Oh Seunguk, Pluhar G Elizabeth, McNeil Elizabeth A, Kroeger Kurt M, Liu Chunyan, Castro Maria G, Lowenstein Pedro R, Freese Andrew, Ohlfest John R
Department of Neurosurgery, University of Minnesota, St. Paul, USA.
J Neurosurg. 2007 Jul;107(1):136-44. doi: 10.3171/JNS-07/07/0136.
The purpose of this study was to evaluate the gene transfer capability and tolerability of plasmid DNA/polyethylenimine (PEI) complexes in comparison with adenovirus and naked plasmid DNA in the canine brain.
Plasmid or adenoviral vectors encoding firefly luciferase were injected directly into the cerebral parenchyma of five adult dogs at varying doses and volumes. Serial physical and neurological examinations, as well as blood and cerebrospinal fluid (CSF) analyses, were conducted before and after the surgery for 3 days. Three days after gene delivery, a luciferase activity assay and immunofluorescence analysis were used to test the brain tissue for gene expression.
Injection into the brain parenchyma resulted in gene transfer throughout the cerebrum with every vector tested. Luciferase expression was highest when adenovirus vectors were used. Injection of plasmid DNA/PEI complexes and naked DNA resulted in similar levels of luciferase expression, which were on average 0.5 to 1.5% of the expression achieved with adenovirus vectors. Immunofluorescent microscopy analysis revealed that plasmid DNA/PEI complexes transduced mainly neurons, whereas adenovirus transduced mainly astrocytes. No significant acute side effects or neurological complications were observed in any of the dogs. Mononuclear cell counts significantly increased in the CSF after adenovirus injection and modestly increased after injection of plasmid DNA/PEI complexes, suggesting that a mild, acute inflammatory response occurred in the central nervous system (CNS).
Compared with rodent models that are limited by very small brains, the dog is an excellent preclinical model in which to assess the distribution and safety of emerging gene transfer technologies. In this study, short-term gene transfer was evaluated as a prelude to long-term expression and safety studies. The authors conclude that the viral and nonviral vectors tested were well tolerated and effective at mediating gene transfer throughout a large portion of the canine brain. The nonviral plasmid vectors were less effective than adenovirus, yet they still achieved appreciable gene expression levels. Due to reduced gene transfer efficiency relative to viral vectors, nonviral vectors may be most useful when the expressed protein is secreted or exerts a bystander effect. Nonviral vectors offer an alternative means to genetically modify cells within the CNS of large mammals.
本研究旨在评估质粒DNA/聚乙烯亚胺（PEI）复合物与腺病毒和裸质粒DNA相比，在犬脑内的基因转移能力和耐受性。
将编码萤火虫荧光素酶的质粒或腺病毒载体以不同剂量和体积直接注射到5只成年犬的脑实质中。在手术前后3天进行系列体格和神经学检查，以及血液和脑脊液（CSF）分析。基因递送3天后，使用荧光素酶活性测定和免疫荧光分析检测脑组织中的基因表达。
将每种测试载体注射到脑实质中均导致整个大脑的基因转移。使用腺病毒载体时荧光素酶表达最高。注射质粒DNA/PEI复合物和裸DNA导致相似水平的荧光素酶表达，平均为腺病毒载体所达到表达水平的0.5%至1.5%。免疫荧光显微镜分析显示质粒DNA/PEI复合物主要转导神经元，而腺病毒主要转导星形胶质细胞。未在任何犬中观察到明显的急性副作用或神经并发症。腺病毒注射后CSF中的单核细胞计数显著增加，注射质粒DNA/PEI复合物后略有增加，表明中枢神经系统（CNS）发生了轻度急性炎症反应。
与受非常小的大脑限制的啮齿动物模型相比，犬是评估新兴基因转移技术的分布和安全性的优秀临床前模型。在本研究中，短期基因转移作为长期表达和安全性研究的前奏进行评估。作者得出结论，所测试病毒和非病毒载体耐受性良好，并且在介导犬脑大部分区域的基因转移方面有效。非病毒质粒载体比腺病毒效率低，但仍实现了可观的基因表达水平。由于相对于病毒载体基因转移效率降低，当表达的蛋白质分泌或发挥旁观者效应时，非病毒载体可能最有用。非病毒载体为在大型哺乳动物的CNS内对细胞进行基因修饰提供了一种替代方法。
