High efficiencies of gene transfer with immobilized recombinant retrovirus: Kinetics and optimization
High efficiencies of gene transfer with immobilized recombinant retrovirus: Kinetics and optimization
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DOI：
10.1021/bp010039n
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发表时间：
2001-07-01
影响因子：
2.9
通讯作者：
Andreadis, ST
中科院分区：
文献类型：
--
作者：
Bajaj, B;Lei, P;Andreadis, ST
我们使用数学建模和实验相结合的方法来研究逆转录病毒转导的限速步骤。对表面结合的纤维连接蛋白(FN)进行研究，并确定最大限度提高基因转移效率的条件。我们的结果表明，纤维连接蛋白辅助的基因转移(FAGT)是病毒在FN涂层平板中孵育的时间和温度的强烈作用。基因转移在短时间内急剧增加，在中间时间达到最大值，最终由于逆转录病毒活性的丧失而下降。病毒的最大转导效率和达到转导效率的时间随病毒孵育温度的降低而增加。根据温度和靶细胞类型的不同，与传统的转导(TT)相比，基因转移的初始速率提高了3-10倍，最大转导效率提高了2-4倍。有趣的是，聚丙稀烯(PB)通过抑制逆转录病毒与FN的结合，以剂量依赖的方式抑制FAGT。与传统的转导相比，FAGT的转导效率是集中逆转录病毒库存的10倍以上。基因转移与含病毒介质的浓度成正比，在所测试的浓度范围内没有饱和迹象。这些结果表明，重组逆转录病毒的固定化可以得到合理的优化，从而获得高效的基因转移到原代细胞，并改善基因治疗在人类疾病治疗中的前景。
We used a combination of mathematical modeling and experiments to investigate the rate-limiting steps of retroviral transduction. on surface-bound fibronectin (FN) and identify the conditions that maximize the efficiency of gene transfer. Our results show that fibronectin-assisted gene transfer (FAGT) is a strong function of the time and temperature of virus incubation in FN-coated plates. Gene transfer increases sharply at short times, reaches a maximum at intermediate times, and eventually declines as a result of loss of retroviral activity. The maximum transduction efficiency and the time at which this is attained increase with decreasing temperature of virus incubation. Depending on the temperature and the type of target cells, the initial rate of gene transfer increases by 3- to 10-fold and the maximum transduction efficiency increases by 2- to 4-fold as compared to traditional transduction (TT). Interestingly, Polybrene (PB) inhibits FAGT in a dose-dependent manner by inhibiting binding of retrovirus to FN. In contrast to traditional transduction, FAGT yields higher than 10-fold transduction efficiencies with concentrated retrovirus stocks. Gene transfer is directly proportional to the concentration of the virus-containing medium with no sign of saturation for the range of concentrations tested. These results suggest that immobilization of recombinant retrovirus can be rationally optimized to yield high efficiency of gene transfer to primary cells and improve the prospect of gene therapy for the treatment of human disease.