ONGOING RESEARCH LINES
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
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
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, 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; Stijn Berendsen
Key collaborators: Andries van der Meer; Nael Nadif Kasri; Dirk Lefeber; 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)
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, Karlien Coene, Antonia Ribes, Stefan Kölker, Blair Leavitt
Funded by: EJPRD