title: Persistent and high-level expression of human liver prolidase in vivo in mice using adenovirus.
abstract: Human liver prolidase, a metal-dependent dipeptidase, is being tested as a potential catalytic bioscavenger against organophosphorus (OP) chemical warfare nerve agents. The purpose of this study was to determine whether persistent and high-levels of biologically active and intact recombinant human (rHu) prolidase could be introduced in vivo in mice using adenovirus (Ad). Here, we report that a single intravenous injection of Ad containing the prolidase gene with a 6× histidine-tag (Ad-prolidase) introduced high-levels of rHu prolidase in the circulation of mice which peaked on days 5-7 at 159 ± 129 U/mL. This level of prolidase is ~120 times greater than that of the enzyme level in mice injected with Ad-null virus. To determine if all of Ad-prolidase-produced rHu prolidase was exported into the circulation, enzyme activity was measured in a variety of tissues. Liver contained the highest levels of rHu prolidase on day 7 (5647 ± 454 U/g) compared to blood or any other tissue. Recombinant Hu prolidase hydrolyzed DFP, a simulant of OP nerve agents, in vitro. In vivo, prolidase overexpression extended the survival of 4 out of 6 mice by 4-8h against exposure to two 1× LD(50) doses of DFP. In contrast, overexpression of mouse butyrylcholinesterase (BChE), a proven stoichiometric bioscavenger of OP compounds, protected 5 out of 6 mice from DFP lethality and surviving mice showed no symptoms of DFP toxicity. In conclusion, the results suggest that gene delivery using Ad is capable of introducing persistent and high levels of human liver prolidase in vivo. The gene-delivered prolidase hydrolyzed DFP in vitro but provided only modest protection in vivo in mice, delaying the death of the animals by only 4-8h.
authors.affiliation: Division of Biochemistry, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
authors.forename: Vineela
authors.initials: V
authors.lastname: Aleti
authors.forename: Gireesh B
authors.initials: GB
authors.lastname: Reddy
authors.forename: Kalpana
authors.initials: K
authors.lastname: Parikh
authors.forename: Peethambaran
authors.initials: P
authors.lastname: Arun
authors.forename: Nageswararao
authors.initials: N
authors.lastname: Chilukuri
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000931:Antidotes / Q000378:metabolism / Q000627:therapeutic use; D002619:Chemical Warfare Agents / Q000378:metabolism / Q000633:toxicity; D004150:Dipeptidases / Q000235:genetics* / Q000378:metabolism* / Q000627:therapeutic use; D005260:Female; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007531:Isoflurophate / Q000378:metabolism / Q000633:toxicity; D008099:Liver / Q000201:enzymology; D051379:Mice; D009943:Organophosphorus Compounds / Q000378:metabolism / Q000633:toxicity; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D014018:Tissue Distribution
chemical_list: D000931:Antidotes; D002619:Chemical Warfare Agents; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; D007531:Isoflurophate; D004150:Dipeptidases; C022449:proline dipeptidase