Schambach Axel, Swaney William P, van der Loo Johannes C M
Department of Experimental Hematology, Hannover Medical School, Hannover, Germany.
Methods Mol Biol. 2009;506:191-205. doi: 10.1007/978-1-59745-409-4_14.
Successful retroviral gene transfer into hematopoietic cells has been demonstrated in a number of small and large animal models and clinical trials. However, severe adverse events related to insertional muta-genesis in a recent clinical trial for X-linked severe combined immunodeficiency reinforced the need to develop novel retroviral vectors with improved biosafety. Improvements include the use of self-inactivating (SIN) vectors as well as improvements in vector design. This chapter describes the basic design of gamma-retroviral and lentiviral SIN vectors that utilize a split-packaging system and includes a description of the various cloning modules frequently used in the design of such vectors that impact biosafety, titer, and transgene expression. In addition, this chapter describes the methods used for high titer vector production using calcium phosphate transfection both at research scale and at large scale for clinical application using a closed system bioreactor.
在许多小型和大型动物模型以及临床试验中，已证实逆转录病毒基因可成功导入造血细胞。然而，在最近一项针对X连锁严重联合免疫缺陷的临床试验中，与插入诱变相关的严重不良事件强化了开发具有更高生物安全性的新型逆转录病毒载体的必要性。改进措施包括使用自我失活（SIN）载体以及改进载体设计。本章描述了利用拆分包装系统的γ逆转录病毒和慢病毒SIN载体的基本设计，并介绍了在这类载体设计中常用的各种影响生物安全性、滴度和转基因表达的克隆模块。此外，本章还描述了在研究规模和使用封闭系统生物反应器的临床应用大规模生产高滴度载体时使用磷酸钙转染的方法。
