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

Stimulating angiogenesis by gene transfer approaches offers the hope of treating tissue ischemia which is untreatable by currently practiced techniques of vessel grafting and bypass surgery. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2) are potent angiogenic molecules, making them ideal candidates for novel gene transfer protocols designed to promote new blood vessel growth. In this study, an ex vivo gene therapy approach utilizing cell encapsulation was employed to deliver VEGF and FGF-2 in a continuous and localized manner. C(2)C(12) myoblasts were genetically engineered to secrete VEGF(121), VEGF(165) and FGF-2. These cell lines were encapsulated in hollow microporous polymer membranes for transplantation in vivo. Therapeutic efficacy was evaluated in a model of acute skin flap ischemia. Capsules were positioned under the distal, ischemic region of the flap. Control flaps showed 50% necrosis at 1 week. Capsules releasing either form of VEGF had no effect on flap survival, but induced a modest increase in distal vascular supply. Delivery of FGF-2 significantly improved flap survival, reducing necrosis to 34.2% (P < 0.001). Flap vascularization was significantly increased by FGF-2 (P < 0.01), with numerous vessels, many of which had a large lumen diameter, growing in the proximity of the implanted capsules. These results demonstrate that FGF-2, delivered from encapsulated cells, is more efficacious than either VEGF(121) or VEGF(165) in treating acute skin ischemia and improving skin flap survival. Furthermore, these data attest to the applicability of cell encapsulation for the delivery of angiogenic factors for the treatment and prevention of tissue ischemia.

Inducing host acceptance to encapsulated xenogeneic myoblasts.

BACKGROUND: Cell encapsulation holds promise for the chronic delivery of recombinant proteins such as erythropoietin. Encapsulated xenogeneic mouse C2C12 myoblasts display long-term survival in the central nervous system whereas they do not in the subcutaneous tissue, suggesting that encapsulation only partially prevents affector and effector mechanisms of the host immune response. Transient immunosuppression with FK506 at the time of subcutaneous implantation leads, however, to their long-term survival. The nature of this acceptance was further investigated in this report. METHODS: Fischer rats were rendered unresponsive to encapsulated murine C2C12 myoblasts secreting mouse erythropoietin by either a 1- or 4-week initial treatment of FK506. To examine the extent of xenograft acceptance, animal were challenged with a second implant 9 weeks after the initial implantation. RESULTS: Challenging animals treated only 1 week with FK506 led to rejection of both primary and secondary implants. Animals administered FK506 for 4 weeks accepted both implants over the period investigated. However, these animals rejected unencapsulated xenogeneic cells injected at a later time, highlighting the requirement of the polymer membrane for immune protection. Developed unresponsiveness to encapsulated xenogeneic myoblasts lasted over extended periods (at least 7 months), in the absence of both immunosuppression and stimulating xenoantigens. CONCLUSIONS: These findings reveal that host acceptance of encapsulated but not unencapsulated xenogeneic myoblasts can be developed in the subcutaneous tissue after transient FK506 immunosuppression. This may have direct clinical relevance as it enables capsules to be replaced without additional immunosuppression, facilitating long-term cell-based therapies.

Long-term host unresponsiveness to encapsulated xenogeneic myoblasts after transient immunosuppression.

BACKGROUND: Encapsulating cells prevents the immune destruction of allogeneic cells in the subcutaneous site as well as allogeneic and xenogeneic cells in the central nervous system. However, when encapsulated xenogeneic cells are implanted s.c., they may be subject to rejection by the host. METHODS: Murine C2C12 myoblasts engineered to secrete mouse erythropoietin (mEpo) were used to evaluate the response of control versus FK506-treated xenogeneic recipients (Fischer rats) to encapsulated myoblasts implanted in the s.c. site. RESULTS: Encapsulated C2C12 mEpo cells were rapidly eliminated in immunocompetent Fischer rats. Devices transplanted into nude rats induced a sustained increase in the hematocrit, associated with an extended viability of the encapsulated cells. Short-term immunosuppression with FK506, for periods lasting either 1, 2, or 4 weeks after implantation, permitted the long-term survival of encapsulated C2C12 mEpo cells in Fischer rats. Animals increased their hematocrits to more than 70% and maintained these levels for 13 weeks, independent of the duration of FK506 treatment. Unencapsulated C2C12 mEpo cells injected i.m. in immunosuppressed animals were rejected over this same period. CONCLUSIONS: Encapsulation alone cannot protect xenogeneic myoblasts from immune destruction in the s.c. site. These results highlight the importance of combining the technique of cell encapsulation with transient immunosuppression to achieve long-term survival of xenografted myoblasts in a peripheral immunoreactive site.

Lentivirus-mediated Bcl-2 expression in betaTC-tet cells improves resistance to hypoxia and cytokine-induced apoptosis while preserving in vitro and in vivo control of insulin secretion.

betaTC-tet cells are conditionally immortalized pancreatic beta cells which can confer long-term correction of hyperglycemia when transplanted in syngeneic streptozocin diabetic mice. The use of these cells for control of type I diabetes in humans will require their encapsulation and transplantation in non-native sites where relative hypoxia and cytokines may threaten their survival. In this study we genetically engineered betaTC-tet cells with the anti-apoptotic gene Bcl-2 using new lentiviral vectors and showed that it protected this cell line against apoptosis induced by hypoxia, staurosporine and a mixture of cytokines (IL-1beta, IFN-gamma and TNF-alpha). We further demonstrated that Bcl-2 expression permitted growth at higher cell density and with shorter doubling time. Expression of Bcl-2, however, did not inter- fere either with the intrinsic mechanism of growth arrest present in the betaTC-tet cells or with their normal glucose dose-dependent insulin secretory activity. Furthermore, Bcl-2 expressing betaTC-tet cells retained their capacity to secrete insulin under mild hypoxia. Finally, transplantation of these cells under the kidney capsule of streptozocin diabetic C3H mice corrected hyperglycemia for several months. These results demonstrate that the murine betaTC-tet cell line can be genetically modified to improve its resistance against different stress-induced apoptosis while preserving its normal physiological function. These modified cells represent an improved source for cell transplantation therapy of type I diabetes.

A gene therapy approach to regulated delivery of erythropoietin as a function of oxygen tension.

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.