Long-term erythropoietin expression in rodents and non-human primates following intramuscular injection of a replication-defective adenoviral vector

Erythropoietin (Epo)-responsive anemia is a debilitating complication of chronic renal failure and human immunodeficiency virus (HIV) infection that effects more than 150,000 Americans. Patients with Epo-responsive anemias are currently treated with repeated injections of recombinant human Epo. In the studies described in this report, we have examined the safety and efficacy of using a single intramuscular (i.m.) injection of replication-defective adenoviral vectors (RDAd) encoding Epo for the treatment of Epo-responsive anemias in both mice and non-human primates. Our results demonstrate that there is a threshold dose of virus (2.5-8 x 10(7) pfu/gram of body weight) which is required to obtain long-term Epo expression and polycythemia in both species. A single i.m. injection of mice with 10(9) pfu of an RDAd encoding murine Epo (AdmEpo) resulted in elevations in hematocrits from control values of 49 +/- 0.9% to treated values of 81 +/- 3%, which were stable for more than 1 year. Similarly, a single i.m. injection of a monkey with 4 x 10(11) pfu of an RDAd-encoding simian Epo (AdsEpo) resulted in elevations of hematocrits from control levels of 40% to treated levels of > or =70%, which were stable for 84 days. Intramuscular injection of monkeys with AdsEpo appeared to be safe in that we did not detect abnormalities in chest X-rays, serum chemistries, hematologic, or clotting profiles (apart from elevated hematocrits) or organ histologies during the 84-day time course of the experiment. Taken together, these results suggest the feasibility of using i.m. injection of RDAd for the treatment of Epo-responsive anemias in humans.     

Control of erythropoietin delivery by doxycycline in mice after intramuscular injection of adeno-associated vector

We reported previously that controlled expression of a foreign gene in response to tetracycline derivative can be accomplished in mice by the autologous transplantation of retrovirus-modified muscle cells. Although regulated systemic delivery of therapeutic proteins from engineered tissues has potential clinical application, the transplantation of muscle cells is not currently feasible in humans. Several studies have shown that a single injection of adeno-associated virus (AAV) vectors into mouse muscle results in long-term expression of reporter genes as well as sustained delivery of proteins into the serum. Because this method is potentially applicable clinically, we constructed an AAV vector in which the expression of the mouse erythropoietin (Epo) cDNA is modulated in response to doxycycline. The vector was injected intramuscularly in normal mice. We observed that hematocrit and serum Epo concentrations could be modulated over a 29-week period in response to the presence or absence of doxycycline in the drinking water of these animals. Thus, a regulated gene expression cassette can be incorporated into a single AAV vector, such that intramuscular injection of the vector allows sustained and regulated expression of a desired gene.     

Aerosol-mediated delivery of recombinant adenovirus to the airways of nonhuman primates

At present, it is conceivable that gene therapy of the cystic fibrosis airway epithelium is possible using the direct transfer of a functional human cystic fibrosis transmembrane conductance regulator (CFTR) gene to a wide variety of patients' tracheo-bronchial cells. Here we describe a novel approach (aerosolization) to deliver a replication-deficient adenovirus carrying the CFTR gene (Ad.CFTR) to the airways. Results obtained in vitro and in Rhesus monkeys suggest that the delivery of recombinant adenovirus as an aerosol is feasible and is not associated with severe toxicity after single or double administration depending on the Ad.CFTR dose. This study supports the concept of aerosolization as a delivery method for adenovirus-mediated lung gene therapy.     

In utero cardiac gene transfer via intraplacental delivery of recombinant adenovirus

BACKGROUND: The relationship among the maternal, placental, and uniquely shunted embryonic circulation was explored to provide access to the embryonic cardiovascular system in utero. Manipulation of gene expression in the developing heart would be particularly useful for studying the effects of altered gene expression on cardiac development and in the etiology of congenital cardiac anomalies. METHODS AND RESULTS: Dye studies demonstrated that intraplacental injection allows direct access to the embryonic cardiac and systemic circulation. To evaluate the efficacy of cardiac gene transfer using this approach, replication-deficient recombinant adenoviral vectors encoding luciferase or beta-galactosidase as reporter genes were injected intraplacentally into embryonic day (E)12.5 murine embryos, an age at which the mass of the heart was observed to be large compared with other organs. Embryos were assayed for transgene expression at E15.5 and at birth. Survival rates at these times were similar among vector-injected and control groups. At E15.5 and at birth, luciferase activity within the heart was 9- and 23-fold higher, respectively, than in the remainder of the embryo, although levels of expression were generally lower at birth than during embryonic life. Beta-galactosidase expression was observed within all regions of the embryonic heart and was localized to approximately 15% of atrial and ventricular cells. CONCLUSIONS: Intraplacental delivery of adenovirus at embryonic day 12.5 results in somatic gene transfer to the murine embryonic heart, which persists at least until birth. The combination of intraplacental injection to directly access the fetal coronary circulation and injection at E12.5 when the mass of the heart is large compared with other organs results in transgene expression in cardiac cells. Intraplacental injections early in embryonic life may thus be useful to study the effects of temporal manipulation of gene expression on cardiac development and disease.     

Autoantibodies to human recombinant erythropoietin in patients with systemic lupus erythematosus: correlation with anemia

OBJECTIVE: To investigate the existence of circulating autoantibodies to erythropoietin (EPO) in sera from patients with systemic lupus erythematosus (SLE), and to correlate their presence with anemia and clinical activity. METHODS: Ninety-two consecutive patients with SLE, 80 patients with rheumatoid arthritis, and 42 normal individuals were studied. The patients with SLE were categorized into 3 groups according to hemoglobin (Hgb) level: group A (45 patients with Hgb > 12 gm/dl), group B (26 patients with Hgb 10.1-12 gm/dl), and group C (21 patients with Hgb < or = 10 gm/dl). In all patients with SLE, the disease activity was evaluated using the European Consensus Lupus Activity Measurement scale. Antibodies to EPO were detected using an enzyme-linked immunosorbent assay and purified recombinant human EPO as antigen. The specificity of the method was evaluated with homologous and cross-reactive inhibition assays. RESULTS: Antibodies to EPO were found in 15.2% of the SLE patient sera. The distribution of these antibodies among the 3 groups of SLE patients was as follows: 8.8% (4 of 45) from group A, 15.4% (4 of 26) from group B, and 28.6% (6 of 21) from group C. The prevalence of antibodies to EPO in patients with severe anemia (group C) was statistically significantly higher compared with patients without anemia (chi(2) = 4.31, P < 0.05). Patients with antibodies to EPO had higher disease activity scores (P < 0.005) and lower levels of the C4 component of complement (P < 0.05) compared with patients without antibodies to EPO. CONCLUSION: In this study, the presence of antibodies to EPO in the sera of SLE patients is demonstrated for the first time. The presence of these antibodies is associated with severe anemia and active disease.     

Cisplatin delivery by biodegradable polymer implant is superior to systemic delivery by osmotic pump or i.p. injection in tumor-bearing mice

The use of biodegradable polymer implants to deliver cisplatin was compared with delivery by systemic injection and by osmotic pump. Drug levels in the tumor were found to be higher than those in the blood and kidney when the drug was delivered using the polymer implant. In contrast, for the other two delivery methods blood and kidney cisplatin levels were greater than those in the tumor. It has been previously shown that tumor response, in terms of growth delay, was greatest when drug was delivered by polymer implant and least when treatment was by osmotic pump.     

Systemic delivery of interleukin 10 by intramuscular injection of expression plasmid DNA prevents autoimmune diabetes in nonobese diabetic mice

We previously demonstrated that intramuscular plasmid injection serves as a useful method of long-term systemic delivery of cytokines. In the present study, we assess intramuscular DNA injection as a means of systemically delivering interleukin 10 (IL-10), a cytokine with immunosuppressive properties, and preventing the progression of autoimmune diabetes in the nonobese diabetic (NOD) mouse, an excellent model for human insulin-dependent diabetes mellitus (IDDM). We injected IL-10 expression plasmid (pCAGGS-IL10) or a control pCAGGS plasmid into the muscles of NOD mice twice at 3 and 5 weeks of age. IL-10 was detectable by ELISA in the sera of mice injected with pCAGGS-IL10 for more than 2 weeks after the injection. Although the severity of insulitis at 13 weeks of age was not improved by the intramuscular injection of pCAGGS-IL10, the incidence of diabetes was markedly reduced in NOD mice injected with pCAGGS-IL10 as compared with those injected with pCAGGS or as compared with nontreated NOD mice. These results show that the progression of autoimmune diseases in mice can effectively be suppressed by intramuscular DNA injection, and suggest that this method is potentially applicable to the treatment of human autoimmune diseases.     

Long-term infusion of L-asparaginase--an alternative to intramuscular injection?

Nine children of the ALL-REZ BFM 87 and 90 trial received L-Asparginase (L-ASP) as a continuous infusion for 48-72 hs (i.e. 25 therapy cycles). Seven patients had had an allergic reaction towards an i.m. application (i.m., 29 therapy cycles). Two further patients got L-ASP initially as continuous infusion. The i.m. applications were carried out 19 times with Erwinia and 10 times with E. coli-Asparaginase, the continuous infusions 15 times with Erwinia and 10 times with E. coli-Asparaginase. In case of four patients continuous infusions of the same L-ASP type (E. coli or Erwinia) was well tolerated, after there had been an allergic reaction after i.m. application. Allergic reactions after i.m. application occurred during 10 courses as local painful erythema, during five courses as urticaria, during four courses as a general exanthema during one course as difficult breathing and during a further course as drop in blood pressure. After continuous infusion of L-ASP urticaria and difficult breathing occurred once and a transient exanthema two times. There was no anaphylactic reaction in any case. These data show that i.m. application of L-ASP causes no life-threatening side effects but allergic reactions (local pain and swelling) which clearly impaired general condition. Continuous infusion is a pharmacologically equivalent alternative with less impairment of the patients' general condition.     

Efficient induction of protective anti-malaria immunity by recombinant adenovirus

The immunogenicity of a previously constructed replication-defective recombinant adenovirus expressing the CS protein of Plasmodium yoelii was compared with that of irradiated sporozoites. We found that immunization of BALB/c mice with a single dose of this recombinant adenovirus induced a much greater CS-specific T-cell response compared with immunization with irradiated sporozoites. More importantly, we found that this recombinant adenovirus induces similar or higher levels of protective immunity than those induced by irradiated sporozoites, eliciting an appreciable resistance to malaria infection.     

Mapping of the active site of recombinant human erythropoietin

Recombinant human erythropoietin (rHuEPO) variants have been constructed to identify amino acid residues important for biological activity. Immunoassays were used to determine the effect of each mutation on rHuEPO folding. With this strategy, we could distinguish between mutations that affected bioactivity directly and those that affected bioactivity because the mutation altered rHuEPO conformation. Four regions were found to be important for bioactivity: amino acids 11 to 15, 44 to 51, 100 to 108, and 147 to 151. EPO variants could be divided into two groups according to the differential effects on EPO receptor binding activity and in vitro biologic activity. This suggests that rHuEPO has two separate receptor binding sites. Mutations in basic residues reduced the biologic activity, whereas mutations in acidic residues did not. This suggests that electrostatic interactions between rHuEPO and the human EPO receptor may involve positive charges on rHuEPO.     

Characterization of the immune response after local delivery of recombinant adenovirus in murine pancreas and successful strategies for readministration

The pancreas is an ideal organ for adenoviral gene therapy because of the high level of gene transfer that can be achieved and because of the many diseases that can potentially be treated using this technology. In this report, we characterize the immune response to direct pancreatic injection of adenovirus and we overcome some of the limitations it imposes by using immunosuppression. Direct injection of recombinant adenovirus into the pancreas leads to the production of neutralizing antibodies and to sensitized splenocytes which engage in increased cytotoxic, lymphoproliferative, and cytokine release activity when reexposed to adenovirus. Transgene expression is transient and the vector cannot be readministered. Deletion of CD4+ T helper cells improves expression over time (40% of pancreatic cells express transgene at day 28 vs. 5% in controls), and allows the vector to be readministered in the pancreas, albeit, inefficiently, when compared to naive animals. Similarly, blockade of CD40 ligand, which preserves the CD4+ T helper cell population, also improves expression over time (30% of pancreatic cells express transgene at day 28), and allows the vector to be readministered. With both approaches, neutralizing antibodies are decreased and the remaining splenocytes do not engage in activated immune responses. Thus, local delivery of the adenoviral vector induces a systemic response that prevents pancreatic readministration, even with direct injection. Blockade of CD40 ligand and T helper cell depletion are transient regimens that induce systemic immunosuppression. Until the development of newer strategies that selectively suppress adenoviral immune responses, these are viable alternatives for enhancement of pancreatic adenoviral delivery.     

Autologous blood transfusion for pulmonary and mediastinal surgery in 144 patients. The effectiveness of recombinant erythropoietin injection

Preserved autologous transfusions have been performed for elective pulmonary and mediastinal surgery to prevent the adverse effects of homologous transfusions. Autologous blood was collected preoperatively from 144 patients. The collected blood volume ranged from 400 to 1,600 ml with a mean volume of 544 ml. In four patients with benign diseases, 1,200 to 1,600 ml of blood was collected using 3,000 U of intravenous recombinant human erythropoietin (rh-EPO) administered every other day. One hundred twenty-three of these patients (85 percent) did not require a homologous transfusion. In the 84 patients undergoing either a pneumonectomy, lobectomy, or segmentectomy, 68 (81 percent) avoided homologous blood exposure. A patient with rh-EPO who bled 2,000 g during surgery received an autotransfusion of only 1,400 ml and his postoperative course was uneventful. Preserved autologous blood collected after rh-EPO injections is an effective method for minimizing homologous blood transfusions in pulmonary and mediastinal surgery.     

Thrombopoietin (TPO) knockout phenotype induced by cross-reactive antibodies against TPO following injection of mice with recombinant adenovirus encoding human TPO

Adenovirus vectors have emerged as potent agents for gene transfer. Immune response against the vector and the encoded protein is one of the major factors in the transient expression following in vivo gene transfer. A single injection of an adenovirus encoding human thrombopoietin (TPO) into mice induced transient thrombocytosis, followed by a chronic immune thrombocytopenia. Thrombocytopenic mice had anti-human TPO Abs of the IgG2a and IgG1 isotypes. Thrombocytopenic mice sera neutralized more efficiently human than murine TPO, and exhibited no detectable anti-murine TPO Abs. Despite their low affinity for murine TPO, anti-TPO Abs induced a TPO knockout-like phenotype, i.e., low number of marrow megakaryocytes and of all kinds of hemopoietic progenitors. Hybridomas derived from a thrombocytopenic mouse revealed cross-reactivity of all of the secreted anti-TPO Ab isotypes. Mice subjected to myelosuppression after virus injection showed that anti-human TPO of IgG1 and IgG2a isotypes disappeared. Thus, sustained human TPO production was responsible for platelet elevation for at least 5 mo. Compelling results showed that elevated IgG2a/IgG2b ratios are always associated with thrombocytopenia, whereas low ratios are associated with tolerance or normal platelet counts. Finally, we hypothesize that in humans some chronic thrombocytopenia associated with a low TPO plasma level are due to anti-TPO Abs.     

Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis

A simplified method for generating recombinant adenovirus was developed by using liposome-mediated co-transfection and by directly observing for the cytopathic effect caused by recombinant adenovirus in transfected 293 cells. This approach avoided difficulties associated with calcium-phosphate precipitation and agarose overlays for plaque assays. The ease of generating recombinant adenovirus was considerably improved. Analysis by PCR of DNA samples from the supernatant of the cell cultures with the cytopathic effect was also developed, which made identification of any newly generated recombinant virus rapid and specific.     

Encapsidated adenovirus mini-chromosome-mediated delivery of genes to the retina: application to the rescue of photoreceptor degeneration

First (DeltaE1/E3) and second (DeltaE1+DeltaE2/E3/E4) generation adenovirus (Ad) vectors have been shown previously to be of limited use in the treatment of human genetic diseases due to the induction of a host cytotoxic T-cell mediated immune response against virally expressed genes. In addition, a limited cloning capacity of approximately 8 kb does not cater for the incorporation of large upstream sequences essential for regulated tissue-specific expression or inclusion of multiple gene-expression cassettes. In this study we have exploited our recently developed Ad-based vector, the encapsidated adenovirus mini-chromosome (EAM) from which all of the viral genes have been deleted. EAMs contain only the inverted terminal repeats required for replication and five cis -acting Ad encapsidation signals necessary for packaging. We have shown previously that EAMs can efficiently transduce a variety of cell types in vitro. In this study we demonstrate that EAMs can transduce and rescue cells from the neurosensory retina in vivo. EAM-mediated delivery of the beta subunit of cyclic GMP phosphodiesterase (PDE) cDNA to mice affected with retinal degeneration (rd) allows prolonged transgene expression and rescue of rod photoreceptor cells. RT-PCR analysis from the injected retina indicates that transgene products are present for at least 18 weeks post-injection. Both the alpha and beta subunits of PDE could be detected up to 90 days postnatal in EAM-injected rd retina by western analysis. A maximal PDE activity of 150 nm/min/mg was detected at 33 days postnatal. Examination of outer nuclear thickness showed significant differences up to 12 weeks post-injection. These results demonstrate an improved level of rescue over first-generation adenoviral vectors and suggest the possibility of successful EAM-mediated treatment of some retinal diseases in humans.     

Enhanced sensitivity of mdx mice to intramuscular injection of compound 48/80

Species-specific differences in the inflammatory response, specifically with regard to mast cells, have been proposed to explain the phenotypic variation among dystrophin-deficient humans, and mdx mice (Gorospe et al., 1994). To test this hypothesis we have intramuscularly injected a mast cell secretogogue into both dystrophin-negative mdx and dystrophin-positive normal mice. Mast cell activity was determined by measuring the activity of mast cell tryptase, while creatine kinase activity was used to determine the course of muscle damage in vivo. Area of damage around the injection site was measured at autopsy, and used as an indication of relative sensitivity to the secretogogue effect of compound 48/80. Mdx mice exhibited more damage in response to intramuscular injection than normal control mice. In addition, mdx mice showed a substantial increase in plasma tryptase activity, followed by a large increase in muscle creatine kinase activity. On the other hand, dystrophin-positive normal controls injected with 48/80 liberated little CK or tryptase activity. These results are consistent with the hypothesis that species-specific differences in mast cell activity, or sensitivity to mast cell products could account for the variation in pathology seen in dystrophin-deficient animals.     

Chicken adenovirus (CELO virus) particles augment receptor-mediated DNA delivery to mammalian cells and yield exceptional levels of stable transformants

Delivery of genes via receptor-mediated endocytosis is severely limited by the poor exit of endocytosed DNA from the endosome. A large enhancement in delivery efficiency has been obtained by including human adenovirus particles in the delivery system. This enhancement is probably a function of the natural adenovirus entry mechanism, which must include passage through or disruption of the endosomal membrane. In an effort to identify safer virus particles useful in this application, we have tested the chicken adenovirus CELO virus for its ability to augment receptor-mediated gene delivery. We report here that CELO virus possesses pH-dependent, liposome disruption activity similar to that of human adenovirus type 5. Furthermore, the chicken adenovirus can be used to augment receptor-mediated gene delivery to levels comparable to those found for the human adenovirus when it is physically linked to polylysine ligand-condensed DNA particles. The chicken adenovirus has the advantage of being produced inexpensively in embryonated eggs, and the virus is naturally replication defective in mammalian cells, even in the presence of wild-type human adenovirus.