Genomic stability of self-inactivating rabies.
Genomic stability of self-inactivating rabies.
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DOI：
10.7554/elife.83459
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发表时间：
2023-11-03
期刊：
影响因子：
7.7
通讯作者：
Tripodi M
中科院分区：
文献类型：
--
作者：
Ciabatti E;González-Rueda A;de Malmazet D;Lee H;Morgese F;Tripodi M
跨突触病毒载体提供了基于突触连接获得神经元遗传通路的手段，并且是解剖神经回路功能的必要工具。其中，逆行性单突触ΔG-Rabies已广泛应用于神经科学研究。最近开发的Δ G狂犬病病毒的工程版本，无毒的自我灭活（SiR）病毒，允许神经回路的长期遗传操纵。然而，狂犬病病毒的高突变率带来了风险，即针对SiR基因组中关键遗传调节元件的突变可能会出现，并将其恢复为典型的ΔG-Rabies。最近在SiR批次中鉴定了此类回复突变体。为了解决这些突变的起源，发病率和相关性，我们研究了SiR在体外和体内的基因组稳定性。我们发现，“回复突变体”突变是罕见的，只有当SiR在体外广泛扩增时才积累，特别是在TEV蛋白酶活性水平不足的次优生产细胞系中。此外，我们证实SiR-CRE与典型的ΔG-Rab-CRE或回复突变-SiR-CRE不同，是无毒的，并且在长期实验期间体内不会出现回复突变。
Transsynaptic viral vectors provide means to gain genetic access to neurons based on synaptic connectivity and are essential tools for the dissection of neural circuit function. Among them, the retrograde monosynaptic ΔG-Rabies has been widely used in neuroscience research. A recently developed engineered version of the ΔG-Rabies, the non-toxic self-inactivating (SiR) virus, allows the long term genetic manipulation of neural circuits. However, the high mutational rate of the rabies virus poses a risk that mutations targeting the key genetic regulatory element in the SiR genome could emerge and revert it to a canonical ΔG-Rabies. Such revertant mutations have recently been identified in a SiR batch. To address the origin, incidence and relevance of these mutations, we investigated the genomic stability of SiR in vitro and in vivo. We found that “revertant” mutations are rare and accumulate only when SiR is extensively amplified in vitro, particularly in suboptimal production cell lines that have insufficient levels of TEV protease activity. Moreover, we confirmed that SiR-CRE, unlike canonical ΔG-Rab-CRE or revertant-SiR-CRE, is non-toxic and that revertant mutations do not emerge in vivo during long-term experiments.