AAV9-CBh-mCherry

Cell-selective gene expression is a key element of many adeno-associated virus (AAV) vector-based gene therapies, and efforts to achieve this have so far focused on AAV capsid engineering, cell-specific promoters, or cell-specific enhancers. Recently, researchers have shown that the AAV9 capsid exerts differential effects on constitutive promoters of sufficient magnitude to alter cell-type gene expression in the rat central nervous system. For AAV9 vectors, chicken β-actin (CBA) promoter-driven gene expression exhibited dominant neuronal gene expression in the rat striatum. Surprisingly, for otherwise identical AAV9 vectors, the truncated CBA hybrid (CBh) promoter shifted gene expression to striatal oligodendrocytes. Furthermore, insertion of six glutamic acid residues immediately after the VP2 start residue shifted CBA-driven cellular gene expression from neurons to oligodendrocytes. Conversely, insertion of six alanines in the same AAV9 capsid region reversed CBh-mediated oligodendrocyte expression back to neurons without altering the entry of the AAV9 capsid into oligodendrocytes. Given the dominance of AAV9 in ongoing clinical trials and AAV capsid engineering, this AAV9 capsid-promoter interaction reveals a previously unknown novel contribution to cell-selective AAV-mediated gene expression in the CNS.

Here, in otherwise identical AAV9 vectors, the CBA and CBh promoters exhibited distinct cellular gene expression patterns. When the AAV9-CBA-mCherry vector was injected into the rat striatum, 88.4% ± 1.6% of mCherry-positive cells colocalized with NeuN (Figures 2S-2V and 2A′), and only 10.4% ± 2.1% of mCherry-positive cells colocalized with Olig2 (Figures 2W-2Z and 2A′). In sharp contrast, AAV9-CBh-mCherry transgene expression colocalized with NeuN (Figures 2B′–2E′) in 46.3% ± 4.6% of mCherry-positive cells (Figure 2J′), while of the remaining mCherry-positive cells, 37.8% ± 4.9% colocalized with Olig2 (Figures 2F′–2J′). Given that the capsids were identical in both cases, it was inevitable that the AAV9 vectors acquired the same cellular pathways and were delivered to the nucleus of neurons and oligodendrocytes in the rat striatum. As both promoters could support gene expression in striatal neurons and oligodendrocytes, the differences in cellular gene expression patterns suggest that the AAV9 capsid exerts a significant effect on promoter activity in different CNS cell types.

Figure 1. Representative Confocal Images of AAV9 with mCherry Expression Driven by a CBA or CBh Promoter. (Powell S K, et al., 2020)