Human retinal organoids lacking MYCN

Abstract

Purpose : Models of retinal development can advance biological understanding and treatment of inherited and congenital retinal disease. MYCN is a critical factor in CNS development and has been implicated in several childhood and adult cancers. High intrinsic MYCN expression in human cones precursors (CPs) suggests that MYCN sensitizes human CPs to retinoblastoma initiation in response to RB1 loss. Here we generate MYCN knockout human iPSCs with and without RB1 knockout and produce retinal organoids (HROs) to study the role of MYCN in retinal development and retinoblastoma initiation.

Methods : RB1^+/+ or RB1^-/- derivatives of WTC11-based GNAT2-GFP cone reporter iPSCs harboring an EGFP-P2A cassette preceding the GNAT2 coding sequence (Bai et al., 2023), were electroporated with plasmids expressing SpCas9 and gRNA sequences targeting the second exon of MYCN. Transfected iPSCs were purified and single cell clones were isolated. Clones were sequenced to identify MYCN frameshift mutations and assess off-target mutations. HROs were generated from MYCN knockout iPSCs (MYCNKO) and unedited sister clones via previously published methods (Bai et al, 2023).

Results : Four iPSC clones from GNAT2-GFP /RB1^+/+ iPSCs and two clones from GNAT2-GFP/RB1^-/- iPSCs with biallelic small deletions predicted to induce translational frameshifts followed by premature stop codons were isolated. All frameshifts were identified distal to the transcription start site in exon 2 of MYCN. MYCN protein was detected in unedited but not in MYCN knockout iPSCs by western blot. Early MYCNKO HROs are viable and are in culture alongside MYCN unedited organoids.

Conclusions : Cone reporter MYCNKO iPSC clones have been produced in RB1^+/+ or RB1^-/- backgrounds and were competent to initiate human retinal development in vitro. Future studies will assess how MYCN loss affects generation of retinal cell types and retinoblastoma initiation.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.