AAV-compatible MiniPromoters with selectivity for bipolar, horizontal, and Müller glia cells.

Abstract

Purpose: Small promoters that drive cell-type restricted gene expression in the eye are important tools for a wide variety of basic and clinical research, and may be critical for future gene therapies. In this work, we have focused on the development of such promoters for use in recombinant adeno-associated virus (rAAV). Here we tested the hypothesis that ocular-restricted Pleiades Promoters, which were developed using single-copy site-specific knock-in to the mouse genome, would retain selectivity when used in rAAV.

Methods: Promoters were: bioinformatically designed (with one from the literature); DNA synthesised (or isolated from previous constructs); and cloned into a new “plug and play” rAAV genome plasmid driving either icre (improved cre) or emGFP (emerald GFP), without or with WPRE (a transcript stabilizing element). Recombinant AAV2/9 virus was produced, and 50 μl at 10EE13 genome copies/mL were injected into the temporal vein of either P0 (post natal, day of birth) or P4, B6129F1 hybrid mice. For icre-containing virus, the injected mice carried the Gt(ROSA)26Sor^tm1Sor allele, which responds to cre by permanently rearranging the genome to express lacZ under control of the ubiquitous ROSA promoter. Expression was analysed at P21 or P56 using whole mount or cryosections for beta-gal detection of lacZ, and cryosections for epifluorescence or anti-GFP immunofluorescence detection of emGFP.

Results: We tested 21 experimental viruses carrying 13 different MiniPromoters designed from 13 genes. Three MiniPromoters were tested with both icre and emGFP, and three were tested with and without WPRE. All MiniPromoters, or their source genes, had previous data consistent with ocular expression. Of the experimental viruses tested, 16 (76%) showed ocular expression. Of the MiniPromoters tested, nine (69%) showed ocular expression. Positive results were independent of whether the reporter was icre or emGFP, and independent of the presence of WPRE, although WPRE strengthened expression independent of MiniPromoter or reporter. This work has resulted in new AAV-compatible MiniPromoters with selectivity for bipolar, horizontal, and Müller glia cells.

Conclusions: This data supports the conclusion that the Pleiades MiniPromoters represent a rich resource of human-DNA promoters, compatible with rAAV, able to express in the eye, and thus particularly useful for clinical applications.