Abstract
Purpose :
Genome editing represents a potentially powerful tool to treat inherited retinal disorders. However, DNA sequence alterations during the repair process after DNA double strand break (DSB) induction are manifold and uncontrolled so far. This represents a serious safety issue in the development of therapeutic strategies. The purpose of this study was to characterize the DNA sequence alterations following a DSB repair on the X chromosome in photoreceptors and RPE cells after AAV mediated gene transfer.
Methods :
Two month old mice ((B6J.SV129-Rpgrtm1stie) were used. This mouse line contains an Isce-I target site downstream of the RPGR-ORF15 gene on the X chromosome. A total of 4 different AAV-vectors were applied including AAV2/5 and AAV2/8 that contain the ISce-I endonuclease linked by a T2A linker with the GFP cDNA under the control of either a tissue specific Rhodopsin kinase (RK) or a CMV promotor. One AAV2/8.RK vector also contained a template DNA of the target region, in which the I-SceI site was replaced by the Hind III for screening purposes. Fifty-six days after subretinal injection, eyes were harvested, retinae isolated, and the GFP-positive cell population enriched by FACS. PCR fragments were subjected to T7 surveyor assay and subsequently Sanger sequenced to detect DNA sequence changes at the target site.
Results :
GFP expression was observed in all injected eyes.Specific PCR of the target region and subsequent surveyor assay revealed DNA-repair activity in all injected eyes and none in the control eyes. Following Sanger sequencing of PCR clones, we observed a high frequency of small insertions and deletions as well as single nucleotide substitutions (between 10 and 30% of all clones), most often located in the 5’ region of the target site. Larger DNA sequence modifications were also found, but to a much lesser extent. In retinae injected with an all-in-one vector containing the template in addition to the nuclease, we detected replacement of the I-SceI site by the HindIII site with low frequency, indicating the presence of HDR at the target site.
Conclusions :
We successfully induced in vivo genome editing in photoreceptors and RPE cells following AAV-mediated gene transfer. Both, NHEJ and HDR were detectable. These data represent the basis for further studies regarding the occurrence of DNA sequence changes at target sites in retinal neurons in vivo.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.