Base editing approach for in vitro and in vivo evaluation of therapeutic targets in USH2A

Abstract

Purpose : Mutations in the USH2A gene are major causes of Usher syndrome and retinitis pigmentosa. Gene therapy for USH2A is challenging due to its large, 15.6 kb coding sequence. The purpose of this study is to evaluate the efficiency of adenine and cytosine base editors for the reversion of pathogenic mutations in USH2A. The frequency of off-target score and bystander edits was evaluated in vitro and in vivo.

Methods : For the USH2A SNVs reported in the LoVD and HGMD databases, candidate guide RNAs were identified that placed the target mutations within positions 2-13. PAM variants used were NGG, NGA, NG, NNGRRT, and NNNRRT. HEK293T stable cell line were generated by integrating tandem arrays of USH2A mutations into the AAVS1 site to facilitate testing. Editing outcomes, including bystander edits, were determined using EditR and CRISPResso2 software. CRISPOR in silico prediction algorithm was used for off-target analysis. For one mutation, c.11864 A>G, p.W3955*, a humanized knock-in mouse was developed. Dual-AAV split-intein viruses carrying the base editor and gRNA were delivered into the subretinal space of W3955* mice

Results : 11% of mutations were theoretically targetable with either ABE7.10/8 or CBE3.9max using the PAM variants above, and 35 sites were selected for empiric testing. In vitro, A-base editors (ABEs) 7.10 and 8 showed A:T to G:C editing efficiencies up to 69.9% and 89.4%, respectively (N= 24 sites). C base editors (CBEs) could induce C:G to T:A edits with an efficiency ranging from 1.9-59.5% (N=11 sites tested). ABE8 showed higher efficiency with less indel creation and a broader editing window compared to CBE. Off-target rates for the editors were low, and most predicted sites were either intronic or intergenic. In vivo, subretinal injection of dual AAV9 ABE base editor into humanized USH2A mice achieved efficient correction in 32±4% of all retinal cells, determined by NGS.

Conclusions : High editing efficiency was observed in vitro and in vivo. A split-intein, AAV-based editor delivered in vivo into an USH2A mouse model provided evidence that an A-base editor is active and efficient in mammalian photoreceptors. These results support the strategy of using base editing as a promising therapeutic strategy for mutation reversion in USH2A patients and in other inherited retinal diseases caused by mutations in large genes.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Nucleotide conversion rates in whole mouse retina for c.11864 A>G site