Abstract
Purpose: :
MicroRNA (miRNA) hairpins are short RNA molecules that regulate gene expression by repressing mRNA translation of hundreds of partially homologous mRNA transcripts. Although miRNA technology is mainly used in RNA interference (RNAi) applications, it is possible to use the target sequences that miRNAs recognise and bind to as a regulatory mechanism to restrict gene expression in cell types where the corresponding miRNAs are absent. We employed photoreceptor- and RPE- specific miRNA target sequences in an AAV2/8 vector expressing GFP as a proof of principle experiment to assess the efficacy of this technique in the murine retina in vivo.
Methods: :
Target sequences of miRNAs that are highly expressed in the photoreceptors but not in the RPE and visa versa were identified. We selected one photoreceptor-specific and one RPE-specific miRNA for our experiments. We established the corresponding target sequences of those miRNAs and inserted, separately at different vectors, each target sequence at the 3’UTR of an AAV-CMV-GFP vector plasmid. Each target sequence was inserted in four tandem repeats. High-titre AAV2/8 particles were produced for each of the RPE-targeting and photoreceptor-targeting vectors. Equal amounts of virus were subretinally injected in wild-type mice and GFP expression was assessed three weeks after delivery by histology.
Results: :
In the eyes injected with the AAV2/8 vector driving GFP expression from the cassette bearing the photoreceptor-specific miRNA target sequences, GFP expression was observed only in the RPE cells indicating potent downregulation of the transcript in photoreceptor cells. Similarly, the eyes that received the AAV2/8 vector driving GFP expression from the cassette with the RPE-specific miRNA target sequences, GFP expression was observed only in the photoreceptor cells.
Conclusions: :
Tissue-specific regulation of expression is essential for restricting expression of genes whose ectopic expression might have adverse effects in surrounding tissues. Tissue-specific promoters have been devised but expression is often "leaky" without providing tightly restricted expression in the tissue of interest. miRNA target sequences provide a stringent and robust mechanism of regulating expression in the retina and could be used in addition to tissue-specific promoters as an additional "safety regulatory element" or on their own to restrict expression in the photoreceptors or in the RPE.
Keywords: gene transfer/gene therapy • retina