scAAV1-GFP

Sensorineural hearing loss is one of the most common disabilities worldwide. This prevalence requires effective tools to study the molecular roles of cochlear cells. A prominent and effective vector for expressing genes of interest in research models is adeno-associated virus (AAV). Here, researchers produced AAV to drive green fluorescent protein (GFP) expression in transplanted neonatal mouse cochleae. Treatment with eeyarestatin I, tyrphostin 23, or lipofectamine 2000 did not result in increased transduction, however, the self-complementary vector design resulted in a significant increase in GFP-positive cells compared to single-stranded controls. Similarly, self-complementary AAV2 vectors demonstrated enhanced transduction efficiency compared to single-stranded AAV2 when injected across the posterior semicircular canal in vivo. Self-complementary vectors of serotypes AAV1, 8, and 9 also demonstrated robust GFP transduction in cochlear cells in vivo, although these vectors were not directly compared to single-stranded vectors. These findings suggest that second-strand synthesis may be the rate-limiting step for AAV transduction of cochlear tissue and that self-complementary AAVs can be used to effectively target large numbers of cochlear cells both in vitro and in vivo.

Cochleae were removed from P0-P1 mice, cultured overnight, and then treated with Lipo, EERI, Tyr23, or vehicle. After liposome packaging or processing, AAV1 particles containing CMV promoter-driven green fluorescent protein (GFP), which are either single-stranded (ssAAV1-CMV-GFP) or self-complementary (scAAV1 -CMV-GFP). The total number of GFP-positive cells in the organ of Corti as well as GFP-positive hair cells coexpressing parvalbumin were quantified. ANOVAs for each of these measures revealed a significant main effect of treatment for total GFP+ cells, but no main effect of treatment for GFP positive hair cells. Similar to the observations for AAV2, the main effect of treatment appears to be entirely attributable to the enhanced transduction observed in the self-complementing group (Figure 1).  Post-hoc comparisons showed that explants receiving scAAV1-CMV-GFP had significantly more GFP-positive cells in the sensory epithelium compared with explants receiving ssAAV1-CMV-GFP (Figure 1F). However, self-complementing AAV1 differed slightly from self-complementing AAV2 in that it did not significantly increase the number of GFP and parvalbumin double-positive hair cells (Figure 1G). Likewise, neither lipofection nor treatment with EERI or Tyr23 had any effect on the total number of GFP-positive cells.

Figure 1. Self-complementarity increased transduction efficiency of AAV1-mediated GFP expression in the organ of Corti. (Casey G, et al., 2020)