hey everyone I’d like to welcome you to the second retina Australia webinar for 2023 the latest in research from retina Australia Grant recipients first I’d like to begin by acknowledging the traditional owners and custodians of the land on which we meet today the peoples of the kulin nation I also pay my respects to their Elders past and present my name is Sally Turnbull I’m the administrative officer for Retina Australia and I will be the facilitator of this webinar today as a participant your camera and microphone will be switched off throughout there will be an opportunity for you to ask questions once both our speakers have finished their presentations if you have any queries during the proceedings you can type a question to me in the Q and A section our first Speaker this afternoon is associate professor Rick Liu from the Center for Eye research here in Melbourne associate professor Lou completed a PhD at the Kaohsiung Medical Institute Medical University in Taiwan and carried out post-doc training at the O’Brien Institute and Center for Eye research Australia, CERA here in Melbourne he has extensive experience in designing and developing Innovative gene therapy approaches to prevent And Delay blindness from major eye diseases especially for neovascular eye diseases and IRDs with projects continuously funded by the NH and MRC since 2012. associate professor Liu is currently a principal investigator and leads a genetic engineering Research Unit at CERA his recent research work has extended into the fields of Next Generation gene therapy focusing on refining these applications to revolutionize ophthalmic care with the latest genetic Technologies this afternoon Rick is going to speak on rna-based editing strategies as potential Therapeutics for inherited retinal dystrophies so I’ll hand over to Rick thank you so much Rick
thanks Sally uh let me share my slides
Hope you can see my slide
so uh so begin uh before I I talk to about science uh first I would like to thank you for an invitation to this webinar this is a great opportunity for us to share our latest uh research to the Retina Australia Community also I like to take this opportunity to thank Retina Australia for funding this project with us the funding was the really first uh financial support to this project it actually make the project fund zero to one and in the last two years we’ve been had a lot of progress about this project and making these two become more viable and then potentially we can harness this tool to treat various retina uh inherited retinal disease so before I start with the slides I like to tell you about a bit more about my why I’m interested in the in this disease and gene therapy particular for for for this area so this is Harry so a lot of you probably already know him so he was born with a genetic disorder called Usher syndrome type one which is born with impact is hearing and gradually impact his vision so Harry was also a very first patient I ever meet so when I so when I saw him I just feel we should do something to Harry because he’s still young so he can so if we can do something for him so he don’t need to live in in the dark in his future so that that’s why when you start to invest our time invest our energy into the solution for it treating inherited retinal disease so not only Harry there’s many kids suffering
uh with the genetic disease those of those of the genetic disease often it caused by genetic mutation or or genetic fault into a certain Gene so those of genes contribute to uh various visual functions. Since the 80s about 300 Genes have been discovered associated with inherited retinal disease and why is really exciting for this discovery and also this discovery also leads to latest breakthrough uh gene therapy strategy to treat uh type one type of inherited retinal disease called Luxturna which has been proved by uh at US FDA in 2017 to treat a RPE65 associated inherited retinal disease called LCA so basically in this gene therapy scientists harnessed a non-pathogenic virus called AAV so AAV are able to carry a normal copy of the RPE65 Gene and through the injection into the retina so in fact the retinal cell we call RPE cell and produce normal RPE65 protein to maintain the RPE function and eventually prevent the further receptor degeneration so this is quite exciting so followed by Luxturna’s proof of concept
success into the clinical there is many many uh gene therapy has been in the clinical trial using a similar approach so those approaches similar to Luxturna they also use same AAV virus to carry a gene to different gene to address different mutation in indication so that why are this is really exciting so apart from Luxturna we will have more this type of gene therapy to address different genes for the various inherited retinal diseases however that’s not the case for AAV because such as a Luxturna or some of genes are quite small they are able to pack into the virus but some of the IRD Genes
are very huge and that you can see similar like this AAV is such a small virus you won’t be able to pack the large gene into the AAV which means the tradition gene therapy similar to Luxurna
approach won’t work for some of the IRD genes. Indeed if you look through the around 300 genes associated with
retinal degeneration so you will find there’s over
45 percent of genes are just about beyond the packaging capacity already too big for the AAV packaging capacity so particularly like Usher 2A PCDH15 which is the mutation Harry carries
with this gene or ABC4 which accounts for the Stargardt’s disease which are quite high incident Gene associated to IRD however this gene until now this still cannot be used with the same approach like Luxturna so it means we need another approach to address those of the mutations which cannot be packed into the single AAV or traditional AAV gene therapy so writing the genetic code with the latest gene editing technology might be a way to go. So since 2014 our lab has been really interested in this technology because we believe this technology has got really powerful potential which can offer another opportunity to also to correct a genetic mutation or notation and also offer a once off cure for those
suffering from a genetic disorder. So CRISPR-CAS 19 editing is one of the really latest and also really promising technology. So this technology was initially introduced by Professor Jennifer Doudner and Emmanuelle Charpentier in 2012. So this technology
they found from the bacteria is a bacterial immune system so this immune system is acting as an immune guard to prevent the virophage infection to a virus so both repurposed these
systems called CRISPR system into a biotechnology so that allows us to efficiently and accurately edit the human genome with high efficiency and even more safety compared to the past technologies like TALEN or other technology.
I’ll give your simple explanation about this technology so this technology is comprised of the two components the first component we call CAS9 enzyme which is acting as molecular scissors so this CAS9 enzyme can be guided by a small piece of RNA we call Guide RNA so that Guide RNA can guide this this big guide molecular scissor through the human genome to search through the specific area to find a bit that is complementary to the Guide RNA so and then this CAS9 will acts as scissors
to cut the double strand DNA to become two pieces so this cutting will then initiate a code DNA repair response this is how we repair that so at the same time we could introduce a repair template and then tell cells for DNA repair you can follow by this template to repair DNA so we can use this mechanism to actually
correct the gene mutation via that template it looks quite exciting about this technology indeed when this technology came up there was a
hope that yes we can use it to cure a genetic disorder. However this repair system is very inefficient so we need a better tool and subsequently there’s a few new technologies
based on this crispr Gene editing technology that are more efficient or safer for example base editing. So with base editing from the word you know you
we can use this technology to correct just a single base without introducing a template. So how it does that is by fusing a small enzyme called deaminase to the CRISPR system so via CRISPR system they can accurately see, search and find the region that we are interested in, so that the deaminase can correct a single base from one nucleotide to another nucleotide which is can contribute more than 50 percent of the mutation can be corrected by this sort of technology.
And also later on another technology also came up called
Prime editing so the prime editing is similar to base editing but they use a reverse transcriptase it’s another enzyme and then provide a repair template along with a guide RNA to repair so both technologies are really is excellent to increase the hope and efficacy to address genetic mutation and so with those of two rows so we could actually now edit both of the mutant
DNA in the genome in the cell and then that can lead to production of the normal second message of code RNA and produce a normally functioning protein.
That’s ideally the fundamental way to cure uh genetic Disease by this way but the system is
never perfect for most of us so this system was limited by some of the factors for example in the eye so we have to deliver a CRISPR
system into the eye to to correct the gene in the eye however it’s very difficult because the system is quite big so you won’t be able to put in a traditional AAV and you only can put in like think about other way but currently even the new protocol is still not quite well developed enough to deliver a gene into the retina and also because this is correcting the genome so the system is accurate but sometimes it will close out of target so it might cause some issue with the editing with the other components so that might cause other problems as well so in this case so at the moment we don’t have the perfect tool to actually correctly know can we step down a little bit to correct our Messenger RNA which is more in the middle template product and that RNA still can produce a functional protein in the cell so that maintains cells health and then prevents the progress of the inherited retinal disease. So the idea of editing RNA was first introduced in 2017 by Professor Feng Zhang’s group based at the Board Institute so in that case they identified that apart from the DNA editor they also found it in the bacteria that apart from edited DNA they also have other systems similar systems that edit RNA so they took this tool and then doing the modification similar to DNA editing so they added a simple deaminase to the RNA editor forming what we call an RNA base editor so these RNA base editors are able to see and identify the area which we are interested in and use this ADAR deaminase to change the codon for example they can change A to I, I can become a G to correct the single mutation. So according to the literature overall in
genetics 58% of the mutations out of all mutations are caused by single nucleotide mutation so this approach might mean we can address more than half of the mutations leading to
retinal disease. Another advantage of this system is it is quite small compared to the traditional CRISPR DNA editor so that would allow us to use a virus for long-term delivery into retina and also this RNA base editing is way more efficient than the traditional DNA editor so and more flexible so that might hold a promise as a treatment for
retinal disease so since we received the funding so we are thinking before we move into treating
real disease we wanted to see how efficient is this system so uh in this case we are comparing this is called CRISPR RNA based editor that we created in our lab so in at the same time we also compare to another system called CRISPR
Inspired system so CRISPR systems use bacterial protein to accurately recognize the position to edit but the CRISPR Inspired system is like artificial fusion by human component so this component because it all comes from human so it could be potentially less immunogenic in general so and to demonstrate that editing efficiency we used a red fluorescence as a model to test both systems so in this case we created a red fluorescence protein so you can see the fluorescence shining but we create one of the mutation in in the halfway of this this sequence so put G to A
to a create a stop codon so it means when the protein translates to halfway so they stop here so that so that there won’t be any fluorescence coming so if our desired base editor works they should be able to create one codon to A to G so that allows it to continue produce the protein in the end so that would be forming a fluorescent shine again. So in this case we found that CRISPR RNA base editing is able to correct more than 90 percent of the cell and reverse fluorescence which is quite exciting. Unfortunately the
CRISPR Inspired system which is combined by human protein didn’t work so well. Since we we get that sort of exciting data from red fluorescence now we we’re thinking that’s the right time we moved to real disease model to to better prove concept so we employ a mouse model which allows us to do individual validation and then we can use this as a model to test the system in vivo so this mouse we call an Rd12 mouse carries a mutation called RPE65 similar to the human LCA disease and that RPE65 mutant causes the RPE cell degeneration and then overall leads to the retinal degeneration you know so and that was caused by a single mutation C to G and in this case so we design our base editor to target this RPE65 by to reverse that A to G so that we are able to remove the stop codon and that the coding can continue so that it is able to create a normal protein uh in this Rd12 mouse model so in vitro we’re doing this really similar work like we’re doing in the red fluorescence.
so we test that RNA base editor along with the CIRTS which is the CRISPR inspired system so initially we are selecting the right guide RNA so in the guide RNA design so we have to make sure it’s matched to the codon of that which we wanted to correct so basically this is kind of the action tell it’s matched, and you have to make the change in this position and then we designed several different kinds of RNA which are matched and we found particularly
that two are pretty efficient for the CRISPR RNA base editor, one is pretty efficient for CIRTS so after that we are putting this
system into the cell engineered with the mutant RPE65 mutation so and this is pretty complicated so it might be easy just see here so from the for the CRISPR RNA base editor we are able to correct a single base mutation in the position that we want up to around 40 percent and similar to the result from the red fluorescence so CIRTS wasn’t quite as efficient in this position and then so you might be curious – Is that 40 percent
enough? So we subsequently looked at the protein expression so we are actually able to reverse the protein expression even with only reaching a 40 correction so that fits the idea is that you don’t need to correct every protein as long as you can produce a certain amount of protein, that is able to in the cell to drive
the function and prevent cell degeneration
so we showed proof of concept in the RPE65 now we have moved to the next step to do a to address more mutation in the photoreceptors in the bipolar cells, like in our lab we are trying to work on to address like ABC A4 USH2A and also the mutation RS1 which happens in the bipolar cell so just give you a quick idea about the USH2A so we are creating an USH2A mouse model which is similar to humans with quite high incidence of USH2A mutation so with our base editor applied to this model we are able to see around even higher around 70 65 to 70 percent correction however we are
aware once you reach certain higher editing efficiencies you might have some off target along with the area like this so that means you correct the main on target so we call on target but you might have some off target so to address the off targets our lab is also working on a couple of strategies, for example we modified the guide RNA to allow to apart from one mismatch so we also mismatch another off
target area with the codon that we don’t want so that the result also shows we are able to minimize the off target here like compared to the original CRISPR design so once we modified the guide RNA we can substantially remove or reduce off target in this case so and also apart from the modified guide RNA we also are working toward another solution called domain RNA base editor which is we put in so in the tradition CRISPR Gene editor we put this code ADAR RNA base editor effectively in both ends but in this case we wanted to put it in the middle of the CRISPR enzyme so that enzyme can integrate into the big component big guide so they won’t move much so that it would theoretically improve the specific targeting window so and then we’ve been trying in several positions to insert the enzyme and we did find a couple of the positions that works pretty well so and one of the position actually
substantially removes the off target from the USH2A gene
without impacting the on target so we now are pretty excited about this technology so now we are moving into more validation for the other mutations where hopefully we can get a similar result from here So what is next? So it looks like we have a really good tool however uh we still have a long way to go to make this into the clinic for example we need to engineer more safe base editors and we need it to be delivered into as many retinal cells as possible so we have to improve the delivery efficacy and also we have to validate the editing efficacy in the retinal cell
from the patient which will give us more idea about how this system works in
a real retinal cell and that can be employed by the stem cell technology to achieve that so this is my last slide so here I think hopefully I convinced you about using this technology as a potential
treatment for IRD however along with the other technology being invented in the past few years we think we now have more choices to to address the various genetic mutations that are associated with IRDs and then so I think in the end so I think we are not set on one particular technological base, but overall we hope we can develop many many more of this these genetic tools and that patients can choose the more suitable technology to address their mutation and then to manage the disease in
a clinical setting. So that’s it for my talk today so before I close my talk I would like to thank a lot my lab members who have contributed this work particularly Satheesh Kumar who is our PhD student he actually did most of this work and also our collaborators from the Department of Optometry from the Uni of Melbourne and then from West Australian Lions Eye Institute and also the Menzies Institute for all your work
for this this work and also the funding body and thanks for your attention thanks so much Rick it’s so exciting to see these new things um starting to appear I will invite our second guest who is Annie Miller and she is a PhD candidate from the Lions Eye Institute in Perth and she’s been working in ophthalmic research for over four years and has written academic papers and undertaken rigorous research in the field of inherited retinal diseases working dominantly in the field of co-mediated vision losses her job includes the development of Novel treatment options for use in inherited retinal diseases and pre-clinical testing of these treatments in mice models. Annie is passionate about science communication and she regularly communicates her research to high school and University students Annie has a bachelor’s degree in pathology and laboratory medicine and a master of clinical pathology from the University of Western Australia she will complete her PhD in the coming months before commencing her postdoctoral work this afternoon Annie will speak on neuroprotective effect of SAHA in retinitis Pigmentosa and ask the question do time and frequency matter? so I’ll hand over to you Annie thank you so much thanks so much for introduction Sally um so I’ll just quickly share my screen I’ll just be one second okay while we’re waiting for Annie um maybe we could see if Rick if anyone has any questions for Rick um I was going to ask a question Rick um just in um when you talk about introducing the gene therapy um to the eye um and you talk about how it goes into the different cells I’m assuming that when it is introduced into the eye it just goes to the appropriate cell like depending on where the different genes need to go is that right is that how it works yes so so it’s really first we need to identify what mutation has occurred in the patient so that can be like RPE65 which functions actually in the retinal pigment epithelial cells so you have to deliver the right component of the therapy to the RPE cell some of the mutation occurs in the photo receptor so you have to deliver into the photo receptor so uh at the moment so we think for the viral technology we are able to harness the AAV vector by introducing different serotypes so that is able to target the cell uh the retinal cell in a different different retinal cell so so that allows us to Target the right cell and to deliver a gene or our CRISPR base editor into the right cell to correct the mutation or to supplement genes in the right cell
so that would be maximizing the therapeutic efficacy to to dry cell but and also that so that’s also a big challenge for the adjust cell talking about the DNA editing so for DNA editing because it’s quite big so we couldn’t use a virus as a vehicle to deliver so we only can use more most of this today is used particle called particle or nanoparticle those particles might not be well addressed to deliver in the right cell right position so that’s how it makes difficult to to use to apply this technology to the eye but for the other organs might be easier than the eye so that’s why we are thinking of
moving to the RNA base editor then the DNA editor which is small you can put in the virus and it can deliver where you need to yes yeah okay fantastic okay I see Annie back let’s see how we’re going Annie alrighty so thank you for my introduction before Sally um and thank you for obviously retina Australia for providing us with the funds to undertake this uh research over the past um year and a half or so um so today I’ll be presenting a talk about the neuroprotective effects of SAHA
in a couple of models of retinitis Pigmentosa
um so just briefly so our lab focuses on the study of inherited retinal disorders and we especially focus on Cone mediated vision loss um so when it comes to treating inherited retinal disorders there’s lots of uh there’s actually not very many treatments available to patients but there’s lots of different treatments available or Sorry not available um happening in in Vivo in the lab at the moment so if you look at two broad categories we have Gene dependent therapies so those are things that um might involve Gene replacement of a faulty Gene so that’s things like AAV based Gene replacement therapy like Luxturna that Rick mentioned before in his presentation uh which is the only FDA approved therapy for people with inherited retinal disorders we also have gene independent um therapies which are being developed at the moment mainly in a lab basis so there’s lots of kind of different areas to that we might have things like retinal prosthesis we might have things like cell therapies replacement of cells and we also have Pharmaceuticals which might be there to say prevent the cell loss that happens in these diseases so we focus a little bit on these Gene independent therapies and we mainly look at neuroprotection and we work in a group of drugs called HDAC inhibitors
so I’ll go a little bit more into HDAC Inhibitors in just a moment but we’ll really briefly just talk about retinitis Pigmentosa itself so when we have RPM patients the usual initial symptom is a night vision loss and then there’s a peripheral vision loss Associated afterwards so um some people don’t progress towards total vision loss but it does vary depending on the patient It’s associated with mutations in over 90 different genes so it’s very genetically heterogeneous lots of different causes and usually we have a primary rod photoreceptor cell death which is the kind of cause of that initial night vision loss and then eventually we have the cone photoreceptor loss as well which follows which results in the purple vision loss and the total vision loss in some people the unfortunate part about um Retinitis
Pigmentosa is that once the photoreceptor cells have um degenerated they can’t regenerate anymore so once they’re gone they are gone forever in that patient and they will lose their vision
so lots of different Studies have looked at the causes of cell death in RP because that’s also not very well understood when it comes to looking at these causes of cell death there’s lots of different avenues that you can look at we have things like apoptosis which is programmed cell death necrosis which is when a cell is kind of you know falling apart not doing its job very well so it just dies because the body goes okay that’s not viable and then we also have autophagy so these kind of three um three different main areas of cell death are kind of all considered to have a part in RP and potentially um they all kind of act together and this kind of cell death might sit in a bit of a spectrum between these three different areas
so there’s still lots of work on going in this field but about 10 years ago now this study um actually looked at 10 different models of inherited retinal disorders um so some retinitis
pigmentosa some achromatopsia and then a lebers congenital amaurosis model and they identified that there’s quite a few of these non-apoptotic cell death markers that have been upregulated across all of these models so um here we have just a little bit of like a schematic showing some of the things of upregulated when we have a mutation it can cause this accumulation of this cGMP molecule and then this has other effects in the cell that could eventually cause the cell to die so we have say for example if you go down this route we have an increase in our PKG molecule this increase in our HDAC or histone D acetylase molecule eventually goes onto this part molecule and then we have death so it is kind of like a Cascade and all of these might have involvement in that Ultimate Death so what we’ve done in our lab is we focus specifically on HDACs to see if we can modulate that if we can reduce that increased activity is there a way that this could potentially um you know cause a protection of the photoreceptor cells in our models
so here we have just a schematic showing what actually happens with this HDAC molecule and what’s its purpose inside of the cell so we have here a diagram showing our DNA and this DNA is wrapped around these histone proteins so the histone proteins basically allow your DNA to wrap either tighter or looser depending on what the cell needs um and we have our HDACs and our HATs or our hats which modulate whether it is tightly coiled or Loosely coiled so our HDACs will make you know the DNA more tightly coiled this results in transcriptional repression while a histone acetyl transferase, our HAT will result in a more relaxed chromatin the idea of having a HDAC inhibitor come in this type of drug is to actually block the HDAC so you’re allowing for a more relaxed chromatin and more transcriptional activation
so what we look at is um pretty much looking at these HDAC Inhibitors and seeing if these are going to be a viable Gene independent therapy for IRDs
now the particular HDAC inhibitor that I’m going to be talking about today is called SAHA um and it’s an already FDA approved drug that’s been used to treat cutaneous T-cell lymphomas um and it’s been mainly used in actually cancer research for many years uh but there’s a few studies recently that have shown that it might actually have a beneficial effect in neurodegenerative disorders as well as retinitis Pigmentosa and other inherited retinal disorders um and so far they have looked at um a model of rd10 a retinitis pigmentosa
model and they have looked at the effect and it does appear to be neuroprotective in that particular model so as we can see on the right here we have on the left our untreated Rd 10 retinal explants and then if we treat with SAHA we actually have an increase in the number of photoreceptor rows so this is mainly the rod photoreceptors being protected
okay so because of this kind of these previous studies and the fact that HDAC
overactivity seems to be consistent over quite a few different models of inherited retinal disorders we now ask the question can SAHA
improve Vision in our rd1 Mouse model in vivo so our rd1 Mouse is a mouse model of autosomal recessive RP and it has a mutation in the Pde6b gene so this Gene is just involved in Rod photo transduction in this model it’s actually a very very fast degenerative rate so both rods and cones are pretty much lost by the age of about p24 so we see our Rod loss starting at about p8 and this is postnatal day so very very early in life and then your cone loss Will begin around p14 and we’ve just shown a a picture here just showing what a wild type or a healthy outer nuclear layer should look like at 24 days of age in mice and you can see it’s quite thick here it has these cone photoreceptors sitting all through here and then if we look at our rd1 over here you can see how degenerated it is you can’t even discern those cone cells anymore and there’s almost no rods left at all
so to um help answer our question this is the treatment regime that we did for our mice so what we did was we decided to do a p12 injection so we wanted to inject our mice a little bit before that cone loss has actually started to see if we can preserve those cone numbers so we performed an Intravitreal injection and we either did that with a treatment or we did it with a sham injection as a control we then waited four days and then we collected optimotory testing data I’ll explain that just a little bit more in the next slide and then we also collected for a couple of other different experiments we also did some longer term studies where we injected at p12 same thing again either sham injection or SAHA injection and then we also uh then collected optimotory and other experiment data at p24 instead so that’s 12 days after we’ve administered our treatment
okay so for a little bit of background about optimotor testing this is a really cool technique that we uh that we have where basically we can put mice into a like a box and it has four screens around the mouse and what it does is it kind of assesses their visual Acuity because obviously with bias we can’t just ask them to read the letters on a chart we actually have to assess it in more of an objective manner so these screens will have these lines that you can see here and they’ll move around and we’ll assess if the mouse is tracking the lines as it um as the lines are moving have a little video here which would hopefully demonstrate that for you you see the mouse on the platform and then we have some lines appearing on the screen and then if the mouse turns its head it will indicate it’s tracking and it can see the stimulus
so um what we what we saw in our rd1 mice is at in scotopic readings which are your dark adapted readings which assess Your Rod function there was absolutely almost no response because these mice have lost almost all of their rod photoreceptors by this age so this was not very surprising and you can see that our wild type is drastically different compared to every single one of our um rd1 groups we also looked at the photopic optimotory data and these mice at this age do retain a little bit of their photopic or their light adapted visual response although it is still really low compared to Wild type so here we see um the uninjected and Sham injected optimotory testings was a little bit reduced compared to our treated especially this one micromolar SAHA here however this result was not statistically significant so we did notice as well though that a lot of um our a lot of our mice actually appeared to have absolutely no response to the stimulus that is coming around um it might be because potentially the photoreceptors have generated too much at that point but if we remove all of the ones that sit on this zero we can have a look over to the right here and we might have some potential non-responders excluded from our group and we can see that this trend is looking a little bit more um a little bit more likely now although we still don’t see any statistical significance showing that it definitely is increased compared to our controls
um so we also assessed uh histology and looked at some staining of the retinas after they’ve been treated so when we do our histological assessment what we do is we look at four four to six different locations in the retina and we have a nice uh picture here of a retinal section and we have the four different locations that we kind of locate through finding the optic nerve so we have our Central Superior here our peripheral Superior here essential inferior and peripheral inferior so that’s what these different labels here mean now after we injected um we collected four days after and we found that most especially in the central retina there was an improved um outer nuclear layer thickness after treatment we noticed especially that this increase was mainly seen at a lower dose of SAHA because we assessed two different doses both one micromolar and 10 micromolar so at this point it looks a little bit more like our lower dose is actually beneficial while our higher dose might not be
and we also looked at this other marker here called GFAP and GFAP is a active Muller glia marker and this usually indicates a little bit of retinal stress in the retina so in these types of mouse models you’ll usually see quite a lot of this GFAP because there’s a lot going on in the retina a lot of cell death and degeneration so we see that after we treat our mice with one micromolar SAHA there’s actually a decrease in this GFAP staining in our retinas but we don’t see that effect as much when we look at our higher dose our 10 Micro molar SAHA
so when we have a look at these results 12 days after treatment we see that we’ve lost this potential treatment effect um so our scotopic and our photopic optimotory data is all at zero so there’s no response at all they’re completely blind by this point um and then when we assess our other nuclear layer thickness as well we also see no biologically relevant changes at this age so it looks like the treatment effect is lost
so this data looked really really promising um but it’s quite hard to work in a model that generates so quickly because you have to be very precise with it um with your collections and you know if and if we might have missed the treatment window the ideal treatment window by a day or two then we’re not going to see much of an effect so what we wanted to do is we wanted to look at a less severe model as well as our light severe model here we have our rhodopson p23h Mouse and this has a mutation in the rhodopsin gene which is also involved in our Rod photo transduction and this is an autosomal dominant RP model and what happens is this aberrant rhodopsin protein called p23h accumulates in the cell and causes a reduced level of phototransduction um we end up with a much slower rate of degeneration uh by around P35 so 35 days of age in these mice the rod outer segments which is like the top portion of the rod cell they begin to shorten um and then by around two months of age p63 we do see that there is fewer Rod nuclei found so um here we ask can SAHA potentially improve Vision in this less severe rhodopsin Mouse model
um so because we’ve never worked with this particular model in our lab we only just got it in last year we wanted to do a little bit of validation ourselves to check that what we’ve read online in different papers is also what we’re seeing in our Mouse model and we see here this timeline of degeneration we have uh one month and two months which look quite similar to each other and then we start to see around this three month Mark that are out in outer nuclear layer which is Our Kind of Blue um the blue staining is saying to decrease in size um four months and six months do look quite similar as well to three months and we can see here in our figure that there is actually a significant reduction in the outer nuclear layer thickness between two and three months
um we also looked at
electroretinogram responses so this is the ERG response and this basically detects the um cell’s response to a light stimulus we have our scotopic a wave responses so our dark adapted response which looks at the rod photoreceptors mainly and we see that around this p60 mark so that’s two months of age there is a significant drop in our in the mouse’s response to this light stimulus we also see a similar thing for our scotopic B wave at p60 at two months and our scotopic B wave is a little bit more involved in some of the other cells in the retina such as the bipolar amacrine and Muller glia cells we also had a look at the photopic A wave so that’s our cone photoreceptors and their response and we see that this kind of drop in function is a lot later it looks to be more around this four month mark here same thing as well with our photopic B wave
so what we did next is um because we identified that there seems to be quite a decrease in the outer nuclear layer thickness at two months of age we wanted to administer our treatment at this age to see if we could protect this uh this loss so at this point we’re looking a little bit more at rods because by this age the cones are not as affected as the rods and we see that if we apply our drug at two months of age we can and we collect four days after we can actually see an increase in the outer nuclear layer thickness as we can see in the images just here um and we can also see with just the diagram on the right So currently this is our preliminary work we’ve only done a small number of biological replicants but we are increasing them at the moment and we’re seeing a really lovely trend towards the fact that SAHA may be beneficial in this model
um so just to summarize what I’ve talked about so we are looking at this drug SAHA and we’re thinking that it actually does have very um a very good potential neuroprotective effect in both of our models of RP so we noted a significant retention of the outer nuclear layer thickness in both of these models after being treated with the drug um so treatment with SAHA in the rd1 model also appears to reduce the retinal stress marker GFAP but we don’t unfortunately see much of an effect when we look at 12 days after treatment so they seem to be lost by this point looking at ongoing work for this um because it’s still a little bit of work in a pro in in process especially the rhodopsin line data what we want to do is we want to assess the cone numbers after treatment as well especially in the rhodopsin line um so obviously now rd1 Mouse it can be quite hard to do that because you could see in this slide before just how degenerated that retina is um but we also wanted to assess if multiple injections of SAHA will increase that window of time that we can prevent this loss of cells and we also are looking at assessing any neuroprotective effects of our drug in other models of IRD because this overactivity of HDAC
seems to be prevalent in quite a few different models of IRD and especially I’m looking at some achromatopsia models for that purpose
um so just a quick thank you to everybody in my lab who’s contributed to this work and my co-um investigators on this grant so uh Dr Livia Cavahlo and Dr Rabab Rashwan um and thank you all for listening and thank you all for being patient during my technical problems at the start
thank you so much um Annie for that and I’m glad we finally got it to work in the end um I want to thank both our speakers both Rick and Annie and thank you to all your um all of you for coming along um we’ve got a little bit of time left just at the end now for some questions um if you’d like to ask a question you can type it into the Q and A section at the bottom of your screen or you can use the raise hand option at the bottom of your screen and we will respond to those in the order and that which they appear when we say your name we’ll enable your microphone and you’ll be able to ask your question you’ll just need to unmute yourself at your end and then after your question has been answered we’ll turn your microphone off again to raise your hand you can select the option at the bottom of your screen or you can use alt y on the keyboard if you’re on the telephone you can press star 9 and if you’re on the telephone to unmute your microphone you need to press star six and you can also use the unmute button on your screen or just press the space bar alternatively just put a question into the Q a section so if people have questions um we’d love to hear from you and um I’ll while we’re waiting I’ll um I’ll ask a question um so Annie I just wanted to get a sense is the idea just that um because these um effects run out that you’d have to the in in real life this would be an ongoing treatment this would be something that you’d have to keep treating to keep the levels of the drug at a certain level or something so that you would have an effect is that the is that what how that would work yeah so definitely um we see this a lot when it comes to seed treating macular degeneration so a lot of people have to go into the clinic about once a month to get a Top-Up of the drug that they receive by intra vitreal injection because the drug doesn’t last forever once you put it into the eye other studies do look at taking things via say like a tablet which would mean that you don’t have to go into the clinic all the time but it’s less effective in that form um so for that kind of purpose we do look at multiple injections and also looking at some other technology that might you know be able to provides the drug on a continuous basis such as some nanoparticle technologies which are coming up in um in the field at the moment and when you say Nano particle Technologies how would they be administered um so they get administered similarly they can either be done by an intravitreal injection um or a sub-retinal injection which is just a more complex in uh surgery that subretinal one um but then once it’s been injected it can then continuously um Supply it to the retina hopefully okay great um I had another question um you were talking about the different breeds of mice that have these conditions I’m assuming that there are a whole lot of other breeds of mice that have been bred to express the genes from for achromatopsia or um other things is that correct yes that’s correct so we have quite a few Mouse models in our lab at the moment that are achromatopsia lines and that’s actually where I predominantly work and we test a lot of these drugs in those lines too fantastic um okay I’m not sure if anyone’s does anyone else have a question I can keep going but I feel like it should be um other people should have a go Rick yes yeah God question just just extending from your first question so so it looks like uh for HDAC inhibitors you have to give a continuing treatment almost for a whole life so we know that a lot uh inherited retinal disease it’s start retinas going to start to degenerate since they’re very young so HDAC inhibitors actually can be inhibit a lot the the the gene which is contribute to the development so how you can make the balance between not to suppress about the gene contributory development while are you protect the regional degeneration at same time for a very young kids
um so that’s definitely a good question we haven’t um exactly considered all those things because you’re right it’s a very complex drug to provide to somebody because it does lots of different things to the cell there’s different expression at different ages but it’s definitely a sign that we would consider in the future if we were to bring this towards clinical trials or potentially look at it in our Mouse models before we do that thank you thanks I’ve got a couple of questions um Rick has asked um is SAHA an abbreviation and what’s in it um so SAHA is an abbreviation I don’t know the full name because it’s a very sign scientific name um and it’s also a very complex chemical so I can’t exactly tell you the like exactly what’s in it but yes a HDAC inhibitor so that’s basically the kind of purpose sort of function of the drug and another question from Julia Hall um she wants to know is HDAC an expensive treatment for example in its current use in other diseases is um and what would be the dosage comparisons that they use and that you tested for efficacy
um I also haven’t checked the price of that but that’s a very uh good question I would say that it wouldn’t be too expensive because um even when we buy it just to do our stuff in our mice it’s actually quite a cheap drug it wouldn’t be a therapy that would come up something like Luxturna where you’re playing oh where you’re paying you know um hundreds of thousands of dollars for it it would probably be a relatively cheap one well that’s that’s the almost the Holy Grail isn’t it if you’ve got something that works for all the conditions that it doesn’t have to be as specific as something that say Rick is working on it gives a protection against all the conditions definitely the Holy Grail yeah um okay well I don’t know that there’s anybody else if you know if no one else has a question I think we might close there um so I just want to say thank you both very much for your time today and um it’s just so fantastic to hear about these exciting new developments and think about where we’re heading uh in the future um so um thank you to you and thank you to the participants for coming and I hope you found it was informative if you still have more questions please don’t hesitate to contact us and we can try and contact our presenters and put you in touch with them um now when you leave the webinar you’ll be redirected to a very short survey um just so that you can give us some feedback on this session and it gives us really important information about planning for the future so we’ve been recording this session and we’ll send out a copy of that recording in the next or a link to that recording in the next couple of days and we’ll also put it on our YouTube channel and on our website um so uh thank you all very much coming for coming that’s the end of the webinar and enjoy the rest of your day
thank you
Summary of the webinar
A summary of this webinar is available to view here.
Retina Australia is delighted to welcome two of our recent research grant recipients, Associate Professor Guei-Sheung (Rick) Liu and Annie Miller, to present some of the results of their latest research.
RNA base editing strategies as potential therapeutic of inherited retinal dystrophies
Presenter: Associate Professor Guei-Sheung (Rick) Liu, Centre for Eye Research Australia.
Associate Professor Guei-Sheung Liu will share updates from his team’s work into a new class of gene editing technology- RNA base editing for IRDs. RNA base editing is designed to correct single-base mutations in RNA transcripts. His team has designed and validated a newly developed RNA base editor (CRISPR-dCas13e) for the editing of the common single-base mutation found in two types of IRDs. The outcome of the study would be a significant step toward a viable treatment for inherited retinal degeneration by using RNA editing technology.
Neuroprotective effect of SAHA in Retinitis Pigmentosa. Do time and frequency matter?
Presenter: Annie Laura Miller, PhD Candidate and Research Assistant, Lions Eye Institute, Perth
Co-investigator, Annie Miller, will report on this research grant project that was awarded by Retina Australia and completed in 2022.
This study aimed to develop a broad treatment approach for Retinitis Pigmentosa (RP), a genetic, blinding retinal disorder, by testing an FDA-approved HDAC inhibitor (SAHA) to protect photoreceptor cells and preserve visual acuity and daylight vision, regardless of the underlying mutation causing the disease. The ultimate goal was the development of gene-independent treatment strategies that preserve visual acuity and daylight vision in RP patients and several different types of vision loss, benefiting a more comprehensive range of patients.
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October 2023
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May 2023