery of DNA to specific cells of choice. Recently, we developed a synthetic nonviral vector platform system known as LMD (liposome:mu:DNA) that was designed for further modular upgrading with tool-kits of chemical components. First-generation LMD systems were prepared from DC-Chol/DOPE cationic liposomes (DC-Chol=3beta-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol, DOPE=dioleoyl-L-alpha-phosphatidylethanolamine), mu peptide from the adenovirus core and plasmid DNA (pDNA). Here we report attempts to realise peptide-targeted gene delivery that build upon the LMD platform. Our strategy was to prepare novel lipopeptides with a lipid moiety designed to insert into the outer lipid bilayer of LMD particles whilst simultaneously presenting a peptide moiety for cell-surface receptor binding. One main functional peptide sequence was selected (PLAEIDGIELA; tenascin peptide sequence) known to target alpha(9)beta(1)-integrin proteins predominant on upper-airway epithelial cells. This sequence was investigated along with a corresponding control sequence. The syntheses of two classes (A and B) of lipopeptides are reported; the syntheses of class A lipopeptides requires a modification of Mitsunobu chemistry that could be of general utility to facilitate Mitsunobu reactions in other diverse systems. "Targeted" LMD and LD transfections with class A or B lipopeptides exhibit nonspecific peptide enhancements (up to one order of magnitude) over nonlipopeptide control transfections but few specific effects. Specific targeting effects can be seen if the overall LMD or LD particle cationic charge is lowered, but nonspecific effects are never eliminated. Whilst promising, these data now highlight the need for in vivo data and even a new modular, aqueous chemistry for the controlled adaptation of LMD particles in buffer in order for successful peptide-targeted, synthetic, nonviral gene delivery to be realised.

An efficient modification of the Fukuyama-Mitsunobu procedure has been developed whereby primary or secondary amines can be synthesized from alkyl alcohols and the corresponding nosyl-protected/activated amine. Most importantly, the use of the DTBAD and diphenylpyridinylphosphine, as Mitsunobu reagents, generates reaction by-products that can be easily removed, providing a remarkably clean product mixture. This improved technique was implemented in the synthesis of a complex lipopeptide designed to target alpha9beta1-integrin proteins predominant on upper airway epithelial cells.

Amines P 0060The Facile Preparation of Primary and Secondary Amines via an Improved Fukuyama-Mitsunobu Procedure. Application to the Synthesis of a Lung-Targeted Gene Delivery Agent. -The title protocol is characterized by utilization of "clean" Mitsunobu reagents allowing for easy purification of the products. -(GUISADO, C.; WATERHOUSE, J. E.; PRICE, W. S.; JORGENSEN, M. R.; MILLER*, A. D.; Org.