Mandel R J, Rendahl K G, Spratt S K, Snyder R O, Cohen L K, Leff S E
Department of Gene Therapy Applications, Cell Genesys Inc., Foster City, California 94404, USA.
J Neurosci. 1998 Jun 1;18(11):4271-84. doi: 10.1523/JNEUROSCI.18-11-04271.1998.
To achieve local, continuous L-DOPA delivery in the striatum by gene replacement as a model for a gene therapy for Parkinson's disease, the present studies used high titer purified recombinant adeno-associated virus (rAAV) containing cDNAs encoding human tyrosine hydroxylase (hTH) or human GTP-cyclohydrolase I [GTPCHI, the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis] or both to infect the 6-OHDA denervated rat striatum. Striatal TH and GTPCHI staining was observed 3 weeks after rAAV transduction, with little detectable perturbation of the tissue. Six months after intrastriatal rAAV transduction, TH staining was present but apparently reduced compared with the 3 week survival time. In a separate group of animals, striatal TH staining was demonstrated 1 year after rAAV transduction. Double staining studies using the neuronal marker NeuN indicated that >90% of rAAV-transduced cells expressing hTH were neurons. Microdialysis experiments indicated that only those lesioned animals that received the mixture of MD-TH and MD-GTPCHI vector displayed BH4 independent in vivo L-DOPA production (mean approximately 4-7 ng/ml). Rats that received the hTH rAAV vector alone produced measurable L-DOPA (mean approximately 1-4 ng/ml) only after receiving exogenous BH4. L-Aromatic amino acid decarboxylase blockade, but not 100 mM KCl-induced depolarization, enhanced L-DOPA overflow, and animals in the non-hTH groups (GTPCHI and alkaline phosphatase) yielded minimal L-DOPA. Although elevated L-DOPA was observed in animals that received mixed hTH and hGTPCHI rAAV vectors, there was no reduction of apomorphine-induced rotational behavior 3 weeks after intrastriatal vector injection. These data demonstrate that purified rAAV, a safe and nonpathogenic viral vector, mediates long-term striatal hTH transgene expression in neurons and can be used to successfully deliver L-DOPA to the striatum.
为了通过基因替代在纹状体中实现局部、持续的左旋多巴递送，以此作为帕金森病基因治疗的模型，本研究使用了高滴度纯化的重组腺相关病毒（rAAV），其包含编码人酪氨酸羟化酶（hTH）或人鸟苷三磷酸环化水解酶I[GTPCHI，四氢生物蝶呤（BH4）合成的限速酶]或两者的cDNA，来感染6-OHDA去神经支配的大鼠纹状体。rAAV转导3周后观察到纹状体TH和GTPCHI染色，组织几乎没有可检测到的扰动。纹状体内rAAV转导6个月后，TH染色存在，但与3周存活时间相比明显减少。在另一组动物中，rAAV转导1年后证明有纹状体TH染色。使用神经元标志物NeuN的双重染色研究表明，>90%表达hTH的rAAV转导细胞是神经元。微透析实验表明，只有那些接受MD-TH和MD-GTPCHI载体混合物的损伤动物在体内显示出不依赖BH4的左旋多巴产生（平均约4-7 ng/ml）。单独接受hTH rAAV载体的大鼠仅在接受外源性BH4后产生可测量的左旋多巴（平均约1-4 ng/ml）。L-芳香族氨基酸脱羧酶阻断，但不是100 mM KCl诱导的去极化，增强了左旋多巴溢出，非hTH组（GTPCHI和碱性磷酸酶）的动物产生的左旋多巴极少。尽管在接受混合hTH和hGTPCHI rAAV载体的动物中观察到左旋多巴升高，但纹状体内注射载体3周后阿扑吗啡诱导的旋转行为没有减少。这些数据表明，纯化的rAAV，一种安全且无致病性的病毒载体，介导神经元中hTH转基因的长期纹状体表达，并可用于成功地将左旋多巴递送至纹状体。
