Use of high-content imaging to quantify transduction of AAV-PHP viruses in the brain following systemic delivery

Abstract

The engineering of the AAV-PHP capsids was an important development for CNS research and the modulation of gene expression in the brain. They cross the blood brain barrier and transduce brain cells after intravenous systemic delivery, a property dependent on the genotype of Ly6a, the AAV-PHP capsid receptor. It is important to determine the transduction efficiency of a given viral preparation, as well as the comparative tropism for different brain cells; however, manual estimation of adeno-associated viral transduction efficiencies can be biased and time consuming. Therefore, we have used the Opera Phenix high-content screening system, equipped with the Harmony processing and analysis software, to reduce bias and develop an automated approach to determining transduction efficiency in the mouse brain. We used R Studio and ‘gatepoints’ to segment the data captured from coronal brain sections into brain regions of interest. C57BL/6J and CBA/Ca mice were injected with an AAV-PHP.B virus containing a green fluorescent protein reporter with a nuclear localization signal. Coronal sections at 600 μm intervals throughout the entire brain were stained with Hoechst dye, combined with immunofluorescence to NeuN and green fluorescent protein to identify all cell nuclei, neurons and transduced cells, respectively. Automated data analysis was applied to give an estimate of neuronal percentages and transduction efficiencies throughout the entire brain as well as for the cortex, striatum and hippocampus. The data from each coronal section from a given mouse were highly comparable. The percentage of neurons in the C57BL/6J and CBA/Ca brains was approximately 40% and this was higher in the cortex than striatum and hippocampus. The systemic injection of AAV-PHP.B resulted in similar transduction rates across the entire brain for C57BL/6J mice. Approximately 10–15% of all cells were transduced, with neuronal transduction efficiencies ranging from 5–15%, estimates that were similar across brain regions, and were in contrast to the much more localized transduction efficiencies achieved through intracerebral injection. We confirmed that the delivery of the AAV-PHP.B viruses to the brain from the vasculature resulted in widespread transduction. Our methodology allows the rapid comparison of transduction rates between brain regions producing comparable data to more time-consuming approaches. The methodology developed here can be applied to the automated quantification of any parameter of interest that can be captured as a fluorescent signal.

AAV-Mediated CAG-Targeting Selectively Reduces Polyglutamine-Expanded Protein and Attenuates Disease Phenotypes in a Spinocerebellar Ataxia Mouse Model

“…Figure 2A shows a dose-dependent increase of vg copies per cell (vgc/cell) in the cerebellum, as well as the striatum, hippocampus, and liver. As reported previously [24], the striatum and hippocampus showed higher transduction efficiency than the cerebellum and liver. At the lowest dose (1.5 × 10 12 vg/kg), viral transduction was barely detected in the cerebellum, while at the highest dose (1.5 × 10 13 vg/kg), the transduction efficiency ranged from 0.5 vgc/cell in the cerebellum to 2 vgc/cell in the striatum.…”

Pilot Study Assessing the Impact of Intrathecal Administration of Variants AAV-PHP.B and AAV-PHP.eB on Brain Transduction in Adult Rhesus Macaques

“…injection ( Hordeaux et al, 2018 ). Fourth, the specificity of expression being driven by structure or cellular-specific promoters within the vector genome should also be considered a way to control what cell types and under what conditions the transgene is expressed ( Smith et al, 2021 ).…”

Magnetogenetic closed-loop reduction of seizure activity in a rat model of epilepsy

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