RESUMEN
Crigler-Najjar (CN) patients have no bilirubin UDP glucuronosyltransferase (UGT1A1) activity and suffer brain damage because of bilirubin toxicity. Vectors based on adeno-associated virus (AAV) serotype 2 transduce liver cells with relatively low efficiency. Recently, AAV serotypes 1, 6, and 8 have been shown to be more efficient for liver cell transduction. We compared AAV serotypes 1, 2, 6, and 8 for correction of UGT1A1 deficiency in the Gunn rat model of CN disease. Adult Gunn rats were injected with CMV-UGT1A1 AAV vectors. Serum bilirubin was decreased over the first year by 64% for AAV1, 16% for AAV2, 25% for AAV6, and 35% for AAV8. Antibodies to UGT1A1 were detected after injection of all AAV serotypes. An AAV1 UGT1A1 vector with the liver-specific albumin promoter corrected serum bilirubin levels but did not induce UGT1A1 antibodies. Two years after injection of AAV vectors all animals had large lipid deposits in the liver. These lipid deposits were not seen in age-matched control animals. AAV1 vectors are promising candidates for CN gene therapy because they can mediate a reduction in serum bilirubin levels in Gunn rats that would be therapeutic in humans.
Asunto(s)
Síndrome de Crigler-Najjar/terapia , Dependovirus/genética , Terapia Genética/métodos , Vectores Genéticos/farmacología , Glucuronosiltransferasa/deficiencia , Animales , Bilis/fisiología , Bilirrubina/sangre , Síndrome de Crigler-Najjar/genética , Dependovirus/clasificación , Dependovirus/inmunología , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/inmunología , Hígado/efectos de los fármacos , Hígado/patología , Reacción en Cadena de la Polimerasa/métodos , Ratas , Ratas Gunn , Serotipificación , Distribución TisularRESUMEN
BACKGROUND/AIM: Multidrug Resistance Protein 3 (MRP3) transports bile salts and glucuronide conjugates in vitro and is postulated to protect the liver in cholestasis. Whether the absence of Mrp3 affects these processes in vivo is tested. METHODS: Mrp3-deficient mice were generated and the contribution of Mrp3 to bile salt and glucuronide conjugate transport was tested in (1): an Ussing-chamber set-up with ileal explants (2), the liver during bile-duct ligation (3), liver perfusion experiments, and (4) in vitro vesicular uptake experiments. RESULTS: The Mrp3((-/-)) mice show no overt phenotype. No differences between WT and Mrp3-deficient mice were found in the trans-ileal transport of taurocholate. After bile-duct ligation, there were no differences in histological liver damage and serum bile salt levels between Mrp3((-/-)) and WT mice, but Mrp3-deficient mice had lower serum bilirubin glucuronide concentrations. Glucuronide conjugates of hyocholate and hyodeoxycholate are substrates of MRP3 in vitro and in livers that lack Mrp3, there is reduced sinusoidal secretion of hyodeoxycholate-glucuronide after perfusion with hyodeoxycholate. CONCLUSIONS: Mrp3 does not have a major role in bile salt physiology, but is involved in the transport of glucuronidated compounds, which could include glucuronidated bile salts in humans.