Lab Meeting: Adrian Krainer
“You have to take risks—you cannot be doing the same thing that everybody else is doing.”
OF MIRACLES AND MEDICINE
A holiday story
[INT--DAY--BUSY GROCERY STORE]
It’s Christmastime and a scientist is at the grocery store with his wife. His packed cart weaves in and out of bustling aisles, narrowly avoiding the hordes of last-minute holiday shoppers.
His cell phone rings in the frozen goods section. The scientist steels himself, takes a deep breath and picks up. On the other end, a pharma exec informs him that the drug he has been working on for a decade, has just been approved by the FDA—a first in class treatment for a deadly neuromuscular disease. Smiling, the scientist hangs up and heads towards checkout. On the way, he adds several bottles of champagne to the cart for good measure—after all, it is the holidays.
[INT--NIGHT--SCIENTIST’S HOME]
Later that night, hundreds of patients and families send heartfelt emails to the scientist—this community finally has a weapon to combat the paralysis wrought by this devastating disease. The scientist later tells his family over dinner: “to see videos of kids just walking normally—nothing can compare with this feeling.” The camera pans out: leaving the smiling scientist in front of a crackling fire, surrounded by family, holding a glass of bubbly.
[FADE OUT]
The screen fades to black, and cursive white lettering appears: “inspired by true events.”
Dr. Adrian Krainer’s role in the development of Spinraza reads like a movie script—creativity, hard work and a bit of serendipity combined to create a lifesaving medicine for spinal muscular atrophy (SMA) patients. That the drug was FDA-approved on December 23 (2016), only adds to the feel-good timbre of this imagined box office hit.
And yet many details, surrounding the scientist himself, will invariably be glossed over by potential filmmakers. How the native Uruguayan’s self-study of genetics as a teenager inspired him to learn English and study in the US: “My reading started pushing me in the direction of pursuing genetics research…this is when I started thinking seriously of going to the US for my studies” (Q#2). Absent from any future film may also be Krainer’s undergrad work with the great Cathy Squires at Columbia, where he first cut his teeth implementing M13 cloning and Sanger sequencing protocols (Q #2). His rotations as a grad student at Harvard with molecular biology legends like Matt Meselson, Jim Wang, Wally Gilbert and Tom Maniatis (thesis advisor), I doubt would make the final cut (Q #3).
The years of work it took to perfect an in vitro splicing system (Maniatis group) and the subsequent discovery of the lariat pathway, do not make engaging movie content—studio focus groups will deem such footage “too technical” (Q#4). For similar reasons, the decades of work mapping basic mechanisms of RNA splicing done in Krainer’s own group at Cold Spring Harbor won’t be given much screen time—save for a few strategically placed “aha” moments. That Krainer’s inspiration to study SMA was borne from an NIH workshop (Q #4), is perhaps not exciting enough for the big screen—let’s change it to a chance encounter with a patient during a blizzard that has them holed up together in a picturesque ski chalet…
Developing a lifesaving drug is dramatic—certainly movie material. Yet the journey of a scientist is complex, and usually not glamorous. The reality is that Dr. Adrian Krainer—now the St. Giles Foundation Professor at CSHL—is a focused and rigorous scientist. Over several decades, his lab has steadily contributed to our basic understanding of RNA splicing, and the role this process plays in health and disease. Perhaps most importantly, Krainer’s story demonstrates that investment in basic discovery research can translate to profound therapies and cures for patients. His lab’s current work is focused on uncovering more about the nature of splicing, and how to further exploit this knowledge to combat conditions like cystic fibrosis, GI cancers, and pediatric glioma: “I'm excited about all of these projects… I'm certainly hoping that there’s going to be another drug like Spinraza.” (Q#5).
Read: sequels
Below is an interview with Dr. Adrian Krainer from November 2022:
1. What was your first taste of science? Briefly, what about this initial experience drew you in? Who was your first great scientific mentor from childhood?
I have an older brother, who is four years ahead of me. When we were growing up in Uruguay, he decided to become a chemist and completed his studies at the national university. When I started taking science courses in high school, I always had an older brother to go to and ask questions. Sometimes what I learned in school could be a bit tedious but talking to him could make it more interesting, e.g., in chemistry, physics, and math. He certainly influenced me positively. One turning point for me in high school, was when I started reading about Mendel's classic experiments—when we learned genetics. I found the rest of biology or zoology interesting, though very descriptive. But genetics really was like a revelation. There was a quantitative aspect to it, there was just impact on inheritance and all of biology. So, I started reading a bit on my own about genetics, and evolution and the marriage of the two.
My reading started pushing me in the direction of pursuing genetics research. Then I realized if I stayed in Uruguay I didn't really have that path to research, or at least not at that time—I would have gone to medical school straight after high school like in the European system. I started exploring the possibility of going abroad to just train myself to become a researcher, with lots of uncertainties and with a limited understanding of what that meant. Fortunately, I had the opportunities and took advantage of that in college. I did research in the lab of Cathy Squires—this is where I learned molecular biology and recombinant DNA techniques. She was a great mentor because as I was learning to do things in the lab, she was extremely encouraging. And I felt like she had more faith in me than I had in myself, so that kind of gave me some confidence about pursuing research.
My parents were also very supportive. In my family, my generation was the first that had the opportunity to get higher education—the previous generation, meaning my parents and aunts/uncles, because of World War II had to emigrate from Europe and didn't get the chance to pursue higher education. But they were always supportive of it, as many immigrant families are. So both my brother and I went to university, and many of my cousins as well.
2. What was it like going from Uruguay to NYC & Columbia for college? What was the transition like socially and academically?
It was a culture shock in many respects. Spanish is my native language, and I learned French in a bilingual school. But my English was nil. Then around age 17, when I started thinking seriously of going to the US for my studies, I knew I had to learn English. I started taking intensive courses at the Uruguayan American institute. I arrived in the US in 1977 for fall classes [at Columbia]. New York City has always been a great city. But in the late 70s, it was very different from what it is today. There were reasons to be apprehensive. Just arriving at Columbia, I was overwhelmed with this huge campus. But it's an adventure, right? Either I was going to do well, or maybe not, I had no idea. But I did enjoy the experience, and ended up doing well academically. It's a very different system, because in Uruguay in the last two years of high school, you are already kind of choosing a track: medicine, chemistry, law or whatever. So then by the time you start college, you pretty much study your profession. In the US you study all kinds of things—particularly at Columbia, which has a core curriculum. I was taking philosophy and literature and many other non-science courses that I actually did enjoy a lot. I started research in my second or third year, which was wonderful. Then it just became a question of balancing, lab and studying and doing other things. But it was a great experience.
In the Squires lab I was working with a PhD student, and she was trying to clone the different ribosomal operons from E.coli—which was difficult technically, because of the multiple paralogs and tight expression regulation. So I helped clone one of those operons. My first attempt didn't work—I was troubleshooting and had some weird plasmid that didn’t look right. Cathy encouraged me to just forget about it and start over. But my second attempt worked—and Cathy [Squires] was very impressed.
After that I helped set up the M13 phage cloning system for sequencing by the Sanger method, which was all fairly new. It was new technology that Joachim Messing had developed, and then I also did a lot of Sanger sequencing, and so I was just having fun in the lab and we ended up publishing a paper. I wrote a manual to help other people use these techniques in the lab—it was fun to do that. I was putting a lot of hours in the lab. Especially in the summers, when it was incredibly hot, I would often just go into the four degree cold room to cool off a little bit. So, there was the added incentive to stay in an air-conditioned place as opposed to my dorm, which wasn't.
3. As a trainee you worked with many accomplished scientists. Are there any unifying traits among these scientists, in how they run their labs and mentor trainees?
After undergrad at Columbia, I went to Harvard, which was a very small but illustrious department. The Biochemistry and Molecular Biology Department had maybe 15 or 16 groups and two or three Nobel laureates. It was kind of overwhelming, when I first arrived: I met Matt Meselson at the department retreat when I arrived, and I had read about all the classical experiments he had done—so I kind of put these people on a pedestal…same thing with [Tom] Maniatis. I also had a wonderful rotation in [Wally] Gilbert’s lab, even though he was away helping to run Biogen at the time. I met him only at the end of my rotation, but that lab was wonderful.
Each of my grad school rotation experiences was very interesting. I don't think that there was anything unifying. I would say Maniatis had the most influence on me, since I joined his lab as a graduate student. Everyone to some extent, when you run your own lab, you sort of emulate the things that you thought were done right. You may avoid other things that you've observed there as well. I mean, there isn't formal training on how to run a lab—[science] is still a bit of an apprenticeship more than anything else.
4. What has been your scientific high point? — what do you consider to be the most exhilarating discovery or set of discoveries you have been involved in throughout your career?
When I was a graduate student I developed a system to study splicing in vitro. When that worked, it was exhilarating. We started observing strange, unexpected things, which led to the discovery of the lariat pathway of splicing. Discovery is the most fun, when you just find something totally novel: for example, how splicing occurs through branch point intermediates, and so on. Making the observation of an anomaly and then nailing down the mechanism is certainly one of the high points. Some of the other things where we’ve been successful were more directed, like purifying splicing factors. It’s a lot of work, and so when you finally get there that's also exhilarating. But it’s not quite the same as a new discovery—rather it is like solving a well-defined problem. Developing Spinraza is a bit like the latter [problem solving], because it was a very sequential set of findings: we made some observations to explain why splicing is different between SMN1 and SMN2, and then started thinking about how to push exon 7 inclusion to be more efficient. In the end, we exploited these observations to perform a systematic screen for antisense splice modulators—in collaboration with Ionis.
Drug development is different from discovery, because it is very methodical, I would say. So it's a different kind of feeling. That said, everything really worked well for those several years [of Spinraza development], but I didn't want to allow myself to be too hopeful and get set up for crushing disappointment if something went wrong. There were many key milestones: like when the first patient is injected with drug—I really lost sleep over that, hoping that nothing went wrong.
And we had to be very patient until the safety studies were done, and we could begin to see if the drug might be doing something useful in patients. After several years, there was a big high point: when I got a call from Frank Bennett to say that they had unblinded the data from the phase-3 studies for interim analysis. At that point they realized, yes, it's clearly working, both late-stage clinical studies are statistically significant. And then it was just a rush to the finish line to get it to patients, and how quickly the FDA would move to approval. I remember Spinraza was approved on December 23, 2016. I got another phone call about that approval, and obviously that was a really high point as well. But even following these initial studies, it was later shown that if you give Spinraza pre-symptomatically, you can actually prevent the onset of disease. So, to see videos of kids with genetic diagnosis of type I SMA just walking normally—nothing can compare with that feeling.
[When you got that December 23 phone call about Spinraza’s approval, how did you celebrate?]
I was Christmas shopping, getting food with my wife from the supermarket, and I got a phone call from Frank [Bennet]. We were getting champagne anyway, so we bought it and then went home to celebrate. Actually that same evening, I got a chain email from SMA patients and families from all over the world—there were at least 100 of them. They were all so excited to get access to the drug, and it was incredibly moving.
[What made you interested in studying SMA and splicing of SMN1/2 originally?]
It was a bit serendipitous. In 1999 we were studying the features in exons that can influence splicing. Mutations, even nonsense mutations, in these regions can cause exon skipping. Until then, there was a notion that the splice sites are the only key sequences controlling this process—and that only mutations at splice sites can perturb splicing. But there was accumulating data showing mutations in exons can alter splicing as well. The reason is that there are exonic splicing enhancer regions. We were trying to understand the sequence features of these exonic enhancer regions that influence splicing—but it wasn't really for therapeutic purposes at that point. For instance, we were looking at rare mutations, like a nonsense mutation in exon 18 of BRCA1. We demonstrated that SRSF1 binds to the wild-type sequence, but a nonsense mutation abrogates its interaction—it doesn't actually matter if it's a nonsense mutation, it could also be a missense mutation. What matters is how this exonic mutation alters the binding of the protein [SRSF1]. We found that this principle was generalizable. We wrote a review where we compiled data from other alleles, including survival motor neuron [SMN] 1 and 2 genes. In 1999, there was a paper that showed the difference between SMN1 and SMN2 is a C to T change in exon 7. If you put a C in SMN2 you get exon 7 inclusion, and if you put a T in SMN1 you get exon skipping—thus this change is necessary and sufficient to alter splicing and drive SMA.
I was invited to a workshop at NINDS at the NIH about spinal muscular atrophy [SMA], just before that paper was published, and they invited two or three splicing people because what they realized is there's some splicing issue here and they needed help. I'm lucky that I went to that meeting because I realized, this disease converges beautifully with what we've been doing. Here is SMA, all the patients have this paralog gene with the nucleotide change that causes exon skipping, and so we jumped headfirst to try to understand mechanistically how the C to T change impacted splicing: it turned out to be SRSF1-dependent, via the same mechanism in BRCA1 we had been studying. We published a paper actually comparing both [BRCA1 and SMN splicing].
I had a postdoc, Luca Cartegni who initiated that project and then started playing with antisense molecules: our initial approach was actually a bit different than what Spinraza became. Originally, we designed an antisense molecule with an RS-domain peptide [arginine/serine-rich domain that recruits splicing factors] attached to it—this emulated the function of SRSF1. Since endogenous SRSF1 cannot bind to SMN2 exon 7 to promote splicing, we would just bring an activation domain [RS] conjugated to an antisense molecule. This approach worked nicely in the test tube, both for BRCA1 and SMN2. But we also had controls with an antisense oligo [at the same location], only without the RS-domain peptide attached. And those control oligos also surprisingly worked [in promoting exon 7 inclusion in SMN2]— if we had placed the ASO somewhere else on the exon it may have done nothing at all. It just so happened that the first two [locations] that we tested had an effect. So, the therapeutic strategy eventually turned into a simpler idea [using a naked instead of RS-conjugated ASO]. We published a paper with the conjugate approach, and had a patent on it. That prompted Frank Bennett to call me up and suggest that we collaborate. That was in 2004. Ultimately, there was a lot of serendipity that I was just invited to, and participated in, that small workshop [at NIH]. I don't know if we would have started these studies otherwise.
5. What set of research questions or projects has you most excited about coming into lab today?
We [as a lab] are very focused, I have always worked on RNA splicing, from my PhD to postdoc, and in my lab. Within this universe of RNA splicing, we continue to focus on basic mechanisms, because it's an incredibly complicated machinery. There is now impressive structural data from other labs, but there are lots of things we still don't understand. The splicing machinery has to recognize something like 200,000 different introns with a lot of sequence variation. The signals are somewhat general motifs, and yet, there's very high splicing fidelity. The whole logic, the combinatorial logic, of how you get efficient spicing with exquisite fidelity and how this can be regulated, is still poorly understood. We have some basic principles, but we continue to explore this system.
Then we have a section of the lab that that is more focused on splicing in disease—whether it's therapy development, like antisense approaches [ASOs], or whether it's understanding how mis-regulation of splicing contributes to different types of cancer. One can design therapies, but some of this work is also very basic. I'm excited about all of these projects. Obviously at any given time, some things are going better than others. So, you tend to get excited when things are on a roll. For example, right now we're developing antisense approaches for pediatric gliomas that have noncanonical histone mutation drivers, and we have very promising results. So that's one of the areas that I'm quite excited about right now--it's not published yet, but we just resubmitted a paper after revision.
6. Who are a couple up-and-coming scientists (lab < 10 yr old) in your area, or more broadly, whom you think we should watch? Why is their work so exciting to you?
It’s been harder than normal to track up-and-coming scientists during the pandemic, because we’ve had very few in-person meetings. Nevertheless, I recently heard very exciting work presented by young(er) scientists, such as neuroscientist Sergiu Pașca at Stanford, developmental biologist Ana Boskovic at EMBL-Rome, and computational biologist Rhiju Dhas at Stanford. They are probably farther along in their careers than your question specifies. I feel that all three are doing highly innovative research and thinking outside the box in terms of the problems they are studying and how they are approaching them.
7. What is one piece of advice for a young scientist (grad/post-doc) aspiring to have a career in academia, and make some important discoveries?
I think you have to avoid the herd mentality. You have to take risks—you cannot be doing the same thing that everybody else is doing. When it comes to using new technology that lots of people are excited about, you still have to do something novel to distinguish your analysis. Sometimes being a contrarian is good…just taking risks.
Let me put it this way. I've heard many times Jim Watson giving advice on how to be successful in science, but no matter what advice he gives, nobody else is going to discover the structure of DNA. So maybe his personal recipe may not work for anybody else, right?
With the benefit of hindsight, I think taking risks is important, but you can also end up regretting that if things don’t work out. So I think you really have to be passionate about what you're doing. That is absolutely necessary.
I think if you believe [in your work] enough, you're willing to try and when it fails, you try in a different way. You also have the luxury of taking risks when you are young, at least for some period of time. When I started my PhD project, on developing in vitro splicing assays—well we had no idea if that was even feasible. And I didn't know if it was feasible, and there were plenty of reasons why it might not be, but it was certainly worth trying. So it is important to also give yourself enough time to really take your best shot.
I think people also shouldn't obsess too much about publishing everything in Cell, Science or Nature. We unfortunately give too much weight to that. The importance should be how significant and novel the work is—whether you can get it into those journals is a function of many different things.
8. Can you tell us about the founding story and vision behind Stoke therapeutics? What excites you most about the work going on here?
My experience with Ionis was very good, but it required a lot of investment to approve Spinraza: Biogen put a billion dollars to take this to approval. Managing the business and legal interests of both pharma [Biogen/Ionis] and CSHL was also challenging—non-scientific stuff. So, I thought my next idea may be best pursued in the smaller biotech setting. I had what I thought was a potentially good idea, which was to manipulate splicing in order to upregulate gene expression—there are already many ways to downregulate gene expression. In SMA [with Spinraza] we were upregulating the correct isoform. But the new idea was to target non-productive splicing that occurs normally, and make more of the productive isoform instead.
The original idea was intron retention. If a gene has a bunch of introns, they are probably all not going to be utilized equally efficiently. If you pinpoint the least efficient one and promote more efficient splicing at that site, you may get more expression of the functional gene copy.
I wrote an NIH Pioneer Award application for this idea, and I wasn't successful. I was also trying to convince somebody in my lab to work on this and was not getting any traction. Eventually, I had a senior postdoc, Isabel Aznarez, who hadn't worked on antisense, but rather on basic mechanisms of nonsense mediated mRNA decay. But she came to talk to me and said that she was contemplating the idea of going to industry and what if we took this idea and started a company. She was willing to do a lot of the activities to find investors and so on, and so I agreed. At that time [2014], Spinraza was still in clinical trials, but everything was already looking very promising. So we had the credibility that came from that—before Spinraza I don't think anybody would have given us the time of day. So, we picked a particular set of investors, Apple Tree Partners and launched Stoke Therapeutics in 2014.
The original idea was to upregulate gene expression for haploinsufficient conditions or hypomorphic mutations. We called this approach TANGO (targeted augmentation of nuclear gene output), reflecting the dance and music that originated in Argentina and Uruguay—Isabel is also from Uruguay. We searched for genes that naturally produce nonproductive isoforms—but the company then evolved a bit, because what's been more successful in practice is to target poison-exon isoforms, like in Dravet syndrome [haploinsufficient epilepsy disorder].
So Mendelian disorders and rare diseases are on the radar for Stoke. Through transcriptomic analysis you can uncover potential isoforms where you can switch the splicing in order to express more of the wild-type allele. Dravet became the first indication, and it's doing very well in phase-2 trials. I'm very hopeful about that program. The animal-model experiments were also very convincing. So far, the work is still in dose escalation and safety/tolerability. But there are already encouraging indications of efficacy. And so probably in the next year or two, phase-3 trials will be initiated. I'm certainly hoping that it's going to turn out to be another Spinraza.
[On the trend of academic trainees going to biotech/industry]
There are many job opportunities in industry. So people can take risks, and if things go south, it's not difficult for them to find another company to work in. But the trend at the moment is that many PhD students, most perhaps, are inclined to go into biotech or Pharma. Nothing wrong with that. But if all of them go, we will have no academic research. I think these things tend to be cyclical. People are somewhat discouraged from going into academia at the moment, because it's not that easy to get a good position or funding. But if more people are going to biotech, then probably the pressure for people who want to stay in academia will eventually get reduced. Overall, I think it's good that there are more options today for people going into science careers. I believe that things will naturally find a balance.
Rapid Fire Questions
Which areas of science, outside of your direct field, are you most excited about seeing develop in the next 5-10 years?
Although I’m not doing research in the area of aging and longevity, I look forward to seeing the results of current efforts on these topics. Going way beyond my area of science, I’m excited about new discoveries in astrophysics and innovative approaches to renewable energy. And I’m curious about the search for extraterrestrial life, although I’m more skeptical about its existence than most scientists and non-scientists.
Name an overlooked scientist, whom you feel strongly that every grad student (or med student) should know and be able to describe their major findings. What were the killer experiments?
Matt Meselson is certainly a famous scientist, but I wonder how many grad or med students nowadays can describe the classical Meselson-Stahl experiment, i.e., how it was done and how it elegantly demonstrated that DNA replicates semi-conservatively. Or the fact that his lab was the first to purify a restriction enzyme, though unluckily it was one that was not useful for recombinant DNA technology. Another famous scientist, also from my graduate-school department, was Paul Doty; how many students realize that his discovery of DNA renaturation in the early 60s enabled modern molecular-biology techniques we now take for granted, like PCR?
What are you reading in your free time? Favorite movies?
Lately I’ve been enjoying re-reading several Latin American novels from the 60s, 70s, and 80s, e.g., by Mario Vargas-Llosa. Right now I’m reading a very insightful memoir by a former US ambassador to Romania, Jim Rosapepe, and his wife, Sheilah Kast. It’s called Dracula Is Dead. I recently had the pleasure of meeting them, and my ancestors came from that part of the world, Transylvania, so I can relate.
I have many favorite movies, old and new. Some of my favorite directors are Chaplin, Kurosawa, Fellini, Kubrick, Tarantino, Spielberg, and Allen. The three oldest movies on my favorites list are Modern Times, City Lights, and The Great Dictator; they were made 80-90 years ago, but they still elicit laughter and tears, and continue to inspire. Pure genius …