A nematode, which is a tiny roundworm, is proving to be an impressive and quick test species for genetic defects in humans linked to deterioration of eye sight that can lead to blindness.
It has opened up a route to genetic repair and ultimately new treatments for inherited diseases of the retina (IRDs), according to Prof Olivier Blacque of UCD school of bimolecular and biomedical science.
The latest research in this field will feature at one of the biggest global gatherings of eye specialists taking place in 2024 next weekend. Hosted by Fighting Blindness, the Retina International World Congress takes place in Dublin, with a public engagement day on Saturday (June 8th) set to be the highlight for the sight-loss community.
The gathering will feature Irish research availing of new opportunities arising from precision genetics, notably work by Blacque and others under the Retinal Dystrophy in Ciliopathies (RDCilia) project, but will also hear progress on age-related macular degeneration (AMD), a common form of blindness, with significant progress on this being achieved by a team led by Prof Matt Campbell at TCD Smurfit institute of genetics.
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Ciliopathies are a group of disorders associated with genetic mutations encoding defective proteins, which result in either abnormal formation or function of cilia, components of almost all vertebrate cells. Their dysfunction can manifest in different ways including retinal degeneration (and ultimately blindness); renal disease and cerebral anomalies. They are minute hair-like structures extending from the cell body into the fluid surrounding the cell, in which they are adapted for moving the cells through surrounding fluid, such as for food uptake and for sensing the environment.
The nematode Caenorhabditis elegans has excellent advantages for disease research, Blacque says, as it has genes similar to human genes mutated in IRDs, like retinitis pigmentosa (RP). “These genes tend to function in similar ways in a worm versus a human, so we can really try and understand the function of the gene by turning to these simpler systems.”
They can manipulate the worm in any way they want “so we can make the same mutation that humans carry in the worm’s version of its gene. We make our little worm models of human disease mimicking the human patient’s mutations. We need to know if the gene is functional in a worm and does it work normally if it carries the patient mutations,” he adds.
They want to understand how mutations cause vision impairment. Hundreds of genes are linked to many types of vision disorder, which are characterised by levels of severity and clinical features. Different mutations in the same gene often produce a different disease outcome, and, in some cases, a wildly different type of vision impairment.
RDCilia is trying to understand why different mutations cause different forms of vision disorder. This is essential for informing future routes to therapy. It also addresses the problem of not knowing the genetic cause of vision impairment. “When a clinical geneticist examines genes related to sight in a patient with vision loss they are often uncertain if the mutations they find are actually causing the disease or not,” Blacque adds.
While mutations that delete a large chunk of a gene are likely to be disease-causing, many observed mutations are subtle, involving only a small change in the genetic code. These are variants of uncertain significance (VUS). “Unfortunately, such a diagnosis means that the patient cannot receive any available therapies for that gene or access gene-specific clinical trials. To overcome this hurdle, by studying these mutations in an animal model RDCilia hopes to provide data that allows some variants of uncertain significance to be reclassified as disease-causing or benign.”
Since it is difficult, if near impossible, to work with human eye samples, they recreate the human mutation scenario in C. elegans. “We can introduce mutations into the worm to see their effect. That helps us to learn how these mutations are likely affecting the human eye.”
Although only 1mm long and consisting of just under 1,000 cells, it shares much of the same basic biology as humans. With many experimental advantages such as a rapid life cycle, ease of genetic manipulation and low cost, the species is used widely to study disease mechanisms.
One particular advantage for RDCilia is how easy it is to introduce human mutations of interest into the equivalent worm gene. “Using a CRISPR-Cas9 technique, we can change a worm gene’s DNA to include a patient mutation in about three weeks. By way of comparison it would take 6-12 months to do the same experiment in a model such as a mouse.”
While worms don’t have eyes they have similar types of cells to those in the eye but which are used for smell instead. “As in humans the vision impairment genes in worms function in the same part of the cell, a tiny structure called a ‘cilium’. If you put these little worms on a surface and place them a distance away from something like almond essence they will crawl towards it if they smell correctly because they have normal cilia. If they don’t that’s how you know the human mutation that you have introduced into the worm is having an effect.”
They are doing drug screens where they treat worms containing human mutations with up to 10,000 different drugs or compounds to see if any can correct the smell defect. “If we get any hits we will then test the drug for vision correction in a higher animal model system, such as zebrafish. If that also works we are then closer to clinical trials in humans.”
They are looking for the magic bullet to correct a range of ciliopathies. Their IRD work includes looking at cone rod dystrophy and Bardet Biedl syndrome (BBS) where patients have multiple symptoms including loss of vision, primarily RP. “We are investigating why some patients develop full-blown BBS with all the symptoms of vision loss, kidney defects, bone abnormalities, etc, whereas other patients with different mutations in the very same gene get RP on its own.”
Age-related macular degeneration (AMD) progress
With AMD the greatest risk factor is age. “If we all lived to age 120 we would all likely get AMD. However, for some, AMD can start in the 60s and 70s,” says Campbell. “We believe that a major contributor to early-stage AMD are leaky blood vessels in the retina. We have found that in some people blood vessels of the retina are hyper-permeable, with the resulting leakage accelerating damage to the delicate neural tissue at the back of the eye that we see in AMD.”
Having the technology developed in the Science Foundation Ireland/Fighting Blindness-funded EYE-D programme has been ground-breaking as it is generating high-resolution images of the retina’s blood vessels.
They inject a fluorescent dye into a vein in the hand or arm to light up the blood vessels to enable them to take pictures of blood vessel leakage and to quantify it. Their focus is in the tissue of the retina. “Nobody has ever linked the inner retinal blood vessels with AMD ... We are looking at what is termed subclinical leakage, that is before an eye doctor can see it with standard imaging techniques.”
With sufficient patient data they will then be able to identify certain genes connected with the development of these leaky blood vessels. “We are looking for 10-15 molecular targets that we can then develop therapies to see if we can stop these vessels from leaking and causing disease. By identifying patients early, and keeping them at that early stage where people don’t really discern any sight loss, the intent is that they will never progress to intermediate or advanced AMD.”
They are also working with Iowa Eye Bank where they can access postmortem donor tissue. “While the clinical imaging techniques that we have are amazing – they can get down to really good resolution – we need to get to cellular-level resolution and see the shape of the cell, and we can only do this with real tissue,” Campbell says.
Fighting Blindness is organising a public engagement day on Saturday, June 8th, coinciding with the Retina International World Congress at the Dublin Royal Convention Centre. One of the biggest global gatherings of eye researchers this year, it offers a rare opportunity for people with a vision impairment to quiz eye specialists and to hear about the latest advances in treatments and cures. Bookings at fightingblindness.ie
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