Gene therapy for long-term expression of erythropoietin in rats.

Abstract

The injection of recombinant erythropoietin (Epo) is now widely used for long-term treatment of anemia associated with chronic renal failure, cancer, and human immunodeficiency virus infections. The ability to deliver this hormone by gene therapy rather than by repeated injections could provide substantial clinical and economic benefits. As a preliminary approach, we investigated in rats the expression and biological effects of transplanting autologous vascular smooth muscle cells transduced with a retroviral vector encoding rat Epo cDNA. Vector-derived Epo secretion caused increases in reticulocytes, with peak levels of 7.8-9.6% around day 10 after implantation. The initial elevation in reticulocytes was followed by clinically significant increases in hematocrit and hemoglobin for up to 11 weeks. Ten control and treated animals showed mean hematocrits of 44.9 ± 0.4% and 58.7 -3.1%, respectively (P < 0.001), and hemoglobin values of 15.6 ± 0.1 g/dl and 19.8 ± 0.9 g/dl, respectively (P < 0.001). There were no significant differences between control and treated animals in the number of white blood cells and platelets. Kidney and to a lesser extent liver are specific organs that synthesize Epo in response to tissue oxygenation. In the treated animals, endogenous Epo mRNA was largely down regulated in kidney and absent from liver. These results indicate that vascular smooth muscle cells can be genetically modified to provide treatment of anemias due to Epo deficiency and suggest that this cell type may be targeted in the treatment of other diseases requiring systemic therapeutic protein delivery.Erythropoietin (Epo) is a 30-kDa glycoprotein hormone that serves as the primary regulator of red cell production in mammals (1, 2). The therapeutic potential for Epo in the treatment of anemia associated with renal failure was demonstrated initially by its administration to anemic uremic rats and sheep (3, 4). The availability of recombinant human Epo provided a major advance in the treatment of anemia in renal failure patients receiving dialysis (5). The attendant dangers of transfusion therapy were eliminated and the quality of life of these patients was significantly improved. This treatment, given two or three times weekly, raises hematocrit and hemoglobin levels and improves cardiovascular status (2, 6).Adenoviral vectors have been used to achieve in vivo Epo gene transfer (7,8). Studies of Epo gene transfer using transplantation of transduced cells have included myoblasts in mice (9, 10) and smooth muscle cells in rats (11). Vascular smooth muscle cells provide an attractive target tissue for gene therapy and have been studied by several investigators (12-17). These cells are easily obtained and cultured and can be efficiently infected with replication-defective retroviral vectors and returned to the donor by seeding in natural or synthetic blood vessels (11)(12)(13)(14)18 Cell Culture, Transduction, and Transplantation. Ecotropic PE501 and amphotropic PA317 retrovirus packaging cell lines (20, 23), NIH 3T3 th...

Long-term production of erythropoietin after electroporation-mediated transfer of plasmid DNA into the muscles of normal and uremic rats

“…On the other hand, continuous Epo delivery by 400 g of pCAGGS-Epo resulted in the down-regulation of endogenous Epo induction (P = 0.42) even after phlebotomy, consistent with previous reports by us 6 and another group. 14 These data indicate that a sufficient dose of pCAGGS-Epo down-regulated the endogenous Epo production for as long as 11 weeks and that erythrocytosis induction was mediated by vector-encoded Epo secretion, although there is another possibility that increased endogenous Epo synthesis occurred in phlebotomized animals but overall levels of Epo protein, endogenous plus ectopic, were not detected because of Epo clearance or excretion. The rats were immediately killed after this phlebotomy experiment.…”

Sustained gene expression in transplanted skin fibroblasts in rats

“…33 However, we showed that the same promoters in retroviral vector constructs permitted stable gene expression for several months in transduced hematopoietic stem cells 34,35 and smooth muscle cells. 16,18,22 These data suggested tissue-specific vector inactivation. When fibroblasts are in a quiescent state the choice of promoter for the transduced gene was a major determinant for long-term in vivo gene expression.…”

“…15 Smooth muscle cells are present within the vasculature as a multilayered mass of long-lived cells in proximity to the circulation and have been investigated as targets for gene therapy. [16][17][18][19][20][21][22] In vivo gene expression from transduced keratinocyte implants has also been studied. [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.…”

Enhanced engraftment of EPO-transduced human bone marrow stromal cells transplanted in a 3D matrix in non-conditioned NOD/SCID mice

“…Other than cell death, this effect may be due to a suppression of the endogenous production of EPO. 12 However, when cells were vehicled by a 3D scaffold, the increased level of hematocrit was maintained for a much longer period of time, up to 14 weeks. Cells recovered from the implants, after the hematocrit had returned to a normal level, were able to grow in vitro and to secrete EPO although at an efficiency of 50%.…”