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Liz Pavone: Hello! I'm Liz Pavone, Manager of Content and Science Communications at Flagship. I'm here with two lead voices from Flagship-founded Ring Therapeutics, the Founder Avak Kahvejian and the CEO Tuyen Ong. We've brought Avak and Tuyen together following Ring’s presentation of exciting nonhuman primate data at the American Society of Gene and Cell Therapies Annual Meeting.

We are going to dive into why that data was so exciting for the field of genetic medicines, as well as get a glimpse at where Ring started to understand how the company made such rapid progress in developing a novel gene delivery platform.

For those unfamiliar, Ring was founded in 2017 to unlock the full potential of gene therapy by leveraging a little known class of viruses called anelloviruses that are found throughout the human body and coexist with our immune system.

Welcome, Avak! Welcome, Tuyen!

So let's start at the beginning. Avak, I'm going to turn to you to tell us about the founding of Ring to really take us back to those early days.

Avak Kahvejian: A few years ago, at Flagship, we asked ourselves whether there were safe viruses, nonpathogenic viruses, living in and among us. And if so, could that represent the ideal vector for genetic medicines? And so we embarked on what we call an exploration where we looked through computational databases, and then also started to do prospective sequencing, and happened upon the largest family of viruses that had never been discovered.

Liz Pavone: You founded the company then in 2017, after realizing that there was, you know, potential here?

Avak Kahvejian: Exactly. We realized that if we have a new way of getting genetic payloads into the human body, and if these are safer, potentially redosable, and more broadly applicable than the existing tools we had that it could represent a huge opportunity in the field of genetic medicines.

Liz Pavone: So Tuyen, turning to you, when you joined, as CEO — you know, Avak really described the really early days — where was the company at that point?

Tuyen Ong: What we really had to do is just build the foundational science behind anelloviruses. One of the challenges initially was really trying to find and in vitro system that allowed us to produce and propagate the anelloviruses for the study. And so, we worked on a MOLT-4 system that actually allowed us to create the world's first synthetic wild-type of an anellovirus.

Subsequent to that, or prior to that, rather, what we actually had to do was to, as Avak pointed out, develop the AnelloScope. This allowed us in essentially to enrich and recover sequences of anellovirus sequences from different tissues. Really starting out at using instead of Msan and breaking down some of the human host cell DNA, and then generating rolling cell coamplification to be able to allow us to actually fish out anellovirus sequences from different tissues.

So we, as a result of that, really have been the only company working on anelloviruses in the entire world. Some of the world's first that we are extremely proud about is one, you know, synthetically producing the anelloviruses, I mentioned before, discovering it and learning about its structure. We've subsequently also understood more about its immune profile and its ability to be redosed, which culminated in the groundbreaking data that we presented at ASGCT.

Liz Pavone: Maybe, Tuyen, the question you can answer for me, too, is this is such an undertaking, developing all of this new technology, really, starting from the very basic science research, why undertake the development of a new vector for gene therapy?

Tuyen Ong: Having worked on gene therapies as well as rare disease. It was painfully obvious to see patients that were highly disappointed when they were not able to participate in clinical trials, given their pre-existing, neutralizing antibodies. And

then really, the heightened hope of the one and done with gene therapies. And you know, there were many occasions where patients were disappointed in both regards. And so, I always thought that there must have been a better solution. You know, there needed to be a better viral vector that allowed us to be able to hit different tissues of interest and deliver its payload and really, truly realize the promise of genetic medicine.

And then, really, as we look at genetic medicines, it really hasn't fulfilled that one and done promise. I mean, we need to have viral vectors that are redosable, are titratable in so many different ways. Even genetic diseases that occur in, you know, in early sort of infancy or in tissues where there's a high turnaround and durability is lacking.

Liz Pavone: Both of you have mentioned that no one else is working on anelloviruses. Why? Why is that? Why isn't anyone else working this space?

Tuyen Ong: Yeah, I think Ring Therapeutics really is pioneering work on anelloviruses in the human virome. In this field a lot of the virologists are really looking to associate viruses with disease. Many have tried and failed, including the [Harald] zur Hausen, who famously connected HPV to cervical cancer. And really, as the research built, and essentially sort of embarked upon a journey to correlate anelloviruses to disease and ultimately sort of failed, the field started, you know, started losing interest in anelloviruses.

And I think to Ä¢¹½ÊÓƵAPP’s credit and to Avak’s credit, it really stayed within the study of the human virome, stayed with the study of anelloviruses. And the more and more we learned about them, the fact that they are commensal, they don't cause disease; they've essentially evolved inside of our bodies. And so that's really given us really unique insights in regards to tropism. They reside in different tissues. We've been able to genomically sequence and identify them in different body systems. But they also persist. And so there's a Dutch author that has followed two patients up over a 30 year timeframe, and showed that their core anellome sequences pretty much remained stable over that 30 year timeframe. And that again, was the genesis of: Could these anelloviruses actually be unique in regards to — can they be reducible? And so nobody had actually achieved this with a viral vector before. And so really, I think it's a testament, really, to the longevity and the just this sustained sort of approach that Ä¢¹½ÊÓƵAPP has in developing its companies.

Liz Pavone: Tuyen you had mentioned, I think, an aspect of the Ring platform, the AnelloScope. Can you maybe describe a little bit what that is, how it works, and maybe kind of talk about, moreover, the broader platform and all the component parts.

Tuyen Ong: Sure, maybe I'll start with a broad platform initially. So, what we've essentially done is be able to develop genomic tools to genomically sequence anelloviruses from different tissues and organs of the body. We initially started quite broad, but now have positioned down to the cellar level. So we use RNA-Seq to determine anello-positive sequences in different tissues. From that, what we've been able to do is to feed that into a machine learning backbone to allow us in the future to determine where those sequences come from in terms of tissues as well as their tropism.

From there, we take those genomic sequences, as Avak pointed out, we're able to generate using our Anello-manufacturer platform to be able to generate vectors. Within that, we're extremely excited by another platform that we have within that which is AnelloBricks. This is essentially using in vitro assembly. So we generate recombinantly off one protein. So this is the capsid protein of the anellovirus, and 60 of those protein monomers form the capsid itself. We're able to take that and essentially form a capsid around a genetic payload. Either DNA, for example, with gene therapies, mRNA, or even potentially gene editing therapeutic payloads down the line as well.

So we're extremely excited by this end-to-end platform. Just because of the sheer sort of potential around the ability to generate different anelloviurse. We've been able to again proof-of-principle show, we can encapsulate different payloads and then now really be able to target different diseases from that. So it's a really powerful compounding platform, as well as a very modular system that's programmable to different patient diseases.

Avak Kahvejian: One of the interesting things when we first started on this was the fact that the DNA sequences that we were dealing with were, were generated for looking at human variation. They weren't generated to hunt for these viruses. And what the implication of that was, is the sample preparation that was used was to actually isolate long, very long, linear pieces of DNA so you could sequence human chromosomes. As a byproduct, some of these sequences, the viral sequences, were caught up and analyzed.

But when we wanted to actually look for these viruses more deliberately, we had to redesign a sample preparation methodology. We had to redesign a DNA enrichment methodology to look for small, single-stranded circular pieces of DNA. When we did that, that was what started that AnelloScope. The power of the AnelloScope was it suddenly opened up a much richer view of these viruses than we had before. And that's why that diversity was open to us, because we really rejiggered the way the DNA was prepared and amplified and then sequenced.

Liz Pavone: Avak, maybe you can take us then to describe the AnelloVectors, at least the early ideas of what an AnelloVector might have been.

Avak Kahvejian: After we did a lot of this early work, and some of this foundational work on understanding the natural composition of the of the anellovirus family certain interesting things emerged. One was, as Tuyen mentioned, that the capsid was formed by a monomeric protein, a single protein that self assembles to create a shell, and that, furthermore, that shell seemed to be nonimmunogenic.

And so for us, it became the important piece that if we could master the creation of these shells, and then replace the insides of the virus, not with viral genomes, but instead, with a payload or a cassette that encoded for a therapeutic protein of interest. We could generate a vector, a genetic medicine.

And so we embarked on a variety of methods to do that. Since we were the first in the world to try to make these things, we had to develop the methodologies from scratch, as Tuyen mentioned. Which cell types do we use? How do we deconstruct the various elements of the of the virus so that we can make the capsid, make the payload, have the two come together so that we have a fully formed vector with the payload inside and the capsid outside. All of these were major, major R&D efforts to get to where we are today and to get to the results that you're seeing today at ASGCT.

Liz Pavone: And Tuyen, maybe you can describe from your perspective what sort of milestones you saw in in the development of the vectors.

Tuyen Ong: I think we've had some significant world’s first, as Avak pointed out. You know, there was a very little, and still, in this present day, little understanding around anelloviruses. In fact, Ring is the only organization that's actually generating data. And we've had some landmark publications.

The first initial discovery, I would say, is the synthetic production of anellovirus. What that ultimately led to was really understanding the structure. We again blew that structure up, understanding that it was made up of difference in the monomeric proteins, and being able to assembly a capsid around that.

And as we continue generating data around the structure, it dawned upon us that the structure obviously drove not only tropism, but could be the answer to why the virus is immune stealthy. And so one of our hypotheses at the time, and still somewhat of a prevailing hypothesis, is really to look at a shear or canyon hypothesis that those pentameric structures actually block antibody neutralizing antibody binding.

So we continued working along that stream, really continuing to build out our immunogenicity data. We worked with Johns Hopkins and Ben Larman really to express surface antigenic peptides from about 800 anellovirus sequences compared to 32,000 protein sequences, and showed that anelloviruses are actually less antigenic than other viruses.

So all of this data really gave us a lot of confidence and excitement that we could actually redose anellovectors. And to subsequent to that and what we presented at ASGCT is that we've been able to inject mice intravenously, target the liver, and repeat that in a sort of repeat fashion 21 days later.

We continued to build upon the mouse data and injected nonhuman primates, using an intravitreal route of administration. Again, showing that we not only could we measure DNA as well as RNA from repeat injection, using the intravitreal route of administration

Liz Pavone: Avak, maybe you can describe why that data is so important. Why is this big for genetic medicines?

Avak Kahvejian: Well, well, first of all, in the field of genetic medicines, getting genetic medicines into the body into the right place has been a major challenge. Whether it's RNA or DNA, right now, many of the indications or applications we're pursuing are relegated to the liver, or maybe the muscle, or maybe lymph nodes. That's because the genetic payloads, those programs, and those nucleic acids have to get into the cytosol of a cell or to the nucleus of the cell, if, in the case of DNA. And that's no small feat. We've been using lipid nanoparticles. We've been using viral based deliveries, mostly AAV based technologies.

Having the ability to do this in different tissues. Number one, unlocks new disease areas. Having the ability to repeat dose, unlocks new disease areas, but also helps prolong the effects of these genetic medicines. And of course, reducing the immunogenicity alleviates or promises to alleviate the safety issues and the untoward effects of using these kinds of delivery methodologies.

So, having a completely new way of getting genetic medicines into different cell types is a major major breakthrough for the field. And this is the first demonstration that there is a new way of delivering genetic medicines. And because it's the full package — having been able to make it, having been able to put a payload inside it, and then having it delivered to an non human primate, which is a very, very good surrogate for how these things will behave in humans. So that to me is a really major milestone for the company, the technology, and for genetic medicines broadly.

Liz Pavone: Could you also comment, too, on I mean, from listening to you both, it's interesting, Ring really started from scratch with anelloviruses. Can you comment a little bit on the timeline to go from doing basic science research to now developing a new vector?

Tuyen Ong: The rate of progress has been phenomenal. When you look back at the history of AAV development. The first AAV, adeno-associates virus, was discovered about 50 years ago. And then, taking that, what we saw was that from vectorization to NHP data took about 15 years. And so this was really achieved with a huge scientific force of researchers.

We at Ring are the only, you know, entity, institution, or organization in the entire world working on this. It is a huge sense of pride for us where we've been able to actually develop the vector and take that into NHP data in about a four year timeframe. What we've essentially been able to do is take an obscure virus, vectorize it, engineer a genetic payload into that EGFP as a reporter, and show that we can dose in mice, dose in NHP. And to do that, not only in in a single injection, but to do so that there's a dose correlation. So that higher for the vector material, entire titer material. We've shown greater DNA and RNA expression. We've shown that we can repeat those in both animal systems 21 days later, with again higher titrate to pull DNA and RNA expression as a result from that.

Liz Pavone: Then, looking forward, you know, this isn't the end of Ring's journey at this point, s what's next after this data readout? What can we expect from Ring?

Tuyen Ong: Yeah, we're immensely excited. And also with the reception of the data that was presented at ASGCT. And so really, our next steps is really starting to plan R&D enabling studies. We've identified a number of different clinical programs that would highly differentiate where another vectors can show tropism, the ability to redose. And so, looking forward to enter in the clinic very soon.

Liz Pavone: So then, I have to ask, with partnerships being so dominant in the biotech community, is that something that Ring plans to pursue?

Tuyen Ong: I think the opportunity for the platform is incredible. And we think we can look at partnerships in so many different ways from discovery of vectors, and that allow us to get the different tissues that currently are unattainable with AAV, for example. The ability to maybe kind of add a pre-existing payload to some of our vectors and establish programs. This really, truly does allow us to realize the potential of the platform and be able to make that bigger impact to patients

Liz Pavone: Avak, from a Flagship perspective, you've founded more than a dozen companies but what is it about Ring that you're so excited about?

Avak Kahvejian: Well, look, I think Ring exemplifies the Flagship pioneering model of asking a bold, open-ended question, allowing the exploration process to reveal the potential and reveal scientific opportunities to us. Having the audacity to actually experiment and try things that may have never been tried before and then letting the data guide whether or not we should be pursuing the opportunity and in what direction we should take it. It is, it is really one key, and it's a great example of how Flagship does what it does and why we do what we do. To build completely new bioplatforms with outlier technologies, and with a with a bold mindset to bring a new class of therapeutics to the world.

Liz Pavone: This has been such a fantastic conversation. Thank you both for offering your perspectives. For more, please visit flagshippioneering.com.