Long-term doxycycline-regulated secretion of erythropoietin by encapsulated myoblasts

We developed an ex vivo gene therapy approach for the regulated delivery of therapeutic proteins based on the implantation of encapsulated, genetically engineered C2C12 myoblasts. We investigated doxycycline-based regulation of gene expression to modulate the secretion of erythropoietin (EPO) from encapsulated myoblasts in a mouse model. An autoregulatory tet-off system provided high induction levels with low basal expression in the noninduced state. Stable C2C12 clones constitutively secreted between 25 and 50 IU mouse EPO/10(6)cells/24 hours in the on-state. The clone C15, selected for its in vivo survival characteristics, displayed a desirable secretion profile when encapsulated. Devices released 5 IU EPO per capsule in the on-state, with EPO levels being undetectable upon the addition of doxycycline (dox). Capsules subcutaneously implanted in DBA/2J mice demonstrated a tightly regulated secretion of EPO through up to four on-off cycles during a period lasting 40 weeks. Hematocrits could be modulated between basal levels (40-50%) and elevated levels (70-90%) through the presence or absence of dox in the drinking water. Hematocrit returned to normal levels, paralleling the kinetics observed following capsule explantation, 6 to 8 weeks following dox administration to polycythemic mice. The results of this study suggest that encapsulation and implantation of a tet-off regulated C2C12 cell clone represents a safe method for the controlled long-term delivery of proteins in vivo.