Faculty Bibliography

Effective delivery of secreted proteins by gene therapy will require a vector that directs stable delivery of a transgene and a regulatory system that permits pharmacologic control over the level and kinetics of therapeutic protein expression. We previously described a regulatory system that enables transcription of a target gene to be controlled by rapamycin, an orally bioavailable drug. Here we demonstrate in vivo regulation of gene expression after intramuscular injection of two separate adenovirus or adeno-associated virus (AAV) vectors, one encoding an inducible human growth hormone (hGH) target gene, and the other a bipartite rapamycin-regulated transcription factor. Upon delivery of either vector system into immunodeficient mice, basal plasma hGH expression was undetectable and was induced to high levels after administration of rapamycin. The precise level and duration of hGH expression could be controlled by the rapamycin dosing regimen. Equivalent profiles of induction were observed after repeated administration of single doses of rapamycin over many months. AAV conferred stable expression of regulated hGH in both immunocompetent and immunodeficient mice, whereas adenovirus-directed hGH expression quickly extinguished in immunocompetent animals. These studies demonstrate that the rapamycin-based regulatory system, delivered intramuscularly by AAV, fulfills many of the conditions necessary for the safe and effective delivery of therapeutic proteins by gene therapy

Stable delivery of a therapeutic protein under pharmacologic control was achieved through in vivo somatic gene transfer. This system was based on the expression of two chimeric, human-derived proteins that were reconstituted by rapamycin into a transcription factor complex. A mixture of two adeno-associated virus vectors, one expressing the transcription factor chimeras and one containing erythropoietin (Epo) under the control of a promoter responsive to the transcription factor, was injected into skeletal muscle of immune-competent mice. Administration of rapamycin resulted in 200-fold induction of plasma Epo. Stable engraftment of this humanized system in immune-competent mice was achieved for 6 months with similar results for at least 3 months in a rhesus monkey

FKBP ligand homodimers can be used to activate signaling events inside cells and animals that have been engineered to express fusions between appropriate signaling domains and FKBP. However, use of these dimerizers in vivo is potentially limited by ligand binding to endogenous FKBP. We have designed ligands that bind specifically to a mutated FKBP over the wild-type protein by remodeling an FKBP-ligand interface to introduce a specificity binding pocket. A compound bearing an ethyl substituent in place of a carbonyl group exhibited sub-nanomolar affinity and 1,000-fold selectivity for a mutant FKBP with a compensating truncation of a phenylalanine residue. Structural and functional analysis of the new pocket showed that recognition is surprisingly relaxed, with the modified ligand only partially filling the engineered cavity. We incorporated the specificity pocket into a fusion protein containing FKBP and the intracellular domain of the Fas receptor. Cells expressing this modified chimeric protein potently underwent apoptosis in response to AP1903, a homodimer of the modified ligand, both in culture and when implanted into mice. Remodeled dimerizers such as AP1903 are ideal reagents for controlling the activities of cells that have been modified by gene therapy procedures, without interference from endogenous FKBP

Systemic delivery of specific therapeutic proteins by a parenteral route of administration is a recognized practice in the management of several gene defects and acquired diseases. As an alternative to repetitive parenteral administration, gene therapy may provide a novel means for systemic delivery of therapeutic proteins while improving patient compliance and therapeutic efficacy. However, for gene therapy to be an efficacious and safe approach to the clinical management of such diseases, gene expression must be tightly regulated. These investigations demonstrate precise in vivo control of protein expression from cells that are engineered to secrete human growth hormone (hGH) in response to stimulation by rapamycin. The cells were implanted intramuscularly into nu/nu mice and stimulated by intravenous or oral administration of rapamycin. In vivo experiments demonstrate that the activity and pharmacokinetics of rapamycin determine the level of serum hGH that result from the engineered cells. In addition, responsiveness of the cells to rapamycin, number of cells implanted, hGH expression kinetics, and the pharmacokinetics of hGH itself, also influence the circulating levels of hGH after rapamycin stimulation. Controlled manipulation of several of these parameters, either independently or in combination, allows for precise regulation of circulating hGH concentration in vivo

Gene therapy was originally conceived as a medical intervention to replace or correct defective genes in patients with inherited disorders. However, it may have much broader potential as an alternative delivery platform for protein therapeutics, such as cytokines, hormones, antibodies and novel engineered proteins. One key technical barrier to the widespread implementation of this form of therapy is the need for precise control over the level of protein production. A suitable system for pharmacologic control of therapeutic gene expression would permit precise titration of gene product dosage, intermittent or pulsatile treatment, and ready termination of therapy by withdrawal of the activating drug. We set out to design such a system with the following properties: (1) low baseline expression and high induction ratio; (2) positive control by an orally bioavailable small-molecule drug; (3) reduced potential for immune recognition through the exclusive use of human proteins; and (4) modularity to allow the independent optimization of each component using the tools of protein engineering. We report here the properties of this system and demonstrate its use to control circulating levels of human growth hormone in mice implanted with engineered human cells

The effect of various enzyme inhibitors and receptor antagonists on antigen (ovalbumin)-induced changes in pulmonary hemodynamics (arterial pressure, capillary pressure, and arterial and venous resistance), fluid filtration, and airway reactivity were monitored for 60 min in recirculating Ringer's-perfused, actively sensitized lungs. Bolus ovalbumin (30 micrograms) injection into the pulmonary artery produced initial (3 min postovalbumin) increases in pulmonary arterial pressure of 68 +/- 9% above baseline, which were followed by secondary increases (143 +/- 45% above baseline) at 30 min postovalbumin. Ovalbumin challenge also caused initial increases in pulmonary capillary pressure, arterial resistance, and venous resistance within 3 min after administration (100 +/- 34%, 51 +/- 10%, and 221 +/- 77% above baseline, respectively), which were further elevated at the end of the 60-min experimental period (292 +/- 74%, 66 +/- 29%, and 559 +/- 61% above baseline, respectively). Ovalbumin-induced increases in intratracheal pressure (771 +/- 142% above baseline) peaked at 3 min postchallenge and gradually returned towards baseline. Ovalbumin-induced changes in lung weight increased gradually over the perfusion period (3.5 +/- 1.0 g above baseline at 60 min postovalbumin). Antigen-induced changes in pulmonary arterial pressure, intratracheal pressure, and lung weight were abolished by pretreatment with the histamine1-receptor antagonist, pyrilamine (1 microM). The cyclooxygenase inhibitor, indomethacin (1 microM), potentiated antigen-induced secondary increases in pulmonary arterial pressure, intratracheal pressure, and lung weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigen challenge of actively sensitized guinea pigs produces airway eosinophilia, airway hyperreactivity, and late-phase bronchoconstriction. The recruited eosinophils are thought to be important cells in the development of the airway hyperreactivity and the late-phase bronchoconstriction. However, the functional abilities of these eosinophils have not been determined in response to antigen challenge. The purpose of this study was to describe the characteristics of superoxide anion release from airway eosinophils obtained 24 h after ovalbumin challenge of actively sensitized guinea pigs. Eosinophils were collected by bronchoalveolar lavage. The total bronchoalveolar lavage eosinophil count was 17- to 27-fold greater in sensitized, ovalbumin-challenged guinea pigs (9.30 +/- 0.11 x 10(6)/guinea pig) than in unsensitized guinea pigs (0.35 +/- 0.07 x 10(6)/guinea pig) or sensitized, saline-challenged guinea pigs (0.56 x 10(6)/guinea pig; n = 2). The increase in eosinophils was due to increased lavage leukocyte count and increased eosinophil differential. Eosinophils were isolated on a Percoll-plasma discontinuous gradient. Two populations of eosinophils were collected, one at the 1.093 g/ml gradient step and one at the 1.107 g/ml gradient step. Unstimulated or phorbol myristate acetate (PMA)-stimulated superoxide anion release was measured by the reduction of ferricytochrome c. Unstimulated superoxide anion release from both eosinophil populations of challenged guinea pigs (4.50 +/- 2.37 and 4.07 +/- 1.48 nmol from 1.093 and 1.107 g/ml eosinophils, respectively) was 6- to 7-fold greater than superoxide anion release from eosinophils of control guinea pigs (0.74 +/- 0.43 and 0.56 +/- 025 nmol from 1.093 and 1.107 g/ml eosinophils, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo endotoxin infusion produces neutrophil-mediated acute lung injury and increases superoxide anion release from phorbol myristate acetate (PMA)-stimulated blood neutrophils collected 18-24 hours after the infusion. Because the turnover time of circulating blood neutrophils is only 6-8 hours, it was hypothesized that the prolonged increase in superoxide anion release from peripheral blood neutrophils is associated with increased superoxide anion release from bone marrow neutrophils. To test this hypothesis, two doses of Escherichia coli endotoxin (5.0 and 0.5 micrograms/kg) were infused into chronically instrumented awake sheep. Blood and bone marrow neutrophils were collected 24 hours after the infusion, and superoxide anion release from unstimulated and PMA-stimulated neutrophils was measured in vitro. Endotoxin infusion produced an increase in pulmonary microvascular permeability, in intravascular activation (degranulation) of blood neutrophils, and in circulating blood neutrophils 24 hours after the infusion. High-dose endotoxin (5.0 micrograms/kg; n = 4) increased superoxide anion release from unstimulated peripheral blood neutrophils (2.25 +/- 0.38 times baseline [p less than or equal to 0.05]) and from peripheral blood neutrophils stimulated with 10(-9) M PMA in vitro (1.46 +/- 0.55 times baseline). Low-dose endotoxin (0.5 micrograms/kg; n = 5), on the other hand, did not alter superoxide anion release from peripheral blood neutrophils. Bone marrow neutrophils could not be isolated reproducibly after high-dose endotoxin because of leukoaggregation. Bone marrow neutrophils were isolated after low-dose endotoxin infusion. Stimulation of these cells with 10(-9) M PMA in vitro resulted in a two- to fourfold increase above control release (p less than or equal to 0.05). Increased superoxide anion release from both peripheral blood and bone marrow neutrophils occurred in the absence of circulating endotoxin, as measured by a Limulus assay. These results show that the prolonged increase in superoxide anion release from peripheral blood neutrophils is associated with an increase in the superoxide anion release from bone marrow neutrophils. Furthermore, the recruitment of affected bone marrow neutrophils into peripheral blood may explain the increased superoxide anion release from blood neutrophils 24 hours after endotoxin infusion

Phorbol myristate acetate (PMA, 10(-7) M) activation of adherent neutrophils (PMNs) led to a markedly attenuated release of superoxide anion (O2-) per cell when PMNs were activated at high density (2.85 fmol O2-/PMN at 2 million in 0.1 ml) in comparison with cells activated at low cell density (12.0 fmol O2-/PMN at 250,000 in 0.1 ml). This 'autoregulatory' phenomenon was not due to a defect in the superoxide anion assay employed, to a differential adherence of neutrophils at high vs. low density, or to substrate (cytochrome c) or cell stimulus (PMA) limitation. It was associated with an inhibition of apparent NADPH oxidase activity and a leftward shift (toward a lower level of activation) in the activation profile of PMNs (as determined by FACS analysis using PMNs preloaded with 2'7'-dichlorofluorescin diacetate in which H2O2 production results in the production of the fluorescent product 2'7'-dichlorofluorescein intracellularly). Other aspects of the neutrophil activation response including arachidonic acid mobilization, phospholipid metabolism, and perhaps phosphatidylinositol turnover were also attenuated when PMNs were activated at high cell density. Studies with cells in solution, cells treated with cycloheximide, and cells treated with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid suggest that PMN contact with a surface, neutrophil protein synthesis, and an increased surface expression of the heterodimer CD11b/CD18 on PMNs all were not required for autoregulation. Finally, morphometric and morphologic examination of PMNs activated at low vs. high density revealed histologic and structural correlates associated with the attenuated PMN activation response of cells triggered at high cell density. We conclude that multiple structural and functional aspects of the PMN activation response are modulated by cell density and suggest that this property is important both in the physiologic control of neutrophil activation and in the design of in vitro assays of the neutrophil activation response

It is well established that activation of neutrophils within the pulmonary circulation produces acute lung injury in which adherence of neutrophils to endothelial cells is an obligatory step in the mechanism of injury. The effects of in vivo activation of neutrophils on the in vitro responses of these cells to stimulation have not been determined, although such information may be important in understanding how different etiological factors may interact to produce infection or acute respiratory failure. By using an assay to sequentially measure superoxide anion (O2-) release from adherent neutrophils stimulated with phorbol myristate acetate (PMA), we measured the in vitro activation response of peripheral blood neutrophils isolated before and 24 h after infusion of zymosan-activated plasma (ZAP; or untreated plasma as a control), air bubbles, or PMA in awake, instrumented sheep. Each of the three inflammatory agents produced an increase in lung microvascular permeability characteristic of acute lung injury; control plasma did not. For the in vivo ZAP experiments, stimulated O2- release in vitro by using PMA was approximately 50% lower (P less than 0.05) for neutrophils isolated 24 h after the in vivo infusion (4.3 +/- 0.8 nmol/500,000 cells) than before (8.1 +/- 0.2 nmol/500,000 cells). For the air emboli or PMA in vivo experiments, there were no changes in neutrophil activation responses in vitro. Similarly, infusion of control plasma did not result in reduced neutrophil O2- release. These results show that alterations in the inflammatory potential of neutrophils may occur in vivo and that such alterations appear to be dependent on the mechanism and agent by which lung injury is produced