Genomic stability of self-inactivating rabies.

Area of Science:

• Neuroscience
• Molecular Biology
• Virology

Background:

• Transsynaptic viral vectors are crucial for studying neural circuits.
• Retrograde monosynaptic ΔG-Rabies virus is a widely used tool.
• Engineered self-inactivating (SiR) rabies virus allows long-term genetic manipulation but carries a risk of reversion.

Purpose of the Study:

• To investigate the genomic stability of the self-inactivating (SiR) rabies virus.
• To determine the origin, incidence, and relevance of revertant mutations in SiR.
• To confirm the non-toxicity and in vivo stability of SiR-CRE.

Main Methods:

• In vitro and in vivo genomic stability assays of SiR.
• Analysis of mutation accumulation under varying amplification conditions.
• TEV protease activity assessment in production cell lines.
• Toxicity and long-term stability assessment of SiR-CRE compared to controls.

Main Results:

• "Revertant" mutations are rare and primarily accumulate during extensive in vitro amplification.
• Suboptimal production cell lines with insufficient TEV protease activity increase mutation risk.
• SiR-CRE demonstrated non-toxicity, unlike canonical ΔG-Rab-CRE or revertant-SiR-CRE.
• Revertant mutations did not emerge in vivo during long-term experiments.

Conclusions:

• The genomic stability of SiR is high under standard conditions.
• Careful optimization of in vitro production, particularly TEV protease levels, minimizes reversion risk.
• SiR-CRE is a safe and stable tool for long-term neural circuit manipulation in vivo.