title: Long-term doxycycline-regulated secretion of erythropoietin by encapsulated myoblasts.
abstract: 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.
authors.affiliation: Division of Surgical Research and Gene Therapy Center, Lausanne Medical School, Lausanne, Switzerland.
authors.forename: Birgit
authors.initials: B
authors.lastname: Sommer
authors.forename: Christopher
authors.initials: C
authors.lastname: Rinsch
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Payen
authors.forename: Bruno
authors.initials: B
authors.lastname: Dalle
authors.forename: Bernard
authors.initials: B
authors.lastname: Schneider
authors.forename: Nicole
authors.initials: N
authors.lastname: Déglon
authors.forename: Annie
authors.initials: A
authors.lastname: Henri
authors.forename: Yves
authors.initials: Y
authors.lastname: Beuzard
authors.forename: Patrick
authors.initials: P
authors.lastname: Aebischer
mesh_terms: 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
chemical_list: D011994:Recombinant Proteins; D004921:Erythropoietin; D004318:Doxycycline