Ultramicroscopy as a novel tool to unravel the tropism of AAV gene therapy vectors in the brain

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.

Optimization of adeno-associated viral vector-mediated transduction of the corticospinal tract: comparison of four promoters

“…In contrast to our study using AAV1, one of the lentivirus studies reported low tropisms for astrocytes using the mPGK promoter [61]. A study that used the human PGK promoter with AAV serotypes 6, 9, and rh10 reported predominant expression in neurons and a limited number of astrocytes and oligodendrocytes [62,63]. In addition, we found that the mPGK promoter is a strong promoter in terms of transgene expression in the sensorimotor cortex; mPGK promoter was superior over the hCMV and sCAG promoters.…”

CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer’s Disease

“…We aimed to directly inject the AAV-vector-packaged SW1 gRNA and Cas9 into the hippocampus of Tg2575 mice, which have multiple copies of human APP SW . We packaged Cas9/gRNA into exo-AAV1 (for in vitro experiments, as AAV1, but not AAV9 serotype, transduces neurons efficiently in vitro 14 ) and exo-AAV9 vectors (for in vivo injections, as AAV9 serotype is highly efficient in transducing neurons after stereotactic injection in mice 15 ).…”

Correlated MRI and Ultramicroscopy (MR-UM) of Brain Tumors Reveals Vast Heterogeneity of Tumor Infiltration and Neoangiogenesis in Preclinical Models and Human Disease

“…Mouse brains were fixed after perfusion with 4% PFA for 24 h and stored in PBS at 4°C in the dark. For UM-analysis whole brains were optically cleared using the FluoClearBABB protocol (Schwarz et al, 2015; Alves et al, 2016; Breckwoldt et al, 2016). The protocol is based on benzyl alcohol/benzyl benzoate clearing in combination with a basic pH.…”