Jeffrey Hughes, Ph.D. and Nancy Cheng, Ph.D. (Burroughs Welcome Foundation)
Non-viral vectors will be developed for the selective delivery of genes to malignant cells. The vector will exploit the increased requirement of rapidly growing cells for more nutrients by attaching a nutrient-ligand onto the vector (liposome). The vector additionally will have a positively charged lipid to enhance nucleic acid binding along with a novel pH sensitive surfactant. The role of the surfactant is to increase the amount of nucleic acid escaping the endosome and correspondingly increase the transfection efficiency. The vector system will be evaluated first in an animal model of cancer. Preliminary studies will be carried out using a marker gene (beta-galactosidase) with later experiments using a gene encoding for cytosine deaminase. Cytosine deaminase can catalyze the conversion of the innocuous agent 5-fluoro cytosine to the anti-cancer agent 5-fluorouracil. By selective delivery of this gene to only cancer cells the therapeutic index of 5-fluorouracil can then be increased.
In this proposal the development of a soft pH sensitive surfactant will be evaluated. This agent will become active at endosomal pH, has membrane disrupting effects, and will be inactivated before reaching the lysosome. For this agent to work, the surfactant must enter the cell by endocytosis. To accomplish this, the soft surfactant will be incorporated into liposomes that have been shown to be enter cells in this manner. The novelty of this delivery system stems from the soft pH sensitive surfactant (SPS). The synthetic features of this route include a pH sensitive region (imidazole), the lipophilic moiety (dodecanol), and the enzymatically cleavable connector (2-bromopropionyl bromide). The SPS will be incorporated into a liposomal nucleic acid carrier with other lipid components. A spacer (polyethylene glycol 3,000) will be attached between the ligand and liposome to increase natural ligand: receptor interactions and a positively charged metabolically cleavable cationic lipid 1,2, dioleoyl-3-trimethylammonium-propane. Liposomes will be prepared in the HAL facility by hydration of a dried lipid layer. After the liposomes are formed, they will be extruded through a polycarbonate membrane to a final size of 100 nm. The N-hydroxylsuccinamide esters of the ligands will be reacted with the performed liposomes. Free ligand will be removed by gel chromatography. The experimental approach will identify various receptor mediated pathways that will be optimal for delivery of genes to malignant cells. Once the best delivery system is chosen, animal studies will be conducted with a plasmid encoding for cytosine deaminase and, if successful, the progression into human studies on the GCRC could proceed rapidly, probably within 4 years.
- Hughes JA, Avrotskaya Av, Juliano RL. Oligonucleotide transport across membranes into cells: effects of chemical modifications. In, Delivery Systems for Antisense oligonucleotide therapeutics. Oxford, CRC Press, 1994.