Expanding the CRISPR Toolkit

Mammoth Biosciences is developing next-generation CRISPR products using alternatives to the Cas9 enzyme to read and write genetic code. The company, co-founded by Nobel laureate and CRISPR co-inventor Jennifer Doudna, is applying the technology broadly beyond therapeutics to include not only diagnostics, but agriculture, environmental monitoring, and biodefense. We spoke Trevor Martin, co-founder and CEO of Mammoth Biosciences, about the use of CRISPR as a diagnostic tool, the advantages alternatives to Cas9 may offer, and the company’s recently announced alliance with Vertex. Since recording this interview, Mammoth entered into a strategic collaboration with Bayer to use its CRISPR systems to develop in-vivo gene-editing therapies. That deal includes a $40 million upfront payment and more than $1 billion in potential milestones.

Daniel Levine: Trevor. Thanks for joining us.

Trevor Martin: Yeah. Thanks for having me.

Daniel Levine: We’re going to talk about Mammoth, CRISPR as a diagnostic tool, and your partnership with Vertex to develop in vivo gene editing therapies. Let’s start with CRISPR, which is a bacterial defense mechanism that’s being harnessed to really revolutionize science and medicine. What is CRISPR and what is its significance in the realm of human health?

Trevor Martin: That’s a great question. CRISPR is really at the forefront of this kind of new wave of what we call engineering biology and it’s a tool that allows us to actually change DNA and RNA. So, you can think about it as this kind of tiny programmable molecular machine that we can use to actually go into a cell or even outside of a cell and actually remove or add or alter DNA, or now, there’s a lot of excitement around RNA sequence editing as well. And the way this works is that the CRISPR protein can actually be programmed by giving it what’s called guide RNA. These are RNA molecules that basically we’re very good at designing and synthesizing, and we can do these with intelligent design so that they target a certain section of the DNA or RNA that we want to edit, and it doesn’t allow the protein to go to other areas that we don’t want it to edit. So, an example could be that there’s a disease that is caused by a certain mutation in some gene. You can design this guide RNA for the CRISPR system—most commonly Cas9 is probably one people have heard of—and it would then go to that sequence in the genome, it would bind there, and it would actually then change the sequence to potentially cure that disease. And that’s what’s really exciting about what CRISPR can be used for among other things.

Daniel Levine: People who have heard of CRISPR have probably heard it in conjunction with Cas9. What is Cas9 and what does it do?

Trevor Martin: Yeah, so Cas9 was one of the first CRISPR systems that people have been excited about and used to do this type of editing, for example, and really fundamentally it’s this enzyme that can act as a pair of molecular scissors that cuts the DNA at a specific location in the genome so that then you could either just use the natural repair of that cut to change something about the genome, or you could add DNA or remove DNA, or now people are combining things like Cas9 with other proteins that maybe turn a gene on, turn a gene off, or modify a gene in some other way.

Daniel Levine: There’s been some efforts to develop a toolkit of other enzymes that can cleave nucleic acid as an alternative to Cas9. What are the limits of Cas9 and why are these alternative enzymes needed?

Trevor Martin: Yeah, so I think what’s exciting is that—I mean Cas9 is a great tool, to be clear—but there are some limitations around it, especially what it could do, for example in vivo. And that’s one of the things we’ve pioneered at Mammoth Biosciences is, for example, this field of ultra-small CRISPR systems. These are proteins that are actually just physically smaller than proteins like Cas9, which can be quite large and that large size of things like Cas9 can mean that it’s actually difficult to put them in the widest variety of delivery methods possible. So, one example would be something like AAV vectors, which are very popular delivery method in vivo and many of the Cas9 proteins just exceed the payload limit of those vectors. What that means is that it can be difficult to use these types of delivery technologies to actually get the CRISPR system where it needs to go in the cells and the tissues, especially when you need to deliver other things as well, like the guide RNA and any other things that you want to deliver in there. With these ultra-small, or just physically smaller systems that we’ve pioneered at Mammoth, it can really solve a lot of those problems around delivery in vivo. Some other examples are things like the targetability of the proteins. So, when we say that something like Cas9 is programmable, there are some rules around where it could act actually be targeted in the genome and that’s defined, for example, by the pam sequence. That’s a restriction on where exactly it could go. You can think about it as it can only go to certain zip codes in the genome. It can’t go to every single zip code. So, we have proteins that, relative to other systems, can target way more sites in the genome and that can really open up more editable sites. Those are some examples of things that the novel systems that we’ve developed at Mammoth can really help unlock as well.

Daniel Levine: I think most of our listeners who are familiar with CRISPR think of it in terms of being a therapeutic tool. We’re going talk about that, but before we do, Mammoth initially focused on CRISPR as a diagnostic tool. What’s the potential for CRISPR as a diagnostic and what advantages does it offer over other types of tests?

Trevor Martin: So, fundamentally Mammoth has really been all about this kind of next generation of CRISPR and as we were starting the company, one of the things that we had invented early on that was really exciting is that by looking at the diversity of all these different CRISPR systems and just really taking an agnostic approach to what they’re capable of, we had been able to develop this really cool property that was enabling a new type of diagnostic test. So, as you mentioned early on in the company, this is one of the first things that we started commercializing—this new diagnostic capability of certain CRISPR systems. And at the highest level, the way this kind of diagnostic capability of the CRISPR systems works is that again, you’re using CRISPR as a programmable technology, but now instead of programming it to target some location on a genome that you want to edit, you’re programming it to target some sort of sequence that you want to detect. As an example—obviously we’re in the middle of a pandemic still—would be targeting SAR’s COVID-2 sequence. And the way you would do that is now you design your guide RNA to be something that’s unique to SAR COVID-2 and not found in say the flu or the human genome, other things you might find in a nasal swab or throat swab. And then let’s say this is all happening in some tube or some swab, what you can do is that for certain proteins with these special kinds of diagnostic properties, if they find their target and they successfully recognize what that guide RNA is targeting the protein to, then they don’t just cut the sequence they’re bound to, which is kind of what you use for classic editing with Cas9. They’ll not stop there and they’ll actually cut many more single stranded DNA or RNA molecules in that solution. And that is a way of reading out the signal that they successfully found there, for example, the SAR V2 target. And that’s a very powerful concept that you can use to build these new types of CRISPR based diagnostic tests.

Daniel Levine: Well, what specific diagnostic test is Mammoth developing and where are you in that process?

Trevor Martin: So, we’ve done a lot of work and we’ve been very fortunate to work with some great collaborators, for example, on a SAR COVID-2 diagnostic. We’ve had collaborations with organizations like the NIH and GSK and Agilent and Hamilton. We even received an EUA approval for one of the first generations of this CRISPR-based diagnostic chemistry that really demonstrated that this chemistry can go toe to toe with these technologies that have been out there and established for decades, like PCR. So, it’s been a really highly accelerated time of development of these technologies that are really just coming onto the stage. And it’s been exciting to see how much they can really be comparable with gold standard methods like PCR. And we have some really great papers that are available on our website as well where we outline how these CRISPR-based diagnostic tests work and also, even more recently, outline how you can use CRISPR-based diagnostics for things like variant detection.

Daniel Levine: So, are you looking at this for genetic diseases?

Trevor Martin: Yeah. Beyond things like Sars-Covid-2 and infectious disease, you could definitely imagine leveraging this type of technology to diagnose things like genetic disease. I think one of the powers of CRISPR is its specificity and looking at things like SNPs or variants, whether that’s for an infectious disease like COVID or beyond. I think that’s one of the really powerful aspects of CRISPR.

Daniel Levine: At the end of October, you announced a partnership with Vertex to develop in vivo gene editing therapies for two genetic diseases using Mammoth’s next generation CRISPR systems. Can you walk us through the terms of that deal?

Trevor Martin: So, we’re really excited to partner with Vertex on these kinds of in vivo therapies. Obviously, as you probably saw, we can’t reveal anything about the targets, but we received an upfront payment of $41 million and we’re eligible for $650 million in future payments on, you know, success. I think more importantly though, it’s a really great example of how at Mammoth we are building this platform technology right across therapeutics and diagnostics. That means that there’s so many diseases that we could tackle and really have a huge impact on patients. And that means that in addition to the work that we’re doing internally, it’s really important to us that we partner with other organizations that are extremely patient focused as well and could help us make sure that this technology is really impactful in diseases that we wouldn’t enter otherwise. I think that’s a big part of what we’re excited about here as well—yeah, just accelerating the advance of this technology to patients.

Daniel Levine: With regard to the partnership with Vertex, what is Mammoth’s role? What is Vertex’s role?

Trevor Martin: We can’t go into too much detail there, unfortunately, but I think at a high level, what we’re excited about is we’re bringing next generation CRISPR technologies and behind that this whole protein discovery engine where we’re constantly building on this proprietary toolbox of Cas proteins including things like Cas14 and CasΦ, and working with an organization like Vertex that has this really deep clinical expertise and experience going to patients, I think, is a really powerful combination.

Daniel Levine: One of the things Mammoth is providing as part of this agreement is its ultra-compact Cas enzymes. What’s the specific significance of being able to deliver these ultra-compact enzymes in an in vivo gene editing product?

Trevor Martin: I think it goes back to a lot of the delivery aspects, for example. Having a much smaller system can just open more doors to what’s possible on the delivery side and obviously that’s something we’re really excited about enabling with our internal products and for the therapies we build with partners as well.

Daniel Levine: And what are the implications for being able to deliver a CRISPR gene editing product in an in vivo therapy rather than an ex vivo? What’s the impact in terms of cost, production, delivery, and the ability to treat a broader range of diseases?

Trevor Martin: It’s a great question. I think some of the things at a high level are what if you could deliver the therapy in a single package or a single payload, so that’s AAV or something else. That is a very powerful concept that would potentially have a huge impact in the in vivo space. I think also it creates a lot of flexibility for the type of cargo you’re delivering broadly in vivo. As I mentioned at the beginning, there’s a lot of development now. It seems like every month, there’s some sort of new technique where you’re leveraging the CRISPR systems and conjunction with additional functionality, whether that’s activating or inhibiting or based editing or otherwise. So, I think they can leave a lot of additional payload capacity for things like that. Those are some examples of what could be enabled.

Daniel Levine: Mammoth is looking to partner across a broad range of applications of the technology beyond human healthcare. Is the Vertex agreement the first in human therapeutics?

Trevor Martin: The vertex agreement is our first public human therapeutics partnership. As I mentioned, we are really excited that we have this platform that can be used for a variety of different applications, obviously within human healthcare, therapeutics and diagnostics, but as you mentioned beyond it as well, right in agriculture, biomanufacturing, all sorts of areas where basically you want to be able to engineer biology. So there, we definitely want to make sure that we’re working with organizations that are also similarly product focused to really make sure this technology can have the full impact that we know it can. Yeah, that’s really exciting.

Daniel Levine: And what’s the plan in terms of pursuing other therapeutic applications? And do you expect to do this only through partnerships or is the company considering pursuing any indications on its own?

Trevor Martin: Yeah, we’ll definitely be pursuing indications internally, and we’re very excited about that. The internal pipeline is core to what makes Mammoth Mammoth because that’s really what’s exciting about this type of platform technology is it means we can really take a lead on driving therapies to patients in addition to working with partners. So, in terms of next steps there in the future, we’ll definitely have more information available about what our internal pipeline targets are and things like that. But right now, we don’t have that public.

Daniel Levine: In addition to the funding Mammoth will get through the Vertex partnership, it completed a $150 million venture financing in September. How far will existing funding take you and what’s the path to revenue?

Trevor Martin: Yeah, so we’re very fortunate to have really supportive long-term investors. Most recently we did a series D for $150 million that was led by Redmile Group. And what I think is exciting there is that really does give us the capital base to start really doubling down on both our diagnostic products and bringing those to market and also beginning to move our therapies further along towards the clinic and patients, both by ourselves and with partners. So, in general, obviously biotech’s expensive and we really are in this for the long haul. So, what we focus on the financing side is really just the long-term health of the company over the next 10 years.

Daniel Levine: Trevor Martin, co-founder and CEO of Mammoth Biosciences. Trevor, thanks so much for your time today.

Trevor Martin: Yeah. Thanks for having me.

This transcript has been edited for clarity and readability.