A Gene Therapy Approach to Regulated Delivery of Erythropoietin as a Function of Oxygen Tension

Abstract

Current therapy for several forms of anemia involves a weekly regime of multiple subcutaneous injections of recombinant human erythropoietin (hEpo). In an effort to provide a physiologically regulated administration of erythropoietin, we are developing cell lines genetically engineered to release hEpo as a function of oxygen tension. C2C12 cells were transfected using a vector containing the hEpo cDNA driven by the hypoxia-responsive promoter to the murine phosphoglycerate kinase gene. In vitro, these cells showed a threefold increase in hEpo secretion as oxygen levels were shifted from 21% to 1.3% oxygen. To test in vivo response, C2C12-hEpo cells were encapsulated in a microporous membrane and implanted subcutaneously on the dorsal flank of DBA/2J mice. On average, serum hEpo levels in animals exposed to 7% oxygen were two-fold higher than values seen in their control counterparts kept at 21% oxygen. Similar studies employing rats confirmed that hEpo delivery is regulated as a function of oxygen tension. These results suggest the feasibility of developing an oxygen-regulated, encapsulated cell-based system for hEpo delivery.

Improvement of mouse β-thalassemia upon erythropoietin delivery by encapsulated myoblasts

“…19 The levels of hormone released at the explantation time are comparable with the pre-implantation values, confirming the long-term stability of in vivo transgene expression driven by the PGK-1 promoter in C2C12 cells and the stability of the transgene amplification conferred by the methotrexate amplification procedure. 19 The tissue reaction to the implant was mild as a result of adequate polymer biocompatibility and long-term immunoprotection.…”

“…Changes in cytokines and hypoxia related to the anemia may be responsible for the lower frequency of encapsulated C2C12 cell survival in ␤-thalassemic mice, in contrast to the excellent survival of similar encapsulated cells in control mice. However, the increase in hematocrit 2-4 weeks after implantation of capsules in the majority of ␤-thalassemic mice 19 suggests that the initial hypoxia may not be the major stress factor for C2C12 cell survival.…”

“…19 The expression of recombinant mEpo by encapsulated myoblasts in ␤-thalassemic mice and the effects on the ␤-thalassemic phenotype are described in this study.…”

Delivery of FGF-2 but not VEGF by encapsulated genetically engineered myoblasts improves survival and vascularization in a model of acute skin flap ischemia

“…25 The PGK promoter has also been shown to up-regulate the expression of transgenes under its control in response to hypoxia. 26 Control cells employed during the study were either unmodified C 2 C 12 myoblasts or C 2 C 12 myoblasts transfected with the empty expression vector pPI-Neo-DHFR ( Figure 1a). To obtain secretion of VEGF, C 2 C 12 myoblasts were transfected with either pPI-VEGF 121 -Neo-DHFR or pPI-VEGF 165 -Neo-DHFR constructs (Figure 1b, c).…”

“…This technique has permitted the systemic delivery of erythropoietin to animals in both a continuous and regulated fashion. [25][26][27] Cell encapsulation has also been employed to locally administer neurotrophic factors (CNTF and GDNF) locally in the central nervous system, both in pre-clinical and clinical studies, for the treatment of neurodegenerative disorders. [28][29][30] During reconstructive surgery, skin flaps are employed to cover wounds with poorly vascularized recipient beds where the damage is too extensive to permit repair using a skin graft.…”