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

Abstract

A number of inherited and acquired serum protein deficiencies including hemophillas A and B, diabetes meilitus, and the erythropoletin-responsive anemias are currently treated with repeated subcutaneous or intravenous infusions of purified or recombinant proteins. The development ofan in vivo gene-tansfer approach to deliver physiologic levels ofrecombinant proteins to the systemic circulation would represent a sgnifiant advance in the treatment of these disorders. Here we describe the construction of a replicationdefective adenovirus (AdEFlhEpo) contini the human erythropoietin (hEpo) cDNA under the transcriptional control of the cellular elongation factor la (EFla) promoter and the 4F2 heavy chain (4F2HC) enhancer. Neonatal CD-1 and adult SCiD mice injected once intramuscularly (i.m.) with 107 to 109 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 inflaatory 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.Erythropoietin (Epo) is a 30-kDa glycoprotein synthesized in adult renal cells that functions as the major regulator of mammalian erythropoiesis (1). Serum human Epo (hEpo) levels are controlled transcriptionally in response to tissue hypoxia or anemia (2, 3). The secreted protein increases erythrocyte production by stimulating the proliferation and preventing the apoptosis of erythroid precursors including erythroid colony-forming units and erythroid blast-forming units (4). Most patients with end-stage renal disease treated with hemodialysis (5) and many patients with human immunodeficiency virus treated with 3'-azido-3'-deoxythymidine (6) suffer from severe anemias resulting from inappropriately low levels of serum hEpo. These patients are currently treated with repeated (two or three times per week) subcutaneous or intravenous infusions of recombinant hEpo (5, 6).The Epo-responsive anemias represent an excellent model system for studies designed to develop an in vivo gene transfer approach for the treatment of serum protein deficiencies. The hEpo gene (7) and cDNA (8) have been cloned and hEpo functions in multiple mammalian species including mice (9). There is a sensitive ELISA assay that can be used to distinguish hEpo from murine Epo (10). Most importantly, the hematocrit is an easily measured physiologic assay of serum Epo levels (9).Previous studies have demonstrated that skeletal myoblasts, genetically engineered to produce secreted proteins in vitro, can be used to stably deliver physiologic levels of recombinant proteins to the systemic circulation after intramuscular implantation (11-15). Although effective, such an ex vivo gene therapy approach is both labor int...

Pulmonary inflammation associated with repeated, prenatal exposure to an E1, E3-deleted adenoviral vector in sheep

“…Administration of adenovirus vectors into the trachea, skeletal muscle, thoracic cavity or vasculature of neonatal mice or rats results in transgene expression for several weeks or months. 12,13,[18][19][20][21][22] Subsequent administration of the adenovirus vectors were tolerated and resulted in robust gene expression in some studies 19,20,23 but not others. 18 The relative success of this approach in rodents has been attributed to the relative immaturity of the immune system in the rodent at birth.…”

“…Alternatively, injection into the skeletal muscle or thoracic cavity have been successful in young rodents 12,13,20,22 and may be successful in sheep.…”

“…of adenovirus into skeletal muscle of newborn mice results in prolonged gene expression. 12,21,22 Injection of similar amounts of adenovirus into the thoracic cavity or abdomen or vasculature of newborn rats or mice is sufficient to induce tolerance, prolong gene expression and reduce inflammatory and immune responses to subsequent exposure. 19,20,23 Interestingly, injection of a higher dose (1 × 10 10 p.f.u.)…”

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

“…Long-term expression of therapeutic transgenes is possible by direct gene transfer in vivo either through infection by viruses, including adenoviral 11 and adeno-associated vectors 12,13 or by direct injection of plasmid DNA. 14 Adenoviruses have been employed to continually deliver the angiogenic factors VEGF 165 , 15 FGF-2 (basic FGF), 16 FGF-1 (acidic FGF) 17 and FGF-5 18 in vivo, with each of these molecules stimulating the outgrowth of new vessels at the site of administration.…”

Systemic action of human growth hormone following adenovirus-mediated gene transfer to rat submandibular glands

“…[1][2][3] In addition to being inconvenient, such a mode of protein therapeutics is usually not cost-efficient, can result in the administration of various impurities, and may not result in physiologically adequate patterns of protein replacement in blood. Growth hormone deficiency in children is an excellent example of one such condition.…”