S3.14 The eyes have it: From genetics to gene therapy
Kat: Hello, and welcome to Genetics Unzipped - the Genetics Society podcast with me, Dr Kat Arney. In this episode, supported by the Medical Research Council, we discover how researchers are letting the light shine in, literally, by bringing discoveries about the underlying genetic faults that cause eye diseases all the way through to game-changing clinical trials of gene therapy designed to save sight.
Kat: Before we get started, a quick heads up about a brand new series brought to you by the Genetics Society, Genetics Shambles, a series of live discussions, interviews and podcasts presented by Robin Ince of Cosmic Shambles fame, in association with The Milner Centre for Evolution at the University of Bath.
Available fortnightly from Wednesday, 1st July, there will be 12 episodes featuring in-depth discussions with some of the leading lights of genetics covering topics such as:
Why do diseases affect some people more than others?
Where are we on the development of drugs and vaccines that will help to fight the coronavirus pandemic and other viruses?
What have we learnt from the human genome and how has it helped us to understand inherited traits and improve our medicines?
And what does the future hold for genetic research in both a scientific, and ethical, sense?
Hopefully we’ll be running audio versions of the episodes on the Genetics Unzipped feed, so make sure you’re subscribed so you don’t miss out. Head over the the How To Listen page on GeneticsUnzipped.com or just search for Genetics Unzipped in your podcast app of choice, and check out http://cosmicshambles.com/ for more great science stuff from the Shambles team..
Kat: These ‘unprecedented times’, as I believe we must call them, have put a big dent into everyone’s plans, including teams who had spent months planning a summer full of great events to inspire and inform the public about science.
This month we should have been hopping on the train up to Edinburgh to interview researchers and patients focusing on genetic eye diseases, as part of a fantastic public engagement event organised by the MRC Human Genetics Unit (@MRCHGU) in the Institute of Genetics and Molecular Medicine at the University of Edinburgh (@MRC_IGMM).
But that’s not happening. So, instead we thought we’d bring a little bit of it directly to you. Our stay-at-home roving reporter Georgia Mills has been taking a closer look…
Ken: The last book I read was in 1994. I was sitting with my back to the setting sun in midsummer, and I was able to start reading a novel in June 1994. I finally finished that book in December 1994 and it took me that long. And that was the last paper book I ever tried reading.
Georgia: This is Ken Reid. He’s 60 years old, but when he was 26 was diagnosed with a degenerative eye condition called retinitis pigmentosa.
Ken: When people lose their sight through trauma, that's instant and everything happens and all the changes have to be made in one step. When you lose your sight through a degenerative condition then you are changing constantly and having to cope with different experiences.
I guess it's something that we're all kind of used to just now when we keep talking about "the new normal". In my world, we keep having new normals happening. As something that I had taken for granted, something that I'd adjusted to and developed a new way of coping, then I find that I can't do that anymore and I've got to change again.
Georgia: The idea of losing your vision can be terrifying, in fact in surveys people consistently rank it as their most valued sense, but the reality is that many of us will have to face deteriorating eyesight thanks to genetic eye diseases.
Later we’ll come back to Ken, and find out why he’s given an unusual donation to help research into RP, and we’ll explore the cutting edge of how gene therapy is being used to treat it, and also find out how to grow an eye in a dish! But first, let’s get the lowdown on genetic eye diseases.
Chloe: Genetic causes of eye disease are a huge problem.
Georgia: Chloe Stanton is a scientist in the MRC Human Genetics Unit at the IGMM in Edinburgh, working on genetic causes of eye disease
Chloe: In the UK alone, there are probably around 2 million people affected by visual loss as a result of genetic causes. Of these people, there are probably more than 350 inherited eye diseases affecting them. Of those inherited eye diseases there are probably more than 500 genes causing them.
Some of these conditions are very common and many of us will know people who are directly affected by them. For example, when I was working on age related Macular Degeneration, I spoke to my grandmother regularly. Every single time I spoke to her she would tell me about her friend who lived up the road whose eyes were affected by age related Macular Degeneration.
Every time my gran spoke to me she was very keen that I should hurry up my research and find a cure for Betty who lived up the road.
Georgia: And why is this such a big problem?
Chloe: The eye is obviously very sensitive to impactful changes in how it works. It's a very delicately structured organ. It has a very precise visual cascade that needs to happen exactly in the right way in order to transmit the signal that your eyes are getting. The light hitting the photosensitive cells at the back of the eye and the retina and transmitting that to the brain. You can see that it would easily go wrong and that could be catastrophic for the individual.
Georgia: Macular Degeneration is the most prevalent cause of eyesight loss. The macula is the 'sweet spot' of the retina - that’s the layer of light-sensitive cells at the back of the eye - and it provides our most accurate colour vision.
When the macula breaks down, or degenerates, this central component of vision starts to get blurry, making it difficult to pick out fine details like faces or text. While Macular Degeneration can have a massive impact on your life, it is usually age-related, so it doesn't present itself until around 50 to 60 years old. But some genetic eye conditions strike much, much younger and RP is one of those.
Chloe: Retinitis Pigmentosa affects the retina, which is the photosensitive layer at the back of the eye. The specific cell types that are affected are the photoreceptors, primarily rods in the first instance and later on in the development of the disease, the cone cells.
So, rod cells are the ones that are responsible for vision in dim light. That is why night blindness appears to be one of the earliest stages of the disease.
Georgia: Retinitis Pigmentosa is an umbrella term for a group of conditions all affecting the retina in this way. And there are a lot of genes that can be involved.
Chloe: More than 100 genes have been found to have mutations in Retinitis Pigmentosa patients. So although each gene mutation might be very rare, the number of people affected by it quite rapidly increases. In fact, about 1 in 3,000 to 4,000 individuals would be affected by RP.
Georgia: Because there are so many genes involved, this means that RP can be inherited in several different ways.
Chloe: It can be an autosomal dominant condition in which you only need to inherit one faulty gene from one of your parents or in an autosomal recessive way in which you need to inherit a faulty copy of the gene from both of your parents.
Or it can be an X-linked condition, in which case the faulty gene is carried on the X chromosome. In this case, the disorder affects sons more severely than daughters. This is the case for people who are affected by this specific subset of RP caused by the RPGR gene.
Georgia: But since the early 90s, huge advances have been made in identifying some of the genes at play, which has made a big difference to the community.
Chloe: It's so valuable to them to know what is causing their condition. It lets them plan, it lets them make diagnosis so much easier for the next generation. But we have to bear in mind that RP is caused by over 100 genes. There are about 3,000 known mutations for RP, so we still don't know all the genetic causes of the disease. We're developing more knowledge all the time about this.
It's even more complicated than that because different mutations have different levels of severity, different rates of progression.
Georgia: But even with a diagnosis, there’s still not much we can actually do when someone finds out that they have this.
Chloe: That's the awful thing about it. At the minute, there aren't really any ways of curing the disease at the moment.
Georgia: Chloe Stanton from the MRC Human Genetics Unit in Edinburgh. So let’s go back to Ken. He was diagnosed with RP at just 26 years old - but even then, it wasn’t completely unexpected.
Ken: I can now recognise events and incidents going way right back, certainly to when I was a teenager, possibly even earlier. One of the early ones I remember was I always had very poor night sight and couldn't understand how people could do things in the dark.
Going to discos was an absolute -- most people probably don't remember what discos in the 70s were like, but they were just dark. You had this lovely interaction where it was very noisy, it was very dark and there were some flashing lights. I could see nothing and trying to find somebody to dance with was a real torment. I didn't know how people managed it.
So, thinking back through that kind of escapade, I realised that my RP was obviously affecting my sight even at that stage. I remember playing Trivial Pursuit not long after it was first launched and I just couldn't tell the difference between orange and pink. So it wasn't colour blindness, it was colour differentiation.
These things just began to accumulate. An eye test at my work said, "Your eyes don't seem to be doing so well. You probably ought to go and see your optometrist." So I did. They mentioned this condition RP, have you ever heard of it? I had never heard of it. There was no history of it in my family. I was referred from my optometrist to my GP, from my GP to the eye hospital. So I went to the eye hospital and they confirmed the diagnosis and said, "Thanks very much, goodbye."
Georgia: What was that like for you, that moment when you were given this name of what was going on?
Ken: In a way it was two-pronged, really. It was quite a shock to be told that I had this condition I'd never heard of. I couldn't pronounce it. I think when I went home I said, "I've got Retinita Pigmentosis or something like that. There was no internet to go and look it up on back then.
I was told, "You've got this condition, there's nothing we can do. Thanks very much for coming in, goodbye." And I was left on my own.
I was totally bemused on one side. On the other side, because I had all these experiences of my early life where I hadn't been able to see things like other people could, it suddenly made everything make sense.
That was quite a relief, so I wasn't in the total devastation state that I know some people get into with that kind of diagnosis because it really made sense of an awful lot that had been difficult. Then instead of having to try and cope doing things that I really wasn't comfortable doing; I was able to stop.
Many people find this rather odd but I stopped driving that day. I had driven the day before and I actually nearly killed somebody when I was driving the day before my diagnosis. Now I knew why and I knew that I had had a lucky escape. And I knew that I never had to do that again and I haven't. I've never driven since.
It sometimes frustrating, especially right now when public transport is so difficult to use, but basically I have been very comfortable using public transport. I'm happy to just hop on a train or a bus to wherever I need to get to.
Georgia: Ken Reed. So, how are scientists trying to find a potential cure for people like Ken who are suffering from this disease? Well, given that it's genetic, there's one type of intervention that seems particularly appropriate.
Robin: Gene therapy is the introduction of genetic material into the human body to treat disease. It's the most rational way of treating a genetic disorder.
Georgia: This is Professor Robin Ali. He is Director for the Centre of Cell and Gene Therapy at King’s College London.
Robin: In the case of RP, this condition can be caused by defects in over 100 different genes. The rational approach for treating the disease is to introduce a functioning copy of the abnormal gene. This usually involves the engineering of a virus, so as to remove the genetic material of the virus, the virus's own genes, and replace that with a therapeutic gene.
The engineered virus which is no longer able to replicate is then injected into the target organ, in this case into the eye. The virus then infects the target cell, in our case cells in the retina, and introduces the therapeutic gene to those cells that are lacking a functioning copy of that gene.
Georgia: I'm sure I'm not the only one who winces at the idea of an eye injection. What kind of unique challenges are there because this is in the eye?
Robin: The challenge with the gene therapy in the eye is that the eye is a very sensitive organ, the retina is very sensitive tissue. So it requires very precise surgery delivery of the engineered virus.
But actually the eye has more advantages than disadvantages with regard to being a target organ because it's small and it's relatively immune-privileged. We have less of an immune response compared with many other organs.
And one is able to visualise gene delivery - so, delivery of the suspension viral vector - through indirect thermoscopy and so we can localise the therapeutic drug very precisely. So we see that the eye is actually very useful, is a very amenable target organ. That's why so many advances are being made with regard to gene delivery in the eye.
Georgia: Tell me about these advances. Where are we in getting to something that's available?
Robin: To date, there's currently over two dozen gene therapy clinical trials for a range of conditions that affect the retina. In fact, there are many companies now that are involved in developing the technology and taking this right the way through to licensed products.
Just a year ago, one of the first ever gene therapies to reach the market first in the US at least, was in fact a gene therapy for an inherited retinal dystrophy called Leber Congenital Amaurosis, which was a form of early onset, childhood onset retinal dystrophy. That was the very first clinical trial of a gene therapy for RP which we started back in 2007.
Several groups have worked on this because it was regarded by many investigators at the time as being one of the most amenable conditions for gene therapy. We and others demonstrated that it's possible to use gene therapy to restore vision in patients with this particular defect.
One group has gone on to commercialise this and it is now available as a licensed gene therapy product. It is available in the US and also the UK, it was approved by NICE. So that's a very exciting development for the field because we've seen the progression from models through to clinical trials through to licensed products.
That's put a real boost, not only for popular gene therapy but in fact for the whole gene therapy field as we see it entering mainstream medicine.
Georgia: Robin Ali from King’s College London. So gene therapy is looking extremely promising, with treatments actually starting to make it through to the clinic and more coming through the pipeline. But this isn’t the only way that researchers are going about looking for new ways of restoring or saving sight.
Ken Reid, who we heard from earlier, was approached by a scientist named Roly Megaw, a clinical lecturer at the MRC Human Genetics Unit in Edinburgh, with a rather ominous-sounding proposition.
Ken: He asked for volunteers who were willing to give up a pound of flesh to enable the research to progress. I thought, that's the least I can give, I've given a lot more along the way. I said, "Yes, I'm up for that."
Georgia: OK, before you start worrying - it wasn’t an actual pound of flesh! It’s a procedure called a punch biopsy, where a small sample of living skin is taken from a volunteer - but because the top layers of your skin are actually dead, the sample has to go quite deep. It’s not the most comfortable thing in the world, but as Ken explains, he felt it was worth it.
Ken: I went along to Roly's clinic in the Edinburgh Eye Pavilion. The message had said he just needed some skin. I thought, "That's alright, just a wee scrape off my arm or something, nothing too much." I thought it would be just superficial skin.
But he said, "Okay, I'll need to give you a local anaesthetic." I said, "It's okay, you're just going to take a bit of skin." He said, "No, I'm not just taking a bit of skin." And it went quite deep and there was a fair bit of blood as well.
He took it off the inside of my forearm. There is still a scar there, it was that deep and that significant. I will carry a mark of it probably forever. He cauterized it and put a plaster on it and said, "That's fine". It just sounded like a really brilliant bit of research going on. It made a huge amount of logical sense to me and so I was all for it.
It's still only research and it's got a long way to go before it becomes treatment but as a potential treatment, I think it sounds really optimistic. So yes, I was more than happy to let him get his scalpel into my arm.
Georgia: So why does someone trying to treat an eye condition need someone’s skin? Well, it's all about trying to understand exactly how RP actually works, the molecular nuts and bolts. I'll let Roly himself explain.
Roly: I work on a particular gene known as the Retinitis Pigmentosa GTPase Regulator gene or RPGR for short. Much like all the genes that result in RP, not much is known about the function of RPGR.
If we are to develop a treatment for RPGR related RP, we need to understand what the protein does. So my research is focused on understanding its function within the photoreceptor cell.
Georgia: Right, and how are you doing that? RPGR, is it quite hard not to say it like a pirate every time?
Roly: Absolutely. One method I've used is using induced pluripotent stem cells to try to model the disease. So we are able, due to work that began 50 years ago by John Gurdon in Cambridge which was continued by Professor Ian Wilmut up in Edinburgh, who managed to clone Dolly the Sheep, right through to a Japanese scientist called Shinya Yamanaka.
Through that 50-year period of research we now are able to reprogramme adult cells to become stem cells. Therefore patients and their unaffected relatives are able to give us their tissue, be it skin or blood, and we are able to reprogramme their adult cells into stem cells.
The great thing about stem cells is that they are multipotent. When I say that, what I mean is that they have the potential to differentiate into any cell type in the body. So I was able to recruit patients with RPGR mutations from NHS Lothian. They very kindly gave me their pound of flesh. They gave me a skin biopsy which I was able to reprogramme into stem cells.
I was then able to use differentiation protocols that had been developed by different labs around the world. I've been able to grow little mini eyes in a dish, some of which have RPGR mutations - the ones that the patients have given me - and some of which do not have RPGR mutations.
Georgia: So now you’ve got these mini-eyes in a dish, how are you investigating them?
Roly: Initially I looked at these mini eyes with a powerful confocal microscope and saw that there was too much actin in them when RPGR is mutated. Actin is the main constituent of the cell skeleton.
I then carried out protein screens and slightly more focused experiments to get a better understanding of exactly which other proteins RPGR interacts with, and therefore how it controls this cytoskeleton. What I want to understand is what other proteins are RPGRs interacting with in order to carry out its function.
So I carried out a screen of 650 proteins to see whether any of them were dysregulated in the cells that were derived from the RPGR mutant stem cells, and I was able to identify a couple of proteins that were dysregulated.
That led me down a path where I further probed that and I was able to find out a little bit more about RPGRs function. So I think it's regulating this skeleton within the cell and the function of that skeleton regulation is as yet undefined, but my work continues.
What I've now done is I've moved into an animal model because as valuable as those stem cells are, the little eye that you form is still very much a primitive eye and RP is a generative condition, it often takes several years to manifest.
So I've now developed mouse models of human mutations which begin to lose their sight as they get older. I'm using a fully formed eye now, to try to better understand exactly what role the RPGR is playing.
Georgia: Amazing. And what do these little eyes look like? Are they eyes staring up at you from a dish?
Roly: Thankfully, no. They are tiny, they are microscopic. What you essentially do is you float a little colony of stem cells off the bottom of the dish and they form a little ball of cells. You then give them certain growth factors which push that ball of stem cells towards a frontal brain fate, so they become more like frontal brain cells.
Then this tiny ball of frontal brain cells has this inherent ability to form these outpouchings, these little outpouchings of eyes. So from one little ball of cells, you might get three or four little eyes forming.
They then sort of invaginate in on themselves and form this double-layered retina, which is microscopic, you can't see it. Thankfully, they don't blink up at you, they just look like little blobs floating around the place.
Georgia: It sounds incredibly complicated. You're taking something and deprogramming it, telling it not to be the thing it was before and then reprogramming it again into something else. So how difficult is this and how often does it work?
Roly: It took a lot of research. As I mentioned there were three scientists beforehand. To get where we are now took a massive amount of work. Actually now, it's really very simple.
All you do is overexpress four genes that are crucial to stem cells retaining their stem-like fate and you overexpress those in adult cells and they can then just reprogramme into stem cells. Then as far as differentiating them into the eyes is concerned, again that is incredibly simple. A couple of growth factors which push them towards the brain and then it almost takes care of itself.
These sort of little bunch of brain cells just know how to form an eye and they know that whenever it starts to produce these eye-like cells, these retina-like cells, they organise themselves, they fold in on themselves to form this little three dimensional mini eye. It really is quite incredible, the inherent ability of these cells to do what they are supposed to do.
Georgia: I did speak to one of your participants and it sounds like you need quite a lot of skin?
Roly: Yes, I would describe it as a small punch biopsy, but the person who is being punched with the biopsy clearly describes it otherwise. But yes, it's a couple of millimetres around. It requires a little bit of local anaesthetic and it requires a little stitch at the end to close the wound.
From that, we then have an unlimited supply of stem cells. So although it was a huge undertaking from the patients, it's so valuable because we can then use these cells forever more in the lab to research this blinding condition.
One of the huge benefits of using stem cells is not only are you using human cells, which obviously are more replicable of disease, it also means you don't have to use animals.
Obviously, we want to reduce the number of animals we are using in our research but certain things just unfortunately require an animal model. The advantage of using a mouse is we have all the imaging equipment that you would have whenever you go to see an eye doctor or an optician.
We have that in the lab adapted to using with the mouse. So we can carry out retinal fundus imaging, we can carry out an OCT image which is a very common and clinical tool to look at eye disease. We can carry out electrophysiology on these mice. So really we get a much better impression about how these mutations affect the health of the photoreceptor.
So whilst we are trying our best to use less and less animal models as we go on, some things just require the animal to be used.
Georgia: Right, so this is still work that's ongoing?
Roly: Yes, absolutely. The mice I've made, I haven't yet published them but they are giving us a lot of good insights into exactly what this RPGR protein is doing. It's regulating the cytoskeleton, but we are starting to get an idea by using this mouse.
Georgia: Roly Megaw there. With all these developments on the way, what’s the outlook for people like Ken who are waiting for a cure?
Roly: If my work led to a drug in ten years, I would be absolutely delighted. It's impossible to predict. We just have to keep plugging away in the lab, keep plugging away in the clinic and then hope that eventually we'll find a drug that does work.
Robin: In the 20 plus years, 25 years I've been working on developing gene therapy for Retinal Degeneration, we've seen huge advances. I think we couldn't imagine how far we could come in 25 years.
I remember when I first started, we were working out ways to deliver genes to the retina and we were pleased if we saw just one or two cells that had taken up a virus and maybe expressing a gene for a couple of weeks.
We are now able to rescue dozens of different animal models highly effectively. It's just a matter of time until this technology can be applied as effectively to humans. So I would say it is a matter of time and money, but we shouldn't underestimate the amount of time and money that is required.
Ken: I have no expectations of anything. I am 60 years old; I have been diagnosed for 34 years now, been blind for 30 years, registered blind. I don't expect to see again. But I have a daughter who is a carrier, I've got x-linked RP, so she's a carrier of the condition.
She hasn't yet got children but if she has a son, there's a 50 percent chance that my grandson would be affected and would grow up like me. I am confident that he will not grow up like me.
There are other people with x-linked RP and other forms of RP as well, who will be able to benefit from this sort of treatment. Maybe when the time comes for them they will be able to get an effective treatment that will prevent them from growing blind. So it's not for me, it's for the next and subsequent generations.
Georgia: And for those people who are going to lose their sight or know someone who is dealing with it right now, Ken has been there, done that and he has some advice.
Ken: The practical advice is; learn to touch type. Being able to interact with modern technology and to communicate with the world is just so important. The, the philosophical advice is it's a life sentence, not a death sentence. So get out and live the life and enjoy it.
Kat: Ken Reid there, and before him you heard Robin Ali from Kings College London, and Roly Megaw and Chloe Stanton from the MRC Human Genetics Unit, in the Institute of Genetics and Molecular Medicine at the University of Edinburgh.
And thanks very much to Georgia Mills for reporting, to Dee Davison at the IGMM for all her help setting the interviews up, and to the MRC for supporting this episode.
The MRC Human Genetics Unit is organising an public event for people living with genetic eye conditions for the MRC Festival of Medical Research on 15 June 2021 - a date for your diaries next year.
And as we heard, genetic eye conditions affect so many people, so you can support groups that fund vital research into saving and restoring sight like Retina UK and RNIB.
Resistance is female
Kat: And finally, it’s time for a quick look at what’s in the latest episode of the podcast from Heredity, the journal of The Genetics Society.
The arms race between the highly toxic rough-skinned newts of North America and the garter snakes that prey upon them is a literal textbook example of evolution in action. However, it appears that a piece of the genetic puzzle underpinning this interaction has been overlooked, until now.
In this episode, Kerry Gendreau from Virginia Tech and Michael Hague at the University of Montana discuss their recent work showing that resistance to the paralysing newt toxin TTX in garter snakes is sex-linked, and the implications this has for a system that is taught to almost every biology student.
Michael: So, it sort of suggests that females might be under selection to evolve increased resistance to the toxin in the newts because they are eating the newts, and males might not be favoured, they might be disfavoured to have these resistant mutations.
And in fact, these mutations in the sodium channel that confer increased resistance, they occur in a really important part of the channel that's really important for the normal baseline functioning of these voltage gated sodium channels in muscle tissue, right? They are really important for propagating action potentials.
It turns out that if you have these TTX resistant mutations, you're a really resistant snake, your sodium channels are kind of screwed up and they don't work as well and your muscle tissue doesn't work as well. So we think there's potentially a cost to this resistance.
The males might be favoured to have low levels of resistance to that cost in the toxic newts, whereas the females might be favoured to have these mutations because they can eat these really toxic newts. So it sort of changes how we think about selection on males versus females.
Kerry: So the reason why the sex linkage of these genes is significant is that sex-linked genes are inherited differently than autosomal genes. So being sex-linked has a lot of implications for evolutionary dynamics.
For one thing, sex-linked genes tend to evolve faster. There's a number of hypotheses surrounding why that is. It could be because of increased mutation rates on sex chromosomes. It also could be because selection pressures are different.
Females only having one copy means that one copy is always going to be expressed and always going to be exposed to selective pressures. This can increase the efficiency of purifying selection and it can also increase the efficiency of positive selection.
What that might mean is that there could be stronger selective pressure on the females versus the males because they are only expressing one copy.
Kat: You can find the full interview in the latest Heredity podcast - just search for Heredity in your favourite podcast app, or follow the link to the podcast page.
That’s all for now. Thanks very much to our stay-at-home roving reporter Georgia Mills. Next time we’ll be tackling the thorny topic of epigenetics - so get ready to pimp your genome.
In the meantime, you can find us on Twitter @geneticsunzip and please do take a moment to rate and review us on Apple podcasts - it really makes a difference and helps more people discover the show.
Genetics Unzipped is presented by me, Kat Arney, and produced by First Create the Media for The Genetics Society - one of the oldest learned societies in the world dedicated to supporting and promoting the research, teaching and application of genetics.
You can find out more and apply to join at genetics.org.uk Our theme music was composed by Dan Pollard, and the logo was designed by James Mayall, transcription is by Viv Andrews and production was by Hannah Varrall. Thanks for listening, and until next time, goodbye.
