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Characterization of a new mouse model for Crigler-Najjar Syndrome Type I and possible therapeutic approaches

Severe unconjugated hyperbilirubinemia may lead to neurologic defects and/or death, if untreated. It can be caused by a physiological delay in UGT1A1 gene expression, mutations in the gene [resulting in the Crigler-Najjar Syndrome Type I (CNI)], or other concurrent causes. Current clinical practice to control hyperbilirubinemia consists of intensive phototherapy. However, CNI patients are finally treated with liver transplantation, as phototherapy becomes less effective with aging. Alternatively, gene therapy is a promising approach for CNI, but existing protocols are unsatisfactory for clinical transfer. Likewise, the mechanisms affecting bilirubin neurotoxicity are still partially ill-defined. A huge spectrum of neurological damage caused by similar levels of unconjugated bilirubin suggests the presence of different genes and/or mechanisms modulating bilirubin neurotoxicity. Two ABC transporters were reported to be potentially important in bilirubin transport in the brain, namely Mdr1 and Mrp1. The objectives of this thesis are the determination of the Mdr1 and Mrp1 role in the in vivo modulation of bilirubin neurotoxicity, and the optimization of gene therapy protocols using a relevant mouse model of the CNI. Experiments with Mdr1-/-/Ugt1-/- double knockout (DKO) mice exposed Mdr1 as a relevant modulator of bilirubin neurotoxicity. A temporary phototherapy treatment rescued survival of 50% of single Ugt1-/- KO mice, while all DKO animals died. DKO animals had increased cerebellar bilirubin levels, supporting the survival differences. Additionally, Mdr1a expression was upregulated by bilirubin. On the contrary, Mrp1-/-/Ugt1-/- DKO showed that Mrp1 did not affect bilirubin neurotoxicity. To improve gene therapy protocols, different liver-specific constructs were tested, as well as different time points of vector administration and routes of delivery, using adeno-associated viral vectors (AAV) expressing the hUGT1A1. Intravenous administration of AAVs to adult males emerged as the most efficient protocol, with plasma bilirubin levels undistinguishable from WT animals for at least 15 months.

Crigler-Najjar Syndrome Type I, ABC transporters, bilirubin, gene therapy, neonatal hyperbilirubinemia

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