Tropism-modified AAV vectors for neonatal gene transfer

氨基酸紊乱是以主要代谢途径中的遗传缺陷为特征的疾病家族。对于大多数这些综合征，目前的治疗仅限于急性发作的支持性护理，以及饮食限制以避免利用有缺陷的代谢途径。这些策略的长期成功在不同疾病之间差异很大。由于这些疾病中的许多是由肝酶中的单基因缺陷引起的，因此它们是针对肝脏的新生儿基因转移策略的有吸引力的靶点。腺相关病毒（AAV）已经成为一种有前途的载体，其可以在动物模型中实现长期肝表达。然而，其用于新生儿基因转移至肝脏的临床潜力受到体内多个器官的非特异性转导以及治疗后新生儿肝脏快速生长对转导细胞的稀释的限制。AAV靶向肝脏的效率的提高可以增加递送至肝脏的载体的有效剂量，同时减少不需要的肝外转导。在这个提议中，我们假设可以修改AAV的结合特性以满足这些目标。我们将创建针对肝脏特异性受体的双特异性靶向构建体，并使用噬菌体文库展示技术来寻找可能靶向的新生儿肝脏特异性受体。然后，我们将检查这些修饰对体外肝和非肝组织的AAV转导的影响。最后，我们将检查AAV静脉注射到新生小鼠后生物分布和表达持续性的变化。AAV对新生儿肝脏的嗜性的改善将提供一种工具，其将广泛适用于主要影响肝脏的一系列儿科代谢疾病，包括氨基酸疾病。此外，本提案中提供的技能和互动将是从博士后研究过渡到独立调查员职业生涯的宝贵帮助。

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.