Article: article from journal or magazin.
Long-term doxycycline-regulated secretion of erythropoietin by encapsulated myoblasts.
Publication types: In Vitro ; Journal Article ; Research Support, Non-U.S. Gov'tPublication Status: ppublish
We developed an ex vivo gene therapy approach for the regulated delivery of therapeutic proteins based on the implantation of encapsulated, genetically engineered C(2)C(12) 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 C(2)C(12) 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 C(2)C(12) cell clone represents a safe method for the controlled long-term delivery of proteins in vivo.
Animals, Cell Survival, Clone Cells, Doxycycline/pharmacology, Erythropoietin, Recombinant/genetics, Erythropoietin, Recombinant/secretion, Female, Gene Therapy/methods, Genetic Engineering, Hematocrit, Kinetics, Mice, Mice, Inbred DBA, Muscle, Skeletal/cytology, Muscle, Skeletal/drug effects
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