hEPO Levels Research Articles

Maximizing the Translational Yield of mRNA Therapeutics by Minimizing 5'-UTRs.

The 5'-untranslated region (5'-UTR) of mRNA contains structural elements, which are recognized by cell-specific RNA-binding proteins, thereby affecting the translation of the molecule. The activation of an innate immune response upon transfection of mRNA into cells is reduced when the mRNA comprises chemically modified nucleotides, putatively by altering the secondary structure of the molecule. Such alteration in the 5'-UTR in turn may affect the functionality of mRNA. In this study, we report on the impact of seven synthetic minimalistic 5'-UTR sequences on the translation of luciferase-encoding unmodified and different chemically modified mRNAs upon transfection in cell culture and in vivo. One minimalistic 5'-UTR, consisting of 14 nucleotides combining the T7 promoter with a Kozak consensus sequence, yielded similar or even higher expression than a 37 nucleotides human alpha-globin 5'-UTR containing mRNA in HepG2 and A549 cells. Furthermore, also the kind of modified nucleotides used in in vitro transcription, affected mRNA translation when using different translation regulators (Kozak vs. translation initiator of short UTRs). The in vitro data were confirmed by bioluminescence imaging of expression in mouse livers, 6 h postintravenous injection of a lipidoid nanoparticle-formulated RNA in female Balb/c mice. Luciferase measurements from liver and spleen showed that minimal 5'-UTRs (3 and 7) were either equally effective or better than human alpha-globin 5'-UTR. These findings were confirmed with a human erythropoietin (hEPO)-encoding mRNA. Significantly, higher levels of hEPO could be quantified in supernatants from A549 cells transfected with minimal 5'-UTR7 containing RNA when compared to commonly used benchmarks 5'-UTRs. Our results demonstrate the superior potential of synthetic minimalistic 5'-UTRs for use in transcript therapies.

Safety Evaluation of Lipid Nanoparticle-Formulated Modified mRNA in the Sprague-Dawley Rat and Cynomolgus Monkey.

The pharmacology, pharmacokinetics, and safety of modified mRNA formulated in lipid nanoparticles (LNPs) were evaluated after repeat intravenous infusion to rats and monkeys. In both species, modified mRNA encoding the protein for human erythropoietin (hEPO) had predictable and consistent pharmacologic and toxicologic effects. Pharmacokinetic analysis conducted following the first dose showed that measured hEPO levels were maximal at 6 hours after the end of intravenous infusion and in excess of 100-fold the anticipated efficacious exposure (17.6 ng/ml) at the highest dose tested.24 hEPO was pharmacologically active in both the rat and the monkey, as indicated by a significant increase in red blood cell mass parameters. The primary safety-related findings were caused by the exaggerated pharmacology of hEPO and included increased hematopoiesis in the liver, spleen, and bone marrow (rats) and minimal hemorrhage in the heart (monkeys). Additional primary safety-related findings in the rat included mildly increased white blood cell counts, changes in the coagulation parameters at all doses, as well as liver injury and release of interferon γ-inducible protein 10 in high-dose groups only. In the monkey, as seen with the parenteral administration of cationic LNPs, splenic necrosis and lymphocyte depletion were observed, accompanied with mild and reversible complement activation. These findings defined a well-tolerated dose level above the anticipated efficacious dose. Overall, these combined studies indicate that LNP-formulated modified mRNA can be administered by intravenous infusion in 2 toxicologically relevant test species and generate supratherapeutic levels of protein (hEPO) in vivo.

Sustained intrathecal therapeutic protein delivery using genetically transduced tissue implants in a freely moving rat model

Systemic delivery of therapeutic proteins to the central nervous system (CNS) is challenging because of the blood-brain barrier restrictions. Direct intrathecal delivery is possible but does not produce stable concentrations. We are proposing an alternative approach for localized delivery into the CNS based on the Transduced Autologous Restorative Gene Therapy (TARGT) system. This system was previously developed using a gene therapy approach with dermal tissue implants. Lewis rat dermal tissue was transduced to secrete human EPO (hEPO). TARGT viability and function were retained following cryopreservation. Upon implantation into the rat cisterna magna, a mild inflammatory response was observed at the TARGT-brain interface throughout 21-day implantation. hEPO expression was verified immunohistochemically and by secreted levels in cerebrospinal fluid (CSF), serum, and in vitro post explant. Detectable CSF hEPO levels were maintained during the study. Serum hEPO levels were similar to rat and human basal serum levels. In vitro, the highest hEPO concentration was observed on day 1 post-explant culture and then remained constant for over 21days. Prolonged incubation within the cisterna magna had no negative impact on TARGT hEPO secretion. These promising results suggest that TARGTs could be utilized for targeted delivery of therapeutic proteins to the CNS.

Transduction of human EPO into human bone marrow mesenchymal stromal cells synergistically enhances cell-protective and migratory effects

Human bone marrow mesenchymal stromal cells (hBM-MSCs) are a promising tools for cell therapy. However, the poor viability of the transplanted cells is a major limiting factor. Human erythropoietin (hEPO) has been extensively studied in non-hematopoietic tissues for its neurotrophic, anti-oxidant, anti-apoptotic, and antiinflammatory effects. In this study, we evaluate whether transduction of the hEPO gene into MSCs provides protection and affects their migration. hBM-MSCs transduced with the hEPO gene (EPO-MSCs) stably secreted high levels of hEPO (10 IU/ml) with no alteration of their mesenchymal phenotype. MSCs were also treated with 10 IU rhEPO, an amount similar to what was secreted by EPO-MSCs, to generate 10U-MSCs. Protection against H2O2-induced oxidative stress and staurosporine-induced apoptosis was registered for both EPO-MSCs and 10U-MSCs, but the protective effects were higher for the EPO-MSCs than for the 10U-MSCs. EPO-MSCs had significantly higher migration rates compared to MSCs and 10U-MSCs. We confirmed that the intracellular signaling of ERK1/2 was higher in the EPO-MSCs than 10U-MSCs. This data demonstrates that the endogenous expression of EPO may efficiently initiate the ERK1/2 signaling pathway, resulting in synergistic effects on the production of neurotrophic factors. Thus, EPO-MSCs are a good candidate for cell therapy in ischemic and neurodegenerative diseases.

Long‐term transduction of miniature pig parotid glands using serotype 2 adeno‐associated viral vectors

Previously, using an adenoviral vector, we showed that miniature pigs could provide a valuable and affordable large animal model for pre-clinical gene therapy studies to correct parotid gland radiation damage. However, adenoviral vectors lead to short-term transgene expression and, ideally, a more stable correction is required. In the present study, we examined the suitability of using a serotype 2 adeno-associated viral (AAV2) vector to mediate more stable gene transfer in the parotid glands of these animals. Heparan sulfate proteoglycan was detected by immunohistochemistry. beta-galactosidase expression was determined histochemically. An AAV2 vector encoding human erythropoietin (hEpo) was administered via Stensen's duct. Salivary and serum hEpo levels were measured using an enzyme-linked immunosorbent assay. Serum chemistry and hematological analyses were performed and serum antibodies to hEpo were measured throughout the study. Vector distribution was determined by a quantitative polymerase chain reaction. Transgene expression was vector dose-dependent, with high levels of hEpo being detected for up to 32 weeks (i.e. the longest time studied). hEpo reached maximal levels during weeks 4-8, but declined to approximately 25% of these values by week 32. Haematocrits were elevated from week 2. Transduced animals exhibited low serum anti-hEpo antibodies (1 : 8-1 : 16). Vector biodistribution at animal sacrifice revealed that most copies were in the targeted parotid gland, with few being detected elsewhere. No consistent adverse changes in serum chemistry or hematology parameters were seen. AAV2 vectors mediate extended gene transfer to miniature pig parotid glands and should be useful for testing pre-clinical gene therapy strategies aiming to correct salivary gland radiation damage.

Extended Transgene Expression From a Nonintegrating Adenoviral Vector Containing Retroviral Elements

We studied the effects of specific retroviral elements in a first-generation serotype 5 adenoviral (Ad5) vector, AdLTR(2)EF1alpha-hEPO. This vector contains 858 base pair (bp) [251-bp envelope sequence plus 607-bp long-terminal repeat (LTR)] from Moloney murine leukemia virus (MoMLV), upstream of the human elongation factor-1alpha (EF1alpha) promoter and human erythropoietin (hEPO) cDNA, with the LTR sequence downstream of the polyadenylation signal. We compared expression of AdLTR(2)EF1alpha-hEPO with corresponding expressions of two conventional Ad5 vectors, AdEF1alpha-hEPO and AdCMV-hEPO, in vivo in submandibular glands in rats. Both the conventional vectors yielded low serum hEPO levels by day 7, and little change in hematocrits. In contrast, after receiving AdLTR(2)EF1alpha-hEPO, the rats showed elevated hEPO levels and hematocrits for 1-3 months. In vitro studies showed that the integration efficiencies of all the vectors were similar (approximately 10(-3)). Approximately 0.1% of the vector genomes were present 1 year after delivery in the case of each of the three vectors, primarily as intact linear double-strand DNA. The unique results seen with AdLTR(2)EF1alpha-hEPO are partly because of LTR enhancer activity. However, other cis-acting activity, which is not immunomodulatory but nevertheless influences promoter methylation, appears to be involved. A vector such as AdLTR(2)EF1alpha-hEPO may prove useful in clinical applications in which extended, but not "permanent," transgene expression is desirable.

Recombinant baculovirus-based multiple protein expression platform for Drosophila S2 cell culture

A platform for selective and controllable expression of multiple foreign protein types was developed in insect cell culture. Based on the fact that baculovirus cannot replicate in nonpermissive Drosophila melanogaster Schneider line 2 (S2) cells, S2 cells that stably express human erythropoietin (hEPO) under the control of the S2-derived inducible metallothionein (MT) promoter were infected with three types of recombinant baculoviruses, each of which expressed a different fluorescent protein gene under the control of MT promoter. Addition of copper sulfate as an inducer to infected, stably transfected S2 cells resulted in simultaneous expression of hEPO and three fluorescent proteins. Expression profiles and levels of the three induced fluorescent proteins were similar in all single infected cells. Importantly, expression profiles and levels of hEPO were similar in both non-infected and infected cells, indicating that baculovirus expressed recombinant proteins do not adversely affect expression of host cell recombinant proteins. Expressions of the three fluorescent proteins were able to be selectively regulated by altering combination ratios of the three types of recombinant baculoviruses. Collectively, these data indicate that the baculovirus/stably transfected S2 cell system can be successfully used to express multiple foreign proteins in a controlled and selective manner without the burden of additional selection markers. Such a system would be expected to be attractive as a multiple protein expression platform for engineering metabolic or glycosylation pathways.

Sorting of transgenic secretory proteins in miniature pig parotid glands following adenoviral‐mediated gene transfer

Gene transfer to salivary glands for use in treating both systemic and upper gastrointestinal tract diseases shows considerable potential. Numerous studies in rodents demonstrate that salivary glands can secrete transgenic secretory proteins either into saliva, primarily via the regulated secretory pathway (RSP), or into the bloodstream, primarily by the constitutive secretory pathway (CSP). The purpose of the present study was to assess the sorting characteristics of human growth hormone (hGH), a RSP protein, and human erythropoietin (hEpo), a CSP protein, in a large animal model of salivary gland gene transfer, the miniature pig. Recombinant serotype 5 adenoviral (Ad5; 10(11) particles/gland) vectors encoding either hGH (AdCMVhGH) or hEpo (AdCMVhEpo) were administered to both parotid glands of male miniature pigs by intraductal cannulation. The secretion of hGH or hEpo was measured in both saliva and serum on days 3, 7 and 14 following administration. Detailed serum chemistry and hematological analyses were performed, and the presence of serum antibodies to hGH and hEpo was measured. For AdCMVhEpo-treated minipigs vector distribution in multiple tissues was determined by quantitative polymerase chain reaction (QPCR). The RSP protein hGH was secreted entirely into saliva, while the CSP protein hEpo was secreted into both saliva and serum. Most hEpo was found in saliva, but serum hEpo levels were sufficient to significantly increase hematocrit levels in treated animals by approximately 10%. Expression of both transgenes was maximal on day 3 and declined to near background by day 14. The amount of vector found in the targeted glands was 100 x more than in other tissues. Secretion of transgenic hGH from minipig parotid glands occurred principally into saliva via the RSP, as seen in rodents, while hEpo was secreted into both saliva and serum, the latter presumably via the CSP. Even though hEpo secretion into the bloodstream was not to the extent previously observed in rodents, serum hEpo levels were considerable and the hEpo was biologically active. Ad5 vector distribution was highly restricted to the parotid glands with little vector detected elsewhere. While the results in this large animal model support the established notion that salivary gland gene transfer can be used for treating systemic single protein deficiency disorders, they also highlight differences in transgenic CSP protein sorting between rodents and miniature pigs.

Prevention of Immune Responses to Human Erythropoietin in Cynomologus Monkeys (Macaca fascicularis)

Genes and proteins of human origin are often administered to monkeys for research purposes, however, it can be difficult to obtain sufficient levels of the products in vivo due to immunological clearance. In this study, we showed that human erythropoietin (hEPO) induces generation of anti-hEPO antibody in cynomolgus macaques (n=2), although 92% of amino acid residues are common between the human and macaque EPO. The administered hEPO was thus eliminated from the animals. On the other hand, when an immunosuppressant, cyclosporin A (CyA), was administered (6 mg/kg) intramuscularly every other day in combination with hEPO (n=2), no anti-hEPO antibody was generated and high serum levels of hEPO were obtained during administration of hEPO, resulting in an increase in serum hemoglobin levels. No adverse effects associated with CyA were observed. Thus, CyA treatment is useful for prevention of immune responses associated with the administration of human proteins in monkeys.

674. Administration of rAAV2 and rAAV5 to the Parotid Gland of Non-Human Primates

Previous studies from our laboratory showed that therapeutic levels of transgene expression were obtained from mouse submandibular glands after retro-ductal administration of relatively low doses (109 particles/animal) of a recombinant serotype 2 adeno- associated viral vector (rAAV2) encoding human erythropoietin (hEPO). The recombinant hormone was preferentially secreted into the bloodstream rather than into saliva. Serum hEPO levels steadily increased for an 8–12 week period and remained relatively stable until the end of the experiment (54 weeks; Voutetakis et al, PNAS, 2004).

Rapamycin control of transgene expression from a single AAV vector in mouse salivary glands

Salivary glands (SGs) appear to be a useful target site for gene therapeutics. The ability to control transgene expression is essential for clinical application. Previously, in a proof-of-concept study, we have shown that the rapamycin-inducible transcriptional regulation system can regulate protein expression after adenoviral-mediated gene transfer to SGs. To evaluate the potential ability to utilize this regulatory system for long-term control of transgene expression in this tissue, we employed a 'third generation', single adenoassociated serotype 2 viral (AAV2) vector encoding human erythropoietin (hEPO) under the control of a rapamycin-inducible promoter. The vector, rAAV-TF2.3-hEPO (10(10) particles/animal), was delivered to mouse SGs. No detectable increase in serum hEPO or hematocrit levels was observed in the absence of rapamycin administration. However, rapamycin induced elevation of serum hEPO levels, as well as concomitant hematocrit changes, that were dose-dependent, completely reversible, and relatively stable over the course of this study (6 months), with no appreciable change in rapamycin responsiveness. Our results suggest that the rapamycin transcriptional regulation system delivered in a single AAV2 vector to SGs may be valuable for systemic protein replacement applications.

877. Long-Term Transgene Expression with a Second Generation Hybrid AdenoRetroviral Vector

Previously, we reported the construction of a prototype hybrid adenoretroviral vector, AdLTR-luc, which included Moloney murine leukemia virus (MoMLV) elements flanking (|[sim]| 2.7 kb 5|[prime]|; |[sim]| 1 kb 3|[prime]|) transgene cassette (Zheng et al., Nat. Biotech., 2000). Like traditional adenoviral vectors, this hybrid adenoretroviral vector is capable of transducing a wide variety of dividing and non-dividing cell types, but like retroviral vectors AdLTR-luc is able to integrate in the host cell genome resulting in long-term transgene expression. To further develop this unique vector, and improve production efficiency, we modified MoMLV sequences in the original hybrid vector. We report here the construction of a second generation hybrid vector, containing either of two transgenes: human erythropoietin (hEPO) or enhanced green fluorescence protein (EGFP). In addition, the transgene cassette included the human elongation factor 1 alpha (EF1a) promoter. Two resulting vectors, AdLTR2EF1a-hEPO and AdLTR2EF1a-EGFP, used 0.8 kb MoMLV elements (0.2 kb envelope and 0.6 kb LTR sequences in a 5|[prime]| to 3|[prime]|direction) upstream, and 0.6 kb LTR sequence downstream, of the transgene. The selection of these fragments was based on a series of gel shift assays following incubation of nuclear protein extracts with different MoMLV sequences. Production of both vectors was efficient and comparable to that obtained with first generation adenoviral vectors (|[sim]|1012 - 1013 particles/ml by Q-PCR). To evaluate if these constructs were able to integrate into genome, the rat submandibular gland A5 cell line was transduced with AdLTR2EF1a-EGFP at 100 particles/cell. After 10 days, colonies were counted and 8.1% (of 869) exhibited green fluorescence while none of the colonies (|[sim]| 900 each) obtained after similar transduction with two conventional adenoviral vectors, AdCMV-EGFP and AdEF1a-EGFP, exhibited green fluorescence. PCR assays on DNA from the cloned cells transduced with AdLTR2EF1a-EGFP were consistent with possible transgene integration into genomic DNA. To test if these constructs could mediate long-term transgene expression in vivo, AdLTR2EF1a-hEPO was delivered into rat submandibular glands at 109 particles/gland (only one gland targeted). Three of 5 rats showed elevated hEPO and hematocrit levels for two months, and in one rat elevated hEPO and hematocrit levels persisted until the end of the experiment (5 months). Transduction with the conventional adenoviral vectors, AdCMV-hEPO and AdEF1a-hEPO, resulted in elevated hEPO levels for |[sim]| 1 week. PCR assays indicated that after AdLTR2EF1a-hEPO transduction, the EF1a-hEPO sequence was found in extracted DNA at 5 months endpoint from targeted submandibular glands of all three rats expressing hEPO for at least two months. In two of these samples, PCR assays also suggested that an integration event occurred. We detected anti-hEPO antibody in sera from the two rats whose hEPO levels decreased after two months. However, in one rat with persistent high levels of hEPO for 5 months, there was no evidence of anti-hEPO antibody. Together, these results suggest that this second generation hybrid adenoretroviral vector, which is more efficiently produced than the original hybrid vector, can mediate long-term transgene expression in vivo.

The regulated long-term delivery of therapeutic proteins by using antigen-specific B lymphocytes

Memory lymphocytes are important mediators of the immune response. These cells are long-lived and undergo clonal expansion upon reexposure to specific antigen, differentiating into effector cells that secrete Ig or cytokines while maintaining a residual pool of memory T and B lymphocytes. Here, the ability of antigen-specific lymphocytes to undergo repeated cycles of antigen-driven clonal expansion and contraction is exploited in a therapeutic protocol aimed at regulating protein delivery. The principle of this strategy is to introduce genes encoding proteins of therapeutic interest into a small number of antigen-specific B lymphocytes. Output of therapeutic protein can then be regulated in vivo by manipulating the size of the responder population by antigen challenge. To evaluate whether such an approach is feasible, we developed a mouse model system in which Emu- and Iglambda-based vectors were used to express human erythropoietin (hEPO) gene in B lymphocytes. These mice were then immunized with the model antigen phycoerythrin (PE), and immune splenocytes (or purified PE-specific B lymphocytes) were adoptively transferred to normal or mutant (EPO-deficient) hosts. High levels of hEPO were detected in the serum of adoptively transferred normal mice after PE administration, and this responsiveness was maintained for several months. Similarly, in EPO-deficient anemic recipients, antigen-driven hEPO expression was shown to restore hematocrit levels to normal. These results show that antigen-mediated regulation of memory lymphocytes can be used as a strategy for delivering therapeutic proteins in vivo.

Expression of double foreign protein types following recombinant baculovirus infection of stably transfected Drosophila S2 cells

We sought to develop a platform for simultaneous, regulatable expression of double foreign protein types in cell culture. Drosophila melanogaster Schneider line 2 (S2) insect cells that stably express human erythropoietin (hEPO) were infected with a recombinant baculovirus containing the green fluorescent protein (GFP) gene. Since baculovirus cannot replicate in nonpermissive S2 cells, baculovirus infection did not affect cell growth or viability. Expression of each foreign protein was under the control of the inducible metallothionein (MT) promoter. Addition of copper sulfate to infected, stably transfected cells resulted in simultaneous expression of both GFP and hEPO. Induced hEPO expression profile and levels were similar in both control and infected cells, indicating that baculovirus infection also did not affect expression of stably introduced foreign gene. GFP protein levels were regulated by the infection dose of recombinant baculovirus, while hEPO expression remained constant. hEPO levels were much higher (∼30-fold) than GFP, indicating plasmid-based introduced gene copies have higher expression than baculovirus-based introduced genes. These data suggest the baculovirus/stable S2 cell system can be used to produce a major target protein by plasmid-based stable transfection, and assistant proteins by recombinant baculovirus infection. Such a system appears to be very attractive as a multiple protein expression platform for engineering metabolic pathways in cell culture.

Reengineered salivary glands are stable endogenous bioreactors for systemic gene therapeutics

The use of critical-for-life organs (e.g., liver or lung) for systemic gene therapeutics can lead to serious safety concerns. To circumvent such issues, we have considered salivary glands (SGs) as an alternative gene therapeutics target tissue. Given the high secretory abilities of SGs, we hypothesized that administration of low doses of recombinant adeno-associated virus (AAV) vectors would allow for therapeutic levels of transgene-encoded secretory proteins in the bloodstream. We administered 10(9) particles of an AAV vector encoding human erythropoietin (hEPO) directly to individual mouse submandibular SGs. Serum hEPO reached maximum levels 8-12 weeks after gene delivery and remained relatively stable for 54 weeks (longest time studied). Hematocrit levels were similarly increased. Moreover, these effects proved to be vector dose-dependent, and even a dosage as low as 10(8) particles per animal led to significant increases in hEPO and hematocrit levels. Vector DNA was detected only within the targeted SGs, and levels of AAV copies within SGs were highly correlated with serum hEPO levels (r = 0.98). These results show that SGs appear to be promising targets with potential clinical applicability for systemic gene therapeutics.

Optimization of human erythropoietin secretion from MLV-infected human primary fibroblasts used for encapsulated cell therapy.

The transplantation of encapsulated cells genetically engineered to secrete human erythropoietin (hEpo) represents an alternative to repeated injections of the recombinant hormone for the treatment of Epo-responsive anemia. In the present study, the ability of primary human foreskin fibroblasts to secrete high levels of hEpo and the importance of cis-acting elements and infection conditions on transgene expression level were assessed. The transduction efficiency was first evaluated with beta-galactosidase (LacZ)-encoding retroviral vectors derived from the murine leukemia retrovirus (MLV) pseudotyped either with an amphotropic envelope or with the G glycoprotein of vesicular stomatitis virus (VSV-G). Human fibroblasts were then infected with an amphotropic hEpo-expressing retroviral vector, which was modified by insertion of a post-transcriptional regulatory element from the woodchuck hepatitis virus (WPRE) and a Kozak consensus sequence (KZ). Human Epo production was further optimized by increasing the multiplicity of infection and by selecting high producer cells. The survival and the transgene expression of these fibroblasts were finally evaluated in vivo. The cells were encapsulated into microporous hollow fibers and subcutaneously implanted in nude mice. A secretion level of approximately 5 IU hEpo/10(6) cells/day was obtained with the basal vector. A 7.5-fold increase in transgene expression was observed with the insertion of WPRE and KZ elements. Finally, according to the optimization of infection conditions, we obtained a 40-fold increase in hEpo secretion, reaching approximately 200 IU hEpo/10(6) cells/day. The in vivo experiments showed an increase in the hematocrit during the first 2 weeks and elevated levels exceeding 60% were maintained over a 6-week period. These results indicate that primary human fibroblasts represent a promising source for encapsulated cell therapy.

Expression of human erythropoietin transgenes and of the endogenous WAP gene in the mammary gland of transgenic rabbits during gestation and lactation.

An understanding of the expression of transgenes in the mammary gland during gestation and lactation is crucial for the use of transgenic mammals as bioreactors. Here we describe the temporal pattern of expression of the endogenous rabbit WAP gene and human erythropoietin (hEPO) transgenes under the control of rabbit WAP promoter and 3' flanking sequences. The endogenous rabbit WAP gene was expressed throughout gestation including the day of mating, as well as during lactation in transgenic rabbits bearing a minigene construct. In non-pregnant cycling females, WAP expression was found independent of transgenic status; however, WAP expression was not detected in non-cycling females. The significance of this new finding is not clear at present. hEPO mRNA was detected in mammary gland biopsies from pregnant transgenic rabbits only on day 28 of gestation. During lactation, transcripts were present in mammary gland biopsy samples taken on days 0, 7, 14 and 21. A sharp decline in the levels of transcripts was found for an hEPOcDNA gene construct at the end of lactation (day 28). Although the levels of hEPO were too low to allow a conclusion to be drawn regarding temporal or position-dependent expression, this finding may reflect an integration position effect.

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.

Stable delivery of physiologic levels of recombinant erythropoietin to the systemic circulation by intramuscular injection of replication-defective adenovirus.

A number of inherited and acquired serum protein deficiencies including hemophilias A and B, diabetes mellitus, and the erythropoietin-responsive anemias are currently treated with repeated subcutaneous or intravenous infusions of purified or recombinant proteins. The development of an in vivo gene-transfer approach to deliver physiologic levels of recombinant proteins to the systemic circulation would represent a significant advance in the treatment of these disorders. Here we describe the construction of a replication-defective adenovirus (AdEF1hEpo) containing the human erythropoietin (hEpo) cDNA under the transcriptional control of the cellular elongation factor 1 alpha (EF1 alpha) promoter and the 4F2 heavy chain (4F2HC) enhancer. Neonatal CD-1 and adult SCID mice injected once intramuscularly (i.m.) with 10(7) to 10(9) plaque-forming units (pfu) of this virus displayed significant dose-dependent elevations of serum hEpo levels and increased hematocrits, which were stable over the 4-month time course of these experiments. Adenovirus injected i.m. remained localized at the site of injection and there was no evidence of either systemic infection or a localized inflammatory response. These results suggest that i.m. injection of recombinant replication-defective adenovirus vectors may serve as a paradigm for the treatment of human serum protein deficiencies.