Our site specific polymeric drug-delivery program will focus its therapeutic strategy on a concept that we will term, Reverse Gene Therapy (RGT). RGT is defined as the site specific use of a plasmid DNA vector encoding for a pathologic gene, that can be used to beneficially treat other pathologic conditions, by localizing, selectively targeting and controlling its delivery with a nonviral sustained release polymeric system. our first choice for a model gene for RGT will be HERG (A561V), the point mutation responsible for one of the forms of the Long QT Syndrome, a hereditary disorder associated with episodes of ventricular arrhythmias and a risk of sudden death. HERG (A561V) encodes for a defective potassium channel rectifier, and its expression has been demonstrated to result in cell membrane incorporation of HERG (A561V), resulting in prolonged myocardial conduction, through a dominant negative effect, interacting with the wild type HERG potassium channel rectifier. It is of interest in this regard that ibutilide, the short acting Class III agent studied in the previous phase of this program, has the identical pharmacological mechanism, and has been shown in our studies to prevent re-entrant atrial flutter both systemically and when delivered in a site specific manner. Hence, we hypothesize that site specific overexpression of HERG (A561V) in the right atrium in an experimental re-entrant pathway should prevent re-entrant atrial flutter in our dog model. Our working hypothesis is that site specific plasmid DNA delivery in a polymeric sustained release preparation can be used to treat re-entrant cardiac arrhythmias. Plasmid DNA delivery will be achieved using sustained release nanoparticles formulated from polylactic-polyglycolic acid (PLGA), with co-incorporated poly-L- lysine(PLL) to enhance DNA delivery. Our cardiac arrhythmia model will be based upon reentrant atrial flutter induced in dogs.
The specific aims of this program renewal are: 1) To investigate the determinants of plasmid DNA-PLGA-PLL nanoparticles. We will focus on the mechanisms governing DNA incorporation into these NP, and transfection in CHO cells and cultured cardiac myocytes (first with reporter studies and then with our model RGT vector). 2) We will investigate the characteristics and mechanisms governing DNA-PLL-NP transfection of the atrial myocardium with respect to site specific localization in an atrial reentry loop, and sustained episomal expression of a reporter construct, nuclear targeted beta-galactosidase. 3) We will perform comparable therapeutic studies with a candidate RGT plasmid vector, HERG (A561V).
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