Abstract
Purpose :
Mutations in the Crumbs homologue 1 (CRB1) gene can cause inherited retinal dystrophies such as Retinitis Pigmentosa (RP) and Leber congenital amaurosis (LCA). It has been established that CRB1 patient derived retinal organoids have shown moderate disorganization of photoreceptor (PR) nuclei and dysregulated gene profiling of Müller glial cells and rod photoreceptors compared to their isogenic controls.
Here, we hypothesize that altering the glucose metabolism by ablating the prolyl hydroxyl domain 2 (PHD2) gene in CRB1 patient human induced pluripotent stem cells (hiPSCs) could inhibit PR death and rescue aberrant phenotypes observed in the patient organoids by increasing the rate of aerobic glycolysis at the expense of oxidative phosphorylation (Warburg effect). This pathway provides PR with more glucose and allows phospholipid synthesis essential for the sustenance and renewal of the PR outer segments.
Methods :
We are generating a homology directed repair (HDR) mediated homozygous PHD2 knockout (KO) in 3 independent CRB1 patient iPSC lines by CRISPR-Cas9 gene editing. The guide RNA (gRNA) targets exon 1 of PHD2 with an aim to disrupt the region ahead of the catalytic domain that is responsible for hydroxylation and degradation of Hypoxia-inducible factor 1-α (HIF1-α), the master oxygen regulator. A single-stranded oligodeoxynucleotide (ssODN) sequence serves as the template for the HDR, introducing a premature stop codon in the open reading frame of PHD2, rendering the protein non-functional. The homozygote KO’s will be verified by sequencing and cells will be analyzed for copy number variations and karyotype stability.
Results :
Preliminary screening of gRNAs show that exons coding for the catalytic domain of PHD2 are unfavorable targets for HDR mediated KO due to their limited size. Therefore, the gRNA targeting coding region of exon 1 is being used to obtain the KO iPS cell lines. Homozygous KO of PHD2 will be validated, following which the CRB1PHD2 cell lines and their corresponding isogenic controls will be differentiated into retinal organoids.
Conclusions :
The retinal organoids will be characterized by immunohistochemistry, scRNA sequencing, qPCR and metabolomics to analyze if the previously observed aberrant phenotypes in the PR are rescued. An improved PR structure is expected to be observed in these organoids due to the modified– glucose metabolism.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.