Abstract
Purpose :
Mutations in Peripherin-2 (PRPH2) are one of the most common causes of inherited Retinal Degeneration. PRPH2 associated disease has autosomal dominant inheritance and so gene correction approaches are a potential treatment strategy. We investigate Adenine Base Editors (ABEs), enzymes which can substitute DNA base pairs without inducing DNA double strand breaks, associated with uncontrolled insertions and deletions, as a potential treatment approach.
Methods :
Human induced pluripotent cells (iPSCs) were generated from a patient heterozygous for a c.623G>A pathogenic variant in PRPH2. IPSCs were validated using immunostaining and were sequenced confirming the mutation. A SpRY-ABE base editor was chosen to correct the G>A base pair change and cloned into a pCMV-T7-ABE8e-n SpRY-P2A-EGFP plasmid. Guide RNAs (gRNA) were designed with the BE-FF web application and the design cloned into pSPgRNA plasmid using the BbsI restriction site. To validate base editing strategy, iPSCs were initially seeded as single cells onto a 24-well plate then lipofected with SpRY-ABE and sgRNA plasmids then expanded. Each colony was screened for editing at a target locus. DNA was collected and sequenced through PCR of PRPH2 exon 2. To understand overall efficiency, patient cells were seeded at 40,000 cells per well, then lipofected with SpRY-ABE and sgRNA plasmids. DNA was collected and target sequence expanded, as above, before being Sanger sequenced and efficiency assessed using EditR software.
Results :
IPSC immunostaining validated pluripotency marker expression. Sequencing confirmed heterozygosity for the c.623G>A PRPH2 mutation. From iPSC screening, two colonies were corrected confirming successful editing (Fig 1). Analysis of efficiency using bulk lipofected cells showed significant A to G editing (p=0.006) using a high concentration ABE and sgRNA (editing efficiency= 9.7%) compared with mock transfected cells (Fig. 2), but no editing at low concentration. Off-target analysis found no significant A to G base pair editing surrounding the target locus.
Conclusions :
We were able to show reasonable editing efficiency of iPSCs carrying the c.623G>A PRPH2 mutation. Further optimization is needed to see if efficiency can be improved. In addition, testing in photoreceptors generated using our organoid model will be needed to understand the actual target cell editing efficiency and likelihood of translational success.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.