Infectivity assessment of recombinant adeno-associated virus (rAAV) and wild-type AAV (wtAAV) exposed to various diluents and environmental conditions.
Infectivity assessment of recombinant adeno-associated virus (rAAV) and wild-type AAV (wtAAV) exposed to various diluents and environmental conditions.
复制标题
暴露于各种稀释剂和环境条件下的重组腺相关病毒 (rAAV) 和野生型 AAV (wtAAV) 的感染性评估。
作者：
Taro Tomono;Y. Hirai;H. Chono;J. Mineno;A. Ishii;M. Onodera;A. Tamaoka;T. Okada
重组腺相关病毒(RAAV)是一种很有前途的基因载体，已被批准作为治疗某些遗传性疾病的基因治疗药物，并正在进行临床试验。为进一步推进食品药品监督管理局新药临床应用研究，必须对rAAV在各种条件下的稳定性进行评价。然而，关于各种rAAV血清型的稳定性的数据很少。我们假设衣壳结构的不同导致了稳定性的不同。为了验证这一假设，将rAAV1、rAAV2、rAAV8和rAAV9四种血清型分别暴露在稀释剂和各种环境条件下，包括紫外线(UV)照射、0.1M氢氧化钠(NaOH)、0.06%次氯酸钠(NaClO)、自来水和70%乙醇(EtoH)。以HeLaRC32细胞转导为稳定性标准，评价处理样品感染性的变化。同时对重组AAV2与野生型AAV2的感染性进行了分析。紫外线照射、NaOH和NaClO暴露后，所有rAAV血清型的活性均减弱。用自来水或70%乙醇处理10天后，rAAV1、rAAV8和rAAV9没有明显的失活，但对rAAV2的活性有影响。此外，rAAV2的感染性没有超过wtAAV2的感染性。这一结果将对使用rAAV进行基因治疗的临床研究具有重要意义。
Recombinant adeno-associated virus (rAAV) is a promising gene delivery vehicle that has been approved as a gene therapy drug for some genetic disorders, and is being evaluated in clinical trials. To further promote clinical research under the Food and Drug Administration Investigational New Drug application, the stability of rAAV must be assessed under various conditions. However, there is scant data concerning the stability of a variety of rAAV serotypes. We hypothesized that the difference of capsid structure causes differences in stability. To investigate this hypothesis, rAAV serotypes (rAAV1, rAAV2, rAAV8, and rAAV9) were exposed to diluents and various environmental conditions, including ultraviolet (UV) irradiation, 0.1M sodium hydroxide (NaOH), 0.06% sodium hypochlorite (NaClO), tap water, and 70% ethanol (EtOH). The changes of the infectivity of the treated samples were assessed by transduction in HeLaRC32 cells as a criterion of stability. The infectivity between recombinant and wild-type AAV (wtAAV2) was also analyzed. The activity of all rAAV serotypes was weakened by UV irradiation and NaOH and NaClO exposure. Treatment for 10 days with tap water or 70% EtOH did not appreciably inactivate rAAV1, rAAV8, and rAAV9, but did affect the activity of rAAV2. Furthermore, the infectivity of rAAV2 did not surpass wtAAV2 infectivity. The results will be important for clinical studies for gene therapy using rAAV.