A PCR-amplified transgene fragment flanked by a single copy of a truncated inverted terminal repeat for recombinant adeno-associated virus production prevents unnecessary plasmid DNA packaging
A PCR-amplified transgene fragment flanked by a single copy of a truncated inverted terminal repeat for recombinant adeno-associated virus production prevents unnecessary plasmid DNA packaging
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用于重组腺相关病毒生产的 PCR 扩增转基因片段，其侧翼有一个截短的反向末端重复序列，可防止不必要的质粒 DNA 包装
DOI：
10.1038/s41434-021-00299-x
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发表时间：
2021
期刊：
影响因子：
5.1
通讯作者：
Okada Takashi
中科院分区：
文献类型：
--
作者：
Adachi Kumi;Tomono Taro;Okada Hironori;Shiozawa Yusuke;Yamamoto Motoko;Miyagawa Yoshitaka;Okada Takashi
重组腺相关病毒（rAAV）用于基因治疗的应用面临某些挑战，包括非载体序列的基因组包装。位于rAAV基因组侧翼的反向末端重复序列（ITR），包括三个反向重复序列区（A、B和C）和非反向重复序列区（D），有助于非载体基因组包装。我们旨在通过比较含有DNA质粒和PCR扩增的转基因的rAAV（包括AD序列的单拷贝（分别为rAAV-pAD/L-AD），其是ITR的截短形式）与野生型ITR基因组（单链和自身互补的AAV; ssAAV和scAAV）的性质来规避该问题。rAAV-pAD/L-AD的包装效率与scAAV相当，但转导效率低于ss/scAAV。值得注意的是，与ss/scAAV相比，rAAV-L-AD减少了质粒骨架包装污染。为了进一步证实该系统的功能性，我们构建了一个含有靶向ATP 5 B的短发夹RNA的rAAV-L-AD-shATP 5 B（rAAV-L-AD-shATP 5 B），并发现它在转染HEK 293 EB细胞后能显著降低ATP 5 B的mRNA水平，表明它是功能性的。因此，我们的系统成功地将L-AD包装到衣壳中，质粒DNA的污染最小，提供了一种新的功能性包装平台，而不会引起质粒骨架的折叠。
The application of recombinant adeno-associated viruses (rAAVs) for gene therapy faces certain challenges, including genome packaging of non-vector sequences. Inverted terminal repeats (ITRs) flanking the rAAV genome, comprising three inverted repeat regions (A, B, and C) and a non-inverted repeat region (D), contribute to non-vector genome packaging. We aimed to circumvent this issue by comparing the properties of rAAV containing DNA plasmids and PCR-amplified transgenes, including a single copy of the AD sequence (rAAV-pAD/L-AD, respectively), which is a truncated form of ITR, with those of wild-type ITR genome (single-stranded and self-complementary AAV; ssAAV and scAAV). The packaging efficiency of rAAV-pAD/L-AD was found to be comparable to that of scAAV, whereas the transduction efficiency of rAAV-pAD/L-AD was lower than that of ss/scAAV. Remarkably, rAAV-L-AD reduced the plasmid backbone packaging contamination compared to ss/scAAV. Furthermore, to confirm the functionality of this system, we generated a rAAV-L-AD harboring a short hairpin RNA targetingATP5B(rAAV-L-AD-shATP5B) and found that it caused a significant decrease inATP5BmRNA levels when transduced into HEK293EB cells, suggesting that it was functional. Thus, our system successfully packaged L-AD into capsids with minimal contamination of plasmid DNA, offering a novel functional packaging platform without causing plasmid backbone encapsidation.
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影响因子：
0.2
作者：
Leng Q;Woodle MC;Lu PY;Mixson AJ
通讯作者：
Mixson AJ
影响因子：
4.8
作者：
Ren, Lili;Ding, Siyuan;Greenberg, Harry B.
通讯作者：
Greenberg, Harry B.
影响因子：
4.6
作者：
Zhou Q;Tian W;Liu C;Lian Z;Dong X;Wu X
通讯作者：
Wu X
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。
影响因子：
--
作者：
Taro Tomono;Y. Hirai;H. Chono;J. Mineno;A. Ishii;M. Onodera;A. Tamaoka;T. Okada
通讯作者：
T. Okada
影响因子：
12.4
作者：
Xie, Jun;Mao, Qin;Gao, Guangping
通讯作者：
Gao, Guangping