Department of Neurology, University of Wisconsin , Madison, Wisconsin, USA.
Department of Biochemistry, University of Florida , Gainesville, Florida, USA.
Mol Ther Methods Clin Dev. 2014 Jan 8;1:4. doi: 10.1038/mtm.2013.4. eCollection 2014.
Intraoperative magnetic resonance imaging (MRI) has been proposed as a method to optimize intracerebral targeting and for tracking infusate distribution in gene therapy trials for nervous system disorders. We thus investigated possible effects of two MRI contrast agents, gadoteridol (Gd) and galbumin (Gab), on the distribution and levels of transgene expression in the rat striatum and their effect on integrity and stability of recombinant adeno-associated virus (rAAV) particles. MRI studies showed that contrast agent distribution did not predict rAAV distribution. However, green fluorescent protein (GFP) immunoreactivity revealed an increase in distribution of rAAV5-GFP, but not rAAV2-GFP, in the presence of Gd when compared with viral vector injected alone. In contrast, Gab increased the distribution of rAAV2-GFP not rAAV5-GFP. These observations pointed to a direct effect of infused contrast agent on the rAAV particles. Negative-stain electron microscopy (EM), DNAase treatment, and differential scanning calorimetry (DSC) were used to monitor rAAV2 and rAAV5 particle integrity and stability following contrast agent incubation. EMs of rAAV2-GFP and rAAV5-GFP particles pretreated with Gd appear morphologically similar to the untreated sample; however, Gab treatment resulted in surface morphology changes and aggregation. A compromise of particle integrity was suggested by sensitivity of the packaged genome to DNAase treatment following Gab incubation but not Gd for both vectors. However, neither agent significantly affected particle stability when analyzed by DSC. An increase in T m was observed for AAV2 in lactated Ringer's buffer. These results thus highlight potential interactions between MRI contrast agents and AAV that might affect vector distribution and stability, as well as the stabilizing effect of lactated Ringer's solution on AAV2.
术中磁共振成像（MRI）已被提议作为一种方法，以优化颅内靶向，并在神经系统疾病的基因治疗试验中跟踪输注物的分布。因此，我们研究了两种 MRI 对比剂，钆喷酸葡胺（Gd）和人血清白蛋白（Gab），对大鼠纹状体中转基因表达的分布和水平的可能影响，以及它们对重组腺相关病毒（rAAV）颗粒完整性和稳定性的影响。MRI 研究表明，对比剂的分布并不能预测 rAAV 的分布。然而，当与单独注射病毒载体相比时，绿色荧光蛋白（GFP）免疫反应显示，在存在 Gd 的情况下，rAAV5-GFP 的分布增加，而 rAAV2-GFP 的分布则没有增加。相比之下，Gab 增加了 rAAV2-GFP 的分布，而不是 rAAV5-GFP。这些观察结果表明，输注的对比剂对 rAAV 颗粒有直接影响。负染电子显微镜（EM）、DNAase 处理和差示扫描量热法（DSC）用于监测 rAAV2 和 rAAV5 颗粒在对比剂孵育后的完整性和稳定性。用 Gd 预处理的 rAAV2-GFP 和 rAAV5-GFP 颗粒的 EM 外观与未处理的样品相似；然而，Gab 处理导致表面形态变化和聚集。在用 Gab 孵育后，包装基因组对 DNAase 处理的敏感性表明颗粒完整性受损，但对于两种载体，Gd 都没有；然而，当用 DSC 分析时，两种试剂都没有显著影响颗粒稳定性。在乳酸盐缓冲液中观察到 AAV2 的 T m 增加。因此，这些结果突出了 MRI 对比剂和 AAV 之间可能影响载体分布和稳定性的潜在相互作用，以及乳酸盐缓冲液对 AAV2 的稳定作用。
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