Genomic stability of Self-inactivating Rabies

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 {Delta}G-Rabies has been widely used in neuroscience research. A recently developed engineered version of the {Delta}G-Rabies, the non-toxic self-inactivating (SiR) virus, represents the first tool for open-ended 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 {Delta}G-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 {Delta}G-Rab-CRE or revertant-SiR-CRE, is non-toxic and that revertant mutations do not emerge in vivo during long-term experiments. HighlightsO_LIRevertant mutations are rare and do not accumulate when SiR is produced in high-TEVp expressing production cell lines C_LIO_LISiR is non-toxic in vivo C_LIO_LIRevertant SiR mutations do not accumulate during in vivo experiments C_LI