Lecture: Clinical Clues in Diagnosis and New Drug Discovery for Inherited Retinal Degeneration

[Amir] Hello, everyone, this is Amir Hajrasouliha. I want to welcome everyone to the site, Cybersight. It’s a pleasure being among the colleagues around the world. I’m Dr. Amir Hajrasouliha, retina specialist at the University of Indiana School of Medicine, Indianapolis, USA. Presenting clinical clues which would help us identify patients with inherited retinal disease followed by a new advancement in treatment of these patients with gene therapy and few potential other methods aiming to help this group of blinding disease. My talk will be followed by new direct discovery in the field of IRD by Dr. Yoshi Imanishi.

My financial disclosure to the talk is listed on this slide.

Inherited retinal disease are a group of clinically and genetically heterogeneous condition which consequently lead to blindness. IRD has a prevalence of 0.1-0.2% in general population. IRD can affect pediatric to adulthood and present different throughout their life. Currently more than 270 genes have been identified as causes of IRD and the list is expanding each year.

Across this group of disease, there is a great variability and degree of visual impairment, the impact of everyday life, disease progression, and the suitability to target intervention. Therefore an early, accurate diagnosis, not only facilitates measures but also enables social, psychological support to be sought at an early stage and may minimize the effect of disease on education and professional choices. IRD can be difficult to diagnose and there are important size of presentation that can be clued to early diagnosis. Here I’m going to view a few of those signs followed by sharing a few cases that showed up in our clinic.

I’m going to start with the presenting signs in the childhood. IRD may present as nystagmus and can be a clue to predict the possibility of inherited retinal disease in the child patient. For example, this seven-month-old boy presented to our clinic complaining of nystagmus. On exam he had fair fix and follow of the light, the horizontal nystagmus was present, and while the anterior chamber, iris, and len was normal he had no family history of any ocular diseases or systemic disorder present.

We saw this in the posterior pole exam which showed pigmentary change in the fovea with drop out of the RPEs throughout the posterior pole. Periphery was unremarkable and there’s no pigmentary changes in the periphery.

The genetic testing revealed a de novo variance of PAX6 mutation in this patient. Of 286 pathological mutation of PAX6, about 90% of them presented aniridia and only 10% of them, like our case, are without aniridia. Which shows isolated foveal hypoplasia or microphthalmia or can present with optic nerve defects. Having a nystagmus without aniridia was the clue to find out the IRD in this patient.

What else can it be? Children might be afraid of dark and stop when they go into a dark room and say they cannot see. For example, this five-year-old Hispanic male with general good health and no past medical history presented to our clinic with poor vision at night and parents was noticing that he was afraid of the dark and couldn’t functionalize very well in a dim light. He first saw our pediatric ophthalmologist and then referred to us because of the retinal findings. He had no family history, born at term, and the best corrected vision was 20/50 in the right and 20/30 on the left.

Posterior pole presentation was diffused peripheral pigmentary change as you can see both in the right and the left eye with loss of the outer retinal layer and only preservation of the central island.

Genetic testing of this patient showed two RDH12 mutation which transforms one from each parents. Mutation of Retinol Dehydrogenase has 12 genes or RDH12 maps on local chromosome 14 and it’s known as LCA13 as well. It accounts for about 4% of autosomal recessive Leber congenital amaurosis. As you know LCA is early onset with severe retinal degeneration that affect 20% of children that attend the school of the blind.

Other clues may be that the patient with proper diagnosis might be a failure in a school exam. This six-year-old boy which was generally healthy with no past medical history, failed a school entry exam and was referred to us for further workup. He had no trauma, no significant past medical history, born at term, and the best corrected vision was 20/40 on the right and 20/100 on the left.

On fundus exam, noted to have these radial stria of the central macula and the optical coherence tomography and geography showed the larger FAZ with drop out of the depocapillary plexus and the schisis and the interretinal separation of the retinal layer in the spoke pattern.

Genetic testing and the clinical diagnosis is consistent with X-linked Retinoschisis with RS1 mutation.

I have also seen a pediatric patient being previously treated with amblyopia or a strabismus being referred to us and diagnosed with IRD. For example, in this case, it’s a seven-year-old boy who presented with vision of 20/100 and 20/20. He was born at term with normal anterior segments and with intermittent XT, previously being treated for amblyopia but last seen last month by a pediatric ophthalmologist and because of the retinal finding referred to us.

On the retinal exam he had the straightening of the blood vessels, displacement temporally of the fovea and probably nonperfusion on the other eye which was confirmed with fluorescein. And geography with multiple branching of the blood vessels even on the contralateral eye and displacement and nonperfusion in the periphery in the right eye.

This was consistent with Familial Exudative Vitreoretinopathy with FZD4 mutation.

Other rational includes in diagnosis is syndromic features and positive family history. For example, someone with Bardet-Biedl syndrome and polydactyly and diabetes would be a referral to our clinic because of a known genetic background in syndrome to be evaluated for IRD.

Presentation in adulthood could also be similar but few clues in my mind would help us with the poor night vision, failing the driving test, or positive family history that can lead us to proper diagnosis of IRD.

Although clues can be helpful, sometimes it’s very challenging and the findings might be very subtle to diagnose the IRD case. I put this case up as an example of it. It was a 19-year-old boy self-referred for evaluation of poor vision. He had always poor vision which had made him being unemployed and depressed and a smoker. And he had claimed that this vision had been bad from early childhood but hasn’t been changed for the past 5-10 years.

On exam he was 20/200 in both eyes with normal anterior segment exam. His posterior pole showed optic nerve drusens with not much of a pigmentary change in the back of the eye. The foveal reflex was somehow preserved. And the left eye was showing similar features of optic nerve drusens with maybe a little of blunting of the foveal reflex but no pigmentary changes in the periphery.

Optical coherence tomography shows smudging of the central ellipsoid line in the right eye, similar finding you see in the left eye with a little bit of a smudging of the ellipsoid line in the back. First seen in geography is showing somehow a bullseye etiologic form with the optic nerve drusen shining up at the ultra fluorescence.

ERG would be helpful in such a case which showed a lack of cone response with preserved rods response, followed by the genetic testing which confirmed the diagnosis of achromatopsia with CNGB3 mutation. Achromatopsia is a congenital autosomal recessive disorder characterized by reduced visual acuity. Sometimes with pendular nystagmus and increased sensitivity to light and photopsia with small central scotoma.

The patient’s visual acuity is usually stable over time but with both nystagmus and sensitivity to light may improve over time which might be the time the patient presents to you, as in our case. ERG is very helpful in detecting while the rod function is normal, there is a poor response in cone function.

As we mentioned, about 0.1 to 0.2% of population has IRD. Frequency of genetic bioinformatics of human gene data suggests that 36% of population in different genetic background has at least one recessive IRD mutation. A value which is probably the highest among the Mendelian conditions in humans. This dark area of these bars is showing the ABCA4 gene mutation. As you can see this is really the highest prevalence among all the other genes. But the question is, why don’t we see more of IRD when 36% of us have at least one gene mutation of autosomal recessive? And the answer is because we have more than 270 genes and the chance of having the autosomal recessive form is much less.

The journey of IRD does not start too long ago. It was only 70 years ago that we diagnosed, we identified DNA as a genetic material pass by. And it takes 25 years to sequence the DNA and another 30 years to be able to PCR. And we had only been able to do the human gene full sequencing for past 20 years. And we had identified 30 years take us to identify 270 genes associated with IRD.

The pace of discovery has been rapid with approximately 50 genes over four years for the past two decades. But we should know that the genetic mutation is not equal diagnosis and vice versa. Clinical finding of most IRDs are rarely pathognomonic of a particular gene mutation. On the other hand, a given genetic mutation may result in a variety of IRD presentation.

For example these three cases that we had, they all looked the same with the present of central island and loss of the periphery but they have all different gene mutation in all of them.

My question to the audience is if you see a patient with IRD, what would you do? Do you do the genetic testing? The first poll is showing you you’re going to do it yourself, the second one you at least going to refer it to some colleague that you think they’re going to do it? Or you’re going to only do it for LCA that you know that there is a treatment option for it? The fourth one, you don’t do it and the fifth one others. If you can answer this poll, I give 10 more seconds for us to finalize the answer.

Oh, interesting. Only 14% of us who are not going to do the genetic testing and about 85% would do it.

I want to put it as a comparison to what ASRS did in 2019 with the exact same poll. About 70% of specialists in the US would have done it and 50% of our international colleagues. And you can see for the past 2-3 years how much more we are getting to know that the genetic testing is reliable and where we want to be. And this is with the help of having the gene therapy for at least one of the diseases that we know which is the LCA with the RPE65 mutation.

But there are some limitations to what we can do. There are currently about 20 active trials of gene therapy for IRD going on. But how much baseline and what’s the limitation of these kind of therapy. One of them is the presence of the pathology. For example, in the X-linked form of RP, there’s too much of damage of the outer retina layer. Would therapy late in the disease would help us to achieve what we want?

The other challenges in the gene therapy would be how much immune response we would have. For example, in the form of the treatment of RPGR, we know that the perimetry would be helpful in identifying the progress of the immunoresponse affected by the subretinal deposits in these patients. While the vision on the best corrected vision on the letter hasn’t changed, the perimetry’s changing showing that we have to come up with new tools to identify the success of the therapy as well as perimetry.

The other things which is challenging is the dissociation between the structure and function. While we thought someone with the achromatopsia which I presented might be a good candidate for treatment because the preservation of the structure, our treatment had shown the improvement had been limited in success. And that might be due to other things like amblyopia and not being able to differentiate the color from early childhood with the pathways of brain not forming.

Other challenges that we have is while we have 20 genes that’s being tested, there are another 250 genes which needs the treatment and here it comes the other approaches that comes into mind and the methods that Dr. Imanishi will discuss about which would help us with the larger group of IRD patients.

There are many other many interesting approaches including the prosthetic retina which has been improved with Argus II. But there is limitation to the availability and cost of those approaches as well.

In summary, I want to point out that although the gene and genetic therapy shows promise, we have to work more and genetic testing is becoming more and more available and acceptable in clinics. And we need to come up with the treatments that can treat more groups of IRD patients.

I want to thank you for your attention and I want to pass my talk to Dr. Imanishi who is going to talk to you more about the new drug discoveries in the field.

[Yoshikazu] In the other half of this webinar, I’ll try to talk about the contemporary drug discovery studies for inherited retinal degeneration. As Dr. Hajrasouliha mentioned there are a number of adeno-associated virus vector for gene therapy that are being developed. Particularly for this topic Dr. Alex Levin covered those stem cell therapy and also gene therapies last year. I’d indicate for those interested, I encourage you to view those recorded webinar.

In my talk today I’ll try to address approaches that can target much for inherited retinal degenerative disorders independent of the gene mutations.

I have no conflicts of interest concerning commercial entities to disclose. And also I want to mention that the most of the therapeutics in these therapies I’m going to mention are currently under clinical development. Therefore, they are not yet approved by FDA for patients with IRDs. But I would like to give you some encouraging evidence that there are certain evidence that those approaches work.

In the majority of inherited retinal degenerations, photoreceptor neurons are most important because those photoreceptors either become dysfunctional or die over time. And there are two types of photoreceptor cells, rods and the cone photoreceptor cells. Those are so important for our vision because we think those photons are received by molecules called visual pigments Rhodopsin or cone visual pigment and absorb some protons by those molecules which lead to cascade of biochemical reactions within the cells. Waiting to the activation of a G protein called transducin and activation phosphodiesterase. And hydrolysis of cyclic GMP to five prime GMP. These changes in the cycle between concentrations are sequenced by cycloGMP gated channels, located to the surface of the cells.

And when people acquire gene mutations to have inherited retinal degenerations, imagine what happened is that rho photoreceptor cells become sick. And so in many cases those rho photoreceptors have become less functional than those in the healthy individuals. Therefore people have a visual impairment. And because of this changes the function of the rho photoreceptor cells, those cells have increased the probability to die. Dependant on the gene mutation, those photoreceptors cells die rapidly or slowly or stay later, those depend on the gene mutations and also environmental causes.

If enough number of photoreceptor cells are lost and then those patients become profoundly blind.

For us to be able to see the light continuously those Rhodopsin molecules need to be activated. This activation requires this 11-cis-retinal, we think the Rhodopsin molecules to be converted into all-trans-retinal. And this all-trans-retinal gets converted, it will turn into vitamin A and then transport it to the neighboring cells such as a retina pigment epithelial cells or mirror retinal cells. We have those vitamin A molecules are converted back into the 11-cis-retinal and then transported back to the photoreceptor cells to conjugate again with apo-opsin molecules to form Rhodopsin. And this continuous recycling process is called the visual cycle and critical for our vision.

Once we understand the biochemical basis of vision, the question arise, is it possible to develop therapies for multiple blinding disorders with inherited retinal degenerations?

For example, in this pathway, there’s a cycle reaction that converts those old trans-retinal molecules into something called bisretinoids. And this process is considered to be detrimental to the vision because these bisretinoid prohibits the functions of retinal pigment epithelial cells and this over accumulation of this molecule is considered to be detrimental. However, for example, a molecule Dr. Hajrasouliha mentioned ALK-001 inhibit this process of the retinal conversion into bisretinoid. Bisretinoid is a conjugate of two old trans-retinal molecules. There’s a therapeutic approach to inhibit this pathway and there’s another approach being developed to inhibit those entrance of vitamin A into the retina. And this can deplete the amount of retinoid or vitamin A molecules with cycling in this pathway.

Another possibility is to inhibit the critical enzymes in the pathway so there’s an approach to inhibit those enzyme called RPE65 to decrease this activity and then decrease the amount of bisretinoid to be produced. Currently there are a number of clinical trials ongoing for the indications such as Stargardt disease. Stargardt disease and age-related macular degeneration and diabetic retinopathy targeting this pathway.

Then the question is once we understand the biochemical defects, we would be able to target those pathways to make a way to the retinal degenerative conditions. I would try to cover those three ideas listed here. First there’s a targeting common pathways promoting photoreceptor degeneration. And metabolic reprogramming/neurotrophic factor ideas. And also preventing the degradation of mutated proteins that are functional but unstable. I’ll start from the first.

As I mentioned earlier on those photoreceptors cells are essential because of this phototransduction cascade. In some cooperation with inherited retinal degeneration patients, people have mutations in the genes and coding the components of this pathway. For example, in the refraction of the eye, autosomal recessive retina pigment or some retina pigment of those patient, mutations are seen in the genes and coding in phosphodiesterases. Also in a small fraction with those IRD patients, mutations can cause over accumulation of this protein called guanylate cyclase. And I looked, as Hajrasouliha mentioned, in a subfraction of the achromatopsia patients, cycloGMP, gated channel genes is mutated. In all those conditions what’s going to happen is that the cycloGMP concentration goes up. For example, when phosphodiesterase cannot hydrolyze cyclicGMP, cyclicGMP goes up. This leads to the over stimulation of a protein called cyclicGMP dependent to kinase or protein kinase G. This stimulation is going to make it detrimental because this will activate that apoptosis of the cone or rod photoreceptor cells.

Therefore a group of investigators led by Dr. François Paquet-Durand, developed a manage to cyclic GMP analogs with the aim to end in protein kinase G, PKG in photoreceptor cells. They synthesized a cycle of GMP analog and the subject that those molecules to survey based assay, basically assessing the survival of rod photoreceptor-like cells derived from the mutant or IRD animal models. And the second step they assessed six molecules to the organotypic explant culture assay to confirm that those molecules can promote the survival of rod and cone photoreceptor cells. And moreover they tested those molecules can act as a calcium signal in cone photoreceptor cells. And any molecule is affect to the calcium signal in rod cone therapy suboptimal because those molecules are potentially targeting the cyclicGMP gated channels that are critical for our vision. And eventually they came up with a molecule called CNO3 and also ribosome formulation TNO3. This formulation facilitate the delivery of this molecule which was the retina.

They tested this molecule; it was an animal model inherited retinal degeneration with a mutation in a phosphodiesterase gene. Those models, of course, this model is called as rd10. And quite encouragingly this molecule count significantly promote the survival of rod photoreceptor cells as shown here. This thick area photoreceptors right here. And this effect as observed throughout the retina as shown by this blue line here compared to the untreated cohort of animals.

There’s encouraging evidence that once we understand the biochemical changes, we will be able to target this particular pathway to delay the photoreceptor degeneration. So the questions are there other common pathways that we can target? One of the pathway could be metabolic pathway involving the glycolysis. Through the rod and cone photoreceptor use glucose as a major nutrient. In the retina pigmentosa glycolysis is compromised both in rod and cone photoreceptor cells. One reason is because in inherited retinal degeneration, rod photoreceptors, especially this region called the outer segment gets shorter and become lost from those cells. And then rod photoreceptor cells cannot interact or effectively with the RP cells which transfer glucose to those cells.

And when rod photoreceptor cells die, those cells cannot secrete a neurotrophic factors called RdCVF which binds to the surface of cone photoreceptor cells, resulting being promoted to glycolysis because of the promoted transfer of glucose to the cell. Therefore this loss of trophic support by rods can lead to compromised glycolysis in cones and eventually death of cone photoreceptor cells. And therefore there’s an idea to deliver this RdCVF factor to the retina by using, for example, adeno associated virus and it’s actually are currently under clinical development. I think those therapy will go into the clinical trial very soon.

Another idea concerns about the deregulation of a gene transcription. This factor called Sirt6 histone deacetylase is known to bind to the gene upstream region of the genes dictating glycolysis. In the presence of this gene Sirt6, the glycolysis associated genes are down regulated or suppressed. But when this Sirt6 function is compromised or removed from the cell those cells can now transcribe those genes very effectively promoting glycolysis.

This idea was taken by Dr. Stephan Tsang’s group who tested this idea using the newly developed PDe6b animal model which demonstrates very rapid photoreceptor degeneration within a matter of two months. But when this Sirt6 gene is removed from the rod and the cone photoreceptor cells, what happens is photoreceptors survived the guinea pig promoted. Along with the improved rod and cone photoreceptor functions as shown by those electrophysiological study. Therefore, metabolic reprogramming is not really particularly specific to the gene mutation because in many IRD, on available animal models, glycolysis is known to be compromised therefore this is potentially up and coming to multiple inherited retinal degenerative conditions.

And last but not least, I would like to cover the topic about preventing the degradation of a mutated proteins. And this approach is stemmed from the fact that a large fraction of inherited human disorders are caused by point mutations in the genes. And for example, when patients have this mutation a wrong amino acid is incorporated into the polypeptide chain of a specific protein, such as rhodopsin phosphodiesterase RPE65 or genes associated with Usher syndrome. And what can happen is that those proteins become partially misfolded and then degraded by the cells using the machine that recorded protein cells. Therefore my group particularly focused on Usher syndrome type III because this disease is characterized by progressive loss of vision as well as hearing. Therefore there might be that window of opportunity for three things to the retinal degeneration of those neurons sensory cells in the system.

We devised a new method for identifying stabilizers of a specific mutant proteins. And this approach it depends on MODC(422-461 that was designed to express a red fluorescent protein which was designed to be degraded by proteasomes. And also another report which is an upcoming one. An N48K mutant protein which is a causative to Usher syndrome type III. This protein was infused with green fluorescent protein. Both of those proteins are very unstable and degraded by the proteasomes. Therefore under normal conditions we don’t see much fluorescence. But if we were to inhibit proteasomes what’s going to happen is that both red and green fluorescence go up. If we can find out a specific molecule that can stabilize a protein and we would be able to see increase in green fluorescence only. We conducted the assay because proteasome inhibitors are known to be toxic to photoreceptor cells. Therefore we wanted to screen out even more of those molecules from the candidate.

This green and red photo we screened for about 50,000 molecules and if we don’t do anything to the cell we don’t see much fluorescence. If we inhibit the proteasome machinery we see increase in both green and red fluorescence. But out of those 50,000 molecules we found that at least 20 or 30 molecules stabilize a CLRN1 protein but also slows the inhibition of the proteasomes. We didn’t pass through those molecules. Fortunately, among 50,000 molecules we are able to find only 1 molecule within that all three. Therefore we worked with a group of many chemists to improve the structure of this molecule. And eventually we came up with this new molecular entity called BF844 which was effective in stabilizing CLRN1 proteins.

We also at the same time developed an animal model of Usher syndrome type III, a mouse model. And unfortunately this mouse model didn’t develop a vision loss but this model developed a significant hearing loss. Therefore we used the hearing loss as a separate way to test the efficacy of this BF844 molecule.

And so for example, this is a mouse model and those mouse models are profoundly deaf therefore the sound pressure level threshold is very high. But the whiter type animals can hear very well, so they can hear very low level sound. And if we give the BF844 molecule to this mouse model, we are able to significantly resolve their hearing by the factor, so their hearing performance was at least 1,000 to 10,000 fold better than the mice that were not given this treatment.

This is a concept of the proteostasis therapy stabilizing the mutant protein. In the case of BF844, we can inhibit the degradation and increase the protein amount and promote the folding of this protein. And there are other potential investigations or therapies being developed. For example, sodium 4-phenylbutyrate is called a chemical chaperone which was tested on a mouse model over LCA, Leber congenital amaurosis, caused by RPE65 mutation. Pharmacological chaperones are being developed for IRDs, for example, caused by rhodopsin mutation. As I mentioned earlier, rhodopsin binds to retina, so retinoid analogs mimicking those structures are potentially effective to stabilize a structure of rhodopsin molecules. Their approach is quite successful in preventing photoreceptor degeneration in animal models.

Also I didn’t mention extensively some of the mutations cause an introduce or stop codon instead of the wrong amino acid. Those are called as nonsense mutation leading to the incomplete polypeptide that will be expressed in the cells. There are efforts to develop a read-through-inducing drugs. Those drugs bind to ribosome and basically they press non stop codon with another amino acid to coding. So they can translate a nonstop codon into amino acid. Only end with a polypeptide to be synthesized. Such approaches are developed for indications such as Usher syndrome type II A and 1C.

Therefore we started from this question: does one size fit all to all the IRDs? The short answer is no. But the field is evolving to develop therapies collectively addressing a wide spectrum of IRDs. And therapies can be potentially tailored in accordance with biochemical or cellular defect that are shared among different IRDs.

With this, thank you very much for your attention. We are open to questions and I’m listing here the email addresses of me and also Dr. Hajrasouliha. If we cannot answer sufficiently your question today, please send us an email so that we’ll be able to answer. Thank you very much for your attention.

[Amir] Thank you, Dr. Imanishi for great talk and going over the discoveries and new modalities of treatment of more patients with IRD. To the registration there were a few questions that I highlighted which was repeated and I thought we might be able to discuss that. One of them was can a bionic eye be used in situation with the retinal degeneration as vision threatening? Yes, as we discussed, there are few modalities of using artificial retina which has been FDA-approved. The clinical use of them are very limited and only few sites are approved to be implanting those. And as the others have been pointed out in their questions, the cost of treatment is also challenging, especially for developing countries.

There was another question is how can this be applied in developing countries? And I think part of Dr. Imanishi’s talk, which is drug discoveries and the modalities that can be used as drug delivery which rather has less complicated approaches and probably more broader audience or patient population that can be treated by it, would help us to lead the way in the treatment options.

[Yoshikazu] Also I would like to address a few of the questions. The question is acetylcysteine will be a new choice of treatment for retinal pigmentosa. And the idea to use acetylcysteine is an antioxidant because this is a precursor of glutathione. And actually when the photoreceptors are known to be the reason with a very high oxygen concentration and so when rod photoreceptors die, it’s considered that the cone photoreceptor cells are exposed to high level of oxygen. N-acetylcysteine is considered to be beneficial and I believe this is under clinical trial run by Johns Hopkins University as Dr. Hajrasouliha mentioned.

[Amir] There is a question. What’s the clinical clue to differentiate between the Stargardt and cone rod dystrophy? This is where we go back to knowing the genetic mutation is not equal to diagnosis and vice versa. As we discussed and we label different diseases as cone rod dystrophy, 1CA, or RP there are many features that are common among the same mutations. ABCA4 can be a cause of rod dystrophy and Stargardts also known cause of it can be ABCA4 as well. Does not mean that other forms of conditions cannot be named as such as well. It is a gray zone of clinical diagnosis versus the genetic diagnosis as well. And as we know more and more about the genes the naming of the disease would be a little bit more difficult to catch up with.

The question is, is any of these drugs have been in the market yet? No. There is no FDA approved drug yet in the market. As we discussed there are some trials with medication. Our site is an active site of recruitment for the ALK-001. It’s a medication which is similar to Vitamin A but it’s trying to reduce the junction infusion of two vitamin A to form a biformid form which is thought to be less toxic in those patients. It’s still on the clinical phase III but not available in the market.

[Yoshikazu] There’s another question. What are those in the pharmacy profession can do to contribute to the field? I believe that people in the pharmacy profession can educate the patient in terms of the potential side effect of the therapies prescribed to those patients. Because, for example, as I mentioned, phosphodiesterase is, for example, inhibited by pharmaceutical such as Sildenilfil. And that’s a side effect of temporal vision defect. So they want to educate in some of those medicines are contraindicated to the inherited retinal degeneration disorders.

[Amir] Thank you all for your attention and as Dr. Imanashi mentioned, if you had any further questions we would be happy to answer them through the email.

[Yoshikazu] Thank you very much.