A high-titer lentiviral production system mediates efficient transduction of differentiated cells including beating cardiac myocytes
A high-titer lentiviral production system mediates efficient transduction of differentiated cells including beating cardiac myocytes
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DOI：
10.1006/jmcc.1999.1035
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发表时间：
1999-11-01
影响因子：
5
通讯作者：
Kedes, L
中科院分区：
文献类型：
--
作者：
Sakoda, T;Kasahara, N;Kedes, L
基于人类免疫缺陷病毒（HIV，慢病毒）1型的载体具有用于基因治疗的许多吸引人的特征，包括使非分裂细胞增殖和长期转基因表达的能力。Mie使用三质粒表达系统通过在丁酸钠存在下瞬时转染人胚肾293 T细胞来产生假型慢病毒区，已知丁酸钠激活HIV的长末端重复定向表达。使用该系统，我们成功地产生了滴度高达2 × 10（8）转导单位/ml（TU/ml）的多功能高滴度慢病毒，并将各种细胞类型中的转导效率提高了7倍至20倍以上。我们证明了这些载体对于非分裂细胞（包括有丝分裂后搏动的大鼠心肌细胞和分化良好的大鼠L 6肌纤维）的有效转导的适用性，虽然基于慢病毒的载体和基于鼠逆转录病毒的载体都有效地转导了培养物中分裂的心脏成纤维细胞和L 6肌成肌细胞，但基于慢病毒的载体也有效地转导了心肌细胞，并产生了100%的滴度。（6.3 +/- 1.2）× 10（5）TU/ml;然而，基于鼠逆转录病毒的载体显示低转导效率，滴度仅达到（8.9 +/-2.1）× 10（2）TU/ml。此外，即使在诱导L 6肌纤维分化后12天，慢病毒介导的β-半乳糖苷酶转导也没有被发现。（β-Gal）在复制成肌细胞中达到的最大表达水平的约30-40%。用鼠逆转录病毒载体转导肌纤维后β-Gal的表达下降到低于1%，在未分化的融合成肌细胞中已经降低的转导水平。这些结果表明，基于慢病毒的载体可以有效地转染分化良好的心肌细胞和分化的肌纤维。这似乎是一种有效的方法，并为心血管疾病的研究和治疗提供了新的工具。(C)北京：科学出版社.
Human immunodeficiency virus (HIV, lentivirus) type-1 based vectors have a number of attractive features for gene therapy, including the ability to transduce non-dividing cells and long term transgene expression. Mie used a three-plasmid expression system to generate pseudotyped lentivirus-based sectors by transient transfection of human embryonic kidney 293T cells in the presence of sodium butyrate, which is known to activate the long terminal repeat-directed expression of HIV. Using this system we successfully generated versatile high titer lentivirus at titers of up to 2 x 10(8) transducing units/ml (TU/ml), and improved transduction efficiency in various cell types from seven to over twenty fold, We demonstrate its applicability of these Vectors for the efficient transduction of non-dividing cells, including post mitotic beating rat cardiac myocytes and well-differentiated rat L6 myofibers, While both lentivirus-based and murine retrovirus-based vectors effectively transduced dividing cardiac fibroblasts and L6 muscle myoblasts in culture, lentivirus-based vectors also efficiently transduced cardiac myocytes and yielded titers of (6.3 +/- 1.2) x 10(5) TU/ml; however murine retrovirus-based vectors showed low transduction efficiency with titers reaching only (8.9 +/- 2.1) x 10(2) TU/ml. Furthermore, even 12 days after induction of differentiation of L6 myofibers, lentivirus-mediated transduction of beta-galactosidase (beta-Gal) at approximately 30-40% of the maximum expression levels achieved in replicating myoblasts, In contrast, the expression of beta-Gal following transduction of the myofibers by murine retrovirus-based vectors fell to less than 1% of an already reduced level of transduction in undifferentiated confluent myoblasts. These results demonstrate that lentivirus-based vectors can efficiently transduce both well-differentiated cardiac myocytes and differentiated myofibers. This appears to be an efficient method and provides a new tool for research and therapy for cardiovascular diseases. (C) 1999 Academic Press.