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Seventeen Minutes of Science: From Bench to Bedside: Using Model Organisms to Find Rare Disease Treatments

Tune in weekly to our virtual series “Seventeen Minutes of Science” every Tuesday at 11am PST / 2pm ET where we go live on Facebook with a new guest each week to talk about how science and biotechnology is woven into their lives for (you guessed it) 17 minutes!

For our 30th episode of 17 Minutes of Science, we were joined by Sangeetha Iyer, a senior scientist at Denali Therapeutics, a biotechnology company focused on the discovery and development of therapies for patients with neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, ALS and others. Sangeetha has a background in neurodegenerative disorders and rare genetic diseases, and before she joined Denali she was working at Perlara BBC where she also focused her research on finding treatments for rare diseases. Recently, Sangeetha helped to published a paper (Repurposing the aldose reductase inhibitor and diabetic neuropathy drug epalrestat for the congenital disorder of glycosylation PMM2-CDG). The findings from this paper have been used in an n of 1 study that has so far been showing positive results for a young girl. Sangeetha will be joining us to talk more about her work in the rare disease field, her recent paper, and the benefits of using model organisms for rare disease research. Learn more about using model organisms in your own research


Sarah Cheesman (Host): [00:00:00] Hello everyone and welcome to 17 Minutes of Silence. My name is Sarah Cheesman, I’m a technical solution scientist at InVivo Biosystems and I’m delighted to be back today where we have a very interesting topic, discovering a better way to treat rare diseases. And my guest is Dr. Sangeetha Iyer, and she is joining us from warmer climates down in the Bay Area today. So we will be discussing her paper that appeared last year in Disease Models and Mechanisms. And the title of this manuscript is Repurposing the Aldose Reductase Inhibitor and Diabetic Neuropathy Drug Epalrestat for the Congenital Disorder of Glycosylation PMM2-CDG. It’s kind of a mouthful. She’s going to tell us all about it because there’s some acronyms in there. Let me go ahead and briefly introduce Sangeetha and then turn it over to her for a moment. She can tell us a little bit more about herself, but she received her Ph.D. in molecular pharmacology from the University of Pittsburgh and then went on to do postdoctoral research UT of Austin. And so she’s had over 10 years of experience in model and assay development and drug screening for human genetic disorders. So in her last role at Perlara, which is where this manuscript originated, Sangeetha developed nematode models of rare disease and conducted several successful screen campaigns for rare disease, one of which is a Phosphomannomutase 2 deficiency, which is what we’re talking about. And these efforts led to a clinical trial with a child, which was successful and is currently being expanded to include other patients. So welcome, Sangeetha. I’d love it if you’d say a few words about yourself.

Sangeetha Iyer (Guest): [00:01:42] Hi, everyone! Sarah, thanks for having me. This is a wonderful opportunity. I’ve been a fan of the 17 Minutes of Science, so it’s great to be here. So to add to what you just said, I started my career with mouse models, worked on xenopus oocytes, drosophila, C. elegans, and now I am still continuing some of my training, so I still work in model and assay development for genetic diseases. I’m currently at Denali Therapeutics and our focus continues to be neurological and rare genetic disorders. So, yeah, but I’m really excited to talk today about some of the work that we did at Perlara with PMM2, and it is a mouthful – possible phosphomannomutase 2 deficiency, but I’ll let you take over.

Sarah Cheesman (Host): [00:02:39] Thank you! And it’s great to meet somebody who has so many model organisms in her quiver, you might just take the prize for everyone we’ve talked to on this show, having that much experience across such a broad, broad range! As we do every time I’ve started my timer. So that will keep us honest on the 17 mark. But you don’t have to worry about it, I will pay attention. So let’s jump in and talk about what inspired you to take an interest in this disease, the PMM2-CDG.

Sangeetha Iyer (Guest): [00:03:10] Right. So while I was at Perlara, we had started working on lysosomal storage disorders to begin with and we had just done some work on one glycosylation disorder that had been on our radar for a little bit was a newly discovered disorder called n kinase 1 deficiency, and coincidentally, we were introduced to the parents of a child with PMM2 deficiency, another congenital disorder of confirmation, actually the most common congenital disorder glycosylation. It was discovered sometime in the nineteen eighties and there’s been a fair amount of work published on it, but surprisingly, no therapeutic opportunities for the disease. Some model system work, but not a whole lot and we felt that it fit very well with our platform of model organisms. Perlara was working with yeast model systems, drosophila as well as C. elegans along with the basic model organism pipeline and on the other hand, we had patient fibroblast, which were also available for PMM2. So PMM2 basically came onto our radar because of Maggie, the girl who has PMM2 disorder. And after meeting with her parents and having some conversations about the utility of our platform, we decided to go ahead and model some of our mutations and see if we could conduct a drug screening campaign. That’s how that program came to be.

Sarah Cheesman (Host): [00:04:49] Ok, so based on the story and persistence of one family who were living through this with their child. And then, so tell us about how you ended up investigating, repurposing the Epalrestat in particular.

Sangeetha Iyer (Guest): [00:05:08] Right, so when Maggie’s parents and we at Perlara embarked on this, we had not heard of a lot of drug screening campaigns for PMM2 deficiency, and one of the reasons we wanted to evaluate a repurposable library drug screening campaign was we wanted to see if there was a fast path to the clinic that was definitely on our radar. It was also a way for us to make some informed decisions for a potential novel chemical screening campaign, should we want to embark on that in the future. When you carry a smaller chemical space, especially with known mechanism of actions or biased mechanism of actions, at least it does give you some information for future path to pursue. So that was the rationale with which we decided to embark on a road on a repurposable library campaign, and surprisingly, we got some really exciting and interesting results that made that path to a fast track to clinic possible.

Sarah Cheesman (Host): [00:06:20] So then tell us, to step back a minute, so you’re using you’re using multiple models and trying to address the question of how to treat this. So two things then, tell us about about the model systems and how they were employed in this? And then also, I’m curious, how many drugs did you actually screen through? How big was that library? How hard was it to find your needle in a haystack, so to speak?

Sangeetha Iyer (Guest): [00:06:43] Right. So we started out with yeast [and since] yeast model systems for PMM2 had not been generated before so our lab generated a number of these model systems. And in yeast, you can create heterozygous compound mutations which is essentially what patients with PMM2 present as. And so we were able to mark several patient mutations in yeast and found those models to have a growth defect, which then made screening possible. In worm models there had been a homozygous null PMM2 model that had been generated. It was larval-lethal so we were not able to use that one for screening. However, we went ahead and with NemaMetrix, now InVivo Biosystems help, we were able to model another, a different patient mutation, one that does not have this severe lack of enzyme activity. So we modeled a patient mutation referred to as the F119L and these worms were viable, [but] did not have a phenotype and so we had to use a bit of a workaround approach to generate a phenotype. And we ultimately, across wombs as well as yeast, the screening campaign that we conducted was approximately, I want to say, 3,000 or so compounds. So in the screening world, 3,000 or so compounds is not a whole lot. It’s a pretty small library. And I would say that at that point for worms, we had already conducted a 50,000 library screen so we knew it was within the realm of possibility for us to go ahead and do a larger screening campaign. But as we alluded to before, that wasn’t necessary.

Sarah Cheesman (Host): [00:08:48] That’s really cool that you had a smaller pool to start with and there were hits in that.

Sangeetha Iyer (Guest): [00:08:53] Yeah!

Sarah Cheesman (Host): [00:08:54] So just curious to go back to the worm model for a second, just for clarification for people listening. Was that the worm carrying the human gene or was there a mutation in the native gene?

Sangeetha Iyer (Guest): [00:09:05] Yes. So it was a mutation in the native gene. So one of the reasons we believed in the power of model organisms specifically for rare monogenic diseases was because when you have a single gene ortholog and one that has high similarity to what one might encounter in humans, you can model the same mutation as you see in humans, in those model organisms. So for PMM2 specifically, the worms do have an ortholog. It is not referred to PMM2, it’s actually referred to as F52B11.6. It’s a sequence name, but the the protein itself is 54% identical to what you see, what you observe in humans. And most importantly, the common mutation sites were identical. They were conserved in the worm ortholog as well. So we that’s how we engineered these specific point mutations associated with human disease in the worm gene.

Sarah Cheesman (Host): [00:10:11] Ok. Wow, that’s powerful. So even at a 50% similarity, but the key was the conservation in the really important parts, important domains of the protein.

Sangeetha Iyer (Guest): [00:10:20] That’s right.

Sarah Cheesman (Host): [00:10:21] Yeah. That’s so powerful to hear those stories, it brings to light, the connection of the thread of life. So amazing to think about the genetic code in that regard. So now you’re armed with with a candidate that performed well in the model organism, in the yeast, in the lab, and then you moved back to Maggie. So can you tell us about what happened then with this candidate?

Sangeetha Iyer (Guest): [00:10:49] Right. So I’ll give you a little bit more information about the screen itself and how we landed in epalrestat and then maybe we’ll talk about what happened with the patient fibroblasts. So in our initial screening campaign between yeast and worms, we found several molecules of some plant origin, so to speak, right? And we recognized that all the thing that wasn’t common to all of these structures that came out as hits from the screening campaign was that they all appear to have some level of aldose reductase inhibitor activity. So aldose reductase is another enzyme in the polyolar glycosylation pathway. And we found that several of the molecules that we were pulling out all possessed this particular activity. That finding, or that realization, is what led us to hone in eventually on epalrestat for its effect on PMM2 enzyme activity. And so we tested a number of these aldose reductase inhibitors, some of which are available through different vendors. And we recognized that more of them actually continue to increase PMM2 enzyme activity. So let’s go back to the disease for a minute. So PMM2, phosphomannomutase 2 deficiency, arises due to lack of function in the PMM2 enzyme, which is important in the glycosylation pathway. So when you have the dysfunctional enzyme, you have buildup of molecules, macromolecules that are abnormally glycosylated, and they don’t get to go through the appropriate cellular recycling mechanisms, which in turn results in ER stress, cellular stress, so to speak, which then has all of its ripple effects in the clinical manifestations of the disease. So really, until the time that we did this work, nobody had discovered that you could boost PMM2 enzyme activity through some other artificial shunt pathway, which is essentially what our model organism screens were telling us, that there was another way to increase PMM2 activity. And while this was true, we identified that this finding was true, for yeast as well as worm protein, we weren’t certain that this would translate to the human enzyme as well. Which is where the patient fibroblast came in. So once we confirmed that these molecules were functioning, were rescuing the disease defect in yeast and worms by increasing the PMM2 activity, we turned our attention to the patient fibroblasts, which were known to have defective enzyme activity, exposed them to the drug, and determined whether enzyme activity was increased in patient fibroblast as well. And that’s where the final piece of the puzzle came together.

Sarah Cheesman (Host): [00:13:49] Where those Maggies fibroblasts are more generic line?

Sangeetha Iyer (Guest): [00:13:55] So we did both. So Coriell is a non-profit bio repository for patient fibroblast, [so] we were able to obtain some PMM2 patient lines from there. We also worked with Dr. Eva Morava at the Mayo Clinic who had access to Maggie’s fibroblast and tested the finding in those as well. And in both cases, we were able to show that PMM2 enzyme activity was in fact increased when exposed to epalrestat.

Sarah Cheesman (Host): [00:14:26] So how did it feel when you realized specifically Maggie’s fibroblasts were responding to this?

Sangeetha Iyer (Guest): [00:14:33] I don’t think one word necessarily captures, but there was a mixture of surprise, elation, disbelief even! I think part of this journey has definitely been an evolution in terms of how we feel. It’s been amazing to see some of the impact that this progress has had on Maggie.

Sarah Cheesman (Host): [00:15:00] So tell us about that. Once you’ve had this encouraging suite of results and then how you chose to move ahead and how that worked?

Sangeetha Iyer (Guest): [00:15:08] Right, so once we had these findings, we spoke with Maggie’s parents, we spoke with Dr. Eva Morava, who at that point had begun collaborating with us on this particular project, and we started discussing if there were opportunities to actually treat Maggie with the drug. And this is the part of the reproposing story that is incredibly powerful because we found out that epalrestat was not patent protected in the US or anywhere else. It is actually a generic [drug] in other parts of the world. But having been on the market for over 20 years, for another indication, there was a wealth of safety data associated with the use of epalrestat** in humans. And so the doctor, Eva Morava, really led this effort along with Maggie’s parents and Ethan Perlstein, the CEO of Perlara. They put together an n of 1 IND application, which was submitted to the FDA. It was approved because of the trove of safety data that was available [for] epalrestat and the data that he had generated substantiating that epalrestat increased PMM2 enzyme activity in a variety of modern systems. And so this package really led to the FDA approving the use of epalrestat in Maggie, provided we were able to reconfigure the dosing or the formulation necessary for a trial. And so, really, it was the efforts of Ethan Perlstein, Maggie’s parents, Holly and Dan Carmichael and Dr. Eva Morava, who spearheaded that effort. And now Maggie’s been on the drug for close to nine months.

Sarah Cheesman (Host): [00:17:06] Wow. And tell us, how is she doing?

Sangeetha Iyer (Guest): [00:17:10] There are several reviews of Maggie online. And for those who are interested, you should definitely take a look because seeing is believing. And I think that was very much the part of the process that was rewarding for me, because until then, we are definitely seeing this in theory, we’d seen this in cells and model organisms. But to see a drug have an impact, a positive impact, on the child, in a child was just incredibly powerful. So prior to being on epalrestat Maggie had limited verbal skills, she had some motor difficulties as well. So not very coordinated, not able to be independent in some of those abilities, so to speak. And having been on epalrestat, she has steadily gained weight. Her communication skills have really blossomed, prior to being on epalrestat, Maggie’s mom, obviously parents are able to communicate with their children no matter what, but after being on epalrestat, Maggie’s mom was able to have conversations with Maggie, which was a huge leap in her abilities. And her motor coordination skills have also steadily improved and there are some great videos of her using a bicycle to get around. It’s just incredibly powerful, very rewarding to watch.

Sarah Cheesman (Host): [00:18:48] Wow! That was our timer. That was 17 minutes, like that, poof! And I just want to say thank you for sharing this remarkable story of what we always lump into that category of “bench to bedside” and to think of all the different models along the way that made this possible. I hope that we can post some of the videos you’re talking about to link to this conversation we’re having today, so anybody who stops by might be able to see that and share in that excitement that you’re expressing. I would love to see them. Sangeetha, it’s been such a treat. Thank you so much for for telling us about this story and all the best as you’re continuing to work in this really important, rare disease space.

Sangeetha Iyer (Guest): [00:19:32] Thank you. Thank you so much for having me. And thank you for the opportunity to spotlight some of the amazing work with worms and repurposable strategies.

Sarah Cheesman (Host): [00:19:44] We look forward to more stories like that. And thanks everyone for tuning in. And we will see you next week.


About The Author

InVivo Biosystems

InVivo Biosystems provides essential services to help pharmaceutical, nutraceutical, biotechnology companies and academic research institutions around the globe accelerate their research and drug development efforts. An expert in CRISPR genome editing, InVivo Biosystems creates custom genome edited C. elegans and zebrafish models to enable aging and other disease studies. In addition, InVivo Biosystems provides in-vivo analytical services to produce data and insights for companies that need to make go/no-go decisions quickly in early-stage development of new compounds.

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