2025 Theses Doctoral

Precision Engineering of Retinal Therapeutics: miRNA Modulation and Prime Editing for Inherited Diseases

Lopes da Costa, Bruna

Inherited retinal diseases (IRDs) are a group of genetic disorders characterized by the progressive degeneration of the retina, leading to vision loss and eventual blindness. This vision loss severely impacts patients’ quality of life and imposes a significant societal burden. The genetic diversity of these diseases complicates the development of effective therapies, and the majority of IRDs currently have no viable treatments, underscoring the urgent need for novel therapeutic strategies. This thesis explores both gene/mutation-specific and gene-agnostic approaches to treat IRDs, aiming to provide new insights into disease mechanisms and therapeutic options.

In Chapter 2, a gene-agnostic approach was examined by engineering retinal metabolism through modulating miR-181a/b expression in retinitis pigmentosa. Our findings demonstrated distinct expression patterns of miR-181a/b during disease progression, with downregulation in retinal pigment epithelium (RPE) cells suggesting a protective response, while upregulation in the neural retina appeared to contribute to disease progression. We also observed compensatory regulation between miR-181a/b-1 and miR-181a/b-2 in RPE cells. Transient downregulation of miR-181a/b in RPE cells led to improved cell morphology, retarded photoreceptor degeneration, and reduced aerobic glycolysis in RPE cells. These results highlight the therapeutic potential of modulating miR-181a/b and underscore the significance of compensatory miRNA mechanisms in developing miRNA-based treatments for IRDs. Importantly, our findings support the notion that modulating aerobic glycolysis in RPE cells could help protect photoreceptors from degeneration in retinitis pigmentosa. By reducing glycolytic activity in RPE cells, we may enhance glucose availability for photoreceptors, presenting a promising gene-agnostic approach to treat IRDs.

Chapters 3 and 4 shifted focus to a gene/mutation-specific approach targeting 𝘗𝘙𝘗𝘏2 c.828 splice site mutations, which are associated with various IRDs. Using prime editing (PE) technology, we introduced three 𝘗𝘙𝘗𝘏2 splice site mutations (c.828+1G>A, c.828+2T>C, and c.828+3A>T) into human induced pluripotent stem cells (hiPSCs). These mutations activated a cryptic splice site and resulted in 29 base pairs of intron retention, producing a mutant transcript. Correcting the c.828+1G>A mutation in a homozygous knock-in hiPSC restored the canonical 𝘗𝘙𝘗𝘏2 transcript and reduced the amount of mutant transcript. Additionally, hiPSC-derived retinal organoids with the c.828+1G>A mutation showed expression of the mutant transcript and exhibited shorter photoreceptor outer segments. These results provide molecular and preliminary morphological evidence of 𝘗𝘙𝘗𝘏2-related disease and highlight the potential of PE technology for correcting𝘗𝘙𝘗𝘏2 c.828 splice site mutations in IRDs. This work sets the stage for further optimization of gene-editing techniques and the development of effective delivery systems, ultimately aiming to translate these findings into viable clinical therapies.

In summary, this thesis explores both gene-agnostic and gene/mutation-specific strategies for treating IRDs, with a particular emphasis on engineering retinal metabolism through miRNA modulation and precision genome editing with PE. The findings presented here hold the potential to significantly improve the lives of patients affected by IRDs, addressing a critical unmet clinical need and offering hope for transformative treatments in the future.