Description
Heterozygous changes within a mutation hotspot in RP1 are associated with autosomal dominant retinitis pigmentosa (adRP). Homozygous or compound heterozygous variations outside of this region are associated with autosomal-recessive retinitis pigmentosa (arRP). We have developed hiPSC-derived retinal organoid (RO) models of adRP-RP1 harbouring a common variant (p.Gln686*, KI) and an RP1 knock out (KO) model of arRP-RP1 to allow for disease modelling.
CRISPR-Cas9 editing in control hiPSCs was used to generate both models. Homology-directed repair (HDR) was used to knock in the heterozygous adRP associated RP1 variation c.2056C>T p.Gln686 and non-homologous end joining (NHEJ) used to generate the RP1 KO, a homozygous stop-gained frameshift deletion in exon 2 (c.85-88del, p.Thr30Ilefs49) predicted to lead to nonsense-mediated decay. hiPSC lines (WT, KI and KO) were differentiated into ROs using an established protocol. One way ANOVA was used for statistical analysis.
All iPSC cell lines were shown to be pluripotent, able to undergo trilineage differentiation and genomically stable. Both RO models showed shorter photoreceptor outer segments when compared to the isogenic control. The outer segments of the KI iPSC-ROs were significantly shorter at three time points than those of the control ROs. This was also seen in IHC staining of rhodopsin in each line.
Our results show that both the KI and KO organoids show differences to the isogenic control. The phenotype of the KO retinal organoids appears more severe than that of the KI organoids, which aligns with the less severe phenotype of patients with RP1-associated adRP. This is the first time these diseases have been modelled in human retinal organoids and show that these can be a valuable tool to study this disease further.
Lay Abstract
Mutations in the gene retinitis pigmentosa 1 (RP1) cause retinitis pigmentosa (RP). This can either be a dominant or recessive. Mutations within a certain region of the gene cause dominant RP and mutations outside of this cause recessive RP. We have modelled both forms of the disease using retinal organoids (ROs), to look for differences.
The RP1 gene was edited using CRISPR-Cas9. Using this we engineered ROs containing a common mutation associated with dominant RP (c.2056C>T p.Gln686) called a knock-in (KI), and to engineer a mutation linked to a recessive mutation (c.85-88del, p.Thr30Ilefs49), called a knock-out (KO).
These two models were shown to be stable. Both showed shorted photoreceptors when compared to the unmodified ROs at multiple time points. This was more pronounced in the recessive disease model.
Our results show that both the KI and KO organoids show differences compared to the unmodified. The KO organoids appear more severely affected than that of the KI organoids, which is the same as in patients. This is the first time these diseases have been modelled in ROs and show that these can be a valuable tool. These models will allow for more studies examining disease mechanism and to test possible treatments.
| Lay Title | Modelling retinitis pigmentosa associated with RP1 |
|---|---|
| Role | PhD Student |