Avian sarcoma leukosis virus receptor-envelope system for simultaneous dissection of multiple neural circuits in mammalian brain

Pathway-specific gene delivery is requisite for understanding complex neuronal systems in which neurons that project to different target regions are locally intermingled. However, conventional genetic tools cannot achieve simultaneous, independent gene delivery into multiple target cells with high efficiency and low cross-reactivity. In this study, we systematically screened all receptor–envelope pairs resulting from the combination of four avian sarcoma leukosis virus (ASLV) envelopes (EnvA, EnvB, EnvC, and EnvE) and five engineered avian-derived receptors (TVA950, TVB S³, TVC, TVB ᵀ, and DR-46TVB) in vitro. Four of the 20 pairs exhibited both high infection rates (TVA–EnvA, 99.6%; TVB S³–EnvB, 97.7%; TVC–EnvC, 98.2%; and DR-46TVB–EnvE, 98.8%) and low cross-reactivity (98%), with no observed cross-reaction. Finally, by expressing three receptor types in a single animal, we achieved pathway-specific, differential fluorescent labeling of three thalamic neuronal populations, each projecting into different somatosensory areas. Thus, we identified three orthogonal pairs from the list of ASLV subgroups and established a new vector system that provides a simultaneous, independent, and highly specific genetic tool for transferring genes into multiple target cells in vivo. Our approach is broadly applicable to pathway-specific labeling and functional analysis of diverse neuronal systems. Significance Genetic dissection of multiple neural pathways remains challenging because of the limited number of genetic methods that can be used simultaneously. To overcome this limitation, we used modified avian sarcoma and leukosis virus envelopes and receptors to develop highly orthogonal genetic tools that can achieve expression of different genes in different target cells. From in vitro and in vivo screens, we identified tools that can specifically transfer genes of interest into mammalian neurons via engineered receptors, with minimal unintended interactions. Using this approach, we achieved pathway-specific, differential fluorescent labeling of three thalamic neuronal populations, each projecting into different cortical regions. Thus,