Periodic Reporting for period 1 - RETORNA (Targeting RNA as an approach for treating retinal disease)
Reporting period: 2023-02-01 to 2025-01-31
The present Doctoral Networks project “Targeting RNA as an approach for treating retinal disease” (RETORNA) aims to treat retinal diseases (RD) by using RNA therapies. Among the research objectives, we are studying different types of RNA that are expressed in retinal cells. By observing bioinformatic models, cell and tissue cultures, animal models, and human patients, we will be able to create RNA profiles for different RD. This screening will identify several targets that will be used as therapeutic approaches. Then, selected RNAs will be used to treat different RD, such as macular degeneration and retinitis pigmentosa. Furthermore, the potential economic and social impact of such strategies upon European countries is been studied, and the possibility of commercialization will be assessed.
The RETORNA network is composed of 8 beneficiaries from Spain, Italy, The Netherlands, Germany, Türkiye and Ireland and 9 associated partners subdivided in: 8 world class research laboratories, 1 private biotech company, 2 patient associations, 3 educational institutions and 3 consulting companies.
RETORNA proposes breakthrough research, distributed in 10 individual projects, performed by 10 selected Doctoral Candidates (DCs). Some of the most highly qualified European scientists on the field are supervising these DCs, who will be awarded with a PhD at the end of the program. RETORNA offers an innovative, international, multi/interdisciplinary platform to train young scientists in the field of vision science, enabling the DCs with research and transferable skills, necessary for starting successful careers in academia, industry and others, enhancing their career perspectives and employability.
The RETORNA network is composed of 8 beneficiaries from Spain, Italy, The Netherlands, Germany, Türkiye and Ireland and 9 associated partners subdivided in: 8 world class research laboratories, 1 private biotech company, 2 patient associations, 3 educational institutions and 3 consulting companies.
RETORNA proposes breakthrough research, distributed in 10 individual projects, performed by 10 selected Doctoral Candidates (DCs). Some of the most highly qualified European scientists on the field are supervising these DCs, who will be awarded with a PhD at the end of the program. RETORNA offers an innovative, international, multi/interdisciplinary platform to train young scientists in the field of vision science, enabling the DCs with research and transferable skills, necessary for starting successful careers in academia, industry and others, enhancing their career perspectives and employability.
The RETORNA team aims to understand, diagnose, and treat retinal diseases through a multidisciplinary approach combining molecular biology, stem cell technology, gene editing, computational modeling, and health economics. Key efforts focus on retinitis pigmentosa (RP), age-related macular degeneration (AMD) or diabetic retinopathy (DR), among other disorders, using in silico and in vitro models, retinal organoids, and animal models.
The consortium explores the roles of microRNAs, long non-coding RNAs, and extracellular vesicles in retinal disease mechanisms, while developing novel RNA-based therapies, including aptamers and circular RNAs targeting pathways like TrkB, PHD2, and AT1R-AngII. CRISPR and prime editing technologies are employed to correct mutations in inherited retinal disorders. Advanced computational tools, including AI-driven aptamer design and deep learning simulations, support therapeutic development. A comprehensive economic analysis underscores the relevance and potential impact of RNA therapies.
In the first periodic report, the consortium created diabetic retinopathy models using iPS cells, confirmed by analyzing the structure and key molecular markers. High glucose exposure led to inflammation, oxidative stress, and cell loss, mimicking patient conditions. The TrkB-aptamer was cloned into the ToRNAdo system for circular RNA expression, achieving high expression in hTERT-RPE1 cells and activating the Akt-pathway.
Using CRISPR/Cas9, homozygous PHD2 knockouts were generated in CRB1 patient-derived hiPSC lines and differentiated into retinal organoids. CRISPR-based tools were developed to correct the Prph2 c.584G>T mutation causing Central Areolar Choroidal Dystrophy (CACD). The effectiveness of CRISPR/Cas9 and prime editing was evaluated in mouse models and human cells. Retinal morphology and physiology analyses in mouse models identified neuropathological events similar to human CACD. mRNA and miRNA profiling revealed key deregulated genes in retinal degeneration. miRNA and lncRNA expression profiles were analyzed in retinitis pigmentosa models, expanding to other retinal models.
An oligonucleotide RNA ligand library was constructed, and an AI-driven algorithm was specifically developed to enable aptamer selection. To support this process, several custom codes and computational algorithms were developed for this aim. Consequently, promising RNA aptamers targeting the AT1R-AngII system were successfully designed and identified. In parallel, a deep learning based model was developed and integrated in physics-based molecular simulations to enhance efficiency and scalability.
Tissue samples from rd10 mice were collected for apoptosis studies and extracellular vesicle extraction, followed by miRNOME analysis. iPS cell lines from RP patients were used to generate organoids for comparative studies. The impact of plasma-derived extracellular vesicles on angiogenesis and epithelial/endothelial dysfunction was investigated. A protocol for isolating RPE cells from porcine eyes was developed, enhancing the understanding of vascular and epithelial barrier function.
These studies are complemented by a systematic literature review examining the economic costs of retinal diseases treatable with RNA therapies. Together, these efforts contribute to a deeper understanding of retinal pathologies and support the development of innovative RNA-based treatments.
The consortium explores the roles of microRNAs, long non-coding RNAs, and extracellular vesicles in retinal disease mechanisms, while developing novel RNA-based therapies, including aptamers and circular RNAs targeting pathways like TrkB, PHD2, and AT1R-AngII. CRISPR and prime editing technologies are employed to correct mutations in inherited retinal disorders. Advanced computational tools, including AI-driven aptamer design and deep learning simulations, support therapeutic development. A comprehensive economic analysis underscores the relevance and potential impact of RNA therapies.
In the first periodic report, the consortium created diabetic retinopathy models using iPS cells, confirmed by analyzing the structure and key molecular markers. High glucose exposure led to inflammation, oxidative stress, and cell loss, mimicking patient conditions. The TrkB-aptamer was cloned into the ToRNAdo system for circular RNA expression, achieving high expression in hTERT-RPE1 cells and activating the Akt-pathway.
Using CRISPR/Cas9, homozygous PHD2 knockouts were generated in CRB1 patient-derived hiPSC lines and differentiated into retinal organoids. CRISPR-based tools were developed to correct the Prph2 c.584G>T mutation causing Central Areolar Choroidal Dystrophy (CACD). The effectiveness of CRISPR/Cas9 and prime editing was evaluated in mouse models and human cells. Retinal morphology and physiology analyses in mouse models identified neuropathological events similar to human CACD. mRNA and miRNA profiling revealed key deregulated genes in retinal degeneration. miRNA and lncRNA expression profiles were analyzed in retinitis pigmentosa models, expanding to other retinal models.
An oligonucleotide RNA ligand library was constructed, and an AI-driven algorithm was specifically developed to enable aptamer selection. To support this process, several custom codes and computational algorithms were developed for this aim. Consequently, promising RNA aptamers targeting the AT1R-AngII system were successfully designed and identified. In parallel, a deep learning based model was developed and integrated in physics-based molecular simulations to enhance efficiency and scalability.
Tissue samples from rd10 mice were collected for apoptosis studies and extracellular vesicle extraction, followed by miRNOME analysis. iPS cell lines from RP patients were used to generate organoids for comparative studies. The impact of plasma-derived extracellular vesicles on angiogenesis and epithelial/endothelial dysfunction was investigated. A protocol for isolating RPE cells from porcine eyes was developed, enhancing the understanding of vascular and epithelial barrier function.
These studies are complemented by a systematic literature review examining the economic costs of retinal diseases treatable with RNA therapies. Together, these efforts contribute to a deeper understanding of retinal pathologies and support the development of innovative RNA-based treatments.
The Consortium aims to significantly impact retinal disease research through its 10 projects. Understanding the role of miR-205-5p and plasma-derived extracellular vesicles in retinal degeneration will provide insights into biomarkers and therapeutic targets, improving diagnosis and treatment. Disease models will serve as pre-clinical platforms for diabetic retinopathy mechanisms and therapy screening.
Advancing CACD pathophysiology by analyzing retinal changes in a novel Prph2 KI/WT mouse model will identify key neuropathological events and molecular pathways, uncovering biomarkers and therapeutic targets. RNA-based therapies evaluation will lay the foundation for novel treatment strategies for CACD and retinal degeneration, contributing to basic research and clinical applications.
Improving gene-editing techniques aims to correct the Prph2 c.584G>T mutation using CRISPR and prime editing, potentially treating inherited eye diseases. Testing these methods in mouse models and human cells will advance gene therapy for vision-related disorders, with success measured through sequencing and flow cytometry.
Developing computational tools will enhance molecular understanding of retinopathies and optimize treatments for retinal degeneration, improving efficiency and accuracy. Proof of concept of increased photoreceptor health after PHD2 gene knockout in CRB1 retinal organoids and Crb1 mutant mice will provide insights into glucose metabolism roles in photoreceptors.
Research on RNA-based TrkB-aptamers aims to develop treatments for retinal degeneration by activating the neuroprotective TrkB pathway and increasing cell survival. Generating non-coding RNA expression atlases using Next Generation Sequencing will provide a better understanding of molecular processes in retinal conditions, representing a valuable resource for researchers.
Identifying miRNA expression changes will uncover regulatory pathways in retinal degeneration, enhancing understanding of RP’s molecular mechanisms and supporting targeted therapeutic strategies. Continued research, demonstration of efficacy and safety, securing funding, partnerships, scalable production methods, intellectual property protection, international collaboration, and advocacy for supportive policies are essential for success.
Advancing CACD pathophysiology by analyzing retinal changes in a novel Prph2 KI/WT mouse model will identify key neuropathological events and molecular pathways, uncovering biomarkers and therapeutic targets. RNA-based therapies evaluation will lay the foundation for novel treatment strategies for CACD and retinal degeneration, contributing to basic research and clinical applications.
Improving gene-editing techniques aims to correct the Prph2 c.584G>T mutation using CRISPR and prime editing, potentially treating inherited eye diseases. Testing these methods in mouse models and human cells will advance gene therapy for vision-related disorders, with success measured through sequencing and flow cytometry.
Developing computational tools will enhance molecular understanding of retinopathies and optimize treatments for retinal degeneration, improving efficiency and accuracy. Proof of concept of increased photoreceptor health after PHD2 gene knockout in CRB1 retinal organoids and Crb1 mutant mice will provide insights into glucose metabolism roles in photoreceptors.
Research on RNA-based TrkB-aptamers aims to develop treatments for retinal degeneration by activating the neuroprotective TrkB pathway and increasing cell survival. Generating non-coding RNA expression atlases using Next Generation Sequencing will provide a better understanding of molecular processes in retinal conditions, representing a valuable resource for researchers.
Identifying miRNA expression changes will uncover regulatory pathways in retinal degeneration, enhancing understanding of RP’s molecular mechanisms and supporting targeted therapeutic strategies. Continued research, demonstration of efficacy and safety, securing funding, partnerships, scalable production methods, intellectual property protection, international collaboration, and advocacy for supportive policies are essential for success.