Abstract
Purpose :
The concept of activating homologous genes originally not expressed in a particular retinal cell type to compensate for a disease-causing loss of function mutation evolved recently with the advent of innovative CRISPR/Cas9-based technologies. Here, we explore the feasibility of incorporating Cas9 fused to transcriptional activators into recombinant adeno-associated virus (rAAV) vectors for developing gene therapies based on ectopic “gene homologue” activation in photoreceptors.
Methods :
We used nuclease deficient Cas9 (dCas9) fused to the hybrid VP64-p65-Rta tripartite activator (VPR) for gene activation. Site-directed mutagenesis and standard cloning techniques were used to generate split versions of the dCas9-VPR fusion. Guide RNA (gRNA) sequences were synthesized and cloned together with dCas9-VPR into pcDNA3 and pAAV2.1 cis vector plasmids. HEK293 and 661W cells were transfected and subsequently harvested for Western blot, immunofluorescence and qRT-PCR analysis.
Results :
The limited packaging capacity of rAAVs does not allow incorporation of the large size Cas9-VPR sequence together with gRNAs into a single rAAV vector genome. To overcome this limitation, we applied the split-intein protein trans-splicing technology. In particular, the split-intein sequences were incorporated at amino acid (aa) position E573 (Truong et al., 2015) or at aa V713 (Chew et al., 2016) of dCas9 allowing for the separate expression of the N- and the C-terminal parts of dCas9. After co-transfection in HEK293 cells, we found a reconstitution efficiency of 56.9 % ± 2.1% (n=4) for the dCas9 variant split at V713 and a significantly lower efficiency of 33.3 % ± 2.1 % (n=4) for the version split at the E573 position. Based on this result, rAAV constructs with dCas9-VPR split at V713 were cloned and shown to result in full-length dCas9-VPR after co-delivery to HEK293 cells. Co-transfection of 661W cells with the two rAAV plasmids harboring the split-intein dCas9-VPR parts and specific gRNAs resulted in a 2475-fold increase in transcript levels. This result could be confirmed by immunocytochemistry on transfected 661W cells.
Conclusions :
This study shows the successful and efficient reconstitution of split-intein dCas9-VPR constructs encoded by rAAV vectors in retinal cells. These promising results pave the way for future rAAV-based dCas9-VPR gene therapy to treat inherited retinal diseases.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.