Intrathecal delivery of AAV9 into the subarachnoid space has been shown to transduce spinal cord and brain and be less affected by preexisting antibodies, which are lower in cerebral spinal fluid. Still, efficiency of transduction needs to be improved. Recently, we identified a new capsid from a library selection in mice, called AAV-F, that allowed robust transduction of the spinal cord gray matter after lumbar injection. In this study, we test transduction of spinal cord by AAV-F (n = 3) compared to AAV9 (n = 2), using a reporter gene, in cynomolgus monkeys after lumbar intrathecal injection. Using an automated image analysis (IA) approach to sensitively quantitate reporter gene expression in spinal cord, we found that AAV-F capsid mediated slightly higher transgene expression (both in percentages of cells and intensity of immunostaining) in motor neurons and interneurons, in the lumbar and thoracic regions, compared to AAV9. Interestingly, although AAV-F mediated higher transgene expression in spinal cord, the number of genomes in spinal cord and periphery were on average lower for AAV-F than AAV9, which suggest that lower numbers of genomes were able to mediate higher transgene expression in spinal cord with this capsid. In contrast, dorsal root ganglion transduction efficiency was lower for AAV-F compared to AAV9 on average. Interestingly, we also observed transduction of Schwann cells in sciatic nerve in two nonhuman primates injected with AAV-F, but none with AAV9. Overall, our data demonstrate the utility of automated IA for quantitation of AAV transduction in the spinal cord and the favorable on-target:off-target transduction profile suggests that the AAV-F capsid be considered for gene therapy applications focused on treating the spinal cord after intrathecal delivery.

中文翻译：

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AAV9 变体衣壳 AAV-F 介导鞘内给药后非人灵长类脊髓中广泛的转基因表达

已证明将 AAV9 鞘内递送至蛛网膜下腔可转导脊髓和大脑，并且受先前存在的抗体影响较小，这些抗体在脑脊髓液中含量较低。尽管如此，转导效率仍需提高。最近，我们从小鼠文库选择中发现了一种新的衣壳，称为 AAV-F，它允许在腰椎注射后对脊髓灰质进行稳健的转导。在这项研究中，我们测试了 AAV-F ( n  = 3) 与 AAV9 ( n = 2)，使用报告基因，在食蟹猴腰椎鞘内注射后。使用自动图像分析 (IA) 方法灵敏地定量脊髓中的报告基因表达，我们发现 AAV-F 衣壳在运动神经元和中间神经元中介导了略高的转基因表达（细胞百分比和免疫染色强度），在与 AAV9 相比，腰椎和胸椎区域。有趣的是，尽管 AAV-F 在脊髓中介导了更高的转基因表达，但 AAV-F 的脊髓和外周基因组数量平均低于 AAV9，这表明较少数量的基因组能够介导脊髓中更高的转基因表达这个衣壳的线。相反，与 AAV9 相比，AAV-F 的背根神经节转导效率平均较低。有趣的是，我们还在两种注射 AAV-F 的非人灵长类动物中观察到雪旺细胞在坐骨神经中的转导，但没有注射 AAV9。总的来说，我们的数据证明了自动化 IA 对脊髓中 AAV 转导定量的效用和有利的在靶：脱靶转导概况表明 AAV-F 衣壳可考虑用于以治疗脊髓为重点的基因治疗应用鞘内分娩后。