AAV6-CMV-mCherry

Recombinant adeno-associated virus (AAV)-mediated therapeutic gene transfer into the dorsal root ganglia (DRG) is an effective and safe method for the treatment of chronic pain. However, AAV with various constitutively active promoters results in transgene expression primarily in neurons, whereas glial cells are resistant to AAV transduction in the peripheral nervous system. Here, researchers evaluated whether in vivo satellite glial cell (SGC) transduction in the DRG can be enhanced by the SGC-specific GFAP promoter and by using shH10 and shH19, which are engineered capsid variants with Müller glia-prone transduction. The results showed that injection of AAV6 and AAVshH10-GFAP-EGFP selectively induced EGFP expression in SGCs, while injection of AAVshH10-CMV-EGFP or AAVshH19-CMV-EGFP into DRGs produced in vivo transduction similar to AAV6-CMV-EGFP, all showing efficient transduction of sensory neurons without significant transduction of glial cell populations. Co-injection of AAV6-CMV-mCherry and AAV6-GFAP-EGFP induced transgene expression in neurons and SGCs, respectively. These results indicate that the GFAP promoter, rather than capsid tropism, determines selective gene expression in SGCs following intraganglionic AAV delivery in adult rats. A dual AAV system (one with the GFAP promoter and the other with the CMV promoter) can efficiently selectively express transgenes in neurons but not SGCs.

The researchers tested the specificity of anti-GFP and anti-mCherry antibodies in detecting individual fluorescent proteins. To this end, HEK293 cells were infected with the AAV6-GFAP-EGFP or AAV6-CMV-eCherry. Cell lysates 48 hr after infection were analyzed by Western blots with anti-GFP or anti-mCherry. This resulted in the expression of individual proteins of the expected size, with no cross-reactivity observed for either the anti-GFP or anti-mCherry antibodies (Figure 1a). In the co-delivery experiments performed in vivo, dual AAV vectors composed of AAV6-GFAP-EGFP and AAV6-CMV-mCherry mixed at a 1:1 ratio were injected into L4/L5 DRG. Immunofluorescent analyses of DRG sections at 5 weeks after injection revealed EGFP-expressing SGCs and mCherry-expressing neurons without evidence of coexpression (Figure 1b,c). Quantitative estimation of transduction rates (4 DRG) showed 40%±8% EGFP+ SGC rings and 34%±11% mCherry+ neuron soma. These results indicate that this dual AAV strategy successfully drives selective expression in DRG neurons versus SGCs using a GFAP promoter to express one transgene in SGCs while using CMV promoter to target another transgene expression only in neurons.

Figure 1. Dual AAVs for selective transduction to neurons versus SGCs. (Xiang H, et al., 2018)