Genomic stability of Self-inactivating Rabies

Abstract

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, 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 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.HighlightsRevertant mutations are rare and do not accumulate when SiR is produced in high-TEVp expressing production cell linesSiR is non-toxic in vivoRevertant SiR mutations do not accumulate during in vivo experiments

“Self-inactivating” rabies viruses are susceptible to loss of their intended attenuating modification

“…If the viruses used for the transsynaptic tracing experiments in the original paper were actually de facto first-generation, ∆G viruses like the SiR samples that we analyzed, how could the authors have found, in postmortem tissue, cells labeled by SiR-CRE that had survived for weeks? The answer may simply be that, as we have shown in Chatterjee et al (21) and again here (Figure 3), and as the original authors also report in their new preprint (49), a preparation of first-generation rabies viral vector expressing Cre can leave a large fraction of labeled cells alive for at least months, in contrast to similar ones encoding tdTomato (21) or EGFP (4). Similarly, Gomme et al found long-term survival of some neurons following infection by a replicationcompetent rabies virus expressing Cre ( 62)).…”

“…Indeed, in response to our posting a preprint of an earlier version of this manuscript in February 2019 (46), the authors of the original paper have recently posted a new preprint in which they report that they are now able to make mutation-free SiR virus and that this intact SiR virus is noncytotoxic when used to directly infect cortical neurons (47). This is a good start but does not change anything about our findings, conclusions, and predictions.…”

“…It remains to be seen to what degree an NPEST or SiR virus that spreads transsynaptically is cytotoxic, not only to transsynaptically-labeled cells but also to the starting cells, which need to express both G and TEVP to allow replication and spread of the virus. This is not a trivial point, as G is toxic when overexpressed (66), and the original authors' finding that cultured cells rapidly lost TEVP activity (49) suggests that TEVP expressed at sufficient levels may be toxic as well.…”

All-viral tracing of monosynaptic inputs to single birthdate-defined neurons in the intact brain

“…From our experience, it appears that the rate at which this occurs differs based on the concentration and amount of virus that the cells are exposed to ( Lavin et al., 2020 ), their cell type, and the circuit in which targeted cells are embedded. However, with recent bioengineering efforts, the limitation of rabies-mediated cytotoxicity can now be overcome and long-term functional studies enabled, though additional viruses and/or transgenic animals are required to achieve this ( Ciabatti et al., 2017 , 2020 ; Chatterjee et al., 2018 ). Another limitation, also independent of the targeting approach, is the lack of knowledge regarding how RABVs spread to input cells.…”

All-viral tracing of monosynaptic inputs to single birthdate-defined neurons in the intact brain

“…From our experience, it appears that the rate at which this occurs differs based on the concentration and amount of virus the cells are exposed to (Lavin et al, 2020), their cell type, and the circuit that targeted cells are embedded in. However, with recent bioengineering efforts the limitation of rabiesmediated cytotoxicity can now be overcome, and thus long-term functional studies enabled, although additional viruses and/or transgenic animals are required to achieve this (Ciabatti et al, 2017(Ciabatti et al, , 2020Chatterjee et al, 2018). Another limitation, also independent of the targeting approach, is the lack of knowledge regarding how rabies viruses spread to input cells.…”