scAAV9-Syn-GFP

Current barriers to the use of adeno-associated virus serotype 9 (AAV9) in clinical trials for the treatment of neurological diseases are high expression of the virus in many non-target tissues, such as liver and heart, and lack of cell specificity within the central nervous system (CNS) when using ubiquitous promoters, such as human cytomegalovirus (CMV) or chicken-β-actin hybrid (CAG). To enhance targeting of transgene expression in CNS cells, self-complementary (sc) AAV9 vectors, scAAV9-GFP vectors carrying neuronal Hb9 and synapsin 1, and nonspecific CMV and CAG promoters were constructed. Studies have shown that the synapsin 1 and Hb9 promoters specifically target neurons in vitro, although they are 10-fold less potent than CMV. In vivo analysis of mouse tissues after cisternae delivery of scAAV9-GFP vectors indicated that the synapsin 1 promoter has a clear advantage over the two Hb9 variants in targeting neurons throughout the brain, as the Hb9 promoter primarily drives gene expression within motor-related regions of the brainstem.

To evaluate the suitability of the SYN1 and Hb9 promoters for in vivo gene transfer compared to CMV and CAG in the traditionally used scAAV9 vectors, wild-type mice on postnatal day 1 were injected via cisternae with 1 × 10¹⁰ viral genomes containing five titer-matched scAAV9-GFP vectors containing CMV, CAG, SYN1, Hb9cmv, and Hb9e promoter sequences. CNS tissues were perfused with paraformaldehyde (PFA) and harvested 3 weeks after injection for immunohistochemical analysis. Immunostaining of spinal cord sections with anti-NeuN antibodies demonstrated that the CMV, CAG, and SYN1 promoters, but not the Hb9cmv or Hb9e promoters, had efficient CNS targeting when used at the same dose (Figure 1). In the spinal cord, scAAV9-CMV-GFP transduction resulted in the highest number of GFP-positive cells and the strongest fluorescence levels in the lumbar and cervical regions (Figure 1a, i), whereas CAG and SYN1 were significantly weaker promoters (Figure 1c). Furthermore, unlike CMV (Figure 1b, j), scAAV9-Syn-GFP targeted CGRP-positive motor neurons within the lumbar spine with lower efficiency (Figure 1f, h). Surprisingly, neither ventral horn neurons of animals treated with scAAV9-Hb9cmv nor scAAV9-Hb9e expressed GFP, although several GFP-positive interneurons (up to 10 per area) were detected within the dorsal horns of the lumbar spinal cord (Figure 1g).

Figure 1. Intracerebrospinal fluid delivery via cisterna magna of scAAV9-GFP results in differential gene delivery efficiencies in the mouse spinal cord depending on the use of the promoter. (Lukashchuk V, et al., 2016)