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Efficient Long Lasting AAV Gene Therapy in a Mouse Model of Crigler-Najjar Syndrome Type I : Coming of Age?

The Crigler-Najjar Type I Syndrome is a rare monogenic disease (incidence around 1/10^6 births) caused by Uridine-diphosphate- glucuronosyltransferase (UGT)1A1 enzyme deficiency, which is responsible for bilirubin conjugation in the liver. There is no permanent cure and hyperbilirubinemia is associated with lifelong risk of kernicterus, that produces serious neurological damage leading to death if untreated. At present, CNSI patients are temporarily treated with daily intensive phototherapy, severely affecting their social life. However, the effectiveness of phototherapy is reduced with the age. The only effective therapy is liver transplantation, feasible only in a restricted number of cases and associated with significant morbidity and mortality. It requires lifelong immunosuppression and involves substantial risks and costs. We aim to set up an effective and long lasting therapy for the definitive cure of the disease, based on the use of AAV vectors, that could promptly be translated to CNSI patients. To this aim, we performed neonatal and adult transduction in a mouse model of Crigler-Najjar Type I Syndrome developed in our laboratory with AAV vectors carrying the hUGT1a1 cDNA under the control of a liver-specific promoter. We first tested the strength of different liver-specific promoters in vitro and in vivo, and showed that the alpha-1-antitrypsin promoter with the ApoE enhancer was the most efficient one. A single i.p. neonatal injection (AAV8-AAT-hUGT1a1 at P4) resulted in a therapeutically significant decrease of plasma bilirubin (~2.5 mg/dL at 13 months). We then performed i.p. neonatal injection (AAV8-AAT- hUGT1a1 at P4), followed by the i.v. injection of AAV9-AAT-hUGT1a1 at P60. This protocol proved to be more efficient than a single neonatal injection, as plasma UCB levels were very close to wt (~1 mg/dL at 13 months after injection). A single adult injection (P60, iv injection) was even more effective, with plasma UCB reaching concentrations undistinguishable from wt levels even after 13 months. However, this procedure was effective only in males, as plasma UCB in females increased over time, reaching values similar to control mutant mice levels. We detected a humoral immune response against the transgene in females. The response was not dependent on the transduction and ectopic expression in APC, as the response was similar with constructs containing a miRNA142- sponge in sense or antisense orientation. miRNA 142 is specifically expressed in APC, and the presence of the sponge completely abolishes expression of the mRNA. Motor coordination abilities of all treated mice were normal. The presence of a cellular immune response will be assessed. We conclude that neonatal injection in both genders and adult injection of males promote immunological tolerance to Ugt1a1 and results in a very efficient long-term correction of the disease, while adult injection in females results in an immune response against the transgene with a failure to improve the hyperbilirubinemic condition.

Crigler-Najjar Syndrome Type I, gene therapy, UGT1A1, mouse model, AAV

DOI: 10.1016/S1525-0016(16)35596-4