Tropism-modified AAV vectors for neonatal gene transfer

氨基酸紊乱是一类以主要代谢途径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和饮食限制，以避免利用有缺陷的代谢途径。这些策略的长期成功与否因疾病而异。由于许多这些疾病是由肝酶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诱人目标。腺相关病毒（AAV）已成为一种有前途的载体，可以在动物模型中实现长期的肝脏表达。然而，新生儿基因转移到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导致转导细胞稀释的限制。提高AAV靶向肝脏的效率可以增加载体传递到肝脏的有效剂量，同时减少不必要的肝外转导。在这个提议中，我们假设可以修改AAV的结合特性来满足这些目标。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寻找可能靶向的新生儿肝脏特异性受体。然后，我们将在体外研究这些修饰对肝和非肝组织中AAV转导的影响。最后，我们将检测新生小鼠静脉注射AAV后生物分布和表达持久性的变化。新生儿肝脏AAV趋向性的改善将提供一种工具，广泛适用于一系列主要影响肝脏的儿科代谢性疾病，包括氨基酸紊乱。此外，在这个建议中提供的技能和互动将是一个宝贵的援助，从博士后研究过渡到职业生涯作为一个独立的研究者。

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.