We propose to test the feasibility of applying """"""""cage"""""""" compounds to enhance treatment with antisense and plasmid based genetic therapies. Enhancement will be achieved by protecting antisense oligonucleotides from enzymatic degradation and by localizing antisense and plasmid bio- activity with light. In this hypothetical scheme, DNA degradation and bio-activity is blocked by biochemical modification with a """"""""caging"""""""" compound on the DNA phosphate backbone. Reactivation is achieved by """"""""uncaging"""""""" the DNA with exposure to light. We propose to test this hypothetical scenario and its potential in developing optically activated genetic therapies using simple biochemical tests, and bioassays for antisense activity and green fluorescent protein expression. We will test three hypotheses: l) light-reversible alterations in oligonucleotide and plasmid DNA structure can be achieved with caging, 2) caging protects oligonucleotides and plasmid DNA from degradation by nucleases, and 3) caged oligonucleotides and plasmid DNA can be re-activated with light after delivery to cells. Except as shown in our preliminary data, blocking nuclease degradation or temporarily inactivating plasmid DNA by the use of caging compounds has not been previously described. Post delivery activation of """"""""caged"""""""" DNA with a pulse of light is an untested method of preserving antisense oligonucleotides and achieving targeted control of expression with antisense or plasmids in vivo.
The commercial application of gene therapy is enormous. For example, Pulmozyme(TM) is an 80 million dollar a year product, which is only 20% effective in treating the symptoms of cystic fibrosis. The ability of achieve tissue specific (by uncaging) delivery of oligonucleotide or other genetic based therapy could represent a market greater that 10 times this amount.
| Das, M; Bouchey, D M; Moore, M J et al. (2001) Hypoxia-induced proliferative response of vascular adventitial fibroblasts is dependent on g protein-mediated activation of mitogen-activated protein kinases. J Biol Chem 276:15631-40 |
