Abstract
Purpose :
Oculocutaneous albinism type 2 (OCA2) is a rare genetic condition due to recessive mutations in
OCA2 gene and results in pigmentation defects of the skin, hair, and eyes. We have previously
shown that induced pluripotent stem cells (iPSCs) derived from OCA2 patients successfully
recapitulate pigmentation defects when differentiated in vitro into retinal pigment epithelium
(RPE). We used CRISPR-Cas9-based homology-dependent repair of a mutant allele in
iPSCs derived from an OCA2 patient. The purpose of
our study was to create an isogenic pair of iPSCs for studying OCA2 related pigmentation
defects in the human RPE.
Methods :
All human iPSC work was approved by the NIH Institutional Review Board, protocol # 11-E1-0245 (NCT01432847). iPSCs were derived from OCA2 patients with compound heterozygous mutations. Two sgRNAs were designed, targeting chromosome 15 (NG_009846.1). The donor template used for HDR replaced the mutation c.1211C>T (NM_000275.3) in exon 12. The OCA2_iPSC colonies were transfected using Lipofectamine ™ followed by single-cell cloning in the presence of Puromycin to positively select GFP+ colonies. Selected iPSC clones were transfected with a Cre recombinase-expressing plasmid followed by treatment with Geneticin to select for GFP-negative colonies resulting from recombination and excision of the cassette containing GFP and neomycin. The GFP-negative iPSC colonies were then expanded and analyzed using flow-cytometry to confirm the complete lack of GFP expression. Sanger sequencing was performed on GFP-negative iPSC colonies to confirm correction of the mutation (OCA2-IC_iPSC).
Results :
We successfully replaced the c.1211C>T mutation in exon 12 of OCA2 with the wild type sequence as confirmed by Sanger sequencing. The isogenic iPSC pair exhibited normal human karyotype and expression of pluripotency markers like NANOG, OCT4, SOX-2, SSEA4, TRA-1-60, and TRA-1-81. The OCA2-IC_iPSC when differentiated towards RPE exhibited normal pigmentation whereas the OCA2_iPSC derived RPE exhibited partial pigmentation.
Conclusions :
Our work establishes proof of principle for CRISPR-mediated gene editing as a strategy for pigment rescue in OCA2. Furthermore, we introduce OCA2 patients and corrected isogenic cell lines as valuable tools for in vitro disease investigation and drug discovery.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.