Human MiniPromoters with Restricted-Retinal Expression when Docked in the Mouse Genome Show the Same Restricted Expression when Delivered in Adeno-Associated Virus (AAV)

Abstract

Purpose: The development of small promoters that can drive cell-type restricted gene expression will be critical for the future success of human gene therapy. In this work, we have focused on the development of such gene-therapy promoters for the retina. First, we tested the hypothesis that the human Pleiades MiniPromoters selected for expression in the brain, would be a rich source of new promoters for the retina. Second, we tested the hypothesis that brain-restricted promoters such as the Pleiades Promoters, which were developed using single-copy site-specific knock-in to the mouse genome, would function predictably when used in adeno-associated virus (AAV). We provide data in support of both hypotheses.

Methods: Promoters were bioinformatically designed, generated by fusion PCR from human BACs, cloned to drive reporters EGFP and/or lacZ, and targeted 5’ of Hprt in mouse embryonic stem cells from which mice were derived; as described in Portales-Casamar et al., P.N.A.S., 2010. Expression analysis was in male mice at embryonic day 12.5 or adulthood. Recombinant AAV plasmids containing MiniPromoters driving hGFP were packaged in AAV2(Quad Y-F) viruses (Petrs-Silva et al., Mol Ther., 2011) and injected intravitreally into four-week old C57BL/6J mice.

Results: We have developed 18 new MiniPromoters capable of driving expression in the brain when docked single-copy site-specifically in the mouse genome. In addition, we have further characterized the expression pattern of 15 MiniPromoters. Together we have identified 17 new MiniPromoters with eye expression. Of these, three with restricted expression in the ganglion-cell layer were tested driving expression from an AAV genome and found to maintain that restricted expression.

Conclusions: We conclude that the Pleiades MiniPromoters, which were initially selected for brain expression, represent a rich resource of promoters able to express in the retina. We further conclude that either the computational method of MiniPromoter design, and/or the characterization of those promoters using single-copy site-specific in the mouse genome, renders them unusually suitable for use in AAV and perhaps other viruses. Finally, the MiniPromoters are all human-DNA sequence and so should be particularly useful for clinical applications.

Keywords: 538 gene transfer/gene therapy • 533 gene/expression • 531 ganglion cells