Development of AAV8-mediated gene therapy clinical trial for Crigler-Najjar syndrome type I: optimization of liver-specific expression cassette. N. Pastore1, E. Nusco1, A. Auricchio1,2, N. Brunetti-Pierri1,2 1) Telethon Institute of Genetics and Medicine, Naples, Italy; 2) Department of Translational Medicine, Federico II University of Naples, Italy.

   Crigler-Najjar syndrome type I is a severe inborn error of bilirubin metabolism due to deficiency of the liver-specific UDP-glucuronosyltransferase encoded by the UGT1A1 gene. Current therapy relies on phototherapy to prevent life-threatening elevations of serum bilirubin but liver transplantation is the only permanent treatment. Gene therapy has the potential to provide a safer and a definitive cure for this severe disease. Adeno associated viral (AAV) vectors derived from serotype 8 have recently shown very promising results for liver-directed gene therapy of patients with hemophilia B. These encouraging results may pave the way towards AAV8-mediated gene therapy for inborn errors of liver metabolism, such as Crigler-Najjar syndrome type I. As a first step towards the development of a clinical trial for this disorder, we focused on the design and optimization of the AAV8 expression cassette. Towards this goal, we injected intravenously the Gunn rats, the animal model of Crigler-Najjar syndrome type I, with an AAV8 vector expressing the human UGT1A1 under the control of the liver-specific thyroxine-binding globulin (TBG) promoter. A significant and sustained 60% reduction of baseline serum bilirubin levels was detected only at the high dose of 7.4x10e13 genome copies (gc)/kg. Next, we achieved a 50% reduction of baseline serum bilirubin with 2.7x10e13 gc/kg of a vector encoding the UGT1A1 under the control of the synthetic, liver-specific LP1 promoter. Interestingly, the injection of another vector bearing the codon optimized UGT1A1 cDNA (cohUGT1A1) under the control of the LP1 promoter resulted in complete normalization of serum bilirubin levels at the further lower dose of 1.1x10e13 gc/kg. This vector also resulted in higher levels of hepatic UGT1A1 protein normalized for the vector genome copy number of injected animals. In summary, our study shows that the AAV8-LP1-cohUGT1A1 results in higher hepatic transgene levels and sustained correction of hyperbilirubinemia in the Gunn rats. These pre-clinical data may form the basis for the development of a gene therapy trial for liver-directed gene therapy of Crigler-Najjar syndrome type I.