Therapeutic liabilities of in vivo viral vector tropism:: Adeno-associated virus vectors, NMDAR1 antisense, and focal seizure sensitivity
Therapeutic liabilities of in vivo viral vector tropism:: Adeno-associated virus vectors, NMDAR1 antisense, and focal seizure sensitivity
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DOI：
10.1006/mthe.2002.0701
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发表时间：
2002-10-01
影响因子：
12.4
通讯作者：
McCown, TJ
中科院分区：
文献类型：
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作者：
Haberman, RP;Criswell, HE;McCown, TJ
N-甲基-D-天冬氨酸（NMDA）受体为局灶性癫痫的基因治疗提供了一个潜在的靶点。为了测试这种方法，我们将729 bp的NMDA受体（NMDAR 1）cDNA片段以反义方向克隆到腺相关病毒（AAV）载体中，其中表达由四环素关闭调节启动子（AAV-tTAK-NR 1A）或巨细胞病毒（CMV）启动子（AAV-CMV-NR 1A）驱动。用重组AAV-tTAK-NR 1A感染原代培养的皮层神经元后，膜片钳研究发现最大NMDA诱发电流显著降低，表明NMDA受体数量减少。类似地，将AAV-tTAK-NR 1A（RI）输注到大鼠颞叶皮质中显著降低了V层锥体细胞中的NMDAR 1样免疫反应性。当将AAV-tTAK-NR 1A载体输注到大鼠下丘皮质的癫痫敏感部位时，癫痫敏感性在4周内显著增加。然而，AAV-CMV-NR 1A载体的丘输注引起相反的效果，癫痫敏感性显著降低。随后将编码由四环素关闭启动子驱动的绿色荧光蛋白（GFP）的载体（AAV-tTAK-GFP）和编码由CMV启动子驱动的β-半乳糖苷酶的载体（AAV-CMV-LacZ）共输注到丘中，转导了仅部分重叠的不同神经元群体。因此，抑制性中间神经元与初级输出神经元的不同转导比率可能解释了癫痫发作的不同影响。虽然AAV载体衍生的NMDAR 1反义可以在体外和体内影响NMDA受体功能，但启动子相关的嗜性差异显著改变了这种基于受体的基因治疗的生理结果。
The N-methyl-D-aspartic acid (NMDA) receptor provides a potential target for gene therapy of focal seizure disorders. To test this approach, we cloned a 729-bp NMDA receptor (NMDAR1) cDNA fragment in the antisense orientation into adeno-associated virus (AAV) vectors, where expression was driven by either a tetracycline-off regulatable promoter (AAV-tTAK-NR1A) or a cytomegalovirus (CMV) promoter (AAV-CMV-NR1A). After infection of primary cultured cortical neurons with recombinant AAV-tTAK-NR1A, patch clamp studies found a significant decrease in maximal NMDA-evoked currents, indicative of a decrease in the number of NMDA receptors. Similarly, infusion of AAV-tTAK-NR1A (I RI) into the rat temporal cortex significantly decreased NMDAR1-like immunoreactivity in layer V pyramidal cells. When AAV-tTAK-NR1A vectors were infused into the seizure-sensitive site of the rat inferior collicular cortex, the seizure sensitivity increased significantly over a period of 4 weeks. However, collicular infusion of AAV-CMV-NR1A vectors caused the opposite effect, a significant decrease in seizure sensitivity. Subsequent collicular coinfusion of vector encoding green fluorescent protein (GFP) driven by the tetracycline-off promoter (AAV-tTAK-GFP) and vector encoding beta-galactosidase driven by the CMV promoter (AAV-CMV-LacZ) transduced distinct neuronal populations with only partial overlap. Thus, differing transduction ratios of inhibitory interneurons to primary output neurons likely account for the divergent seizure influences. Although AAV vector-derived NMDAR1 antisense can influence NMDA receptor function both in vitro and in vivo, promoter-related tropic differences dramatically alter the physiological outcome of this receptor-based gene therapy.