Impact of Erythropoietin Production by Erythroblastic Island Macrophages on Homeostatic Murine Erythropoiesis.

Erythropoietin (EPO) is an essential hormone for erythropoiesis, protecting differentiating erythroblasts against apoptosis. EPO has been largely studied in stress or pathological conditions but its regulatory role in steady state erythropoiesis has been less documented. Herein, we report production of EPO by bone marrow-derived macrophages (BMDM) in vitro, and its further enhancement in BMDM conditioned with media from apoptotic cells. Confocal microscopy confirmed EPO production in erythroblastic island (EBI)-associated macrophages, and analysis of mice depleted of EBI macrophages by clodronate liposomes revealed drops in EPO levels in bone marrow (BM) cell lysates, and decreased percentages of EPO-responsive erythroblasts in the BM. We hypothesize that EBI macrophages are an in-situ source of EPO and sustain basal erythropoiesis in part through its secretion. To study this hypothesis, mice were injected with clodronate liposomes and were supplied with exogenous EPO (1-10 IU/mouse) to evaluate potential rescue of the deficiency in erythroid cells. Our results show that at doses of 5 and 10 IU, EPO significantly rescues BM steady state erythropoiesis in mice deficient of macrophages. We propose existence of a mechanism by which EBI macrophages secrete EPO in response to apoptotic erythroblasts, which is in turn controlled by the numbers of erythroid precursors generated.

Delivery of human erythropoietin gene with an adeno-associated virus vector through parotid glands to treat renal anaemia in a swine model.

Anaemia is a common complication of chronic kidney disease, for which there is presently no adequate treatment. The delivery of human erythropoietin (hEPO) cDNA to salivary glands reportedly increases red blood cell counts, haematocrit (HCT) and haemoglobin concentration, representing a potential new method of renal anaemia treatment. However, no studies have examined the effects of this method in an animal model of renal anaemia. Here we established a miniature pig animal model of renal anaemia through continuous feeding with adenine. In these animals, we delivered the AAV2hEPO gene to the parotid glands through Stensen's duct. As a control, we transferred AAVLacZ. Enzyme-linked immunosorbent assay was used to detect hEPO in serum and saliva. Red blood counts and serum biochemistry were used to evaluate how hEPO gene administration affected renal anaemia. Compared with the control group, we found increased hEPO concentrations in parotid saliva and serum, respectively, at 2 and 6 weeks after AAV2hEPO administration to the anaemic animals. HCT and haemoglobin were also increased after AAV2hEPO was delivered; most serum indicators of renal damage were not changed over the time span of the experiment, suggesting the adenine-induced kidney damage had not been completely reversed. However, blood urea nitrogen and B2 microglobulin levels showed small but significant improvement. Overall, our present findings suggest that adeno-associated virus 2 (AAV2)-mediated gene transduction of hEPO via the parotid gland is a promising potential alternative therapy for renal anaemia.

Evaluation of the combined effects of pegylation and glycosylation on the stability of erythropoietin using a stability-indicating SE-HPLC.

Recombinant human erythropoietin (rhEPO) is a commonly used biopharmaceutical for the treatment of anemia-associated disorders. Epogen; glycosylated erythropoietin (G-EPO) has short half-life and poor stability. Pegylated Epogen (Peg-G-EPO) was introduced to the market to overcome these limitations. The combined effects of pegylation and glycosylation on the stability of Peg-G-EPO was studied. Determination of Peg-G-EPO in the presence of its degradation products was achieved using SE-HPLC. The assay was validated according to ICH guidelines over concentration range of 50.00-320.00 µg/mL (r 0.9999). A mobile phase of 50 mM phosphate buffer (pH 6.5) with 300 mM sodium chloride and 20% ethanol was employed. Isocratic elution was carried out at 0.5 mL/min over run time of 30 min. Peg-G-EPO was found stable towards mechanical agitation only at low concentrations while it was stable towards repeated freeze/thaw; regardless of the concentration. Effect of temperature and pH were also investigated and Peg-G-EPO was found stable within narrow ranges. Results indicated formation of small molecular weight and very high molecular weight aggregates that have been filtered-off the column. Although Peg-G-EPO was found relatively more stable than its non-pegylated but glycosylated version, results indicated the need for careful stability-assessment of Peg-G-EPO.

Delivery of hypoxia and glioma dual-specific suicide gene using dexamethasone conjugated polyethylenimine for glioblastoma-specific gene therapy.

Gene therapy has been considered a promising approach for glioblastoma therapy. To avoid side effects and increase the specificity of gene expression, gene expression should be tightly regulated. In this study, glioma and hypoxia dual-specific plasmids (pEpo-NI2-SV-Luc and pEpo-NI2-SV-HSVtk) were developed by combining the erythropoietin (Epo) enhancer and nestin intron 2 (NI2). In the in vitro studies, pEpo-NI2-SV-Luc showed higher gene expression under hypoxia than normoxia in a glioblastoma-specific manner. The MTT and caspase assays demonstrated that pEpo-NI2-SV-HSVtk specifically induced caspase activity and cell death in hypoxic glioblastoma cells. For in vivo evaluation, subcutaneous and intracranial glioblastoma models were established. Dexamethasone-conjugated-polyethylenimine (PEI-Dexa) was used as a gene carrier, since PEI-Dexa efficiently delivers plasmid to glioblastoma cells and also has an antitumor effect due to the effect of dexamethasone. In the in vivo study in the subcutaneous and intracranial glioblastoma models, the tumor size was reduced more effectively in the pEpo-NI2-SV-HSVtk group than in the control and pSV-HSVtk groups. In addition, higher levels of HSVtk gene expression and TUNEL-positive cells were observed in the pEpo-NI2-SV-HSVtk group compared with the control and pSV-HSVtk groups, suggesting that pEpo-NI2-SV-HSVtk increased the therapeutic efficacy in hypoxic glioblastoma. Therefore, pEpo-NI2-SV-HSVtk/PEI-Dexa complex may be useful for glioblastoma-specific gene therapy.

Bringing the science of proteins into the realm of organic chemistry: total chemical synthesis of SEP (synthetic erythropoiesis protein).

Erythropoietin, commonly known as EPO, is a glycoprotein hormone that stimulates the production of red blood cells. Recombinant EPO has been described as "arguably the most successful drug spawned by the revolution in recombinant DNA technology". Recently, the EPO glycoprotein molecule has re-emerged as a major target of synthetic organic chemistry. In this article I will give an account of an important body of earlier work on the chemical synthesis of a designed EPO analogue that had full biological activity and improved pharmacokinetic properties. The design and synthesis of this "synthetic erythropoiesis protein" was ahead of its time, but has gained new relevance in recent months. Here I will document the story of one of the major accomplishments of synthetic chemistry in a more complete way than is possible in the primary literature, and put the work in its contemporaneous context.

Convenient and reproducible in vivo gene transfer to mouse parotid glands.

OBJECTIVES: Published studies of gene transfer to mouse salivary glands have not employed the parotid glands. Parotid glands are the likely target tissue for most clinical applications of salivary gene transfer. The purpose of the present study was to develop a convenient and reproducible method of retroductal gene transfer to mouse parotid glands. METHODS: The volume for vector delivery was assessed by infusion of Toluidine Blue into Stensen's ducts of Balb/c mice after direct intraoral cannulation. Recombinant, serotype 5 adenoviral vectors, encoding either firefly luciferase or human erythropoietin (hEpo), were constructed and then administered to parotid glands (10(7) vector particles/gland). Transgene expression in vivo was measured by enzyme activity (luciferase) or an enzyme-linked immunosorbent assay (hEpo). Vector biodistribution was measured by real-time quantitative (Q) PCR. RESULTS: The chosen volume for mouse parotid vector delivery was 20µL. Little vector was detected outside of the targeted glands, with both QPCR and luciferase assays. Transgene expression was readily detected in glands (luciferase, hEpo), and serum and saliva (hEpo). Most secreted hEpo was detected in saliva. CONCLUSION: These studies show that mouse parotid glands can be conveniently and reproducibly targeted for gene transfer, and should be useful for pre-clinical studies with many murine disease models.

Epo delivery by genetically engineered C2C12 myoblasts immobilized in microcapsules.

ver the last half century, the use of erythropoietin (Epo) in the management of malignancies has been extensively studied. Originally viewed as the renal hormone responsible for red blood cell production, many recent in vivo and clinical approaches demonstrate that various tissues locally produce Epo in response to physical or metabolic stress. Thus, not only its circulating erythrocyte mass regulator activity but also the recently discovered nonhematological actions are being thoroughly investigated in order to fulfill the specific Epo delivery requirements for each therapeutic approach.

Topological transformation of liposomes by a membrane-affecting domain of recombinant human erythropoietin.

Recombinant human erythropoietin (rh-Epo) is well accepted as a hematopoietic drug, but many other pleiotropic properties are currently under investigation. Rh-Epo-induced receptor-mediated signal transductions are accompanied with membrane dynamic processes, which facilitate the activation of individual pathways. However, its direct effect on membrane dynamics is still unknown. In the present study, we have proven the capability of rh-Epo to associate to and transform artificial lipid membranes. Association studies using neutral, negatively, and positively charged liposomes with the native as well as modified rh-Epo were performed and analyzed by transmission electron microscopy and differential scanning calorimetry. By these studies, we demonstrated that rh-Epo has the capability to transform negatively charged unilamellar vesicles into so-called disc-like micelles. Rh-Epo association to the negatively charged head groups via lysine and arginine initiates this transformation. At physiological temperatures, conformational changes within the rh-Epo structure expose a defined amino-acid sequence, which is able to induce the formation of discoid membrane structures. Enzymatic digestion, analysis, and isolation of related peptides by rp-HPLC and characterization by MS/MS enabled the identification of the membrane-affecting domain of rh-Epo (MAD-E) that represents the exposed helix B of rh-Epo. Finally, association studies performed with these peptides confirmed that the MAD-E is responsible for the formation of disc-like micelles. Since this helix B of rh-Epo has recently been supposed to be involved in the activation of neuroprotective pathways, we believe that the membrane-transforming capacity of rh-Epo participates in the proliferative activity of rh-Epo.

A retrievable implant for the long-term encapsulation and survival of therapeutic xenogeneic cells.

The long-term function of transplanted therapeutic cells typically requires systemic immune suppression. Here, we show that a retrievable implant comprising a silicone reservoir and a porous polymeric membrane protects human cells encapsulated in it after implant transplantation in the intraperitoneal space of immunocompetent mice. Membranes with pores 1 µm in diameter allowed host macrophages to migrate into the device without the loss of transplanted cells, whereas membranes with pore sizes <0.8 µm prevented their infiltration by immune cells. A synthetic polymer coating prevented fibrosis and was necessary for the long-term function of the device. For >130 days, the device supported human cells engineered to secrete erythropoietin in immunocompetent mice, as well as transgenic human cells carrying an inducible gene circuit for the on-demand secretion of erythropoietin. Pancreatic islets from rats encapsulated in the device and implanted in diabetic mice restored normoglycaemia in the mice for over 75 days. The biocompatible device provides a retrievable solution for the transplantation of engineered cells in the absence of immunosuppression.

Therapy of anemia in kidney failure, using plasmid encoding erythropoietin.

Numerous studies using erythropoietin (EPO) gene delivery vectors, either viral or nonviral, have shown uncontrolled EPO expression leading to transient or sustained erythrocytosis and, more recently, severe autoimmune anemia. Therefore, there is a need to develop other EPO gene delivery systems that allow sustained and adjustable expression of EPO. We have examined a new approach of delivering plasmid encoding mouse EPO cDNA into mouse skeletal muscle, using an amphiphilic block copolymer. Repeated injections of low doses of block copolymer-EPOcDNA formulations increased hematocrit in a dose-dependent manner for more than 9 months, without any initial overshoot. Low doses of block copolymer-EPOcDNA formulations prevented autoimmune anemia in immunocompetent Swiss mice and prevented or reversed chronic anemia in an acquired mouse model of renal failure. We conclude that repeated injections of low doses of block copolymer-DNA formulations that do not induce (1) inflammation at the injection site, (2) overexpression of EPO, or (3) the production of anti-EPO neutralizing auto-antibodies hold promise for in vivo expression of therapeutic proteins, in particular for systemic delivery.

A "top-down" approach to actuate poly(amine-co-ester) terpolymers for potent and safe mRNA delivery.

Gene delivery is known to be a complicated multi-step biological process. It has been observed that subtle differences in the structure and properties of polymeric materials used for gene delivery can lead to dramatic differences in transfection efficiency. Therefore, screening of properties is pivotal to optimizing the polymer. So far, most polymeric materials are built in a "bottom-up" manner, i.e. synthesized from monomers that allow modification of polymer composition or structural factors. With this method, we previously synthesized and screened a library of biodegradable poly(amine-co-ester) (PACE) terpolymers for optimized DNA delivery. However, it can be tedious and time consuming to synthesize a polymer library for screening, particularly when small changes of a factor need to be tested, when multiple factors are involved, and when the effects of different factors are synergistic. In the present work, we evaluate the potential of PACE to deliver mRNA. After observing that mRNA transfection efficiency was highly dependent on both end group composition and molecular weight (MW) of PACE in a synergistic manner, we developed a "top-down" process we called actuation, to simultaneously vary these two factors. Some of the actuated PACE (aPACE) materials presented superior mRNA delivery properties compared to regular PACE, with up to a 106-fold-increase in mRNA transfection efficiency in vitro. Moreover, when aPACE was used to deliver mRNA coding for erythropoietin (EPO) in vivo, it produced high levels of EPO in the blood for up to 48 h without inducing systemic toxicity. This polymer constitutes a new delivery vehicle for mRNA-based treatments that provides safe yet potent protein production.

Gender differences in serotype 2 adeno-associated virus biodistribution after administration to rodent salivary glands.

Salivary glands (SGs) have proven useful targets for clinical applications of gene therapeutics. In this toxicology and biodistribution study, which conforms to U.S. Food and Drug Administration Good Laboratory Practice regulations, four doses (10(7)-10(10) particles) of a serotype 2 adeno-associated viral (AAV2) vector encoding human erythropoietin were directly administered to the right submandibular gland of male and female BALB/c mice (n = 21 per gender dose group). Control-treated (saline administered; n = 66) and vector-treated (n = 168) animals did not differ in clinical appearance, morbidity and mortality rates, food and water consumption, weight gain ratios, and final weight. Clinical hematology values also were unaffected by AAV2 administration except for parameters influenced by the expression of the recombinant protein (e.g., hematocrit). Mice were killed on days 3, 30, 55, and 92. No major vector-related toxicity was uncovered after complete pathology and histopathology review. However, a significant gender-related difference in vector biodistribution was revealed by quantitative polymerase chain reaction. In male mice vector (group receiving 10(10) particles/animal) effectively transduced, and was primarily confined within, the SGs (i.e., approximately 800 times more copies in SGs than in liver; day 3) and long lived. In contrast, in female mice, SG transduction was less efficient (260-fold less than in males; day 3) and short lived, and vector was disseminated widely via both the bloodstream (SG:liver copy ratio, approximately 1) and saliva (30-fold greater than in males). The observed vector biodistribution is likely due to differences in AAV2 receptor targets and structural differences affecting SG integrity. Sexual dimorphism is a factor of major significance that could potentially affect gene therapy clinical applications in SGs.

Hypoxia-regulated expression of attenuated diphtheria toxin A fused with hypoxia-inducible factor-1alpha oxygen-dependent degradation domain preferentially induces apoptosis of hypoxic cells in solid tumor.

Tumor cells in hypoxic areas of solid tumors are resistant to conventional chemotherapy and radiotherapy and thus are obstacles of cancer therapy. We report here the feasibility of applying hypoxia-regulated expression of diphtheria toxin A (DT-A) for killing hypoxic tumor cells. The expression vector was constructed to express DT-A fused with hypoxia-inducible factor-1alpha (HIF-1alpha) oxygen-dependent degradation (ODD) domain under the control of vascular endothelial growth factor gene promoter and contain erythropoietin mRNA-binding protein (ERBP)-binding sequence downstream of the DT-A/ODD sequence. In vitro ubiquitination assay showed that DT-A/ODD, but not DT-A, was ubiquitinated as efficient as HIF-1alpha under normoxic conditions in a von Hippel-Lindau- and oxygen-dependent manner. DT-A/ODD exhibited a comparable translation inhibitory activity to DT-A. ERBP-binding sequence was effective in stabilizing mRNA under hypoxic conditions in various cell types. Transfection of the vector expressing DT-A/ODD into high-metastatic Lewis lung carcinoma (3LL) A11 cells resulted in induction of apoptosis independently of hypoxia, probably due to its extreme toxicity. However, transfection of the vector expressing attenuated DT-A(W153F)/ODD or DT-A(H21A)/ODD resulted in a hypoxia-dependent induction of apoptosis. Liposomal gene transfer of the vector encoding DT-A(W153F)/ODD induced apoptosis in hypoxic, but not in normoxic, areas of solid tumors established by A11 variant cells with higher resistance to hypoxia-induced apoptosis and inhibited the growth of hypoxic tumors established by 3LL-P29 cells. These results suggest that hypoxia-regulated expression of attenuated DT-A(W153F)/ODD fusion protein is potentially of use for killing hypoxic tumor cells with minimizing the damage to normoxic normal tissues.

Recent advances in erythropoietic agents in renal anemia.

Red cell production in chronic kidney disease is usually too low to maintain a normal haemoglobin, and thus anaemia develops in a large proportion of patients. The ability to stimulate erythropoiesis in the bone marrow by the use of therapeutic agents has only been possible in the last 20 years, initially with recombinant human erythropoietin (epoetin), and later darbepoetin alfa. Many new agents are, however, in clinical development, and these include CERA, Hematide, and HIF stabilisers, in addition to the imminent launch of biosimilar epoetins. The main issue with biosimilars is the unknown risk of immunogenicity. CERA is a large molecule, approximately twice the size of epoetin, which was created by integrating a single polymer chain into the erythropoietin molecule. CERA has a much prolonged half-life, and Phase II and III clinical trials have investigated administration of CERA every 3 or 4 weeks. Hematide is derived from original research on the erythropoietin-mimetic peptides, and is in Phase II of its clinical trial programme. Again, this compound is being investigated as a once-monthly administration. The HIF stabilizers are orally-active inhibitors of the enzyme that degrades hypoxia-inducible factor (prolyl hydroxylase), and this leads to upregulation of erythropoietin gene expression. Other strategies for stimulating erythropoiesis, briefly described in this review, are at an earlier stage of development. This is an exciting and rapidly developing area of scientific and translational research.

Computational and nonglycosylated systems: a simpler approach for development of nanosized PEGylated proteins.

Cysteine PEGylation includes several steps, and is difficult to manage in practice. In the current investigation, the cysteine PEGylation of erythropoietin analogs was examined using computational and nonglycosylated systems to define a simpler approach for specific PEGylation. Two model analogs (E31C and E89C) were selected for PEGylation based on lowest structural deviation from the native form, accessibility, and nucleophilicity of the free thiol group. The selected analogs were cloned and the expression was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot using Coomassie blue staining and anti-His monoclonal antibody, respectively. PEGylation with 20 kDa mPEG-maleimide resulted in 79% and 82% conjugation yield for E31C and E89C nonglycosylated erythropoietin (ngEPO) analogs, respectively. The size distribution and charge analysis showed an increase in size and negative charge of the PEGylated forms compared with nonconjugated ones. Biological assay revealed that E31C and E89C mutations and subsequent PEGylation of ngEPO analogs have no deleterious effects on in vitro biological activity when compared to CHO-derived recombinant human erythropoietin. In addition, PEG-conjugated ngEPOs showed a significant increase in plasma half-lives after injection into rats when compared to nonconjugated ones. The development of the cysteine-PEGylated proteins using nonglycosylated expression system and in silico technique can be considered an efficient approach in terms of optimization of PEGylation parameters, time, and cost.

Stabilization of bacterially expressed erythropoietin by single site-specific introduction of short branched PEG chains at naturally occurring glycosylation sites.

The covalent attachment of polyethylene glycol (PEG) to therapeutic proteins can improve their physicochemical properties. In this work we utilized the non-natural amino acid p-azidophenylalanine (pAzF) in combination with the chemoselective Staudinger-phosphite reaction to install branched PEG chains to recombinant unglycosylated erythropoietin (EPO) at each single naturally occurring glycosylation site. PEGylation with two short 750 or 2000 Da PEG units at positions 24, 38, or 83 significantly decreased unspecific aggregation and proteolytic degradation while biological activity in vitro was preserved or even increased in comparison to full-glycosylated EPO. This site-specific bioconjugation approach permits to analyse the impact of PEGylation at single positions. These results represent an important step towards the engineering of site-specifically modified EPO variants from bacterial expression with increased therapeutic efficacy.

Salivary glands as a potential gene transfer target for gene therapeutics of some monogenetic endocrine disorders.

Salivary glands (SGs) exhibit several important features which are also common to endocrine glands: self-containment due to a surrounding capsule, highly efficient protein production and the ability to secrete proteins into the bloodstream. We have hypothesized that SGs are potentially useful as gene transfer targets for the correction of inherited monogenetic endocrine disorders. In the present communication, we extend our studies and attempt to test our hypothesis by comparing the efficacy of two commonly used viral vectors and the resulting serum and salivary distribution of transgene encoded hormones. A low dose (5 x10(9) particles) of either an adenoviral serotype 5 (Ad5) vector encoding the human erythropoietin (hEPO) cDNA or an adeno-associated virus sero-type 2 (AAV2) vector encoding either the hEPO or human growth hormone (hGH) cDNA was administered to individual submandibular SGs of Balb/c mice. Serum and salivary hEPO and hGH levels were determined at defined time points. Two additional recombinant viruses encoding enhanced green fluorescence protein (GFP) were also used (AdGFP and AAVGFP) in order to perform immunohistochemical analyses of transgenic protein localization in SG sections post-administration. AAV2 vectors led to stable gene transfer unlike the results with the Ad5 vectors. Indeed, in one mouse we observed hEPO production for a period of two years after administration of AAVhEPO to SGs. hEPO, which is a constitutive pathway secretory protein, was readily secreted into the bloodstream from the SGs, yielding therapeutically adequate serum levels. Conversely, hGH, a regulated secretory pathway protein, was preferentially secreted into saliva. SGs may be an attractive candidate target tissue for gene therapeutics of some monogenetic endocrine deficiency disorders. At present, AAV2 vectors seem particularly useful for such applications, and transgenes encoding constitutive secretory pathway hormones are more suitable for this application with SGs than those encoding regulated secretory pathway hormones.

Impaired splenic erythropoiesis in phlebotomized mice injected with CL2MDP-liposome: an experimental model for studying the role of stromal macrophages in erythropoiesis.

Erythropoiesis occurs in the presence of erythropoietin (EPO) without macrophages in vitro. In hematopoietic tissues, however, erythroid cells associate closely with stromal macrophages, forming erythroblastic islands via interactions with adhesion molecules. To elucidate the role of macrophages in erythropoiesis, we selectively abrogated stromal macrophages of splenic red pulp of phlebotomized mice by injection with dichloromethylene diphosphonate encapsulated in multilamellar liposomes (CL2MDP-liposome). In the spleen, no erythropoietic activity occurred until 5 days after the treatment. Colony assay revealed that the erythropoiesis was suppressed at the level of CFU-E. The splenic erythropoietic activity gradually developed from day 6 after the treatment, when F4/80+ macrophages began to appear in the red pulp. EPO mRNA was expressed in kidney but not in liver or spleen of phlebotomized mice injected with CL2MDP-liposome, and the serum EPO concentration in these mice was higher than that in phlebotomized mice. These findings suggest that abrogation of stromal macrophages by injection with CL2MDP-liposome impairs the splenic microenvironment for erythropoiesis induced by hypoxic stress, and this may be an excellent experimental model for further characterization of the in vivo role of splenic macrophages in erythropoiesis.

Survival of encapsulated human primary fibroblasts and erythropoietin expression under xenogeneic conditions.

Allogeneic cells are the most attractive source for cell transplantation, as the use of xenogeneic cells is hampered by safety concerns and the use of autologous cells involves practical difficulties. The immune rejection of allogeneic cells can be overcome by physical immunoprotection provided by polymer encapsulation. To study the variability of cell and donor sources, we compared different primary human cells as candidates for gene therapy-mediated delivery of human erythropoietin (hEpo). DARC-3.1 fibroblasts, MDX-01 fibroblasts, and ARPE-19 retinal pigment epithelial cells were encapsulated into polyethersulfone hollow fibers and implanted for 1 month in nude mice as well as in immunocompetent and FK506-immunosuppressed mice to test their in vivo resistance, with the assumption that xenogeneic conditions constitute a stringent model for human application. DARC-3.1 fibroblasts showed the best survival, prompting us to evaluate cell lineages from the same donor (DARC-3.2) or another donor (DARC-4.3 and DARC-4.4). With the exception of DARC-4.3, the remaining three lineages showed comparable survival in immunocompetent C3H and DBA/2J mice. DARC-3.1 fibroblasts were retrovirally engineered with hEpo cDNA, reaching a secretion level of 170 IU of hEpo per 10(6) cells per day. Encapsulated DARC-3.1-hEpo cells led to significantly increased hematocrits in the various hosts and under various transplantation conditions. The present study shows that encapsulated primary human DARC-3.1 fibroblasts are able to survive under xenogeneic conditions and, once engineered with hEpo cDNA, to increase the hematocrit of transplanted mice.

CellMAC: a novel technology for encapsulation of mammalian cells in cellulose sulfate/pDADMAC capsules assembled on a transient alginate/Ca2+ scaffold.

Microencapsulation of desired mammalian cell phenotypes in biocompatible polymer matrices represents a powerful technology for cell-based therapies and biopharmaceutical manufacturing of protein therapeutics. We have pioneered a novel jet break-up-compatible process for encapsulation of mammalian cells in cellulose sulfate (CS)/poly-diallyl-dimethyl-ammoniumchloride (pDADMAC) (CellMAC) capsules. CS and pDADMAC polymerize on a transient ad hoc co-assembled Ca2+/alginate scaffold and form homogenous capsules following dissolution of the alginate core by Ca2+ chelating agents. CellMAC capsules exhibited excellent mechanical properties and showed a molecular weight cut-off between 43 and 77kDa. Chinese hamster ovary cells engineered for constitutive production of the glycohormone erythropoietin reached high viable cell densities when grown inside CellMAC capsules, while specific erythropoietin (EPO) productivities matched those of conventional non-encapsulated control cultures. CellMAC-encapsulated EPO-production cell lines induced increased EPO serum levels when implanted intraperitoneally into mice and provided robust glycoprotein production during standard stirred-tank bioreactor operation. We expect the CellMAC technology to foster advances in therapeutic encapsulation of engineered cell lines as well as manufacturing of protein pharmaceuticals.