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.
Highlights
Small and cell-type restricted promoters are important tools for basic and preclinical research, and clinical delivery of gene therapies
A negative result did not distinguish between lack of capsid transduction, or lack of promoter expression
MiniPromoters that showed desirable expression patterns following neonatal intravenous delivery were studied further. rAAV9 smCBA-emerald green fluorescent protein (EmGFP) was injected at either postnatal day (P) 0 or P4 to evaluate the different cell types of the eye, as it has previously been shown that the development of the blood retinal barrier (BRB) occurring during this window results in expression primarily in different cell types [64]
Summary
Small and cell-type restricted promoters are important tools for basic and preclinical research, and clinical delivery of gene therapies. A major challenge in the development of PAX6 gene therapy is that expression of the endogenous protein is complex, and inappropriate locations and levels of PAX6 could be detrimental [44,45,46,47]. Building on this previous work of ours and others, we present our current strategy for MiniPromoter (minimal human promoter element(s) designed for expression in restricted cell types) design and testing, and the resulting resource. We present promoter development as an overview for each cell type, but only show results in detail for the “recommended” MiniPromoter(s)
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