This PNAS study describes the development of EKV, a humanized dividing cell line that propagates bona fide sporadic CJD (sCJD) prions, and the Human Prion Assay (HPA), a cell-based method that quantifies infectivity with sensitivity comparable to transgenic mouse bioassay while enabling rapid therapeutic screening.
0:00Welcome to Base by Base, the papercast that brings genomics to you wherever you are. Thanks for listening, and don't forget to follow and rate us in your podcast app. So imagine a disease that is um, 100% fatal.
0:12Right. It's a terrifying thought. Yeah, it progresses incredibly rapidly. It attacks the brain, stealing memories, movement, and then ultimately life itself. And the truly chilling part, it isn't caused by a virus.
0:25It's not a bacteria. It is caused by a rogue, misfolded protein of pria. Which completely defies how we usually think about infectious diseases. Exactly. And for decades, scientists have been completely unable to grow the human version of these rogue proteins in a standard laboratory dish.
0:43Like we have been essentially flying blind, and that has totally bottlenecked our search for a cure. It really has. It's been a massive roadblock for the whole field. So as we get into this deep drive.
0:52I want you to ask yourself. What really happens when we finally bridge a decades old gap in neurodegenerative research? And, you know, how could growing human prions in a scalable cell culture completely change your ability to cure them?
1:05It is such a profound question. And um, the answer really has implications that stretch far beyond just one single disease. Because when we can finally observe the pathogen clearly in a controlled environment, Well, we can finally figure out how to dismantle it.
1:21Today we celebrate the work of Akin Nihat, Perinida Aurora, John Collins, Parmjit S. Jatt, and their international research team, who have advanced their understanding of human prion biology by successfully developing a scalable dividing cell model.
1:37It is just a remarkable achievement. I mean, to truly appreciate what this research team pulled off, we need to understand the specific biological anomaly that we are dealing with here. Right, so let's lay the groundwork.
1:48Yeah, so the focus of this research is sporadic kretzfelt jock of disease, commonly referred to as SCJD. It is the most frequent human prion disease globally. I mean, frequent in the context of prime diseases, right.
2:00But still incredibly rare in the general population. Oh, absolutely. Very rare. But as we mentioned, it is uniformly fatal. So what actually makes a normal protein go rogue like this? I think that's the part that's so hard to wrap your head around.
2:11Yeah, the mechanism is really what sets pryons apart from almost every other pathogen out there. We all naturally produce normal cellular prion proteins in our bodies, particularly, you know, throughout the nervous system in the brain.
2:25In the literature, these are designated as PRPC. And they have a specific three-dimensional shape largely made up of these coiled structures called alphaeluses. Right, so they're normally all coiled up and doing their job.
2:36Exactly. But in SCJD, a spontaneous event triggers this normal protein to misfold. It basically reconfigures into this rigid, flat structure that is dominated by beta sheets. Oh, wow. And this disease associated highly resistant form is known as PRPSC.
2:54And, um, the defining characteristic of PRPSE is that it is auto catalytic. Wait, hold on. Let me make sure I'm picturing this right. Auto catalytics. So it isn't reproducing by making copies of itself using DNA or RNA, like a virus would, right?
3:09No, not at all. It doesn't have any genetic material of its own. So it is physically converting the healthy proteins around it. That is the absolute core of it. It acts as a physical template. So when a misfolded PRPSE bumps into a healthy, normally folded PRPC, it literally forces that healthy protein to unravel and refold into the rigid diseased shape.
3:31That is terrifying. So now you have 2 rogue proteins. Right. And then those 2 bump into 2 more, creating four, then eight, it triggers this catastrophic biological domino effect. Wow. The misfolded proteins just clump together into these toxic aggregates, which ultimately leads to massive, irreversible neuronal death.
3:50So the protein itself is the infectious agent. And, you know, looking at the bigger picture here, this specific method of spreading one misfolded protein corrupting others, that mechanism isn't completely isolated to SCJD, is it?
4:03No, it's not. We are increasingly realizing that this mechanistic parallel exists across several major nerd degenerative diseases. We broadly categorize them as proteinopathies. Right, like Alzheimer's.
4:13Exactly. Alzheimer's disease involves the misfolding and propagation of amyloid and tau proteins. Parkinson's disease is driven by the misfolding of alpha synucleine. So they're all kind of following this same rogue template rule book.
4:24They are. And furthermore, just like these human prions, those other proteins seem to form distinct strains. Strains, like how viruses have strains. Sort of, but with proteins, a strain simply means the protein can fold into several different 3D conformations, and depending on the specific shape it takes, it causes entirely different clinical symptoms in the patient.
4:45That is fascinating. Yeah. So if we can map the fundamental rules of how prion strains propagate, we might unlock the underlying rules of Alzheimer's and Parkinson's as well. But to map those rules, you need to be able to actually study the proteins up close.
5:00And from what I understand, that has been the massive historical roadblock here. I mean, if human cells absolutely refuse to grow these prions in a lab. How on earth do you study them? You can't just force a human cell to cooperate.
5:12Historically, researchers just could not do it. When they attempted to infect standard human cell lines with human prions, The infection would simply, while it would just die out after a few cell divisions.
5:23Really? It just wouldn't stick. Yeah. The cells would either clear the misfolded proteins, or they just wouldn't provide the right environment to sustain that domino effect. So to study the disease or to screen potential drugs, scientists were forced to rely on whole animal models, specifically transgenic mice.
5:42And running experiments on a whole living animal is I mean, that's a completely different universe compared to growing cells in a little plastic dish. Oh, the logistical and temporal hurdles are just immense.
5:55You need highly specialized, prohibitively expensive bio safety containment facilities. You have to maintain colonies of these transgenic mice, you know, carefully managing their genetics and their housing.
6:06That sounds incredibly resource intensive. It is. And even then, after you expose the mouse to the pryon. It can take up to 2 years, sometimes 500 days or more just to get the results from a single biological essay.
6:17500 days for one result. Imagine trying to test 1000s of potential drugs when each test takes 2 years. Exactly. not feasible. That sounds like an agonizingly slow way to do science, especially when, you know, patients don't have years to wait.
6:31But what about those rapid diagnostic tests I've read about? Like uh, RTQIC. They seem to deliver results much faster. They do, yeah. And they are brilliant tools for clinical diagnosis. So, RTQYC stands for real-time quaking induced conversion.
6:48Right. It uses a vigorous shaking to literally force the prion proteins to interact and clump together in a test tube. And then we use fluorescent dyes to measure that clumping. But there is a massive caveat here.
7:00Okay, what is it? Those assays measure the biochemical seating activity of the protein, they do not measure actual complex biological infectivity. Okay, let's unpack this for a second. Let me look it this way.
7:12Screening drugs with RTQIC is kind of like testing a fire extinguisher on a photograph of a fire instead of actual flames. Oh, that is a perfect analogy. Right. Because you might see the image distort or wash away, but you aren't measuring your ability to put out a real spreading inferno.
7:28You are measuring the real infectious agent. That analogy holds up perfectly, yes. You are measuring a forced chemical reaction in a tube. Not the incredibly intricate biology of a living breathing cell actually taking in a pathogen, sustaining the replication and slowly getting sick.
7:44Which perfectly explains why so many supposed anti-preon drugs that looked like incredibly promising in those surrogate rapid tests ended up completely failing when they finally made it to human clinical trials.
7:58Exactly. They were putting out the photograph, not the fire. Wow. So the field desperately needed a dividing cell line that could sustainably replicate genuine, infectious human SEJD prions. They needed a system that could be grown infinitely screen rapidly and trusted to actually reflect human biology.
8:17And that is the monumental task this team set out to accomplish. Well, let's get into the methodology then, because what they did here isn't just finding a cell that worked. They basically had to custom build a biological chimera.
8:27They really did. So they started with a cell line known as CEFI. These are mouse brain cells, cataclym energic cells, to be precise. And in the prion research world, Sat 5 cells are actually famous because they are exceptional at harboring and replicating non-human prions like those from sheep or from other mice.
8:45Wait, murine, meaning mouse related. If we are trying to cure a human disease, Why on earth are we starting with mouse brain cells? I know, it seems completely counterintuitive, but the rationale is based on something called cellular cofactors.
8:59Cellular co-factors, meaning what exactly? Well, there are these hidden, unidentified molecules or internal environments within a cell that are absolutely required for pryons to replicate. And whatever those secret ingredients are, standard human cell cultures seem to just lose them when they're grown in a lab.
9:17Oh, interesting. But the mouse cells don't. Right. Sat of 5 mouse cells retain them. So the researchers hypothesize that the sati 5 cells possess the optimal, broad cellular machinery. They have the fully functioning factory.
9:28They just needed to be retooled to process human raw materials. Got it. So the mouse cell is the factory building. But before they could start manufacturing the human protein, they had to clear out the old mouse assembly line, right?
9:40They did. If they simply added the human prion gene, while the endogenous mouse prion proteins were still present, they would experience something called dominant negative inhibition. Okay, think of it like trying to build a complex 3D puzzle, but someone keeps throwing in pieces from a completely different puzzle.
9:57The mouse proteins physically get in the way of the human proteins trying to misfold and lock together. A very clear way to visualize it. Yeah. So to prevent that interference, the team used RNA interference technology, specifically short hairpin RNA, to essentially send a stop order to the cell's genetic factory.
10:16So they just shut down the mouse protein production. Exactly. They silence the mouse PRP expression, creating a knockdown cell line, which they named KDB3. So now they have a blank slate, a functional mouse brain cell factory with the right environment, but 0 pry on proteins of its own.
10:31Then they introduce the human blueprint. They reconstituted these blank slate cells with the human PRNP gene. And specifically, they utilized a human variant that codes for the amino acid Voline at Codon 129.
10:44Code on 129. Yes, and we will explore why that specific genetic address is so vital to the results a bit later. Okay, we'll pin that. And they couldn't just drop the human gene in and hope for the best, right?
10:55They had to give it a, like, microscopic GPS tracker. They did, yeah. To ensure the newly synthesized human prion protein actually trafficked correctly through the marine cell and anchored to the outer cell membrane where it needs to be, they attached a mouse signal peptide to the human sequence.
11:13Wow. So they engineered a truly humanized marine cell. Yes. But even with this brilliantly engineered chimera, it wasn't a guarantee they'd catch the infection, because, as we mentioned earlier, we still don't know exactly what those magical cellular curve factors are.
11:29Right. You're still working on the dark a bit. So they had to play the numbers game. They faced a massive stochastic challenge. The cellular environment is probabilistic. It very slightly from cell to cell, even within the same dish.
11:40Since they couldn't intentionally design the perfect environment, they used iterative single cell cloning. What does that look like in practice? Well, they took their engineered cells, diluted the liquid culture down so drastically that they could isolate 100s of individual single cells in separate wells, and grew them into isolated clonal colonies.
12:00That sounds incredibly tedious. Very. Then they exposed every single colony to human SCJD brain homogenate to see which ones would propagate the disease. It's less like engineering a specific machine and more like planting 100s of seeds in slightly different soils, just waiting to see which one magically sprouts the exact plant you need.
12:20That's a great way to put it. And out of all those colonies, they found one that sprouted. Clone 81 F9. Okay, so they found one. Yes. It reproducibly propagated modest amounts of the disease associated and misfolded protein, but the levels were too low for robust assay, so they didn't stop.
12:35They took 81 f 9 and subjected it to the single cell cloning process again and again. So they were effectively directing the evolution of the cells toward higher and higher susceptibility. Exactly. Iterative evolution in a dish, taking the strongest performers and breeding them, essentially, and that extensive process led them to the 4th generation subclone, which they...
12:59I remember the numbers being pretty crazy, like EKV accumulated 5 to 14 times more of the misfolded PRPSC protein than that earlier 81 F9 generation. Yes, the directed enrichment worked beautifully. The numbers are definitely impressive.
13:13Yeah. But, you know, how do we know they didn't just engineer a cell that naturally pumps out inert, harmless protein clubs? Like, how do we know this EKV cell is producing truly infectious lethal prions?
13:25That is the big question. And this brings us to the most critical validation in the entire study. To prove they had captured genuine infectivity. They had to perform the gold standard in vivo validation.
13:37They took the lysates, basically the disrupted, broken open contents of their infected EKV cells. And crucially, they did this after passing the cells through multiple generations to guarantee absolutely none of the original human brain tissue from the initial exposure was left.
13:51So it's purely the lab grown pryans at this point. Exactly. Then they injected those cellicates directly into the brains of humanized TG 152C mice. Okay, and what happened to the mice? 100% of the mice injected with the cellisate developed terminal prion disease.
14:08Wow, every single one. So it definitely wasn't an anomaly. The consistency was absolute. And the details of how the disease manifested are really what confirmed the success. The mean incubation period was 251 days.
14:20Right. And when the researchers examine the neuropathology under a microscope, looking at the actual tissue structure of the mouse brains, They found widespread spongiosis. Meaning the brain tissue is literally riddled with microscopic holes, making it look like a sponge due to massive cell death.
14:38Yes, exactly. In addition to that evacuation, they observe dense synaptic prion protein deposition, intensely clustered in the thalamus and mid-brain regions. And that specific pattern means something.
14:49It means everything. The specific anatomical pattern of the brain damage and the biochemical signature of the proteins extracted from those mice was functionally indistinguishable from a control group of mice that had been injected with actual raw human SEJD brain tissue.
15:06Oh, wow. So the pryons grown in a plastic dish, and the pryons pulled directly from a human brain act exactly the same in a living organism. Precisely. They captured the true disease. That is just phenomena.
15:18It is. And once they validated that the EKV cells were churning out genuine, infectious pryons, They immediately scaled the technology. They adapted the cells into a 96 well format, creating what they termed the human prion assay, or HPA.
15:32A 96 well played. Instead of managing a massive room full of mice feeding them, monitoring them for a year and a half , You have a little plastic tray you can hold in one hand, running 96 separate experiments simultaneously.
15:43The sheer efficiency is mind blowing. I mean the sensitivity is equally staggeling. The team demonstrated that the HPA could successfully detect SCJD prions from crude human brain homogenates, even when the sample was diluted 100,000 fold.
15:56Yeah, that is finding a microscopic needle in a biological haystack. So how did the math stack up when they pitted this new plate against the old animal models like side by side? They ran a side-by-side endpoint titration.
16:09They use the exact same sample and calculated the infectious tighter using both methods. The gold standard mouse bioassay calculated a tighter of 5.88 log units. Okay. The new EKV human prion assay calculated 5.66 log units.
16:26Wait, they practically mirror each other. But one takes a fraction of the time and resources. Exactly. The mouse bioasso required dozens of animals and took roughly 500 days to reach that conclusion. The human prion essay delivered the exact same quantitative measurement in a matter of weeks.
16:42That is a total paradigm shift for the pace of neurodegenerative science. And I mean, this isn't just about detecting the protein faster. It's about finding a cure. Were they able to use this to test any actual treatments?
16:52Yes, to demonstrate the platform's viability for drug screening, they develop persistently infected lines designated as IEKV. These were cells that maintained a stable, vigorous prion infection over 15 continuous passages.
17:05Wow, so they essentially engineered a renewable, endless source of live human prion infection that they could tap into at any time. An infinitely renewable target. So they have the disease locked in a dish.
17:18Did they try curing it? They did. They treated these persistently infected i.e. KV cells with a known anti-pryon antibody called ICSM 18. And the treatment was a complete success. The established infection was cured, and the cell ceased producing the rogue PRPSC proteins.
17:36Which confirms the ultimate goal of the platform. You can establish the disease in these 96 wells and then systematically drop 1000s of different pharmaceutical compounds into those wells to see which one clears the infection.
17:48Exactly. It replaces unreliable surrogate testing with high throughput, genuine biological infectivity testing. We can finally evaluate experimental therapeutics against the real human pathogen at scale.
17:59That promises to drastically accelerate the drug discovery pipeline for SCJD. It's a phenomenal leap forward. But, you know, biology is notoriously messy, and cutting edge models rarely come without caveats.
18:12What are the limitations here? Where does this new platform fall short? Well, the authors are commendably transparent about the model's current limitations. The most prominent is what the field refers to as the mismatch issue.
18:23Ah, okay. What does that mean? Well, earlier, we noted that the human PR and P gene, they inserted into the EKV cells, specifically coded for the amino acid villene at Codon 129. Right. The genetic address, every human inherits 2 copies of this gene, one from each parent.
18:39Exactly. At code on 129, a person can inherit either messianine or viline. If you have tubilines, your genotype is VV. If you have 2 methenians, you are M. Okay, I'm following. Because the EKV cells were engineered with Velene, they are functional EVV.
18:55The researchers found that while these cells enthusiastically replicated prions from Etsy JD patients who were also VV, or even heterozygis envy, they struggled significantly with the others. Meaning they did not efficiently replicate prians from patients who were methian homozygis or MM.
19:12Let me make sure I'm fully grasping this. Even though the prion protein is fundamentally the same overall structure, just one tiny amino acid difference at a single location means the cell won't catch the infection.
19:22It perfectly illustrates one of the most fascinating biological truths about prion strains. A prion strain doesn't just exist as one rigid shape. It exists as a cloud of slightly different confirmation.
19:35And the host sells genetic makeup, whether it provides methanine or violine at that exact spot, acts as a lock. The prion shape is the key. see. If the key from the MM patient doesn't fit the VV lock of the cell, that specific cloud of rogue shapes cannot take hold and replicate.
19:54So it's a feature of the biology, not a failure of the essay, it proves just how agonizingly specific these protein interactions are. Precisely. Though it does mean the team will likely need to engineer a complementary MM version of these cells so they can screen drugs for all types of patients.
20:09Oh, absolutely. And the team acknowledges that exact necessity. They're already working toward complementary cell lines to bridge that gap. But there was one other vital nuance, the study uncovered regarding occult or hidden infectivity.
20:21Hidden infectivity. Where was hiding? Remember the KDD 3 cells, the blank slate mouse cells where they had deleted the mouse prion protein, but before they added the human gene? Right, the functional factory with no raw materials.
20:33They shouldn't be able to misfold anything. As a stringent control measure, the researchers expose those blank slate cells to infectious human prions, they then passage the cells 6 times. Meaning they let the cells divide, wash them, move them to a new dish, and repeated this 6 times to dilute and wash away any of the initial brain sample.
20:53Exactly. Then, they injected those cells into a mouse. And astonishingly, one of those mice developed terminal prion disease. Wait, if the blank Slade cells couldn't replicate the prion, how on earth did the mouse get sick?
21:07It indicates that a microscopic residual amount of the original infectious human brain homogeneate physically clung to the cells or the plastic dish? You're kidding. Nope. It didn't replicate because the cells lacked the protein to do so, but the original prion survived through 6 rounds of division and transfer.
21:24It's just it's terrifyingly resilient. It highlights the extreme resilience of these proteins, and it serves as a stark warning to researchers. Future drug assays will require extended passaging and rigorous controls to guarantee we are measuring newly generated prions rather than just stubborn leftovers from the initial exposure.
21:42Wow. So when we step back and view the entirety of this research, What's the ultimate takeaway here? By iteratively engineering and blindly selecting highly susceptible dividing cells, this team has established a robust, scalable platform capable of propagating authentic human SCJD prions.
21:59This breakthrough bridges a decades long translational gap in neurology. It transforms an agonizingly slow, animal dependent testing process into a rapid, precise cell-based assay. Which is the exact tool set required to turn biological mysteries into curable diseases.
22:15But, you know, this leaves you with something entirely new to Ponder. We've just discussed how this platform relies on matching the exact genetic lock and key of the prion strain to the cell. If we can engineer cells this precisely, could we one day see a world of truly personalized medicine, a future where a patient's own skin cells were taken, reprogrammed into brain cells, and dropped into these 96 well plates, to find a bespoke customized cure tailored exclusively to the unique misfolded proteins in their specific brain.
22:46The possibilities for tailored therapeutics are just staggering. This episode was based on an open access article under the CCBY 4.0 license. You can find a direct link to the paper and the license in our episode description.
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