Development of novel ciliopathy specific promoter sequences for optimal gene therapy transgene expression in syndromic inherited retinal diseases

Abstract

Bardet-Biedl syndrome (BBS) is a syndromic ciliopathy characterised by progressive retinal degeneration, obesity, polydactyly, renal dysfunction, and cognitive impairment. More than 26 genes have been implicated in the BBS disease pathogenesis, many of which encode components of the BBSome, or associated chaperonin complexes required for ciliary trafficking in photoreceptors. The genetic heterogeneity, large coding sequences, and systemic manifestations of BBS are the main significant challenges for developing retinal gene therapeutics. In addition to the limited packaging capacity of the adeno-associated virus (AAV) vectors and the need for photoreceptor-specific transgene expression. Synthetic promoters are increasingly recognised as critical tools to optimize transgene delivery for BBS gene therapy. Native endogenous promoters have several fundamental limitations in terms of their size, cis-elements distribution and mode of induction, these factors reflected insufficient transcriptional activity. Synthetic promoters, by contrast, can be engineered to achieve spaciotemporal transgene expression levels. These synthetic promoters are capable for driving the transgene’s expression specifically in the target cells with the required levels. Although there are different approaches have been tested to develop human-based synthetic promoters, in this study we utilized a new approach, by conjugating transcription factors binding sites (TFBS) which can enhance the expression of BBS cDNAs expressed from recombinant AAV vectors. Based on the simple-enrichment analysis results, the promoters of BBS genes sequences were significantly enriched with a group of transcription factor response elements (TFREs) such as RFX, SP transcription factors. We designed the sequences of the resulted TFREs into heterotypic clusters to create BBS gene specific synthetic promoters’ library. This library is composed of six different candidate promoter constructs (CPCs) cloned upstream minimal promoters YBTATA and minP. All CPCs activities were tested in HEK293Ts and ARPE-19 cells. The transcriptional activity of the CPCs was compared in ARPE-19 cells during ciliogenesis when BBS genes are active, 24-hr serum-starvation, and in serum-containing condition. During ciliogenesis, some CPCs containing proximal and distal regulatory binding sites showed increased transcriptional activity by driving the green fluorescent protein expression (GFP) in comparison to same CPCs activity in serum-containing media. Our synthetic promoters offer a versatile and powerful means of achieving optimal transgene expression for BBS gene therapy. By combining compact size, cell -type specificity, and potential inducibility, our synthetic promoters addressed critical barriers shown by natural promoters.