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% to 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.
Highlights
Adeno-associated viral (AAV) vectors are commonly used vehicles in gene therapy research for neurological disease
Whilst an evenly distributed green fluorescent protein (GFP) signal could be detected in the C57BL/6J brains, confirming that the ssAAV-PHP.B-CAG-NLS-GFP virus had crossed the blood brain barrier (BBB), a GFP signal was absent from the brains of CBA/Ca mice (Fig. 1B)
Genotyping confirmed that the C57BL/6J mice were positive for the Ly6a variants that have been shown to be permissive for AAV-PHP viruses to cross the BBB whereas the CBA/Ca mice were not (Fig. 1D).[7,8]
Summary
Adeno-associated viral (AAV) vectors are commonly used vehicles in gene therapy research for neurological disease. Many natural and engineered isolates have been described.[1,2] With the advent of more diverse gene therapy vectors, there is an urgent need to develop accurate and rapid quantification techniques that can be applied to measure viral transduction in complex tissues. A Cre-recombination-based AAV targeted evolution (CREATE) platform was used to generate variants of AAV9: AAV-PHP.B,3 and the enhanced form AAVPHP.eB 4 that penetrate the blood brain barrier (BBB) more effectively than AAV9 following systemic delivery. They have improved CNS tropism, resulting in widespread transduction of neurons and glia throughout the mouse brain. The transduction efficiency of these variants is up to 40-fold higher than that of AAV9, the transduction levels can be variable.[3,4,5,6] the transduction capability of AAV-PHP variants has been shown to be both strain- and species-specific.[7,8,9,10,11] In mice, the CNS tropism of AAV-PHP.B and AAV-PHP.eB is absent in some inbred strains e.g. BALB/cJ, and the ability of these viruses to cross the BBB is dependent on single nucleotide variants in the Ly6a gene, which encodes the receptor for the AAV-PHP capsids.[7,8,11] These limitations have been circumvented by the further development of the CREATE platform and the identification of capsid variants that can transduce the CNS broadly, with different biases towards neurons, glia or vascular cells and that cross the BBB for a diverse set of murine inbred strains.[5]
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