UNC Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Mol Ther. 2010 Mar;18(3):570-8. doi: 10.1038/mt.2009.292. Epub 2009 Dec 29.
DNA shuffling and directed evolution were employed to develop a novel adeno-associated virus (AAV) vector capable of crossing the seizure-compromised blood-brain barrier (BBB) and transducing cells in the brain. Capsid DNA from AAV serotypes 1-6, 8, and 9 were shuffled and recombined to create a library of chimeric AAVs. One day after kainic acid-induced limbic seizure activity in rats, the virus library was infused intravenously (i.v.), and 3 days later, neuron-rich cells were mechanically dissociated from seizure-sensitive brain sites, collected and viral DNA extracted. After three cycles of selection, green fluorescent protein (GFP)-packaged clones were administered directly into brain or i.v. 1 day after kainic acid-induced seizures. Several clones that were effective after intracranial administration did not transduce brain cells after the i.v. administration. However, two clones (32 and 83) transduced the cells after direct brain infusion and after i.v. administration transduced the cells that were localized to the piriform cortex and ventral hippocampus, areas exhibiting a seizure-compromised BBB. No transduction occurred in areas devoid of BBB compromise. Only one parental serotype (AAV8) exhibited a similar expression profile, but the biodistribution of 32 and 83 diverged dramatically from this parental serotype. Thus, novel AAV vectors have been created that can selectively cross the seizure-compromised BBB and transduce cells.
DNA 改组和定向进化被用于开发一种新型腺相关病毒（AAV）载体，该载体能够穿透癫痫发作受损的血脑屏障（BBB）并转导大脑中的细胞。AAV 血清型 1-6、8 和 9 的衣壳 DNA 被改组和重组，以创建嵌合 AAV 的文库。在大鼠海人酸诱导的边缘性癫痫发作后 1 天，将病毒文库静脉内（i.v.）输注，3 天后，从癫痫发作敏感的脑区机械分离富含神经元的细胞，收集并提取病毒 DNA。经过三轮选择，将包装有绿色荧光蛋白（GFP）的克隆直接递送至脑内或海人酸诱导癫痫发作后 1 天静脉内。一些在颅内给药后有效的克隆在静脉内给药后不能转导脑细胞。然而，两个克隆（32 和 83）在直接脑内输注后转导了细胞，并且在静脉内给药后转导了位于梨状皮层和腹侧海马的细胞，这些区域表现出癫痫发作受损的 BBB。在没有 BBB 受损的区域没有发生转导。只有一种亲本血清型（AAV8）表现出类似的表达谱，但 32 和 83 的分布与该亲本血清型有很大差异。因此，已经创建了能够选择性穿透癫痫发作受损的 BBB 并转导细胞的新型 AAV 载体。
