The incorporation of an ion channel gene mutation associated with the long QT syndrome (Q9E-hMiRP1) in a plasmid vector for site-specific arrhythmia gene therapy: in vitro and in vivo feasibility studies.

The present studies investigated the cardiac potassium channel missense mutation, Q9E-hMiRP1, for potential use as a gene therapy construct for cardiac arrhythmias. This gene abnormality is one of a number of mutations that can cause the long QT syndrome (LQTS), a hereditary arrhythmia disorder that is associated with sudden death. However, individuals who carry the Q9E-hMiRP1 variant are predisposed to developing the LQTS only after clarithromycin administration. Because the electrophysiologic mechanism of action of Q9E-hMiRP1 (i.e., diminished potassium currents resulting in delayed myocardial repolarization) is comparable to that of class III antiarrhythmic agents, we examined Q9E-hMiRP1 as a candidate gene therapy construct for site-specific treatment of reentrant atrial cardiac arrhythmias. Our rationale was also based on the hypothetical safety of the atrial use of Q9E-hMiRP1 because LQTS characteristically causes ventricular but not atrial arrhythmias. Furthermore, the possible use of clarithromycin to control the conduction effects of overexpressed Q9E-hMiRP1 pharmacologically was another attractive feature. In our studies we investigated the use of two bicistronic plasmid DNA gene vectors with either hMiRP1 or Q9E-MiRP1 and green fluorescent protein (GFP), plus a C-terminus of the hMiRP1 or of the Q9E-hMiRP1 coding region for the FLAG (MDYKDDDDK) peptide. We generated two stable cell lines using HEK293 and SH-SY5Y (human cell lines), overexpressing the genes of interest, confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blots. The expected plasma membrane localization of each overexpressed transgene was confirmed by immunofluorescent confocal fluorescent microscopy using anti-FLAG antibody. Patchclamp studies demonstrated that cells transfected with Q9E-hMiRP1 plasmid DNA exhibited significantly reduced potassium currents but only with clarithromycin administration. A novel plasmid DNA delivery system was formulated for use in our animal studies of the hMiRP1 vectors, which was composed of DNA-anti-DNA antibody-cationic lipid (DAC) heteroplexes. In vitro and in vivo studies using DAC heteroplexes containing anti-DNA antibodies with nuclear targeting capability demonstrated significantly increased transfection compared to naked DNA, and to DNA-cationic lipid complexes. Pig atrial myocardial injections of DAC heteroplexes demonstrated 16% of regional cardiac myocytes transfected using the Q9E-hMiRP1 plasmid, and 15% of cells with the hMiRP1 vector. It is concluded that the present studies support the view that site-specific gene therapy for atrial arrhythmias is feasible using plasmid vectors for overexpressing ion channel mutations that have electrophysiologic effects comparable to class III antiarrhythmic agents.