Abstract
Age-related macular degeneration (AMD) associated with dysfunction of retinal pigment epithelial (RPE) cells is the most common cause of untreatable blindness. To advance gene therapy as a viable treatment for AMD there is a need for technologies that enable controlled, RPE-specific expression of therapeutic genes. Here we describe design, construction and testing of compact synthetic promoters with a pre-defined transcriptional activity and RPE cell specificity. Initial comparative informatic analyses of RPE and photoreceptor (PR) cell transcriptomic data identified conserved and overrepresented transcription factor regulatory elements (TFREs, 8-19 bp) specifically associated with transcriptionally active RPE genes. Both RPE-specific TFREs and those derived from the generically active cytomegalovirus-immediate early (CMV-IE)promoter were then screened in vitro to identify sequence elements able to control recombinant gene transcription in model induced pluripotent stem (iPS)-derived and primary human RPE cells. Two libraries of heterotypic synthetic promoters varying in predicted RPE specificity and transcriptional activity were designed de novo using combinations of up to 20 discrete TFREs in series (323-602 bp) and their transcriptional activity in model RPE cells was compared to that of the endogenous BEST1 promoter (661 bp, plus an engineered derivative) and the highly active generic CMV-IE promoter (650 bp). Synthetic promoters with a highpredicted specificity, comprised predominantly of endogenous TFREs exhibited a range of activities up to 8-fold that of the RPE-specific BEST1 gene promoter. Moreover, albeit at a lower predicted specificity, synthetic promoter transcriptional activity in model RPE cells was enhanced beyond that of the CMV-IE promoter when viral elements were utilized in combination with endogenous RPE-specific TFREs, with a reduction in promoter size of 15%. Taken together, while our data reveal an inverse relationship between synthetic promoter activity and cell-type specificity, cell context-specific control of recombinant gene transcriptional activity may be achievable.
Highlights
The retinal pigment epithelium (RPE) is a multifunctional monolayer of neuroepithelium‐derived cells, flanked by photoreceptor (PR) cells and the choroid complex
Using a combined in silico and in vitro screening approach, we successfully identified active transcription factor regulatory element (TFRE) candidates that could be utilized to construct synthetic promoter assemblies with strong and/or RPE specific expression
The data in this study indirectly serves as a reference for TFREs that should be avoided in constructing PR‐specific promoters
Summary
The retinal pigment epithelium (RPE) is a multifunctional monolayer of neuroepithelium‐derived cells, flanked by photoreceptor (PR) cells and the choroid complex. The recent approval of voretigene neparvovec (Luxturna®) to treat retinal degeneration highlights that gene therapies to disease‐ causing genetic mutations are possible This achievement is partly made possible by the use of adeno‐associated viral (AAV) vectors that can transduce and maintain therapeutic gene expression in non‐ dividing cells (including the retina) with minimal immune responses (Naso et al, 2017). More advanced attempts to create promoters with increased tissue‐ specificity are exemplified via de novo design of synthetic promoters that could mediate gene expression in muscle cells (Li et al, 1999), colorectal cancer cells (Roberts et al, 2017) or liver cells with responsiveness to glucose (Han et al, 2011) These studies involved screening hundreds to thousands of synthetic promoters, which is unfeasible for primary and iPS‐derived cells, such as RPE, with a limited capacity for expansion and which exhibit a particular differentiated morphological state. While this study demonstrates effective construction of promoters for RPE cells, similar approaches could be used to design promoters for applications requiring specific and/or high expression of recombinant genes in other cell types
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