Design of synthetic promoters for controlled expression of therapeutic genes in retinal pigment epithelial cells

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.

Engineering of the CMV promoter for controlled expression of recombinant genes in HEK293 cells

“…This is a very significant and useful finding, as they form the basis of promoter engineering containing enhanced binding sites, [ 37 ] or can be directly utilized as modular building blocks to construct synthetic promoters de novo. [ 25,28 ] We further identified several sub‐optimal TF binding sequences (MYBL1, Oct, E2F) which suggests an immense opportunity for maximizing CMV promoter's transcriptional output. Hundreds of TFRE motif sequence variants can be characterized simultaneously via in vitro use of high‐throughput parallel screening methods, allowing determination of their optimal binding affinity.…”

“…The TSS is indicated with an arrow relative ability of TFREs to activate transcription of recombinant genes in HEK293 cells, we created a set of GFP reporter constructs that contained seven repeat copies of a specific TFRE in series, upstream of a minimal CMV core promoter (-36 to +48 relative to the TSS, containing a TATA box and an Inr motif) as previously described. [25,28] We note that the transcriptional output from a single TF binding site is often insufficient to drive detectable levels of recombinant gene expression. Optimized PEI-mediated transient transfection of plasmid DNA into suspension HEK293 cells yielded a transfection efficiency of ∼ 94% with a cell viability of ∼ 90% at 48 h post-transfection (measured using a vector harboring a CMV promoter).…”

From Bench to Bed: The Current Genome Editing Therapies for Glaucoma

“…Recently, the cell-specific targeting of these gene vectors has greatly advanced to pave the way for successful clinical trials in future. The current strategies of cell-specific targeting include modifying tropism of delivery vectors, designing to target the specific molecular markers and carrying cell-specific promoter in the viruses ( Hulliger et al, 2020 ; Johari et al, 2021 ). A previous study found that due to the high expression of heparin sulfate proteoglycan in RGCs, which mediates attachment to AAV2, AAV2 has a high tropism for RGCs ( Summerford and Samulski, 1998 ).…”

Evaluation of site-specific methylation of the CMV promoter and its role in CHO cell productivity of a recombinant monoclonal antibody

“…[ 14 ]. In addition, this mechanistic understanding may influence design of synthetic promoters [ 15 ] and further the scope of next-generation cell line development [ 16 ].…”