Calcium electrotransfer for termination of transgene expression in muscle

Gene electrotransfer is expanding in clinical use, thus we have searched for an emergency procedure to stop transgene expression in case of serious adverse events. Calcium is cytotoxic at high intracellular levels, so we tested effects of calcium electrotransfer on transgene expression in muscle. A clinical grade calcium solution (20μl, 168mM) was injected into transfected mouse or rat tibialis cranialis muscle. Ca²⁺ uptake was quantified using calcium 45 (⁴⁵Ca), and voltage and time between injection and pulsation were varied. Extinction of transgene expression was investigated by using both in vivo imaging of infrared fluorescent "Katushka" and erythropoietin evaluated by ELISA and hemoglobin. Histology was performed. Electrotransfer of Katushka and erythropoietin yielded significant expression. Maximal calcium uptake occurred after injection of Ca²⁺ before electropulsing using eight high voltage pulses of 1000V/cm. Using these parameters, in vivo imaging showed that transgene expression significantly decreased 4hr after Ca²⁺ electrotransfer and was eliminated within 24hr. Similarly, serum erythropoietin was reduced by 46% at 4hr and to control levels at 2 days. Histological analyses showed muscle damage and subsequent regeneration. Electrotransfer of isotonic CaCl₂ terminates transgenic protein expression in muscles and may be used for contingency elimination of transgene expression.