WHOLE-BODY BIODISTRIBUTION OF AAV-MEDIATED GENE EXPRESSION IN MICE USING MULTI-RESOLUTION 3D CRYOVIZTM IMAGING

• Published in: Cytotherapy (Oxford, England) England), 2024-06, Vol.26 (6), p.S27-S28
• Main Authors: Gargesha, M., Caligiuri, S., Scott, B., Braunscheidel, K., Kenny, P.J., Roy, D.
• Format: Article
• Language: English
• Subjects: peripheral nervous system; sensory ganglia; serial block-face imaging
• ISSN: 1465-3249; 1477-2566
• DOI: 10.1016/j.jcyt.2024.03.046

The aim of this study was to employ CryoVizTM Imaging to characterize 3D expression of retro-orbitally injected AAV-PHP.S-tdTomato in mice. CryoVizTM Imaging is a serial sectioning and block-face imaging technology that acquires large-field-of-view, 3-D microscopic color anatomical and molecular fluorescence volumes from frozen tissue blocks. In vitro assays suggest that AAV-PHP.S-tdTomato, a virus serotype that specifically targets sensory ganglia, leads to robust infection of sensory nuclei and the choroid plexus of the mouse brain. A high-resolution imaging study of the systemic sensory ganglia entering the brain would be beneficial to understand virus-mediated gene transfer. First, in a whole mouse, a standard resolution imaging protocol was used for scouting for interesting “hot spots” of AAV infection. Subsequently, on excised mouse heads, a very high-resolution imaging protocol was employed to further examine these “hot spots”. AAV-PHP.S-tdTomato was injected retro-orbitally into the 4th ventricle of the brain. ∼3 weeks after AAV delivery, mice were euthanized, embedded in a histological medium, and flash frozen in liquid N2 for CryoVizTM Imaging. Mice were imaged at two different resolutions – xy (in-plane) resolution of 10µm (or 4µm) and a z-section thickness of 40µm (or 20µm) for whole mouse (or mouse heads). A dual-band fluorescence filter optimized to detect tdTomato (Excitation peak: 554nm, Emission peak: 581nm) was employed. In whole mice, we found robust AAV transduction (i.e. orange-red tdTomato) in the head and neck region (Fig. 1a-b, red). In excised mouse heads, strong AAV transduction was found in the hindbrain close to the retroorbital injection site, mainly in the vicinity of the 4th ventricle choroid plexus (Fig. 2a, blue oval). In addition, the following regions of the brain anatomy (Fig. 2b, blue oval) also exhibited significant AAV transduction - Trigeminal nerve, Vagus nerve, Glossopharyngeal nerve, and Nodose ganglia. CryoVizTM Imaging has revealed whole body biodistribution of AAV-PHP.S-tdTomato in standard and very high resolutions. These data suggest that intracranial delivery of AAVs results in highly selective transduction in sensory ganglia, while not infecting other tissues or neurons in periphery or in the brain. CryoVizTM Imaging is an important and critical tool for confirming selective transduction of sensory ganglia and identifying ‘ectopic’ expression of transgenes in a whole-body manner.