Abstract
Recombinant adeno-associated viral (AAV) vectors have advanced to the vanguard of gene therapy. Numerous naturally occurring serotypes have been used to target cells in various tissues. There is a strong need for fast and dynamic methods which efficiently unravel viral tropism in whole organs. Ultramicroscopy (UM) is a novel fluorescence microscopy technique that images optically cleared undissected specimens, achieving good resolutions at high penetration depths while being non-destructive. UM was applied to obtain high-resolution 3D analysis of AAV transduction in adult mouse brains, especially in the hippocampus, a region of interest for Alzheimer’s disease therapy. We separately or simultaneously compared transduction efficacies for commonly used serotypes (AAV9 and AAVrh10) using fluorescent reporter expression. We provide a detailed comparative and quantitative analysis of the transduction profiles. UM allowed a rapid analysis of marker fluorescence expression in neurons with intact projections deep inside the brain, in defined anatomical structures. Major hippocampal neuronal transduction was observed with both vectors, with slightly better efficacy for AAV9 in UM. Glial response and synaptic marker expression did not change post transduction.We propose UM as a novel valuable complementary tool to efficiently and simultaneously unravel tropism of different viruses in a single non-dissected adult rodent brain.
Highlights
In the characteristics of the capsid surface, has been identified[9,10]
AAV9 and AAVrh[10] vector stocks encoding the green fluorescent protein (GFP) reporter under control of the chicken β-actin/cytomegalovirus (CMV) hybrid (CAG) promoter (Fig. 1a), that is known for its high neuron-specificity after direct associated virus (AAV) injections to the adult CNS16, were titre-matched
In order to study the pattern of GFP biodistribution in the mouse hippocampus, we analysed direct GFP fluorescence in brain slices from mice injected with AAV9 or AAVrh[10] encoding the GFP protein (Fig. 1c–f)
Summary
Vector-packaging systems comprising approximately ten different natural serotypes are accessible for the generation of AAV gene therapy vectors[11,12], depending on specific interactions of the capsid proteins within transduced cells. This has been reported in several studies and showed dissimilarities in the transduction efficiency of particular AAV serotypes in different cell types and tissues[13]. UM is a novel fluorescence microscopy technique that applies a focused light sheet to illuminate an optically cleared specimen from the side, e.g., a whole rodent brain, perpendicular to the objective This technique achieves excellent resolution at high penetration depths while being non-destructive at the same time. We propose UM as a valuable complementary tool to efficiently unravel viral tropism in non-dissected intact whole organs
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