RESUMEN
Crigler-Najjar syndrome type 1 (CN type 1) is an autosomal recessive disorder characterized by nonhemolytic jaundice resulting from mutations to the gene encoding bilirubin-UDP-glucuronosyltransferase (UDPGT). The Gunn rat is an accurate animal model of this disease because the bilirubin-UDPGT gene in this strain carries a premature stop codon. The primary objective of this study was to complement this deficiency in vivo using liver-directed gene therapy. The efficiency of adenovirus type 5 (Ad5)-mediated gene transfer to the neonatal rat liver was first assessed by intravenous (i.v.) injection of an Ad5 vector carrying a nuclear-localized LacZ gene. An Ad5 vector expressing the cDNA encoding human bilirubin-UDPGT (Ad5/CMV/hUG-Br1) was then generated and injected i.v. into neonatal Gunn rats. Plasma samples were collected and bilirubin levels were determined at regular intervals. Although the mean level of bilirubin in homozygous Gunn rats 1-2 days after birth was already 14.5-fold higher than that of heterozygous siblings, treatment with Ad5/CMV/hUG-Br1 reduced plasma bilirubin to normal levels within 1 week. Plasma bilirubin in the treated homozygous rats remained normal for 4 weeks before gradually climbing to intermediate levels that were approximately half that of untreated homozygotes by 12 weeks. Administration of Ad5-mediated gene therapy to neonatal Gunn rats effectively complemented the deficiency in bilirubin-UDPGT, resulting in substantial reductions in plasma bilirubin over a 3-month period. The efficacy of Ad5-mediated gene therapy in neonates suggests that this approach might be effective against other hepatic disorders, including autosomal recessive deficiencies in lipid metabolism and vascular homeostasis.
Asunto(s)
Síndrome de Crigler-Najjar/terapia , Terapia Genética/métodos , Glucuronosiltransferasa/genética , Adenovirus Humanos/genética , Alanina Transaminasa/sangre , Animales , Animales Recién Nacidos , Bilirrubina/sangre , Síndrome de Crigler-Najjar/enzimología , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Homocigoto , Humanos , Ictericia , Operón Lac/genética , Hígado/metabolismo , Señales de Localización Nuclear , Ratas , Ratas GunnRESUMEN
PURPOSE: The authors have investigated the hypothesis that prostaglandin E2 (PGE2) synthesis is regulated during corneal endothelial wound healing. Previous studies have shown that PGE2 is an important mediator of endothelial mitosis, migration, and differentiation. METHODS: Biosynthesis of PGE2 was investigated in a wound closure model of the cultured rabbit corneal endothelium and in cultures treated with experimental agents. Prostaglandin E2 synthesis was measured by enzyme-linked immunosorbent assay. Pharmacologic experiments were designed to evaluate the contributions of protein kinases, phospholipase A2, and cyclooxygenase to endogenous PGE2 synthesis. RESULTS: Prostaglandin E2 synthesis is increased markedly in response to injury and is proportional to the extent of wounding. Biosynthesis of PGE2 returns to basal levels concurrently with recovery of the injury. Synthesis is dependent on the activities of protein kinase C (PKC), phospholipase A (PLA), and cyclooxygenase. Two forms of cyclooxygenase are present in corneal endothelial cells, and pharmacologic studies indicate that the activity of the COX 2 contributes to injury-dependent PGE2 synthesis. CONCLUSIONS: Prostaglandin E2 synthesis is increased in injured corneal endothelial cells. This synthesis is dependent on the coordinated regulation of PKC, PLA, and cyclooxygenase. Prostaglandin E2 synthesis presents an attractive target for pharmacologic manipulation of endothelial recovery from injury.