Description
Background:
Variants in the CRB1 gene cause severe inherited retinal dystrophies, including Leber congenital amaurosis, retinitis pigmentosa, cone-rod dystrophy and macular dystrophy. CRB1-retinopathies represent the 10th most common cause of early-onset vision loss in the UK, yet no clinically approved treatments currently exist. While AAV-mediated gene therapy has shown promise, limitations including restricted cargo capacity and immunogenicity have driven interest in non-viral delivery strategies. mRNA-based therapies represent a potential alternative, however efficient delivery and functional rescue in retinal systems remain largely unexplored.
Methods:
Day 34 human iPSC-derived CRB1 patient and control (WT) retinal organoids were transfected with lipid-peptide nanocomplex with Peptide Y or ME27 peptide at 0.1µg (0.1) or 0.2µg (0.2) EGFP mRNA. Transfected organoids were imaged at day 35 and EGFP-positive cells were manually quantified.
Results:
In both WT and CRB1 organoids, the nanocomplex incorporating the ME27 peptide with 0.1µg EGFP mRNA (ME27-0.1) was the most efficient condition overall (p<0.01), yielding 31.4 ± 5.12 and 25.3 ± 3.26 EGFP-positive cells in WT and CRB1 organoids, respectively. This was higher than Peptide Y with 0.1µg EGFP mRNA (Peptide Y-0.1), which yielded 13.8 ± 1.59 and 12.3 ± 2.00 cells. In contrast, at 0.2µg mRNA, no significant difference was observed between peptides in either model. In WT organoids, ME27 yielded 18.6 ± 2.86 EGFP-positive cells compared with 10.4 ± 1.17 for Peptide Y (p=0.547), while in CRB1 organoids, ME27 yielded 19.0 ± 3.48 cells compared with 8.43 ± 1.85 (p=0.632).
Conclusion:
These findings demonstrate that lipid-peptide nanocomplexes can successfully transfect human iPSC-derived retinal organoid models. Further studies will investigate CRB1 mRNA delivery to evaluate whether nanocomplex-mediated therapy can rescue the CRB1-associated phenotype in a retinal organoid model.
Lay Abstract
The retina is a light-sensitive tissue at the back of the eye. The CRB1 gene provides instructions that help cells in the retina stick together and maintain the correct layered structure. When CRB1 is faulty, retinal organisation is disrupted, leading to severe sight loss. Faulty CRB1 genes are the 10th most common cause of vision loss in the UK, yet there are currently no treatments. Current research explores treatments that use modified viruses to deliver healthy genetic material into the retina, however this approach may trigger an immune reaction. To reduce this risk, our lab uses modified ‘fat bubbles’, called nanocomplexes, to deliver genetic material.
In this study, we test whether nanocomplexes can successfully deliver genetic material into cells in lab-grown retinas. Successful delivery is confirmed by the production of green fluorescent proteins in the cells. Our results show that this approach can effectively deliver genetic material into lab-grown retinas.
Future work will focus on delivering healthy CRB1 genetic material to the diseased lab-grown retinas to determine whether normal retinal structure can be restored. Ultimately, this research could pave the way for a new treatment, offering hope to the many patients living with inherited sight loss.
| Lay Title | A New Treatment Approach for CRB1-Related Sight loss. |
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| Role | Master Student |