Cellular selectivity of AAV serotypes for gene delivery in neurons and astrocytes by neonatal intracerebroventricular injection

The non-pathogenic parvovirus, adeno-associated virus (AAV), is an efficient vector for transgene expression in vivo and shows promise for treatment of brain disorders in clinical trials. Currently, there are more than 100 AAV serotypes identified that differ in the binding capacity of capsid proteins to specific cell surface receptors that can transduce different cell types and brain regions in the CNS. In the current study, multiple AAV serotypes expressing a GFP reporter (AAV1, AAV2/1, AAVDJ, AAV8, AAVDJ8, AAV9, AAVDJ9) were screened for their infectivity in both primary murine astrocyte and neuronal cell cultures. AAV2/1, AAVDJ8 and AAV9 were selected for further investigation of their tropism throughout different brain regions and cell types. Each AAV was administered to P0-neonatal mice via intracerebroventricular injections (ICV). Brains were then systematically analyzed for GFP expression at 3 or 6 weeks post-infection in various regions, including the olfactory bulb, striatum, cortex, hippocampus, substantia nigra (SN) and cerebellum. Cell counting data revealed that AAV2/1 infections were more prevalent in the cortical layers but penetrated to the midbrain less than AAVDJ8 and AAV9. Additionally, there were differences in the persistence of viral transgene expression amongst the three serotypes examined in vivo at 3 and 6 weeks post-infection. Because AAV-mediated transgene expression is of interest in neurodegenerative diseases such as Parkinson’s Disease, we examined the SN with microscopy techniques, such as CLARITY tissue transmutation, to identify AAV serotypes that resulted in optimal transgene expression in either astrocytes or dopaminergic neurons. AAVDJ8 displayed more tropism in astrocytes compared to AAV9 in the SN region. We conclude that ICV injection results in lasting expression of virally encoded transgene when using AAV vectors and that specific AAV serotypes are required to selectively deliver transgenes of interest to different brain regions in both astrocytes and neurons.

腺相关病毒（adeno-associated virus, AAV）作为一种非致病性细小病毒，是可在体内高效实现转基因表达的载体，在临床试验中展现出治疗脑部疾病的潜力。目前已鉴定出100余种AAV血清型，其衣壳蛋白与特定细胞表面受体的结合能力存在差异，可转导中枢神经系统（central nervous system, CNS）内的不同细胞类型与脑区。本研究筛选了多种表达绿色荧光蛋白（green fluorescent protein, GFP）报告基因的AAV血清型（AAV1、AAV2/1、AAVDJ、AAV8、AAVDJ8、AAV9、AAVDJ9），以检测其在原代小鼠星形胶质细胞与神经元细胞培养物中的感染性。最终选取AAV2/1、AAVDJ8与AAV9，进一步探究其在不同脑区与细胞类型中的嗜性。将各AAV通过脑室内注射（intracerebroventricular injection, ICV）接种于出生0天的新生小鼠体内。于感染后3周或6周，对嗅球、纹状体、皮层、海马、黑质（substantia nigra, SN）与小脑等多个脑区的GFP表达情况进行系统性分析。细胞计数结果显示，AAV2/1感染在皮层层中更为普遍，但相较于AAVDJ8与AAV9，其向中脑的穿透能力更弱。此外，在感染后3周与6周的体内实验中，三种血清型的病毒转基因表达持久性存在差异。鉴于AAV介导的转基因表达在帕金森病（Parkinson’s Disease）等神经退行性疾病中具有研究价值，我们采用诸如CLARITY组织透明化改造技术之类的显微镜技术对黑质进行检测，以筛选出可在星形胶质细胞或多巴胺能神经元中实现最优转基因表达的AAV血清型。相较于AAV9，AAVDJ8在黑质区域中对星形胶质细胞的嗜性更强。本研究得出结论：使用AAV载体通过脑室内注射可实现病毒编码转基因的持久表达，且需借助特定AAV血清型，才能将目的转基因选择性递送至星形胶质细胞与神经元内的不同脑区。