Functional gene delivery using a novel blood-brain-barrier-crossing AAV capsid in rats

Compared to mice, similar genetic tools have been traditionally very limited in rats, such that selective imaging or manipulation of circuits has been challenging. Developing blood-brain barrier (BBB) crossing and cell-type specific AAVs is an important research avenue as these viral vectors allow more precise control over transduction sparseness and coverage of larger target areas upon minimally invasive intravenous delivery (e.g. tail injections). One example of such an AAV is CAP-Mac, previously shown to be highly neuron-specific and to have high transduction efficiency in non-human primates (Chuapoco et al., 2023) as well as in the rat brain when packaged with fluorescent reporters (Chen et al., 2023).In this study, we focused on the functional testing of AAV.CAP-Mac in optogenetic settings in rats. Acute extracellular recordings during light stimulations were used to functionally validate the successful transduction of neurons with channelrhodopsins. Using different viral constructs containing channelrhodopsin and/or fluorescent reporters packaged in AAV.CAP-Mac (hChR2-P2P-mRuby3, hChR2-P2P-mNeongreen, hChR2, mNeonGreen), we tested the cargo-dependency of AAV.CAP-Mac’s BBB crossing and transduction efficiency to optimise channelrhodopsin expression in the brain upon systemic delivery in rats. AAV-delivered channelrhodopsin expression was visualised and quantified using serial two-photon microscopy methods while also characterising AAV.CAP-Mac’s target preference. Screening a selection of constructs and different viral titres, our findings suggest an effect of plasmid size on AAV.CAP-Mac’s transduction efficiency, with hChR2-P2P-mRuby3 providing the highest expression level. These results pave the way for using AAV.CAP-Mac in future behavioural studies in freely moving rats.