Bohn M C, Choi-Lundberg D L, Davidson B L, Leranth C, Kozlowski D A, Smith J C, O'Banion M K, Redmond D E
Department of Pediatrics, Children's Memorial Institute for Education and Research, Northwestern University Medical School, Chicago, IL 60614, USA.
Hum Gene Ther. 1999 May 1;10(7):1175-84. doi: 10.1089/10430349950018166.
Transgene expression in the brain of St. Kitts green monkey, Cercopithecus aethiops sabeus, was studied following injection of a serotype 5 adenoviral vector deleted in E1 and E3. The vector harbored the transgene for Escherichia coli beta-galactosidase (beta-Gal) with the simian virus 40 (SV40) nuclear localization signal under control of the Rous sarcoma viral (RSV) long terminal repeat. Several titers ranging from 5 x 10(7) to 2 x 10(9) plaque-forming units (PFU) in volumes ranging from 5 to 250 microl were injected into the caudate nuclei of 18 monkeys. Monkeys were treated with dexamethasone for 9 days, beginning the day prior to surgery, and were sacrificed at 1 week or at 1, 2, or 3 months. At 1 week, beta-Gal was expressed in thousands of cells, including both neurons and astrocytes. In addition, some dopaminergic neurons in the substantia nigra expressed transgene, suggesting retrograde transport of the vector. At 1 month 162,000+/-68,000 (SEM) or 65,000+/-29,000 beta-Gal-expressing cells persisted in striatum injected with 6 x 10(8) PFU in 30 microl or 5 x 10(7) PFU in 5 microl, respectively. Transgene expression was also observed in one of two monkeys sacrificed at 2 months and in a single monkey sacrificed at 3 months. No transgene expression was observed at 1 month in striatum injected with a higher titer (2 x 10(9) PFU in 100 microl) or more dilute vector (5 x 10(7) PFU in 30 microl). Staining for the major histocompatibility complex II (MHC II) subtype DR showed intense staining in sites injected with a higher vector titer, in which no transgene persisted at 1 month, whereas low to moderate staining was present in sites with high transgene expression. These observations suggest that there is an optimal range of vector titers for obtaining persistent transgene expression from E1E3-deleted adenovirus in primate brain, above which host responses limit transgene stability.
在向圣基茨绿猴（Cercopithecus aethiops sabeus）脑内注射缺失E1和E3的5型腺病毒载体后，对转基因表达情况进行了研究。该载体携带了大肠杆菌β-半乳糖苷酶（β-Gal）的转基因，并带有猿猴病毒40（SV40）核定位信号，受劳氏肉瘤病毒（RSV）长末端重复序列的控制。将5至250微升体积、滴度范围从5×10⁷到2×10⁹空斑形成单位（PFU）的病毒液分别注射到18只猴子的尾状核中。从手术前一天开始，猴子接受地塞米松治疗9天，并在1周或1、2或3个月时处死。在1周时，β-Gal在数千个细胞中表达，包括神经元和星形胶质细胞。此外，黑质中的一些多巴胺能神经元表达了转基因，提示载体的逆行运输。在1个月时，分别向30微升中注射6×10⁸ PFU或5微升中注射5×10⁷ PFU的纹状体中，分别有162,000±68,000（SEM）或65,000±29,000个表达β-Gal的细胞持续存在。在2个月处死的两只猴子中的一只以及3个月处死的一只猴子中也观察到了转基因表达。在向100微升中注射更高滴度（2×10⁹ PFU）或更稀释载体（30微升中5×10⁷ PFU）的纹状体中，1个月时未观察到转基因表达。主要组织相容性复合体II（MHC II）亚型DR的染色显示，在注射了更高载体滴度的部位有强烈染色，但在1个月时这些部位没有转基因持续存在，而在转基因高表达部位则有低至中度染色。这些观察结果表明，从缺失E1E3的腺病毒在灵长类动物脑中获得持续转基因表达存在一个最佳载体滴度范围，高于此范围宿主反应会限制转基因的稳定性。
