Inborn errors of liver metabolism are frequent causes of morbidity and mortality especially in children. For several of these diseases, treatment approaches depend on ,manipulation of the affected metabolic pathway by diet, drugs, vitamin cofactors, enzyme induction, end-product replacement, and alternative pathway activation. Unfortunately, these approaches often remain unsatisfactory especially in the face of illness or catabolism. Ideally, transfer of the normal genes in the liver cells that are defective might restore the metabolic function. The goal of my PhD thesis was to develop gene-based therapeutic strategies to correct a life-threatening inborn error of liver metabolism, Crigler-Najjar syndrome type I (CNI). CNI is a severe inborn error of bilirubin metabolism due to mutations of the uridine diphospho-glucuronosyl transferase lAl (UGTIA1) gene, Affected patients have elevations of serum bilirubin, and they have to spend extended hours under bilirubin lights throughout childhood and adolescence. Despite this therapy, they remain at risk of brain damage when intercurrent infections may increase production of bilirubin above that which can be controlled by the bilirubin light therapy. Thus, patients with CNI often are advised to consider liver transplantation. Therefore, alternative therapies are highly needed to overcome the mortality and morbidity associated with transplantation procedure, and risks of life-long immunosuppression. Gene therapy has the potential to provide a definitive cure for patient with CNI My studies have focused on the development of gene therapy strategies for this disease. First, I investigated in Gunn rats, the animal model for CNI, the efficacy of adeno-associated viral (AA V) vector-mediated muscledirected gene therapy and I found that serotype I AA V vector expressing UOTIAI resulted in expression of UGTIAl protein and functionally active enzyme in injected muscles, and aj 50% reduction in serum bilirubin levels for at least 1 year post-injection. Taken together, these data show that clinically relevant and sustained reduction of serum bilirubin levels can be achieved by simple and safe intramuscular injections. Following initial problems with intravenous injections of AA V2 vector, a major success has been achieved with AA V2/8 vectors for liver-directed gene therapy of hemophilia. Encouraged by these results and by the possibility of achieving full correction of the hyperbilirubineotia with systemic delivery, next I focused on the design and optimization of an AA V2I8 vector for liver-directed gene therapy of CNI. I generated multiple expression cassettes expressing the UGTIAl gene inserted into the AA V2/8 vectors for in vivo testing. The results of these studies showed that AAV2/8 vector with codon optimized UGTlAI gene tender the control of the hepatocyte-specific LP} promoter resulted in improved and sustained correction of hyperbilirubinemia in Gunn rats. Taken together, these data demonstrate the development of an optimal expression cassette for liver-directed gene therapy of CNI and form the preclinical basis for the development of a gene therapy trial for this severe disorder.

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