Transduction of Craniofacial Motoneurons Following Intramuscular Injections of Canine Adenovirus Type-2 (CAV-2) in Rhesus Macaques
Transduction of Craniofacial Motoneurons Following Intramuscular Injections of Canine Adenovirus Type-2 (CAV-2) in Rhesus Macaques
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DOI：
10.3389/fnana.2019.00084
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发表时间：
2019-09-18
影响因子：
2.9
通讯作者：
Sommer, Marc A.
中科院分区：
文献类型：
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作者：
Bohlen, Martin O.;El-Nahal, Hala G.;Sommer, Marc A.
病毒载体介导的转基因在灵长类运动神经元中的可靠表达将提高我们从解剖学和生理学上询问运动系统的能力。因此，我们研究了复制缺陷、早期区域1缺失的犬腺病毒2型(CAV-2)载体在四只恒河猴(Macaca Mulatta)头面部肌肉注射后将荧光蛋白转基因表达到脑干运动神经元的有效性。将载体注射到经手术识别和分离的头面部肌肉中。存活1-2个月后，处死动物，用免疫组织化学方法检测相应运动神经元中的转基因表达。我们发现，CAV-2注射到单个头面部肌肉中的剂量范围类似于10(10)到10(11)个物理颗粒/肌肉，导致了强大的运动神经元转导和免疫组织化学鉴定的荧光蛋白在多个动物中的表达。通过在不同的肌肉中使用不同的效价，通过荧光团的表达和标记的运动神经元位置跟踪所产生的转导模式，我们在两个动物中建立了定性的剂量-反应关系。在一只接受了分布在许多注射部位的非典型高滴度(5.7×10(11)总CAV-2物理颗粒)的动物中，没有检测到转导，这可能是由于报复性免疫反应。我们得出结论，CAV-2载体有望在头面部肌肉注射后对灵长类运动神经元进行基因改造。我们的发现值得重点关注使用CAV-2载体来传递Opsins、DREADD和其他分子探针，以改进基于遗传学的灵长类研究方法。CAV-2转导参数的优化还需要进一步的工作。编码蛋白质的CAV-2载体可以为修饰灵长类中枢神经系统靶向神经元群体的活性提供一条新的、可靠的途径。
Reliable viral vector-mediated transgene expression in primate motoneurons would improve our ability to anatomically and physiologically interrogate motor systems. We therefore investigated the efficacy of replication defective, early region 1-deleted canine adenovirus type-2 (CAV-2) vectors for mediating transgene expression of fluorescent proteins into brainstem motoneurons following craniofacial intramuscular injections in four rhesus monkeys (Macaca mulatta). Vector injections were placed into surgically identified and isolated craniofacial muscles. After a 1- to 2-month survival time, animals were sacrificed and transgene expression was assessed with immunohistochemistry in the corresponding motoneuronal populations. We found that injections of CAV-2 into individual craniofacial muscles at doses in the range of similar to 10(10) to 10(11) physical particles/muscle resulted in robust motoneuronal transduction and expression of immunohistochemically identified fluorescent proteins across multiple animals. By using different titers in separate muscles, with the resulting transduction patterns tracked via fluorophore expression and labeled motoneuron location, we established qualitative dose-response relationships in two animals. In one animal that received an atypically high titer (5.7 X 10(11) total CAV-2 physical particles) distributed across numerous injection sites, no transduction was detected, likely due to a retaliatory immune response. We conclude that CAV-2 vectors show promise for genetic modification of primate motoneurons following craniofacial intramuscular injections. Our findings warrant focused attention toward the use of CAV-2 vectors to deliver opsins, DREADDs, and other molecular probes to improve genetics-based methods for primate research. Further work is required to optimize CAV-2 transduction parameters. CAV-2 vectors encoding proteins could provide a new, reliable route for modifying activity in targeted neuronal populations of the primate central nervous system.