Human MiniPromoters for ocular-rAAV expression in ON bipolar, cone, corneal, endothelial, Müller glial, and PAX6 cells

Abstract

Small and cell-type restricted promoters are important tools for basic and preclinical research, and clinical delivery of gene therapies. In clinical gene therapy, ophthalmic trials have been leading the field, with over 50% of ocular clinical trials using promoters that restrict expression based on cell type. Here, 19 human DNA MiniPromoters were bioinformatically designed for rAAV, tested by neonatal intravenous delivery in mouse, and successful MiniPromoters went on to be tested by intravitreal, subretinal, intrastromal, and/or intravenous delivery in adult mouse. We present promoter development as an overview for each cell type, but only show results in detail for the recommended MiniPromoters: Ple265 and Ple341 (PCP2) ON bipolar, Ple349 (PDE6H) cone, Ple253 (PITX3) corneal stroma, Ple32 (CLDN5) endothelial cells of the blood–retina barrier, Ple316 (NR2E1) Müller glia, and Ple331 (PAX6) PAX6 positive. Overall, we present a resource of new, redesigned, and improved MiniPromoters for ocular gene therapy that range in size from 784 to 2484 bp, and from weaker, equal, or stronger in strength relative to the ubiquitous control promoter smCBA. All MiniPromoters will be useful for therapies involving small regulatory RNA and DNA, and proteins ranging from 517 to 1084 amino acids, representing 62.9–90.2% of human proteins.

Transgene distribution and immune response after ultrasound delivery of rAAV9 and PHP.B to the brain in a mouse model of amyloidosis

“…The rAAV genome plasmid (pEMS2143) used here has also been used previously 56 , 57 and is available from Addgene ( www.addgene.org ). In brief, it is ssAAV-smCBA-EmGFP-WPRE, where ssAAV is single-stranded AAV, smCBA is a smCBA enhancer chosen for ubiquitous expression, 25 , 56 , 57 , 58 EmGFP is used, 59 and WPRE is the 3′ UTR woodchuck hepatitis virus post-transcriptional regulatory element mut6. 60 , 61 For this work, pEMS2143 was packaged into either rAAV9 or PHP.B by the University of Pennsylvania Vector Core (Philadelphia, PA, USA).…”

A systematic comparison of optogenetic approaches to visual restoration

“…Given the demonstrated differences in other promoters specificity in degenerate retinas 61 and when moving to primates, 60 L7-6 appears to be particularly attractive and adds to a growing armory of retinal-cell-population-specific mini-promoters. 30 , 73 , 74 , 75 , 76 , 77 …”

Ectopic insert-dependent neuronal expression of GFAP promoter-driven AAV constructs in adult mouse retina

“…Since AAV vectors currently represent the least toxic and most effective method of gene delivery in vivo , this poses important challenges for future research into cell-based therapies. While several studies have reported Müller glia-specific AAV-based minipromoter constructs derived from genes other than GFAP ( Korecki et al, 2021 ; Lin et al, 2022 ), these too may show ectopic expression when used to overexpress specific genes. In mice, where transgenic reagents such as the GlastCreERT2;Sun1-GFP that can reliably and irreversibly label glia are readily available, prospective genetic lineage analysis should be performed routinely in conjunction with AAV-based manipulations.…”