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

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

Delivery of erythropoietin by encapsulated myoblasts in a genetic model of severe anemia.

BACKGROUND: Existing animal models of anemia inadequately reflect the hematocrit usually present in chronic renal failure (CRF) patients and do not permit long-term treatment studies. The transgenic mouse strain 134.3LC (Epo-TAg(H)) displays a severe chronic anemia resembling that observed clinically during CRF, while displaying an active, normal life span. This phenotype makes it a particularly interesting mouse model for testing erythropoietin (Epo)-based gene transfer strategies. METHODS: Ex vivo gene therapy was employed to administer mouse Epo to homozygous anemic Epo-TAg(H) mice. Encapsulated C(2)C(12) myoblasts genetically engineered to secrete 163 IU mouse Epo/10(6) cells/day were subcutaneously transplanted on the dorsal flank of the mice. Efficacy of delivered Epo was monitored by weekly measurements of animal hematocrit. RESULTS: Most treated homozygous Epo-TAg(H) mice displayed only a transient rise in hematocrit before eventually decreasing to levels as low as 3%. Administering the immunosuppressor anti-CD4+ monoclonal antibody (mAb) to homozygous Epo-TAg(H) mice, beginning at the time of implantation, permitted a rise in hematocrit that remained stable at elevated levels in cases of continued immunosuppression. CONCLUSIONS: Mice having the T antigen insertion in both Epo alleles appeared to develop an immune response to the natural mouse Epo delivered by encapsulated cells. By preventing this reaction using immunosuppression, we demonstrate that encapsulated myoblasts can deliver therapeutic doses of mouse Epo systemically and restore hemopoiesis in a genetic model of severe anemia

Mitochondria! function is impaired in the skeletal muscle of pre-frail elderly

Aging is accompanied by a gradual decline in both muscle mass and strength over time, which can eventually lead to pathologies, such as frailty and sarcopenia. While these two conditions are well characterized, further investigation of the early biological signs present in pre-frail elderly is still needed to help identify strategies for preventative therapeutic intervention. The goal of the present clinical study was to evaluate the level of mitochondrial (dys)function in a well-defined population of pre-frail elderly (>60 years of age). Pre-frail elderly were compared with an age-matched population of active elderly. Muscle mitochondrial function was assessed in vivo using phosphorus magnetic resonance spectroscopy (31P-MRS) and a comprehensive set of biological biomarkers were measured ex vivo in vastus lateralis muscle biopsies. In pre-frail subjects, phosphocreatine recovery was impaired and mitochondrial respiratory complex protein and activity levels were significantly lower when compared with active elderly. Analysis of microarray data showed that mitochondrial genes were also significantly down-regulated in muscle of pre-frail compared to active elderly. These results show that mitochondrial impairment is a hallmark of pre-frailty development and the onset of decline in muscle function in the elderly

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

Long-term doxycycline-regulated secretion of erythropoietin by encapsulated myoblasts

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

Delivery of erythropoietin by encapsulated myoblasts in a genetic model of severe anemia

BACKGROUND: Existing animal models of anemia inadequately reflect the hematocrit usually present in chronic renal failure (CRF) patients and do not permit long-term treatment studies. The transgenic mouse strain 134.3LC (Epo-TAg(H)) displays a severe chronic anemia resembling that observed clinically during CRF, while displaying an active, normal life span. This phenotype makes it a particularly interesting mouse model for testing erythropoietin (Epo)-based gene transfer strategies. METHODS: Ex vivo gene therapy was employed to administer mouse Epo to homozygous anemic Epo-TAg(H) mice. Encapsulated C(2)C(12) myoblasts genetically engineered to secrete 163 IU mouse Epo/10(6) cells/day were subcutaneously transplanted on the dorsal flank of the mice. Efficacy of delivered Epo was monitored by weekly measurements of animal hematocrit. RESULTS: Most treated homozygous Epo-TAg(H) mice displayed only a transient rise in hematocrit before eventually decreasing to levels as low as 3%. Administering the immunosuppressor anti-CD4+ monoclonal antibody (mAb) to homozygous Epo-TAg(H) mice, beginning at the time of implantation, permitted a rise in hematocrit that remained stable at elevated levels in cases of continued immunosuppression. CONCLUSIONS: Mice having the T antigen insertion in both Epo alleles appeared to develop an immune response to the natural mouse Epo delivered by encapsulated cells. By preventing this reaction using immunosuppression, we demonstrate that encapsulated myoblasts can deliver therapeutic doses of mouse Epo systemically and restore hemopoiesis in a genetic model of severe anemia