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ONGOING RESEARCH LINES (click on the title to know more)

Allele-specific invalidation of dominant-negative mutations in PRPH2 underlying CACD 

Central areolar choroidal dystrophy (CACD) is a progressive retinal disorder that mainly affects central vision, and usually starts in adulthood. CACD is caused by autosomal dominant mutations in PRPH2, a gene that encodes a tetraspanin protein present in the photoreceptor outer segments. Based on the hypothesis that (some) PRPH2 mutations act in a dominant-negative fashion, this project is aimed at an allele-specific inhibition of the expression of the mutant protein via antisense oligonucleotides (AONs) or CRISPR/Cas9-based genome editing. The efficacy of the strategies will be assessed in patient-derived cellular models.

 

Responsible PI: Rob Collin

Group members involved: Manon Peeters, Alex Garanto

Key collaborators: Anneke den Hollander, Carel Hoyng, Timo Mulders

Funded by: Donders Institute for Brain, Cognition and Behavior

Antisense oligonucleotide therapy for CEP290-associated LCA

The most frequent genetic cause of Leber congenital amaurosis (LCA) is an intronic mutation in CEP290 (c.2991+1655A>G), accounting for up to 15% of all LCA cases in many Western countries. This mutation is located in intron 26 of the CEP290 gene and creates a cryptic splice donor site that leads to the insertion of an aberrant exon into part of the CEP290 transcripts, and causes premature termination of the CEP290 protein. Antisense oligonucleotides (AONs) are small RNA molecules that are able to interfere with pre-mRNA splicing, and as such have a great therapeutic potential. Our initial work included transfection of AONs into immortalized cells of LCA patients homozygously carrying the intronic CEP290 mutation, in which we were able to show a complete rescue of the splice defect that is associated with this mutation, as well as restoration of protein levels and ciliation. Subsequently, ProQR Therapeutics has initiated a clinical trial, the interim results of which have recently been announced: the AONs appear to be safe and in some subjects led to an improvement of visual function. Current work includes expanding our knowledge on potential off-target effects, as well as measuring long-term efficacy of AON administration in mice.

 

Responsible PIs: Rob Collin and Alex Garanto

Group members involved: Lonneke Duijkers

Key collaborators: Budd Tucker

Funded by: Netherlands Organization for Scientific Research (NWO)

Antisense oligonucleotide therapy for ABCA4-associated Stargardt disease

Over the last few years, in studies supervised by our colleague Prof. Frans Cremers, we have identified numerous mutations in the ABCA4 gene that result in aberrant splicing of ABCA4 pre-mRNA. Bi-allelic ABCA4 mutations underlie Stargardt disease (STGD1), a progressive disorder characterized by central visual impairment that often leads to complete blindness. For ABCA4, there appears to be a strong correlation between the degree of aberrant splicing and the severity of the phenotype. Whereas some of the defects lead to splicing defects in many different cell types, we also identified ABCA4 mutations that appear to exert a retina-specific effect. We discovered that the majority of splice defects caused by the different ABCA4 mutations can be rescued by the administration of specific AONs, each of these having a different mode-of-action depending on the splicing defect. Current work includes the design and development of AON sequences for newly identified ABCA4 mutations, as well as further optimization of effective AONs using stem cell technology, with the incentive to translate effective therapeutic molecules to the clinic.

 

Responsible PIs: Rob Collin and Alex Garanto

Group members involved: Tomasz Tomkiewicz, Lonneke Duijkers, Nuria Suárez-Herrera, Dyah Karjosukarso, Femke Bukkems

Key collaborators: Frans Cremers, Mubeen Khan, Elfride de Baere, Carel Hoyng, Michael Cheetham, Susanne Roosing

Funded by: LSBS, Oogfonds, Stichting Bartiméus Sonneheerdt, Stichting Blindenhulp, Stichting Dowilvo via Uitzicht, Foundation Fighting Blindness USA (PPA and TRAP award), European Commission (StarT), RetinaUK

Delivery of antisense oligonucleotides to the retina

Antisense oligonucleotides (AONs) have shown promising therapeutic potential for inherited retinal diseases. However, little is known about their biodistribution in the eye. In this project, we study how the uptake and biodistribution of these molecules occur within the retina, and how these processes are influenced by the exact chemical composition of the AONs. With this, we aim to further optimize the delivery and increase the efficacy.

 

Responsible PI: Alex Garanto

Group members involved: Irene Vázquez-Domínguez, Lonneke Duijkers, Anita Hoogendoorn

Funded by: Foundation Fighting Blindness USA

Development of novel cellular models to assess therapeutic efficacy

Molecular therapies for inherited retinal diseases offer a wide spectrum of opportunities, ranging from conventional gene replacement to newly available genome editing tools. One of the limitations is that the expression of the genes associated with these diseases is often restricted to the retina. In addition, animal models do not often recapitulate the human phenotype. Therefore, it is challenging to conduct efficacy and toxicity tests for these therapeutic molecules. Within this project, we aim to generate cellular models that allow us to assess molecular and cellular dysfunction, as well as study the functional effect of therapeutic intervention.

 

Responsible PI: Alex Garanto

Group members involved: Edwin van Oosten; Anita Hoogendoorn

Key collaborators: Andries van der Meer; Nael Nadif Kasri; Dirk Lefeber; Rob Collin; Boehringer Ingelheim; Locsense

Funded by: Netherlands Organization for Scientific Research (NWO)

Expanding the use of antisense oligonucleotide therapy for other subtypes of IRDs

In this project, we aim to assess the use of antisense oligonucleotide-based strategies for several genes associated with recessive forms of IRD. One of the objectives is to use AONs for exon skipping for deep-intronic mutations identified in several different genes. The second strategy is to use this technology to delete exons that harbor severe (protein-truncating) mutations without disrupting the reading frame of the transcript to (partially) restore protein function. By employing antisense oligonucleotides, we will specifically take-out mutation-containing exons, and assess whether this leads to restoration of protein and cellular function using cellular models (organoids) or zebrafish.

 

Responsible PIs: Rob Collin and Alex Garanto

Group members involved: Lonneke Duijkers, Anita Hoogendoorn, Irene Vázquez-Domínguez

Key collaborators: Susanne Roosing

Funded by: Macula Fonds, Oogfonds, ANVVB and Stichting Blinden-Penning via Uitzicht, Rotterdamse Stichting Blindenbelangen, Stichting Blindenhulp, Stichting tot Verbetering van het Lot der Blinden, Stichting voor Ooglijders, Stichting Dowilvo, Curing Retinal Blindness Foundation and Foundation Fighting Blindness USA

Gene augmentation therapy for EYS- or PCARE-associated RP

Homozygous or compound heterozygous mutations in EYS or PCARE account for 5-10% and 1-2% of all cases with autosomal recessive retinitis pigmentosa (RP), respectively. This project focuses on understanding the role of the EYS and PCARE protein in the retina, as well as the design of gene augmentation strategies to treat these genetic subtypes of IRD. We have employed a variety of molecular and cell biological techniques, and mutant zebrafish models to gain insight into the physiological role of these two proteins. Current work includes the development and optimization of therapeutic vectors that aim to restore correct protein synthesis and cellular function.

 

Responsible PI: Rob Collin

Group members involved: Tess Afanasyeva, Lonneke Duijkers, Anita Hoogendoorn

Key collaborators: Julio Corral-Serrano, Ideke Lamers, Ronald Roepman, Michael Cheetham, Uwe Wolfrum, Marius Ueffing

Funded by: European Commission (EyeTN), EJPRD (GET-READY)

Therapeutic genome editing for ABCA4-­associated Stargardt disease

Stargardt disease (STGD1) is caused by bi-allelic mutations in the ABCA4 gene. Developments in the field of gene therapy have shown promising results for gene replacement therapies. However, the ABCA4 coding region largely exceeds the capacity of a conventional adeno-associated viral vectors. In the last lustrum, genome editing tools (e.g. CRISPR/Cas9) have become a powerful strategy to edit DNA. In this project, we aim to explore the potential of ABCA4 genome editing for the treatment of STGD1.

 

Responsible PI: Alex Garanto

Group members involved: Anita Hoogendoorn; Irene Vázquez-Domínguez

Key collaborators: Susanne Kohl, Pietro de Angeli

Funded by: Oogfonds, Macula Fonds, LSBS via Uitzicht, Rotterdamse Stichting Blindenbelangen, Stichting Blindenhulp.

Changing rare disorders of the lysine metabolism

This project is an international collaboration to use genetic and stem cell technology to develop new models and treatments for the neurometabolic diseases Glutaric aciduria type I (GA1) and Pyridoxine-dependent epilepsy (PDE).

Responsible PI: Clara van Karnebeek and Alex Garanto

Group members involved: Imke Schuurmans

Key collaborators: Nael Nadif Kasri, Antonia Ribes, Stefan Kölker, Blair Leavitt, Berta Zamora, Dirk Lefeber, Udo Engelke

Funded by: EJPRD (CHARLIE), UMD Catalyst

Treating Sugar Metabolism Diseases

In collaboration with Dirk Lefeber, we are modeling several enzymes involed in congenital disorders of glycosylation. In addition, using molecular therapeutic strategies we are aiming to correct these defects. 

Responsible PI: Dirk Lefeber and Alex Garanto

Group members involved: Sjanie Huang, Eline van de Ven, Daniëlle Swinkels

Key collaborators: Atze Bergsma, Federica Conte, Nael Nadif Kasri, Belén Pérez

Funded by: NWO, UMD Catalyst

Investigating the extracellular vesicle content as a novel marker for Usher syndrome 1b

Extracellular vesicles (EVs) play a critical role in several cellular processes with implications for disease pathogenesis. These vesicles exhibit dynamic changes in content depending on the status of the cells producing them; for example, under stress conditions, EV content adapts to either alleviate the imbalance and reduce cell death, or propagate the imbalance to neighboring cells and influence their fate. In the context of retinal diseases (RDs), where visual impairment results from retinal degeneration, the retinal pigment epithelium (RPE) is the major EV producer. However, the specific role of EVs in RDs remains unclear. We aim to compare EVs from tear fluid and RPE, focusing on a disease where RPE is affected due to mutations in the MYO7A gene, namely Usher syndrome 1b. The primary objective is to identify potential biomarkers and/or pathways associated with this disease.

Responsible Researcher: Irene Vázquez Domínguez

Group members involved: Rob Collin, Edwin van Oosten

Key collaborators: Sander Bervoets, Hedwig Velde, Ronald Pennings, Erwin van Wyk, Dunja Lukovic, and José María Millán

Funded by: Stitching Ushersyndroom and Ireland Usher Foundation via Uitzicht call 2021-22, Stitching Blindenhulp, Stichting tot Verbetering van het Lot der Blinden and Stiching Ooglijders.

Investigating the role of lipids in retinal diseases

Lipids are essential for vision. Yet, significant knowledge gaps remain regarding the function of different lipids in healthy and diseased retinas. This is pertinent given the link between an altered retinal lipid composition and rare causes of blindness like Sjögren-Larsson Syndrome and Stargardt disease, or the more common diabetic retinopathy and age-related macular degeneration. We are developing human-based models to further elucidate the role of lipids in disease and identify novel therapeutic targets. 

Responsible PI: Alex Garanto

Group members involved: Daniëlle Swinkels, Edwin van Oosten, Anita Hoogendoorn

Key collaborators: Michèl Willemsen, Udo Engelke, Fred Vaz

Funded by: LSBS through Uitzicht 2023-17, Stichting blindenhulp, Stichting Het Lot, Rotterdamse Stichting blindenbelangen

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