Sustained delivery of erythropoietin in mice by genetically modified skin fibroblasts.

Abstract

We have examined whether the secretion of erythropoietin (Epo) from genetically modified cells could represent an alternative to repeated injections of the recombinant hormone for treating chronic anemias responsive to Epo. Primary mouse skin fibroblasts were transduced with a retroviral vector in which the murine Epo cDNA is expressed under the control of the murine phosphoglycerate kinase promoter. "Neo-organs" containing the genetically modified fibroblasts embedded into collagen lattices were implanted into the peritoneal cavity of mice. Increased hematocrit (>80%o) and elevated serum Epo concentration (ranging from 60 to 408 milliunits/ml) were observed in recipient animals over a 10-month observation period. Hematocrit values measured in recipient mice varied according to the number of implanted Epo-secreting fibroblasts (ranging from 2.5 to 20 x 106). The implantation of neo-organs containing Eposecreting fibroblasts appeared, therefore, as a convenient method to achieve permanent in vivo delivery of the hormone. We estimated that the biological efficacy of the approach may be relevant for the treatment of human hemoglobinopathies.

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

“…Several studies have shown long-term expression of recombinant Epo, following the transduction of autologous fibroblasts 18 or myoblasts 20 with retroviral vectors or direct injections of either adenoviral vectors [22][23][24] or adenoassociated viral vector. 25 However, until now, only ex vivo transduction of hematopoietic cells, genetically modified by a retroviral vector, has been shown to increase durably hematocrit and to improve the red cell phenotype of ␤-thalassemic mice.…”

“…C2C12 myoblasts were engineered to secrete high levels of mEpo by transfection with pPImEpo-Neo-DHFR vector described previously, 18 in which hEpo cDNA was replaced by mEpo cDNA. Cells were encapsulated in microporous polyethersulfone hollow fibers (10 mm × 0.6 mm), 18,26 and differentiated by placing the capsules in 10% Prolifix (BioMedia, Boussens, France) differentiation medium for 3 days. Each capsule was loaded with 2 × 10 5 cells.…”

“…17 However, the necessary ablation of the hematopoietic stem cells of the recipient mice and the unpredictable in vivo expression of the transgene constitute major drawbacks. Fibroblasts, genetically modified to express recombinant Epo, 18 failed to improve the anemia in homozygous ␤-thalassemic mice (unpublished results).…”

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

“…Cells engineered to secrete one or several angiogenic proteins could be introduced at the site of ischemia to stimulate new vessel growth. Myoblasts 21,22 and fibroblasts 23 have been employed in the past to systemically deliver proteins such as erythropoietin and human growth hormone by direct cell transplantation. Ideally, for an angiogenesisbased therapy, it would be desirable to halt treatment after ischemic tissue is revascularized, as prolonged exposure to angiogenic factors could be deleterious.…”

Sustained gene expression in transplanted skin fibroblasts in rats

“…[23][24][25] Retrovirus-mediated gene transfer into primary skin fibroblasts has provided measurable amounts of therapeutic proteins in animal models, including adenosine deaminase (ADA), 26,27 factor IX, 28,29 ␤-glucuronidase 30,31 and erythropoietin. 32 A major problem emerging from experiments using skin equivalent grafts was the limited time of vector-encoded gene expression once transduced cells were implanted in vivo. We have shown in both rats and dogs that vector-derived gene expression was diminished at between 3 and 4 weeks despite the long-term presence of vector sequences in transplanted cells.…”