Anzalone Prime: An Interview with Prime Editing Developer Andrew Anzalone

Abstract

GEN BiotechnologyVol. 2, No. 2 Asked & AnsweredFree AccessAnzalone Prime: An Interview with Prime Editing Developer Andrew AnzaloneAndrew Anzalone, Alex Philippidis, and Kevin DaviesAndrew Anzalone*Address correspondence to: Andrew Anzalone, Prime Medicine, 21 Erie St, Cambridge, MA 02139, USA, E-mail Address: aanzalone@primemedicine.comPrime Medicine, Cambridge, Massachusetts, USA.Search for more papers by this author, Alex PhilippidisSenior Business Editor, GEN.Search for more papers by this author, and Kevin DaviesGEN Biotechnology, New Rochelle, New York, USA.Search for more papers by this authorPublished Online:18 Apr 2023https://doi.org/10.1089/genbio.2023.29091.aanAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Andrew Anzalone, Co-Founder and Head of Prime Editing Platform at Prime Medicine.Andrew Anzalone is the lead developer of prime editing (PE) and the scientific cofounder and head of the PE platform at Prime Medicine. Founded in 2019, Prime Medicine is one of several biotech companies developing new precision genome editing platforms capable of engineering a variety of base substitutions and other more flexible insertions and deletions.PE is an exciting technology for precision genome editing, because it can in principle engineer any potential nucleotide substitution by performing an ingenious RNA-based chemistry on the double helix itself (Fig. 1).FIG. 1. Programmable correction of genetic mutations with prime editing. In this example, a Prime Editor protein containing a Cas domain and a reverse transcriptase (RT) domain complexes with a prime editing guide RNA, or pegRNA (orange), that contains “search” and “replace” sequences. The Prime Editor complex is programmed to target a gene containing a mutated sequence (red) using the pegRNA's “search” sequence. After locating the mutated sequence, the Cas domain nicks one of the two DNA strands, liberating a 3’ DNA flap. The 3’ flap then pairs to a complementary portion of the pegRNA's “replace” sequence, allowing the RT domain to copy the programmed edit (blue) directly into the gene using the pegRNA's “replace” sequence as a template. Cellular DNA repair processes remove the mutated DNA sequence and incorporate the edited sequence into the complementary strand of DNA, resulting in correction of the mutation in both strands of DNA. (Courtesy: A. Anzalone/Prime Medicine.)In this interview, conducted by GEN Senior Business Editor Alex Philippidis and GEN Biotechnology Executive Editor Kevin Davies, Anzalone discusses the genesis of the PE technology and the clinical programs outlined by his colleagues at Prime Medicine.(This interview has been lightly edited for length and clarity).GEN Biotechnology: Andrew, it has only been about 5 years since your time at Columbia University, where you got your MD and PhD, and the inception of PE technology. How did it all begin?Anzalone: It all started back in my days as a trainee, where I was focused on chemistry and molecular engineering. I was also very interested in medicine and seeing how we can push science forward to help people with diseases. As I went through my training, I had such an enthusiasm for science, working in the laboratory, and this desire to develop therapies. As I moved toward the end of my training, I wanted to look for a research position that would allow me to really translate this science into something potentially meaningful.In the backdrop of all this, of course, was this CRISPR revolution that had started around 2012–2013, with some landmark publications from Jennifer Doudna, Emmanuelle Charpentier, Feng Zhang, George Church, the list goes on and on.As I looked for new opportunities in my research and new directions, I came across some really interesting work in this gene editing field using these CRISPR technologies. And David Liu (Broad Institute) quickly rose up on my list of people of interest. He had just developed a base editing system, which could precisely make single base-pair changes to DNA. Not only did he develop one base editor, but he had also just published a second base editor, about a year apart from each other.I looked at this, and I thought, “Wow! This is really exploding.” This is something that I could have never imagined would be possible 10 years ago, when I had started medical school, learning about all these different genetic diseases. So I saw that as an opportunity to build on what had been done prior. We have this incredible natural system, these CRISPR enzymes that can target, in a programmable way, a DNA sequence, to go and find it in this vast sea of otherwise similar looking DNA sequences.And we could start making more sophisticated machines that could edit the DNA in different ways. Of course, a big challenge was: we have this CRISPR system that can target and find sequences in the genome, but how to change them to exactly what you want was still a bit of a challenge. There was still something to be done to address the second half of what was required for gene editing. That is where the idea came from for how we might develop a new system, which eventually became PE.An earlier report said your idea came on an evening stroll around your New York neighborhood. It is not exactly Kary Mullis popping LSD on a midnight drive through Napa Valley en route to discovering polymerase chain reaction, is it?! Perhaps you can reveal how the real breakthrough came about?Yes, there was no LSD involved! But I do like to do my thinking on walks, when I can do some hard thinking. At some point, a notion occurred to me that these CRISPR systems are so elegant—they have these RNA molecules that are almost like instructions and give them an address for where to go in the genome, to bind to DNA, and then to change that DNA. What a nice opportunity for that programmability to now not just be used to say where to go, but also what to do.In fact, we have already got this RNA sequence that has information in it, bound at the target side in the genome where you want to make a change. Why not just add some instructions on what that change might be? We have got programmability for targeting. What about the programmability for editing? The system is already poised to do that. It already has the right molecules in place. So that was the conception. Obviously, that is just an idea that is not worth much on its own. It was not until I entered the Broad Institute that I really started to get into the details and think about how this might be possible.Did you have to twist David's arm a little bit?I think many people saw the promise in the idea, including David, but also many of the challenges that we would face. There were many questions that, in any one of them, if things did not go as planned, the system would not work so well. There was some optimism, [but] a healthy amount of skepticism as well. It is a bit of a funny story. I had discussed the idea on my postdoc interview with David and many members of the group, and it was quite a colorful discussion, but a really nice one. A few weeks after my interview, I followed up with David and he agreed to allow me to join the laboratory and pursue this project.Initially, it started as a project that only I worked on. I cannot emphasize enough how great an environment David had developed in his laboratory, in terms of learning all the techniques from people. These were all things I had not done before. I had never done genome editing in a human cell before getting to David's laboratory, I had never done high-throughput sequencing. I definitely credit all my laboratory mates there for showing me these things and helping me talk through ideas to make this work.As the project showed more promise, others started to join me on the project. Ultimately the article we published had 12 or so coauthors on it, all of whom made a very important contribution.1 I really do credit the environment that was cultivated there to a lot of the success, and especially the speed of success.Was it smooth sailing that first year or did you have to overcome stumbling blocks?There were at least four challenges that laid in front of us. David refers to it as “four little miracles” that needed to happen for PE to actually work. When I had these little stumbling blocks we tweaked some things, worked on some new designs, and got past each one of them, one at a time until ultimately getting into human cells and making it work. Base editing is excellent at making certain categories of substitution, but PE, in principle, could give you the full repertoire, exploiting all there is to offer from having a nucleic acid sequence template.RNA and DNA share pretty much the same letters. That information is easily exchanged in both directions. The directions that we needed to exchange were from RNA into DNA —that is a process performed by a category of enzymes—reverse transcriptases. They synthesize DNA from an RNA template.Because this template sequence could be changed very simply by changing one base here, another base there, deleting some bases, adding some bases—that template will dictate what sequence goes into the DNA. So, it is easy to swap out one for another and make any edit in principle that you really want in a pretty confined but substantial region of DNA.Your landmark article, published in Nature1 (Fig. 2), was in press when David presented the work for the first time, at the Cold Spring Harbor CRISPR conference in 2019. Do you recall what he said at the end of his talk in his acknowledgment slide?FIG. 2. Prime editing debuted in this 2019 Nature paper by Anzalone and colleagues from the Broad Institute.It was a very meaningful experience for me. David said something along the lines of he is “excited to see what I do in the second year of my postdoc!” It was partially set back by the pandemic, unfortunately, but I think we still got a bit more done in the PE space then.By the time the Nature article was published we are talking toward the close of 2019, were you and David already thinking that this technology would be commercially developed as an independent company?Absolutely. There was a lot of recognition early on, a few months before we published the Nature article, because we were trying all these different things and they were all working at once. We finally developed the system, getting it to the state where it was working in human cells. We could swap out the RNA with a new sequence and make an edit somewhere else in the genome, and we just saw the potential for that. It does not take a lot of effort to look through the types of mutations that cause genetic disease and just tally up the percentage of those we could potentially fix with PE.It is a pretty large number, somewhere close to 90% of the mutations in certain databases such as ClinVar. We immediately recognized the opportunity there and the potential. And David obviously has a lot of experience founding companies and was able to get that going pretty efficiently.What are some of the advantages of PE beyond only nicking the double helix? Another is the expanded range of edits it can give researchers?What would an ideal gene editing system look like? There are probably five factors that many of us have discussed as being really critical. First is programmability. Essentially, this means you can go one place in the genome and then the next day change that location and go somewhere else and do that with ease and quickly.Second would be specificity. You only want to go to the place you want to go, not other places—that has implications for things like off-target editing. The third one is versatility. When you get to that place in the genome, can you make all the different types of changes you might want to make? How broad can that genome editing system be in terms of making a point mutation, deletion, etc.? And then how precisely will it do so at that location? Are you making just that change, or are you making other changes as well? Finally, how efficient is that system? Ideally, every cell that gets treated with these editing systems would make an edit.So these are five different principles that you would look for in an ideal gene editing system. PE, and in many ways all CRISPR systems, is very programmable. It is very straightforward to give a new set of instructions to these CRISPR guide RNAs, to go to a new place in the genome.Say one day, I want to fix this mutation in CFTR that causes cystic fibrosis. On another day, I want to fix this mutation in sickle cell disease. That is a real powerful principle of the CRISPR technology. Now, once you get there, you want to minimize the other things you do besides making the precise edit that you are intending to make. In a PE system, we are using this RNA template to make a change in principle. The fidelity of copying that sequence is very high. So you are very likely to only incorporate that one change and you only make a single break in the DNA.That is really important. When a cell has a double-strand break (this is what the more traditional CRISPR nucleases do), it sets off a lot of alarm bells. It quickly identifies that broken DNA end and stitches it together as fast as possible, without a lot of care for what happens to the sequence when it does that. So you can imagine these two pieces coming together. But little errors happening in that process lead to these small changes, insertions, or deletions of a few bases that often will disrupt a gene.That is just an intrinsic thing the cells will do. Now, if you make only one break in the double helix, you avoid many of the responses in the cell, and that is exactly what PE does. It makes just a single nick to copy DNA sequence onto that one strand of DNA and then other DNA repair mechanisms can incorporate that edit into both strands eventually. But you avoid alternative DNA repair pathways that tend to lead to undesirable outcomes.Another advantage is that at any potential off-target sites in the genome (places where you did not intend that prime editor system or CRISPR system to go to), you are not going to be making double-strand breaks, so it is much less likely to cause damage at those other places in the genome.When Prime Medicine launched in 2019, did you have any doubts about being an active part of building the company? Presumably you could have had your pick of any faculty position or maybe return to medicine?I thought hard about that but actually not that long. I had always been motivated from early days in research. Actually, my first research experiences were in medicinal chemistry at a small pharmaceutical company, so I was always interested in developing therapeutics.Obviously, the Liu laboratory had a lot of success doing science that had the ability to translate into therapeutics in the past, and that was one of the reasons I was very attracted to going to this group. When the opportunity (to join Prime Medicine) arose, I did not think too long. This would be a great opportunity for me. I love the science. I was there when it started. I cannot think of anything better than the possibility of being able to carry that forward to the point of using it for human therapeutics. That is really what drove me to join the company.In the first 6 months, the company's growth was a little slow—the early days of the pandemic. Those presented some challenges, but in the next 1–2 years, we have had an amazing growth period. We have hired up to almost 200 people now and we have been able to tap into the excitement that the community feels about gene editing and about PE in particular. Many staff members who have joined Prime Medicine have worked on gene editing in the past and have experience using PE. They have seen the potential and want to contribute to making it into medicines.Despite the obstacles, the pandemic, and competition and other things, we still had a pretty rapid growth trajectory, and brought in the people to get the job done. I am really proud about the team we have built.Who are some of the key members of your executive team, and is David Liu still actively involved?David is involved. He touches base with us on a weekly basis or so and gives us a lot of feedback on what we are doing. Also, his laboratory is actively working on PE technology development, so things that his group develops and the improvements to the technology often come over to us, and we have the ability to use those and capitalize on those developments.We have an excellent leadership team. CEO Keith Gottesdiener has been in biotech and pharma for several decades. Jeremy Duffield, our chief scientific officer, is also very experienced and an expert in the disease biology. We have put together a great group and have a lot of internal expertise.There are now six other people from David's laboratory at Prime Medicine who have been there from the beginning. All are authors on a PE publication, so this expertise has really helped us accelerate.In 2022, you filed a successful initial public offering (IPO) and released details of the pipeline. Why such an early IPO given how challenging the financial climate was?I think our ability to do the IPO probably signifies one thing—the excitement for the technology and its potential. It was an amazing experience to be part of that process to go public. I had to pinch myself a few times in Times Square in New York City. I have walked through there so many times in my life. I never thought I would be in the Nasdaq building, so it was quite the experience. I am really proud of the team for getting us there!We released our pipeline, and we have many areas that we are looking to work in. We were not sure when we started where PE was going to work or where it would work the best. Maybe there would be some places where it just would not work, and we would not be able to do anything in that area. But the more things we tried, we found things that work through the optimizations and enhancements to the technology that have been developed. Most of the areas where we are trying to do something, we have had a lot of success.Prime Medicine's pipeline is ambitious, featuring a dozen targets including well-known Mendelian diseases, including Duchenne muscular dystrophy and fragile X. What are some of the initial clinical priorities?That is a very nuanced question in the sense that we have the potential to address some of these really well-known diseases that have affected so many people. For many of them, there has not been a great therapeutic option available, so PE offers a really exciting opportunity. But in some cases, there are also challenges associated with delivery of the gene editing systems to the right cells in the body. So we have looked at the landscape and said, “there are certain places where we think we could be really successful early on, because some of these challenges have been addressed, such as delivery, and other places where we think the technology does something really special, but we probably need some other areas to catch up before they can become a possibility.”We have tried to develop a pipeline that has some examples from each of these categories. There shall be places where we can move a lot quicker. For example, ex vivo programs are a lot easier to do in the sense that the delivery challenges are not as prominent, and then certain in vivo programs where delivery has also been established previously.Are you trying to pursue all these programs yourselves or will you be seeking partners to develop some of them?It would be a little unrealistic to say that we are going to take 18 programs forward in the next few years and be able to develop them into medicines for people! I assume there are many solutions to those challenges, whether that be through partnering with other companies, or by prioritization, internally. That is something we will be deciding in the next couple of years.Which of these programs are furthest advanced and what is going to determine which targets Prime Medicine prioritizes?Those questions are still being worked through. The targets where we have had a lot of ability to deliver the gene editing systems to the cells of interest. Again, the ex vivo programs have the ability to get past that a little bit more quickly. Those we can make pretty rapid progress in, and likewise delivery by nanoparticles to the liver. These are also areas where we can move a little faster. That said, we are still paying a lot of attention to those other programs where there is still so much potential, they just may require other delivery solutions.The development of CRISPR in 2012–2013 led to a protracted patent dispute. Is there a possibility something similar could happen in the world of PE?I am certainly not a patent attorney! I will say that I believe Prime Medicine has a very strong position in PE. We filed patents at the Broad Institute very early on that cover the PE system. We have been awarded some of those patents, and we continute to file more patents on both our technology and our therapeutic applications. Additionally, we maintain a strong relationship with David Liu's group and have in-licensed their PE developments as well. So I feel very good about our intellectual property position and the work that we did to establish that position.There is increasing competition in the development of a new generation or new wave of CRISPR-based gene editing tools—PASTE, twin PE and transposases have had a lot of attention. Do you anticipate increased competition in developing these precision editing tools as we go forward?The CRISPR space has always been competitive and genome editing in general has been a very competitive space. Even from the beginning, when the first CRISPR systems were described, there were races to find new effector proteins, new CRISPR systems that could be translated into therapeutics, whether those be smaller Cas systems or others that had other beneficial properties. Similarly, there has been competition to develop the next generation approaches to do genome editing, which has been very healthy, making this technology more effective and ultimately more useful for the development of therapeutics. I think that is a really exciting thing to witness as a scientist and as a member of the community.From the standpoint of Prime Medicine, we are also really interested in continuing to do that development and contributing to that innovation and moving things forward.You are attacking a wide array of genetic disorders including some that are not simple base substitutions. What is it about the platform that allows you to potentially tackle some of these more complicated genetic mechanisms?One of the developments to the PE technology that I have not discussed yet is “dual flap prime editing” or twin-PE. Essentially this is an approach to PE that allows you to replace or delete much larger pieces of DNA. One of the exciting applications for that, in my opinion, is the ability to delete expanded repeats in DNA that are known to cause some of these diseases—Huntington's disease, Fragile X, and Friedreich's ataxia. There is a long list of these (repeat expansion disorders), they are devastating and very challenging to treat. So PE has this second-generation dual-flap version that can potentially be harnessed to address some of these.Beyond that, we are very interested in other larger changes that you could make to a genome, including the insertion of large DNA sequences. With the twin-PE approach, we showed when I was still in David's laboratory that you could use it to variously and efficiently insert short DNA sequences of 30–50 bases that are called recombinase attachment sites. These can be paired up with a class of enzymes called integrases or recombinases. Many scientists have used versions of these to create companies.Once that specific attachment site has been prime edited into a specific location in the genome, these recombinases can take new DNA pieces and insert them there. We have developed the mechanism for targeted integration of large pieces of DNA. We did the proof of concept in the Liu laboratory and we have been able to develop it at Prime Medicine. At the JP Morgan conference, we showed an example of this in T cells, where we could do targeted integration of 3.5 kb DNA at 60% efficiency.All these additional developments on that PE platform are enabling so many new things, and allow us to address new exciting areas that the initial PE system maybe would not have been able to do.There are many other factors in successfully developing a gene editing technology besides optimizing the chemistry, including delivery. To what extent is Prime Medicine exploring other important aspects of building a successful clinical platform?We are very invested in that. We are here to develop medicines, not to do science projects that can only be demonstrated in a tissue culture experiment. Of course, that is the beginning, but it is not the endpoint. As you have mentioned, delivery approaches are required to deliver these therapeutics, as is (bone marrow) conditioning and other associated processes. This is all part of making the medicine, so we are very much thinking about that on the delivery front.We have been thinking a lot about how do we get our gene editing system into cells in vivo, using transient delivery systems that are based on RNA. These are lipid nanoparticle formulations, the same way that many of the COVID-19 vaccines have been delivered.We are thinking about ways that we can get these into other cell types beyond the liver, which is where primarily they have been effective, and then also thinking of viral delivery solutions that can get our gene editing components into even broader sets of cell types, such as the central nervous system. We are attempting to develop a full medicine, not just the gene editing system.Some genetic therapies that have been recently approved have price tags of $2–3 million or more. You went into training to be a doctor. How do you balance the need for these biotech companies to get a return on their huge investments with delivering an affordable therapy for millions of patients around the world?It is an important question. There are these therapies that could be developed for the Western world, and genetic diseases affect everybody and not in the same way. Obviously, these are inherited from family members and (affect) a large percentage of patients. Certain regions of the world have their own sets of genetic mutations that you might have to fix, so developing treatments for everybody—we are going to figure out ways to pay forward and make it accessible to patients.In the near term, my personal view, which is not the most sophisticated, is that that is going to require—just based on the amount of investment and time that it takes to make one of these the first time—that the cost be proportional, so that we can support the growth of this new therapeutic area. My hope would be that over time, we learn how to do this with more ease, with less upfront investment, with more of an ability to swap out a guide RNA, for example, and not have to do an entirely new clinical trial and be able to move from one patient and their mutation to another patient and their mutation with very minimal extra cost. I think, as this world comes into reality [we will see], the possibility of making these genetic systems for more patients at a very affordable price would make sense.I cannot tell you how many times I saw patients come into the hospital for the same problem over and over again, because of their genetic disease. And from a cost standpoint, it might even make sense to treat that patient, and prevent all those recurrent costs, not to mention the quality of their life that would be affected, and most importantly how much of a benefit that would have to them. I think there is a good reason to invest here, and from my personal perspective, I hope that costs can be minimized in the future so that this is not a major restriction.Reference1. Anzalone AV, Randolph PB, Davis JR, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 2019;576:149–157; doi: 10.1038/s41586-019-1711-4 Crossref, Medline, Google ScholarFiguresReferencesRelatedDetails Volume 2Issue 2Apr 2023 InformationCopyright 2023, Mary Ann Liebert, Inc., publishersTo cite this article:Andrew Anzalone, Alex Philippidis, and Kevin Davies.Anzalone Prime: An Interview with Prime Editing Developer Andrew Anzalone.GEN Biotechnology.Apr 2023.81-86.http://doi.org/10.1089/genbio.2023.29091.aanPublished in Volume: 2 Issue 2: April 18, 2023PDF download