The amino acid disorders are a family of diseases characterized by genetic defects in a primary metabolic pathway. For most of these syndromes, current treatment is limited to supportive care for acute episodes, and dietary restriction to avoid utilization of the defective metabolic pathway. The long-term success of these strategies varies greatly between diseases. Because many of these disorders are caused by single gene defects in hepatic enzymes, they are appealing targets for neonatal gene transfer strategies directed at the liver. Adeno-associated virus (AAV) has emerged as a promising vector that can achieve long- term hepatic expression in animal models. However, its clinical potential for neonatal gene transfer to the liver is limited by non- specific transduction of multiple organs in vivo, and by the dilution of transduced cells by the rapid growth of the newborn liver following treatment. Improvements in the efficiency of AAV targeting to the liver could increase the effective dose of vector delivered to the liver, while decreasing unwanted extrahepatic transduction. In this proposal we hypothesize that the binding properties of AAV can be modified to meet these goals. We will create bispecific targeting constructs directed at liver-specific receptors, and use phage library display technology to seek neonatal liver-specific receptors which may be targeted. We will then examine the effects of these modifications on AAV transduction of hepatic and non-hepatic tissues in vitro. Finally, we will examine changes in the biodistibution and persistence of expression following intravenous injection of AAV into newborn mice. Improvements in AAV tropism for the neonatal liver will provide a tool which will be widely applicable to a range of pediatric metabolic diseases that primarily affect the liver, including the amino acid disorders. In addition, the skills and interaction provided in this proposal will be an invaluable aid in the transition from post-doctoral research to a career as an independent investigator.
