Abstract
Purpose :
The P23H mutation in the rhodopsin (RHO) gene represents the most common form
of adRP in North Americans. At birth the TgP23H hRHO pig model of adRP retains many rods
and corresponds to mid-stage human adRP disease. Despite an almost normal number of rods,
untreated TgP23H hRHO pigs never have a rod-isolated ffERG response. We previously
reported successful gene editing using the Rho1-2 meganuclease in this model of adRP, when
Rho 1-2 was packaged in AAV5 and delivered sub-retinally. Efficacy was demonstrated in
reduced rod degeneration and improved rod isolated retinal function (sdOCT, ffERG and IHC).
The purpose here is to evaluate the nature and frequency of the mutations introduced by the
meganuclease Rho1-2.
Methods :
DNA samples from TgP23HhRHO pig retinas, either untreated or treated with Rho1-
2, were examined for induced mutations at the P23H locus. DNA was isolated from tissue
samples from the region encompassing the bleb area created by the subretinal injection. A 228
bp DNA fragment centered on the Rho1-2 recognition sequence was amplified, barcoded for
multiplexing and sequenced on an Illumina MiSeq. Paired-end sequence reads from each
sample were trimmed using Trimmomatic (v. 0.38.1) and joined using fastq-join (v. 1.1.2-806.1)
on usegalaxy.org, then clustered using CD-HIT (v. 4.8.1) to create clusters with 100% sequence
identity. Data were then filtered to retain only clusters with at least two sequences (97.8% of
sequences retained).
Results :
We obtained 778,777 sequence read pairs from 14 samples. In samples from
untreated retinas (n = 4, reads = 331,043) we found no INDELs at the meganuclease target site,
as expected. For Rho1-2 treated retinas (n = 10, reads = 447,734) we found a wide variety of
INDELs, which represented ~2.6% of the reads.
Conclusions :
Our data show that Rho1-2 meganuclease generates a range of INDELs, which
are predicted to have inactivating mutations that truncate or create deletions in the P23H
protein. Given rods represent ~ 2% of the cell bodies in the retina at the examined ages, and
Rho1-2 is only expressed in rods, this suggests that most of the remaining rods carry INDELs in
the P23H transgene, resulting in their long-term survival.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.