GEN Protocols Expert Exchanges: Next Generation Techniques to Develop Genetic Medicines
GEN Protocols Expert Exchanges: Next Generation Techniques to Develop Genetic Medicines
ANJALI SARKAR: Hello, fellow scientists and science lovers. This is Anjali Sarkar, senior editor at GEN and GEN protocols, welcoming you to expert exchanges. Today, I'll be talking to Dietrich Stephan, chairman and CEO at NeuBase Therapeutics. Before we begin, I'd like to take the opportunity to introduce you to Gen Protocols. The need for access to reliable and reproducible technical know-how in the biosciences inspired us at genetic engineering and biotechnology news to develop GEN Protocols, a freely-accessible digital hub for scientific methods where researchers from academia and industry can share and showcase their technical expertise, nurture collaborations, and discuss technical challenges and solutions.
ANJALI SARKAR: GEN Protocols is open for submissions year-round. Together with a rich resource of up-to-date protocols and applications, GEN Protocols brings you expert exchanges where experts discuss the tools and techniques that drive biotechnology. In today's expert exchange, we will be talking to Dr. Stephan about tools and techniques that his company, NeuBase, has used to circumvent limitations of the first generation of genetic medicines to develop a new class of small molecule drugs that target root causes of genetic diseases.
ANJALI SARKAR: Welcome, Dietrich. Before we start on the technologies at NeuBase, what are the top methodological advances, in your opinion, in the entire area of drug discovery and particularly in the area of genetic medicines that you believe will have an impact in the years to come?
DIETRICH STEPHAN: Well, the most obvious thing is the sequencing and availability of the genome and variant databases. Such that the industry can actually design genetic medicines to target certain misbehaving genes. So I know it's obvious, but without that foundation, there would be no precision genetic medicines industry, and so that has to be my first answer. Thereafter, I believe it relates to computational sciences to enable us to house all of that information, and query it, and design medicines that don't have or have minimal off targets engagements across the genome and transcriptome.
DIETRICH STEPHAN: And an extension of that, of course, is machine learning and deep learning to be able to do SAR on these genetic medicines in a manner that is actually feasible. There's just too information for-- too much information for a group of scientists to look at these molecules and optimize them. So I think that the availability of infrastructure like that, machine-readable computer packages that allow simple biologists like me to import data and run predictive analytics on them is the second piece of enabling methodology.
DIETRICH STEPHAN: I would say thereafter, I think high throughput paralyzed manufacturing is critical across all of these new therapeutic modalities. If you think about the challenges, for example, in manufacturing mRNA vaccines to distribute them globally for COVID, or antibody drug conjugates, or oligos, or peptides, all of them, I would say, broadly suffer from manufacturing bottlenecks, especially in the screening phase.
DIETRICH STEPHAN: So I think that improvements on that side of things have been great, and there's a lot of work to be done.
ANJALI SARKAR: Sure. Something I find particularly interesting is how one arrives at the decision as to what exactly is to be drugged. So for example, you find a gene-- in a simple example, you find a gene X with a geno function mutation, and you decide to drug an enzyme that specifically degrades gene X, for example. So could you talk a bit about the methods that are being used to decide what is to be drugged in a set of druggable targets?
ANJALI SARKAR: And what role both AI, as you mentioned, and basic research, and the combination of both are playing in identifying the best targets that can be drugged?
DIETRICH STEPHAN: Well, I'll give you my view on this, and I'm not sure it's the generic answer that everyone can follow. But in my world, what we do is we start with word of causality of the disease. So what is the absolute core trigger of the disease? And in almost every case, it's a gene or genes with variants in it. And so, it's the nucelotide change within the gene that causes the disease or contributes to disease.
DIETRICH STEPHAN: So that's where we always start. And then, with our technology, we then ask the question, what does that mutation do to the gene? In your example, gain a function. It activates overexpression of the gene. And so, what we can do is then say, OK, how can we directly reduce the amount of that result in transfer to protein is the first approach. We want to really address root causality before we go one step adjacent and activate a degrading enzyme, for example.
DIETRICH STEPHAN: So we look at the available technologies to reduce expression. So at the DNA level, that would be either genome editing to edit that mutation out or inhibiting transcription, which is a strategy that we at NeuBase believe we can uniquely do. So we can shut down the gene and root causality. You would then say, OK, well, if we can't address it at the DNA level, let's go to the RNA level and do some RNA silencing either via ASO gap murders or SIs.
DIETRICH STEPHAN: I think once you get to the protein level, then you're talking about trying to degrade the protein, as you had suggested, or try to come at it with an antibody to block its function. And at least, in my opinion, when you get that far downstream from root causality, the world gets really complicated. And you're left grappling around the edges for some handhold. It's very hard to find because protein structures are very complex, and you're often now talking about going one step adjacent to the etiologic protein, which is even one further step removed and potentially degrading a target.
DIETRICH STEPHAN: So the way I think about it is, I always start with root causality, try and grapple with pathogenesis there. And then, if I am forced to because of a lack of technical capabilities, then I'll go one step further downstream until I can grab the target.
ANJALI SARKAR: So before we go on to the NeuBase technology, could you share your thoughts on the lessons that we've learned from the first generation of genetic medicines and their limitations?
DIETRICH STEPHAN: It's so amazing that the entire pharmaceutical industry let's say over the last decade has shifted to address nucleic acids, upstream of proteins. And the reason that I'm so excited about it is, and to state the obvious, proteins are really complicated. They're three-dimensional structures. Until a few months ago, we really didn't know how to model them, and they're dynamic.
DIETRICH STEPHAN: They're constantly moving and changing shapes. And so, trying to throw small molecules at them in a high throughput, random manner is expensive, time-consuming, has a low probability of success. And that's why, historically, it's taken us on average a decade and $2 to $4 billion to develop one drug. And that would be OK except for we have to recoup those costs in the marketplace, and so that's why drug prices are so high.
DIETRICH STEPHAN: And the low probability of success has resulted in a remaining disease burden that's significant out there in the world. So it's expensive, and it's low probability of success. So that was the hope and the promise of the genome. Every disease is genetic, so we were going to sequence the genome, figure out all of the root causes, get that information to patients, and then figure out how to drug at root causality.
DIETRICH STEPHAN: Now, the industry has made great strides largely in drugging RNA, and not so much in drugging DNA. And the simple reason is because the genome has found a way to protect itself. It's called the double helix. So it's closed off from insults because it's precious. It's the root of all life and all heritability, and so, it's sacred and protected. So most of the industry has now targeted RNAs one step closer to real causality and largely drug in single-stranded RNAs because they're accessible to these genetic medicines.
DIETRICH STEPHAN: And so, the beauty of that is, we've proven that genetic medicines work in people. Not only for silencing gain of function, so Alnylam and Ionis have led the world in degrading a gain of function on RNAs to cure diseases. But also replacing RNAs, and so these are the Modernas, and BioNTechs, and [? Lorams ?] of the world that can deliver on RNA when the body needs it. And more recently, we've started trying to address the genome, but it's been largely ineffective.
DIETRICH STEPHAN: So gene therapy to replace genes at the DNA levels, antigenic bases by distribution issues. Gene editing at the very earliest stages, but you're trying to deliver large proteins that are antigenic and can't get past the liver. So if I was to encapsulate the excitement of first generation precision genetic medicines, it's that they work. The downside of them are in five or six key areas.
DIETRICH STEPHAN: Distribution in the body. Generally, they don't want to distribute, so we're left with treating the liver or trying to squirt drug into the brain. The second is tolerability. So these are large. Generally, proteins are negatively-charged molecules that trigger the immune system. So two is tolerability as an issue.
DIETRICH STEPHAN: Three is selectivity. Generally, these genetic medicines can't-- first generation genetic medicines can't see point mutations. Fourth is manufacturability, which we've already talked about. They generally have to go build multi $100 million factories. That's simply not scalable. Durability of effect.
DIETRICH STEPHAN: We've learned from gene therapy that durability is a big deal, and the fifth is true scalability of these platforms. And so, if you look at some of my colleagues that have had great early success, they're left drug and delivery because they can't get past it. And so, scalability is an issue. So those are the five or six negative attributes of these first generation genetic medicines. And so we at NeuBase believe we've solved those.
ANJALI SARKAR: So if you could talk about how you have solved starting at, say, the biodistribution angle of drug delivery. How have you tackled charge of the molecule issues? Shuttling it into the brain? And lately, there's been a lot of excitement about Extracellular Vesicles, EVs. Have you used those? What kind of method-based advances have you made at NeuBase to ensure that the drug goes to the right target, past the liver, and actually reaches both systemic as well as organ-specific targets?
DIETRICH STEPHAN: Anjali, thank you for the question, and I would love to be able to, if we have time, touch on all five of those negative attributes that we engineered around. So I'd be remiss if I didn't mention that the first thing we've done is consciously build this platform to, number one, address all mechanistic roots of causality. So gain of function, loss of function, change of function, mutations.
DIETRICH STEPHAN: And so, in one technology platform, we believe we have the capability to consolidate the nascent and fragmented genetic medicines industry. So with respect to biodistribution, that was the first challenge that we grappled with. And so, at the outset, we said, OK. What is limiting biodistribution of early genetic medicines? And it was really size and charge.
DIETRICH STEPHAN: So if you've got a big molecule, often, you can't haul it into the cells or tissues of interest. And so, it's obvious if you're delivering a big protein, but even some of these SI RNAs are tens of kilodaltons in size. And so, that was the first thing we did, and so we developed a genetic medicine that's fully synthetic but has a minimalistic structure. And so, the actual pharmaco cores are about five kilodaltons in size so significantly smaller than other genetic medicines.
DIETRICH STEPHAN: The second thing that we engineered in was a neutral charge. So why does charge limit biodistribution? In general, if you systemically administer a negatively-charged genetic medicine, it will be cleared by the liver via scavenger receptors and immediately be excreted. And so, you want neutrality of charge to be able to get past those scavenger receptors in the liver, and so, that's what our genetic medicines look like.
DIETRICH STEPHAN: Minimalistic structure negative in charge, and then we said, well, we know that if we simply systemically inject that, it will have almost no in-circulation half life and be renally excreted. And so, we developed a proprietary delivery shuttle that allows it not only to get past the liver, but interact with the plasma membranes. Essentially stick to the plasma membranes of every cell in every tissue in the body and be directly trafficked into the cytoplasm.
DIETRICH STEPHAN: So not only is that a non-cell type specific, tissue type specific, non-species type specific delivery modality, but it bypasses the endosome to a large degree, which is also an issue that's plagued early generation genetic medicines. And so, I know it's easy to say that we cracked the biodistribution problem, but there are many nuances that we had to grapple with in developing the correct construct to do that.
ANJALI SARKAR: Does this method allow for the DNA-based drug or the modified DNA-based drug to just attach to the plasma membrane? Or also get inside the nucleus or inside the mitochondria? And how have you worked that out?
DIETRICH STEPHAN: So it's interesting. So our medicines actually, they look very much like an oligonucleotide. So if you could imagine an oligonucleotide with a backbone and nucleobases. The backbone is made of a synthetic chemistry, so it's not a sugar phosphate backbone like our bodies use or a derivatised sugar phosphate backbone like all of our friends in the genetic medicines industry. This is actually a polyamide backbone, and so it's very similar chemically to [? Novolin. ?] So that gives you an intuitive sense of its neutrality, but also why it's so well-tolerated in the body.
DIETRICH STEPHAN: So we add the delivery shuttle onto it, and those two moieties together-- I can't go into the exact chemistry of the delivery shuttle, but that's proprietary. But the delivery shuttle allows-- conjugated to the oligo, allows the complex to lay down on the surface of the cell on the phospholipid bilayer and form an emulsion with the phospholipid bilayer that is then pulled across the membrane.
DIETRICH STEPHAN: It essentially diffuses across the membrane along the membrane gradient. So there's always a membrane electrical gradient, and that is what holds the oligo delivery shuttle complex directly into the cytoplasm. Now, when it's in the cytoplasm, because these are so small, we can either jettison the delivery shuttle using a cleavable lever if we want to, to make the oligo a monomeric unit, or leave it on.
DIETRICH STEPHAN: And that complex is small enough to diffuse right into the nucleus. And so we've experimented with putting nuclear localization tags onto these things. And they really don't improve efficacy. We have not yet tried to target the mitochondrial genome. It's a great question. And so I don't know if we can get into the mitochondria by simple diffusion or not.
DIETRICH STEPHAN: Certainly, we go right to nuclear pores into the nucleus, and we see target engagement within 24 hours, for example in vivo after a systemic dosing.
ANJALI SARKAR: So the chromatin is coiled up in the nucleus with some regions coiled up more than the others-- euchromatin, heterochromatin. How do you then ensure that these novel drugs that you are developing are able to unfold any tightly packed DNA? And are they-- how are you ensuring that they get to these tightly packed regions of the genome?
DIETRICH STEPHAN: Well, it's such a wonderful question. And you're getting into, again, some of the elegance of how we balance broad biodistribution with selectivity for the cells which express the disease gene. So our strategy has been, get the drug everywhere, into every cell and every tissue if we can, lean on the improved selectivity of this polyamide backbone.
DIETRICH STEPHAN: And I want to bring that up because it's important. Again, if you think about nylon as an imperfect analogy, what you intuitively sense is that it's semi-rigid. And so when the oligo tries to approach a gene, and there's a mismatch between the sequence of the drug and the gene, the oligo can't-- the drug can't bend around the oligo and engage the locus. And because of that, these polyamide backbones have exquisite sequence selectivity, down to the single base.
DIETRICH STEPHAN: So that's helpful and that's making sure that we minimize off-target engagements and adverse events. So I just want to put that chess piece on the table. Now, the third component that you alluded to is, what if we want to drug a gene that's tightly packed into chromatin and deactivated in the certain cell. I don't believe we can put a lock on that gene until that gene is unwound from the histones and begins to be expressed.
DIETRICH STEPHAN: Now, I don't have proof about that. But mechanistically, the way these drugs invade the genome is, as the double helix at 37 degrees kind of breathes, and opens up when it's expressed, and allows RNA polymerase to come through, that's the opportunity of our drugs to take to invade in, bind, and then when the DNA tries to close again, it can't close up, because the binding affinity is so high.
DIETRICH STEPHAN: So you need a breathing locus in order for our drugs to act. But that's not a negative, because if a gene is deactivated in the certain cell, it's almost by definition not able to cause the disease in that cell, because it's not doing anything. It's simply shut off. So I would argue that in most cases, that works to our advantage. If a healthy human cell is doing what it's supposed to do and not expressing a certain mutant gene, there's no reason for us to drug it.
DIETRICH STEPHAN: And If we can't drug it, that's OK.
ANJALI SARKAR: So now that we've talked a bit about biodistribution and getting the drug into the right locus, let's tackle the next point that you talked about-- toxicity. You said that you are targeting them-- all cells. So they're entering all cells. Is there an issue with them staying there? Or are they being cleared in time? And how are you ensuring their clearance?
ANJALI SARKAR: And any toxicity issues that may arise with these drugs, how are these being tackled?
DIETRICH STEPHAN: So just from a PK biodistribution perspective, we've shown in non-human primates that when these drugs are administered systemically, within two hours, they're largely taken up out of the circulation-- so we have an in-circulation half-life of about two hours-- and very quickly taken up by all of the tissues in the body of the non-human primate, including all of the neurons in all of the deep brain structures. So we get across the blood brain barrier.
DIETRICH STEPHAN: And on average, the in-tissue half life appears to be about three months. So it's very durable in tissues. And over time, slowly, the drug is renally excreted intact. And so it's not biodegraded. It's resistant to protease nuclease digestion. So that's the PK biodistribution profile of these molecules. Some of the main features that reduce or enhance tolerability, let's say, is number one, neutral charge.
DIETRICH STEPHAN: So they don't aggregate. They don't trigger the TLR9 system and elicit a Th1 innate response. They don't appear to activate the alternative complement pathway system. And we know they don't trigger an acquired immune response. So from a tolerability perspective, this polyamide backbone, much, again, similar to nylon, appears to have all of the characteristics that one would think of when you think of sutures in the body.
DIETRICH STEPHAN: And you know, it's not the perfect example, because it's slightly different from a chemical perspective. But it's non-immunogenic, it doesn't aggregate, and the semi-rigidity of the backbone reduces off-target effects. And so from a tolerability perspective, we're OK with it being in the body for that long. The only thing that we worry about from a tolerability perspective-- and I mean that sincerely.
DIETRICH STEPHAN: The only thing we worry about is interaction of the drug with identical sequences elsewhere in the genome. And so we spent a lot of time on the bioinformatics up front to design the drugs so they have unique sequence targets.
ANJALI SARKAR: And in your experience with these-- testing these drugs in small and large animal models, there have been no adverse effects to your knowledge, right?
DIETRICH STEPHAN: Not at all. It's interesting. So these first generation compounds we call peptide nucleic acids have been published on extensively. And there's a nice body of literature in high profile peer reviewed journals, like nature journals, that speak to tolerability up to 100-fold the effective doses. And even then, those are transient aggregation issues, for example in the kidney, that resolve themselves when the drug is withdrawn.
DIETRICH STEPHAN: In-house, we've seen no tolerability issues at or above the effective doses. And that's what has given us confidence, together with the pharmacology, to nominate our first clinical candidate scale-up CMC manufacturing. And we'll file our first IND next year, and then scale thereafter.
ANJALI SARKAR: Talking about manufacturing, one of the points that you'd mentioned from the lessons learned from the first generation of genetic medicines is the scalability issue. And now, with precision medicine, some of these genetic medicines will be population-wide-- applicable population-wide. But some would be individual-based, or on all population. So what are the methodological advances that you are using to manufacture them at the scale that they are required while still maintaining all the quality control and et cetera issues that you need in the drug development process?
DIETRICH STEPHAN: So again, that was one of our criteria that we had as a requirement as we built the platform. And so, at the base level, these are small molecules that can be manufactured at the ton scale using established CMOs across the globe in a GMP setting-- very sort of routine manufacturing. Those, then, get snapped together like LEGO blocks using standard synthetic peptide synthesis manufacturing capabilities.
DIETRICH STEPHAN: And once again, this is a-- at this point in history-- a commodity out there in the world. There are any number of CMOs that do GMP peptide synthesis at scale. So when we combine those two, we now have the manufacturing capability to source molecules for any one particular disease without having to build our own factory. But when you think about scalability, single molecules, if you spend enough time with them, you can get them to population-wide scale.
DIETRICH STEPHAN: I mean, again, think of our friends at Moderna and Pfizer. It's a very complex medicine, but they've been able to scale it by focusing on that one medicine. Now, why do we think we have true scalability is because we use the same delivery shuttle for every medicine we build. And so we can put that on rails from a manufacturing perspective. The basic building blocks of the polyamide scaffold are identical.
DIETRICH STEPHAN: And so we can optimize manufacturing of that. And then all we're doing is shuffling nucleobases on the oligo. And then, when you think about that completed construct, the PK and biodistribution of each one of those molecules is almost identical, because it's not driven by the nucleobases on the construct. It's driven by the delivery shuttle. And so that allows us to, going into any development program, already understand, for example, the route, dose, and frequency that we need to move forward within the clinic.
DIETRICH STEPHAN: And so it cuts out a whole bunch of work. We believe that at some point after our first two, or three, or five drugs, that we will have a standard toxicology package that will allow us to streamline our preclinical and phase I tox work, and move more effectively through that, with the only delta being the off-target engagements. So you're getting a sense, not only just from a manufacturing perspective, but from a modularity perspective how we've built in scalability to our company.
ANJALI SARKAR: To enhance their applicability, is it also possible-- are other methods that you are using to combine these drugs for diseases that involve multiple genetic lesions or copy number variants?
DIETRICH STEPHAN: Yes. Ultimately, as a company, we want to go into diseases that are complex genetic diseases, where more than one variant, perhaps in combination with the environment, trigger disease, or cancers where you often have multiple acquired mutations in a tumor that you need to hit. We already know that we can create molecules that engage to specific sequences within the genome, and that they work.
DIETRICH STEPHAN: And we know this from the gene editing work using our technology. So these are combinations of two different drugs held together with a linker, one of which can very rapidly find a locus within the genome, and the second one that adds another layer of selectivity to the targeting that ensures that we have zero off-target edits. And there's no reason that those two drugs could have, for example, a cleavable linker in them that would allow them to target to distant loci within the genome.
DIETRICH STEPHAN: We have not begun development in that area yet, aside from the gene editing application. But conceptually, you just have to make sure you get both drugs into the same cell. And we can do that with these linkers.
ANJALI SARKAR: The other point that you'd mentioned is durability. And you did mention that the clearance is different in different tissues, but it's short and it's cleared intact. But does that mean that multiple doses, multiple administrations of this drug will be required for any of the diseases that you are trying to target? What's the durability of these drugs like?
DIETRICH STEPHAN: Yeah, so from a exposure perspective, we talked about that we believe the in-tissue residence on-- or in-tissue half life is on the order of three months. We believe that durability of the pharmacologic effect is on par with that. So if the drug is present, we believe it's working. And what we are doing is starting with application areas where we want to temporarily drug the genome.
DIETRICH STEPHAN: And in those cases, we want to put a lock on a DNA locus, address root causality, but at some point, either because of cell division or simply because of the k off rate, the drug will come off, and eventually be renally excreted. And so we do envision repeat dosing, let's say on the order of monthly to quarterly, to replenish the pharmacologic effect.
DIETRICH STEPHAN: Now, as we move forward into our gene editing applications-- and I'm happy to share a publication on this topic if you're interested. But basically, the way our drugs do gene editing is they open up the double helix. They sort of pry it open, which is one of the unique features of these high affinity oligos, is they can invade. And when you've created an opening, we allow a DNA guide strand to engage on top of the mutation.
DIETRICH STEPHAN: And without double stranded breaks, we recruit endogenous mismatch repair enzymes to fix the mutation. It's a very different strategy from CRISPR-based strategies. And in that case, that's permanent. And so the cart-- think of a jack where you jack up your car. That will eventually come off and be renally excreted. But now the mutation is permanently fixed.
DIETRICH STEPHAN: In that case, we won't need repeat chronic dosing.
ANJALI SARKAR: Thank you. Thank you, Dietrich. And is there-- that was all the questions I had for you. Is there any other question that I didn't touch upon regarding method-based advances at NeuBase that you'd like to talk about?
DIETRICH STEPHAN: Let's see. So we talked about addressing all causal mechanisms-- gain of function, loss of function, change of function. We talked about being able to do that in a way that's broadly biodistributed, well-tolerated, sequence selective, manufacturable, enduring, and scalable. I think we hit all the points.
ANJALI SARKAR: That brings us to the end of today's Expert Exchange. Thank you, Dietrich, for a very illuminating discussion. A reminder to all scientists among our viewers, GEN Protocols is open for submissions, and we welcome your protocols in all aspects of biotechnology. So until next time, good luck in your research. And goodbye from all of us at GEN Protocols. [MUSIC PLAYING]