rAAV-Compatible MiniPromoters with Restricted Expression in the Retinal ON Bipolar, Müller Glia, and Cone Photoreceptor cells, the Corneal Stroma, and the Endothelial cells of the Blood Retina Barrier

Abstract

Purpose : Over 50% of retinal gene therapies currently in clinical trials are using promoters derived from human genes that restrict expression based on cell type. Such promoters have demonstrated increased efficacy and reduced unwanted off-target effects. However, the field would greatly benefit from a larger collection of restricted, smaller, and stronger human promoters (MiniPromoters) to accelerate the path of many therapies towards clinical trial. Using bioinformatics and in vivo expression analysis, our team has previously developed numerous MiniPromoters, most recently validating a set of promoters for retinal ganglion cells in both mouse and non-human primate (Simpson et al. 2018).

Methods : MiniPromoters either were new bioinformatics-driven designs from genes with therapeutically interesting expression patterns, or were further reduced in size or strengthened from our previous work. All promoters were cloned into a custom rAAV genome plasmid driving EmGFP (emerald GFP), and including an intron, WPRE (transcript stabilizer), and SV40 polyA, and then packaged into rAAV9. All viruses were tested by injection intravenously into newborn mice. A subset of MiniPromoters was further characterized by direct injection into the adult mouse eye, including subretinal, intravitreal, and intrastromal routes. Analysis included GFP immunofluorescent of expression driven by the MiniPromoter compared to the ubiquitous promoter smCBA. For the most promising promoters, co-staining with relevant markers and quantification of epifluorescent signal was also undertaken.

Results : Successful MiniPromoters ranged in size from 986 to 2,484 bp. Highlights include promoters derived from PCP2 for the retinal bipolar ON cells, NR2E1 for the retinal Müller glia, PDE6H for the retinal cone photoreceptors, PITX3 for the corneal stroma, and CLDN5 for the endothelial cells of the blood retina barrier.

Conclusions : Results demonstrate that small-restricted MiniPromoters can be developed for clinically relevant cell types of the eye. All published Pleiades (Ple) MiniPromoters are available to the community through Addgene (www.addgene.org), and unpublished materials are available from Dr. Elizabeth M. Simpson ([email protected]).

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.