Sensitive and precise regulation of haemoglobin after gene transfer of erythropoietin to muscle tissue using electroporation

Electroporation-based gene transfer (electro gene transfer (EGT)) is gaining increasing momentum, in particular for muscle tissue, where long-term high-level expression is obtainable. Induction of expression using the Tet-On system was previously established; however, attempts to reach a predefined target dose - a prescription, have not been reported. We set three target haemoglobin levels (10, 12 and 14 mmol/l, base level was 8.2 mmol/l) and aimed at them by transferring the erythropoietin (EPO) gene to mouse tibialis cranialis (TC) muscle, and varying (1) DNA amount, (2) muscle mass transfected and (3) induction with the Tet-On system. Results showed that (a) using GFP, luciferase and EPO low DNA amounts were needed. In fact, 0.5 mu g of DNA to one TC muscle led to significant Hgb elevation - this amount extrapolates to 1.4 mg of DNA in humans, (b) three prescribers hit the targets with average Hgb of 10.5, 12.0 and 13.7 mmol/l, (c) different approaches could be used, (d) undershooting could be corrected by retransferring, and (e) overshooting could be alleviated by reducing dose of inducer (doxycycline (dox)). In conclusion, this study shows that using EGT to muscle, a preset level of protein expression can be reached. This is of great interest for future clinical use.