Intrathecal long-term gene expression by self-complementary adeno-associated virus type 1 suitable for chronic pain studies in rats.
Intrathecal long-term gene expression by self-complementary adeno-associated virus type 1 suitable for chronic pain studies in rats.
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DOI：
10.1186/1744-8069-2-4
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发表时间：
2006-01-30
期刊：
影响因子：
3.3
通讯作者：
Beutler AS
中科院分区：
文献类型：
--
作者：
Storek B;Harder NM;Banck MS;Wang C;McCarty DM;Janssen WG;Morrison JH;Walsh CE;Beutler AS
鞘内（IT）基因转移是针对脊髓伤害感受机制的一种有吸引力的方法，但所报道的方法实现的基因表达持续时间很短（长达两周），削弱了它们在慢性疼痛环境中的效用。本研究的总体目标是开发 IT 基因转移，产生真正的长期转基因表达，定义为单次载体施用后≥3个月。我们将“IT”给药定义为模拟腰椎穿刺而无创地注射到腰部脑脊液（CSF）中。我们的研究重点是重组腺相关病毒（rAAV），它是最有希望用于临床的载体类型之一。传统的单链 rAAV2 载体在 IT 递送至大鼠后表现不佳。单独或结合反向末端重复序列的修饰，测试了带有血清型 1、3 和 5 衣壳的 rAAV 的假分型。前者改变载体向性，后者允许包装自我互补的 rAAV (sc-rAAV) 载体。结合两种类型的修饰导致将 sc-rAAV2/l 鉴定为在 IT 领域表现优异的载体。每只动物 IT 递送 3 × 10e9 sc-rAAV2/l 颗粒可导致增强型绿色荧光蛋白 (EGFP) 稳定表达 ≥ 3 个月，可通过蛋白质印迹、定量 PCR 以及通过共聚焦显微镜进行的盲法研究进行检测。马尾和脊髓下部的表达最强，前脑中的表达最小。对原位固定的具有完整神经根和脑膜的 SC 进行显微镜检查，发现神经根中存在强烈的 EGFP 荧光。 sc-rAAV1介导稳定的IT转基因表达≥3个月。我们的研究结果支持以下基本假设：用于基因转移的 IT 靶细胞缺乏将单链 rAAV 基因组有效转化为双链 DNA 的机制，并且更倾向于摄取血清型 1 载体而不是 2 型载体。本文提出的实验将为在慢性疼痛的基础和转化研究中利用 IT rAAV 基因转移提供合理的基础。
Intrathecal (IT) gene transfer is an attractive approach for targeting spinal mechanisms of nociception but the duration of gene expression achieved by reported methods is short (up to two weeks) impairing their utility in the chronic pain setting. The overall goal of this study was to develop IT gene transfer yielding true long-term transgene expression defined as ≥ 3 mo following a single vector administration. We defined "IT" administration as atraumatic injection into the lumbar cerebrospinal fluid (CSF) modeling a lumbar puncture. Our studies focused on recombinant adeno-associated virus (rAAV), one of the most promising vector types for clinical use. Conventional single stranded rAAV2 vectors performed poorly after IT delivery in rats. Pseudotyping of rAAV with capsids of serotypes 1, 3, and 5 was tested alone or in combination with a modification of the inverted terminal repeat. The former alters vector tropism and the latter allows packaging of self-complementary rAAV (sc-rAAV) vectors. Combining both types of modification led to the identification of sc-rAAV2/l as a vector that performed superiorly in the IT space. IT delivery of 3 × 10e9 sc-rAAV2/l particles per animal led to stable expression of enhanced green fluorescent protein (EGFP) for ≥ 3 mo detectable by Western blotting, quantitative PCR, and in a blinded study by confocal microscopy. Expression was strongest in the cauda equina and the lower sections of the spinal cord and only minimal in the forebrain. Microscopic examination of the SC fixed in situ with intact nerve roots and meninges revealed strong EGFP fluorescence in the nerve roots. sc-rAAVl mediates stable IT transgene expression for ≥ 3 mo. Our findings support the underlying hypothesis that IT target cells for gene transfer lack the machinery for efficient conversion of the single-stranded rAAV genome into double-stranded DNA and favor uptake of serotype 1 vectors over 2. Experiments presented here will provide a rational basis for utilizing IT rAAV gene transfer in basic and translational studies on chronic pain.
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影响因子：
5.1
作者：
McCarty, DM;Monahan, PE;Samulski, RJ
通讯作者：
Samulski, RJ
影响因子：
5.1
作者：
McCarty, DM;Fu, H;Samulski, RJ
通讯作者：
Samulski, RJ
影响因子：
12.4
作者：
Chao, HJ;Monahan, PE;Walsh, CE
通讯作者：
Walsh, CE
影响因子：
2.7
作者：
Stilwell, JL;Samulski, RJ
通讯作者：
Samulski, RJ
影响因子：
4.2
作者：
Finegold, AA;Mannes, AJ;Iadarola, MJ
通讯作者：
Iadarola, MJ