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 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.
Sommer
Birgit
B
Division of Surgical Research and Gene Therapy Center, Lausanne Medical School, Lausanne, Switzerland.
Rinsch
Christopher
C
Payen
Emmanuel
E
Dalle
Bruno
B
Schneider
Bernard
B
Déglon
Nicole
N
Henri
Annie
A
Beuzard
Yves
Y
Aebischer
Patrick
P
D000818:Animals; D002470:Cell Survival; D002999:Clone Cells; D004318:Doxycycline / Q000494:pharmacology; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D006400:Hematocrit; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D008811:Mice, Inbred DBA; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins
D011994:Recombinant Proteins; D004921:Erythropoietin; D004318:Doxycycline