RT info:eu-repo/semantics/doctoralThesis
T1 Caracterización clínica y análisis de exoma clínico, generación y edición genética de células madre pluripotentes inducidas (iPSC) de pacientes con distrofias hereditarias de la retina asociadas al gen PROM1
A1 Puertas Neyra, Kevin Louis
A2 Universidad de Valladolid. Escuela de Doctorado
K1 Oftalmología
K1 Retina
K1 Genetics
K1 Genética
K1 Células madre pluripotentes inducidas (iPSC)
K1 3201.09 Oftalmología
AB Introduction: Inherited retinal dystrophies (IRD) are one of the leading causes of irreversible blindness worldwide. They are caused by mutations in genes that trigger photoreceptor degeneration and progressive vision loss. IRD affect the quality of life of patients and their families, generating a significant social impact and a constant burden on healthcare resources due to their chronic and progressive nature, especially since the vast majority currently lack of curative treatments. Moreover, their huge phenotypic and genotypic heterogeneity hinders the diagnosis and understanding of their pathophysiology. IRD associated with the PROM1 gene are a representative example of this heterogeneity. In this context, Next Generation Sequencing (NGS) has become an essential tool for studying the molecular diversity of IRD. This technology allows the analysis of specific genomic regions and the sequencing of millions of DNA fragments. Specifically, Clinical Exome Sequencing (CES) analyzes the coding regions of genes relevant to the patient's pathology, increasing the chance of identifying modifier genes. On the other hand, induced pluripotent stem cells (iPSC) have revolutionized the study of IRD pathophysiology through the development of in vitro iPSC-based disease models, particularly when combined with CRISPR/Cas9 gene-editing technology. Additionally, from a therapeutic perspective, iPSC-derived cells serve as a foundation for the development of regenerative medicine approaches.Methods: A series of eight patients with PROM1-associated retinopathies was analyzed. Three patients with the same homozygous variant, c.1354dupT (p.Tyr452Leufs*13), were identified, each exhibiting a different autosomal recessive (AR) phenotype: Cone-Rod Dystrophy (CORD), Retinitis Pigmentosa (RP), and Stargardt Disease Type 4 (STGD4). These target patients, along with a healthy relative, underwent comprehensive ophthalmological examination and CES. Subsequently, iPSCs were generated from each target patient using episomal reprogramming and CRISPR/Cas9. These iPSCs were characterized according to international standards and genetically edited to correct the mutation. Restoration of PROM1 expression in the edited iPSC lines was confirmed by flow cytometry and Western blot (WB) analysis.Results: The target patients carried monoallelic variants in genes associated with the complement system, lipid metabolism, glaucoma, photoreceptor differentiation, and peroxisome biogenesis disorders. The following iPSC lines were generated: [CORD]-FiPSC1-Ep5F-2, [RP]-FiPSC1-Ep5F-10, and [STGD4]-FiPSC1-Ep5F-8, all of which met the required pluripotency and functionality characterization criteria. The edited iPSC lines: [CORD]-FiPSC1-Ep5F-2-GC1-23, [RP]-FiPSC1-Ep5F-10-GC1-18, and [STGD4]-FiPSC1-Ep5F-8-GC1-2 were generated and tested positive for CD133 in both flow cytometry and WB analyses.Conclusions: The homozygous PROM1 variant c.1354dupT (p.Tyr452Leufs*13) is associated with three distinct AR IRD phenotypes: CORD, RP, and STGD4. This pleiotropic effect may be influenced by monoallelic variants in modifier genes; however, further research is required to establish a causal relationship. The iPSC lines derived from the patients are pluripotent and functional; furthermore, the gene-editing machinery successfully restored the ability of PROM1 to encode CD133 in the edited iPSC lines. The generated iPSC lines can be used to model PROM1-associated retinopathies. In addition, after functional validation, the edited iPSC lines and the developed CRISPR/Cas9 system may serve as a foundation for the future development of gene and cell therapies for patients with PROM1-related retinopathies.
YR 2025
FD 2025
LK https://uvadoc.uva.es/handle/10324/82712
UL https://uvadoc.uva.es/handle/10324/82712
LA spa
NO Escuela de Doctorado
DS UVaDOC
RD 20-feb-2026