Development of formulations that enhance physical stability of viral vectors for gene therapy
Development of formulations that enhance physical stability of viral vectors for gene therapy
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DOI：
10.1038/sj.gt.3301527
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发表时间：
2001-09-01
期刊：
影响因子：
5.1
通讯作者：
Wilson, JM
中科院分区：
文献类型：
--
作者：
Croyle, MA;Cheng, X;Wilson, JM
这项研究总结了我们最初的努力，以解决一个问题，这是至关重要的成功的任何多中心基因治疗临床试验-维持载体的活力，在运输和储存在偏远的测试网站。我们已经确定了影响病毒制剂稳定性的处方和加工因素，如选择适当的缓冲系统、冷冻保护剂和储存条件。表达E.将大肠杆菌β-半乳糖苷酶（lacZ）悬浮在复合碳水化合物、环糊精和各种表面活性剂的共混物中。使用293和84-31细胞的X-gal染色来确定所有制备物的感染滴度。在-20 ℃和4 ℃下储存后，磷酸钾缓冲制剂始终保持高病毒滴度。蔗糖、甘露醇和表面活性剂的共混物在4 ℃下35天的滴度损失可以忽略不计。蔗糖和环糊精的制剂在-20 ℃下稳定2年。在蔗糖中冻干并在4 ℃下储存1年的单位剂量病毒制剂中观察到滴度损失可忽略不计，最初因处理损失0.5 log。对冻干蔗糖/甘露醇混合物的研究表明，加工后的病毒回收率与干燥产品的最终含水量直接相关。病毒浓度也在用冻干后滴度损失最小的高浓缩制剂处理后的回收率中起重要作用。总之，可以在25 ℃下运输和储存的冻干制剂为临床试验中病毒载体的分布提供了当前问题的解决方案。
This study summarizes our initial efforts to address an issue that is critical to the success of any multicenter gene therapy clinical trial - maintenance of vector viability during shipping and storage at remote test sites. We have identified formulation and processing factors that influence stability of viral preparations such as selection of appropriate buffer systems, cryoprotectants, and storage conditions. Adenovirus and adeno-associated virus expressing E. coli beta-galactosidase (lacZ) were suspended in blends of complex carbohydrates, cyclodextrins and various surfactants. X-gal stains of 293 and 84-31 cells were used to determine infectious titer of all preparations. Potassium phosphate-buffered preparations consistently maintained high viral titers after storage at-20 and 4 degreesC. Blends of sucrose, mannitol, and surfactant showed negligible loss of titer for 35 days at 4 degreesC. Formulations of sucrose and cyclodextrin were stable for 2 years at -20 degreesC. Negligible loss in titer was observed in unit-dose viral preparations lyophilized in sucrose and stored at 4 degreesC for 1 year after an initial loss of 0.5 log due to processing. Studies with lyophilized sucrose/mannitol blends have shown that viral recovery after processing is directly related to the final moisture content of the dried product. Virus concentration also plays a significant role in recovery after processing with highly concentrated preparations showing minimal loss in titer after lyophilization. In summary, lyophilized preparations that can be shipped and stored at 25 degreesC offer a solution to the current problem of distribution of viral vectors for clinical trials.