David R. Liu, PhD, joined the Harvard University chemistry faculty in 1999 at the tender age of 26 years. When you excel at research through high school, undergraduate, and PhD, postdoctoral fellowships become somewhat superfluous—although Liu regrets skipping his postdoc and does not recommend others bypass such training. In 2005, he was promoted to full professor and appointed an investigator with the Howard Hughes Medical Institute (HHMI).

Although his diverse research interests include phage-directed evolution and small-molecule drug discovery, it is his laboratory’s groundbreaking work in genome editing that has truly garnered the interest of the human gene therapy community. In 2016, Liu’s postdoctoral fellow Alexis Komor, PhD, spearheaded the development of the first base editing technology, which had the ability to install specific base substitutions (C to T, or G to A) without cleaving DNA. Eighteen months later, her colleague Nicole Gaudelli, PhD, developed a complementary adenine (A to G) base editor (both studies were published in Nature). Base editing technology is being commercially developed by Beam Therapeutics.

More recently, the Liu laboratory has described prime editing, which enables the installation of all base substitutions, small insertions, and small deletions by using an RNA rather than a DNA template, as well as the first method for making precise changes to the sequence of mitochondrial DNA.

In this revealing interview, David Liu recalls his first foray into genome editing 20 years ago and discusses recent progress, including exciting preclinical results, in a range of genetic disorders applying base editing. (This interview has been lightly edited for length and clarity; a longer version can be found in GEN’s sister journal, Human Gene Therapy.)

GEN Edge: I would like to start by going back to your first interest in genome editing. I read that you even dabbled in some work in this space 20 years ago?

David Liu: You’ve done your homework, or at least you’ve heard the rumors!

I began my academic career in 1999. One of the first projects we started, when I had just a few graduate students as an assistant professor, was a project we nicknamed the Unifactor 2000, because it sounded futuristic to have 2000 in the title. The purpose of the project was to develop a universal transcription factor. Our idea, in retrospect naive in a number of ways, was to digitally address DNA using triplex formation and recruit a piece of RNA or DNA linked to a DNA-cutting enzyme, transcriptional activator, or transcriptional repressor in a sequence-programmable manner.

To support the feasibility of the Unifactor 2000, we had identified reports that researchers had successfully observed triplex formation, even in cells. And although triplex formation is a well-documented phenomenon, it requires conditions that are mostly mutually exclusive with conditions in cells. That turned out to be the Achilles heel of the project, but we were very excited about the possibility of simply recruiting different effector domains to sites in the genome in a sequence-programmable manner. It’s just that a way to do that efficiently in cells did not really exist in 1999–2000, so probably around the time the Unifactor 2000 project reached its namesake year, we killed the project.

GEN Edge: The arrival of Alexis Komor in your laboratory was hugely important in the development of base editing, but to what degree were you exploring genome editing before that?

Liu: We actually first got into genome editing through my good friend and colleague Keith Joung, who was then a young professor at Mass General Hospital at Harvard Medical School (HMS). A student who was on an MD/PhD track—Vikram Pattanayak—was an HMS student who happened to sit next to Keith at an HMS retreat dinner. They had a conversation, which resulted in Vikram becoming interested in the idea of zinc fingers, their programmability, and the extent to which they were able to bind DNA at designed targets…

We completed that initial project, “Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection,” published in 2011. ¹ That was the first article in the genome editing field we ever published. We then went on to perform similar substrate specificity profiles powered by Darwinian selection on TALENs, also in collaboration with Keith’s laboratory, and then on CRISPR-Cas9 in collaboration with Jennifer Doudna’s laboratory.

So understanding specificity was really the first foray into gene editing for our laboratory and then we quickly transitioned to how to improve some of the key features of gene editing agents as we started to identify their bottlenecks.

GEN Edge: It has been about five years since your seminal base editing report (Komor et al.) in Nature. ² As you look back on the past five years, are you surprised just how fast this technology has taken off?

Liu: I am incredibly surprised! If you would asked me in 2016 how long would it take for people to be able to take engineered gene editing macromolecular machines, deliver them into an animal so that they could correct one base-pair causing a grievous genetic disease like progeria and rescue the symptoms in the animal, I probably would have said, “Optimistically, I hope within 10 or 20 years.”

So much has to go right: you have to develop the machine and have it function with an efficiency and a specificity that does not cause problems in the animal. You have to figure out how to deliver this machine, which has traditionally been one of the challenges in any kind of applied protein engineering. You have to hope that the biology that links the genetics to the disease is rock solid. And of course, you will be trying to forge a path for connecting the biology of the corrected allele to the rescue of the disease, which is not a guarantee either.

It still sounds like science fiction when we think about it. I think it is a testament not only to the hard work and talents of people such as Alexis Komor (Fig. 1) but also really to the entire field. There are many laboratories that are using and improving base editors. (The nonprofit) Addgene says there have been about 11,000 base editing constructs sent out just from our laboratory, and I’m sure thousands more from other laboratories as well.

GEN Edge: There was another base editing article led by Akhiko Kondo, from Kobe University, in 2016. How does their technology differ from or complement yours?

Liu: The Kondo group had been independently working on ways to mutate genomes using deaminases linked to DNA-binding proteins. Several months after our article,² they published their study in Science³ reporting the fusion of a different deaminase to Cas9—it has slightly different properties, because the deaminase has slightly different enzymatic properties.

I think the contributions that we made that may have helped their study included nicking the nonedited strand and fusing the UGI, for which they cited our article—but they may have been thinking along those lines anyway… It established that two totally different laboratories with different deaminases and different interests—the Kondo laboratory’s focus then was agriculture and biofuels—could show that this was a robust approach to making targeted point mutations in the genome.

Second, what happens next in terms of translating the technology into some kind of societal benefit was important. After I gave one of my very first talks on base editing before the article was published, I was approached by (VC firms) F-Prime and Arch to start a company around base editing. My naive initial answer was, “I’m already a co-founder of a gene editing company, Editas, and they should have the opportunity to incorporate base editing into their company.” They said, “We think base editing should be a separate company.” Their argument was that being a different technology would require a different effort to optimize and to apply successfully, and the targets that we would pursue would also be different. Ultimately, I think it was good advice.

Another good decision we made early on was to not try to fragment the base editing field into multiple companies. If our main interest is to bring base editing technology to benefit patients, we should try to get everybody under the same tent. The group that would end up forming Beam Therapeutics reached out to Professors Kondo and Nishida, who published that Science article, and invited them to join forces and be part of the company, which they did. Their own commercialization effort sublicensed their part of base editing to Beam for human therapeutics outside of microbiome applications, while keeping their own ability to develop their base editors for microbiome use.

The important point is base editing did not become multiple companies spending resources trying to compete with each other.

GEN Edge: Your progeria study was just published in Nature⁴ in collaboration with NIH Director Francis Collins. Why is that study so exciting and what are the prospects for translating this study in a mouse model into patients?

Liu: The project was really interesting and had a surprising outcome for several reasons. Among all the various gene editing therapeutics efforts that we are doing in our laboratory or in collaboration with other laboratories, I would have said at the outset that progeria would be toward the challenging end. It is a systemic disease, so it must be addressed, therefore, in vivo. The proximal cause of death is often cardiovascular failure. You are not going to edit the heart ex vivo and then transplant it, presumably. And it is a grievous progressive genetic disease for which there is no treatment that has been shown to greatly extend lifespan. It also was not clear what the window of opportunity would be to correct the disease… it could be that once an animal with progeria is born, its fate is sealed. All of those features—systemic, in vivo, progressive, rapidly degenerating disease—suggested this could be a real challenge.

GEN Edge: But you did it anyway…!

Dr. Liu: Yes. I really credit the graduate student, Luke Koblan, who led the effort and decided to try our brand new A-base editor (ABE), which we had not even published at the time (the development of which was led by Nicole Gaudelli)…⁵

The results were encouraging enough that we started a collaboration with Jonathan Brown at Vanderbilt, who studies vascular biology and disease… We injected the single pilot mouse with AAV encoding this base editor. It is the kind of swing-for-the-fences pilot experiment that is arguably naively optimistic, because it is a stretch to go from patient-derived fibroblasts to a mouse. The split-intein dual AAV system we used to deliver the base editor into the mouse had not even been published at that time.

But they injected the single mouse. We did not have at the time the sophistication or the manpower to do extensive vascular pathology analysis or lifespan analysis. We simply took some of the basic organs such as the liver and the heart and sequenced them. We were surprised to find 20–60% editing in most of the organs that we sequenced.

And when we did an RNA analysis, we saw that the amount of the toxic progerin RNA that results from mis-splicing caused by this silent point mutation had gone down quite a bit and the amount of protein in the Western blot had also gone down in favor of the healthy normally spliced lamin A protein, which was a remarkable outcome.

We got that data shortly before I was invited to give a talk at the NIH.… Right after the talk, Francis [Collins] offered to collaborate and explained that he had this large colony of progeria mice that each has two copies of the human mutated lamin A gene. The fact that they had the human gene really makes the work more therapeutically relevant because in principle the exact same composition of matter AAV9 targeting the human progerin gene that we use to treat these mice could be used in a patient.

GEN Edge: How does this look for potentially trying this in patients?

Liu: We’ve decided to take two approaches. One approach is a recognition that although there are some treatments, including the recently FDA-approved small molecule called lonafarrnib, which works by inhibiting protein farneslyation, which offer patients some benefits in quality of life and lifespans, there has been no treatment, including lonafarnib, that seems to show the kind of lifespan extension and general animal vitality rescue that we observed from directly correcting the root cause of the mutation with a base editor.

Leslie Gordon from the Progeria Research Foundation made compelling arguments that we should take two approaches: we should advance the current treatment in its current form and do the remaining toxicity and biodistribution studies to pave the way for a potential clinical trial of pretty much what we reported in Nature.

In the meantime… we’re hoping to optimize the base editors, AAV, and the timing of the dose to get a deeper understanding of what kind of patients might benefit, and how to offer them the highest ratio of potential benefit to potential risk.

GEN Edge: The other interesting animal model study in base editing last year was from Sek Kathiresan and his team at Verve Therapeutics. They are tackling genetic forms of heart disease but looking at the potential of base editing to treat a much broader set of complex diseases. I wonder whether you are comfortable with that?

Liu: I’m not involved in Verve, although Verve and Beam are co-developing Verve’s base editing therapeutic, which Verve has announced is their lead program. From my vantage point, it is an amazing outcome. They sought to knock down PCSK9. They compared doing so with a Cas9 nuclease and with a base editor, and they found a better outcome with the base editor.

Of course, the really exciting data are that they treated nonhuman primate monkeys with a lipid that delivers a base editor into the liver; it edited quite efficiently the PCSK9 gene, knocking it down. The result is dramatically improved blood parameters, such as serum low-density lipoprotein cholesterol and triglycerides.

The second part of your question is really interesting. It was in my opinion a brilliant choice of target, because there are lots of human genetics around PCSK9 that has established a pretty strong relationship between knocking out the gene and lowering risk of cardiovascular disease. It provides an opportunity—in my opinion—to choose a patient population that is matched to both the medical need and to societal acceptance of gene editing. Of course, there are open questions that, as the field of clinical base editing matures, will help guide the best risk–benefit ratio.

In other words, you can start out by treating patients with very high-risk familial hypercholesterolemia—people who have a very high chance of having heart attacks or strokes or other cardiovascular problems much younger than normal. In that case you can say, even if this is a new experimental therapeutic modality, the potential benefits are worth the potential risks. But if the unknowns begin to be known, and the risks begin to be understood to be acceptable, and the technology is optimized with respect to minimizing off-target editing and delivering efficiently and editing on target efficiently—then as you point out, it does raise the possibility that perhaps not just the most endangered subpopulations of patients, but maybe also a broader set of patients, could benefit.

For that matter, there are other conditions in which there are known disease prevention alleles or disease risk alleles that could be corrected or installed with the base edit. Alzheimer’s disease, of course, has APOE4, which is a serious risk allele. But it also has Icelandic amyloid precursor protein (APP) variant—Ala673Thr—which confers strong protective benefit enjoyed by roughly 1 out of 1,000 or fewer Icelandic people and virtually nobody else. In other words, from the perspective of APP, the vast majority of the human population has the disease-causing allele.

It would be an interesting question: if clinical base editing begins to flourish and is viewed as safe and efficacious, whether society should pursue not just disease-correction alleles or alleles that elevate disease risk like APOE4, but also perhaps disease prevention, like the PCSK9 example or the APP example. There are others—there is a prion mutation that lowers your risk of prion disease, for example.

GEN Edge: There are some 7,000 known Mendelian genetic diseases. What proportion of those genetic diseases is base editing potentially able to help correct? How do you begin to make a bigger dent in tackling not just a few cases but hundreds or thousands?

When Alexis Komor published our article in 2016, the answer was we could only make two kinds of changes—C-to-T and G-to-A—and we only showed that we could do this with canonical spCas9-derived base editors, we could only position the base editing activity window over ∼25% of those pathogenic mutations that were in principle correctable by making one of those two changes.

Thanks to the hard work of many laboratories including those of Keith Joung, Ben Kleinstiver, Osamu Nureki, and our own, a variety of Cas variants have been engineered or evolved that have greatly expanded the flexibility with respect to where you can park a base editor by offering tremendous PAM flexibility. If you crunch the numbers, the math works out that now say 95% of pathogenic transition mutations can be addressed with a base editor. Of course, now C base editors and ABEs, which collectively can cover something like 25–30% of all known pathogenic human gene variants, are widely used.

That still leaves a huge amount of territory, which was one of the inspirations behind our development of prime editing.⁶ Prime editors can make all 12 kinds of base-to-base changes, as well as small insertions and deletions. Although prime editors are much newer and have gone through fewer rounds of improvement—there have only been a couple of dozen articles or preprints published as opposed to several hundred base editor articles at this point. But the community and our laboratory are working really hard on expanding the scope of prime editing the same way that the community expanded the scope of base editing, to hopefully cover a large majority of that enormous pie chart.

GEN Edge: You presented prime editing for the first time at Cold Spring Harbor in late 2019. At the end you acknowledged the first author of the article, Andrew Anzalone, and quipped, “I’m really looking forward to seeing what Andrew can do in the second year of his postdoc!” Has he been as productive as his first year?!

Liu: [Andrew] became one of the founding scientists at a company called Prime Medicine to transform his remarkable work into therapeutics… Andrew is one of the most talented and collaborative people I have ever had the pleasure of mentoring in 22 years. In the second year, he developed some improvements of prime editing that we have not reported yet but hope to soon. He contributed to a number of projects in ways that are hard to imagine. I started to notice that when Andrew presented at group meetings over Zoom, more people than normal would come just to hear him talk. He grew quite a following.

GEN Edge: 2020 was a big year for CRISPR with the award of the Nobel Prize for Chemistry. I’m sure there were some mixed emotions at the Broad, but you put out a nice statement congratulating Jennifer and Emmanuelle. What do you think that award meant for the field of CRISPR and genome editing more broadly?

Liu: I think everybody in the gene editing field, including everybody at the Broad Institute, was really happy and excited to see the seminal work of Emmanuelle Charpentier and Jennifer Doudna recognized by the Nobel Prize in Chemistry. You cannot award prizes to everybody who has contributed to a field and in a sense, the recognition that their contributions—and by association, the contributions of everybody who have really built that field so quickly into one that is already having impact on society—is a wonderful incredibly positive development for the whole field. Their seminal article—the first to combine gene editing with CRISPR—was highly influential for many researchers, including myself. They are richly deserving of this recognition.

The full transcript of this interview can be found in the journal Human Gene Therapy (March 2021).

References:

1. Pattanayak V, Ramirez CL, Joung JK, et al. Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat Methods 2011;8:765–770.
2. Komor AC, Kim YB, Packer MS, et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 2016;533:420–424.
3. Nishida K, Arazoe T, Yachie N, et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 2016;353:aaf8729.
4. Koblan LW, Erdos MR, Wilson C, et al. In vivo vase editing rescues Hutchinson-Gilford progeria syndrome in mice. Nature 2021;589:608–614.
5. Gaudelli NM, Komor AC, Rees HA, et al. Programmable base editing of A · T to G · C in genomic DNA without DNA cleavage. Nature 2017;551:464–471.
6. 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.

The post Feel That Base: An Interview with Base Editing Pioneer David Liu appeared first on GEN - Genetic Engineering and Biotechnology News.

In 2008, I arranged to meet a trio of young executives from a small British start-up called Oxford Nanopore Technologies (ONT) at a genomics conference in San Diego. The trio had just flown in from London but as we ordered our first round of drinks on a hotel patio at sunset, they appeared energized by the bucolic surroundings and the tranquil California weather. Gordon Sanghera, PhD, had taken the reigns as chief executive after a successful stint at a medical devices company. He was accompanied by business executive Spike Willcocks, PhD, and communications chief Zoe McDougall.

Sanghera impressed me with confident stories of how his fledgling company would one day transform the next-gen sequencing (NGS) space. Supporting data were scant but undeniably tantalizing: Willcocks opened his laptop and showed me the schematic models and preliminary results that fueled an almost magical vision for the future of DNA sequencing: a pocket-sized instrument—the MinION—that could read out a DNA sequence in real time by enzymatically chopping a single strand of DNA, like a master chef slicing a scallion, capturing the signature of each nucleotide as it was sucked into the belly of a bacterial nanopore protein.

This was almost too-good-to-be-true, but I admired their ambition coupled with an ability not to take themselves too seriously. I was also intrigued because, when it came to its reputation for cutting-edge life sciences research and commercial spin-offs, Oxford University (my alma mater) usually played second-fiddle to Cambridge, with its legendary history in molecular biology of Crick & Watson, Fred Sanger, and more.

Indeed, the NGS revolution had its roots in Cambridge, as two chemistry professors, Shankar Balasubramanian, PhD and David Klenerman, PhD, both since knighted, launched a company called Solexa. In 2006, Solexa was acquired by Illumina, supplying the sequencing-by-synthesis technology platform that would dominate the NGS space for the next 15 years and counting.

None of that seemed to worry these upstart Oxford entrepreneurs. “We might open up with Monty Python’s ‘And now for something completely different!’” Sanghera said as he anticipated ONT’s arrival on the scene. I had a hunch he wasn’t joking. I summarized our San Diego conversation in my book, The $1,000 Genome, published in 2010.

Two years later, the company’s chief technology officer, Clive Brown, delivered one of the more memorable talks at the Advances in Genome Biology & Technology conference, the marquee science conference in Marco Island, Florida. By this time, ONT’s sequencing strategy had shifted to running an unzipped single strand of DNA through the nanopore, using some sophisticated algorithms to compute the corresponding sequence from the squiggly shifts in electrical resistance.

Brown’s unveiling of the MinION—a two-hour lecture crammed into 17 minutes, delivered with dry British humor (and, yes, a Python reference)—electrified the audience and signaled a new contender in the NGS arena. “So we did a genome,” Brown said nonchalantly, referring to the decoded sequence of the virus PhiX174. (Seven years earlier, he’d led the Solexa team past the same milestone.) On Twitter, scientists positively drooled about the arrival of nanopore sequencing.

However, not everything went to plan in those early years: product launches were delayed by microchip design flaws, deals went awry, and lawsuits inevitably started flying. But ONT weathered the storm, winning droves of loyal fans enamored by the platform’s plug-and-play ease of use and extraordinary portability. Today, the company offers an expanded portfolio of sequencing products and with community support, has greatly improved the accuracy and flexibility of the sequencing platform. Together with rivals PacBio, ONT provides long-read sequencing to complement the short-read technology of market leader Illumina.

To its credit, ONT has lost none of its swagger. Sanghera still likes to make a grand entrance at the company’s customer events, striding on stage accompanied by heavily amplified riffs from The Clash or The Arctic Monkeys. Press speculation says that ONT is finally on course for an initial public offering; it has raised more than $800 million since launch and is valued at around $2 billion.

Top marks

For those interested in the origins and current status of nanopore sequencing, a new article will prove invaluable. “Nanopore sequencing,” written by science historian Lara Marks, is available at What is Biotechnology?

Marks delves into the academic origins of nanopore sequencing, interviewing the pioneers including David Deamer, PhD, (who first sketched the idea for nanopore sequencing in 1989), and Oxford University chemist and ONT co-founder Hagan Bayley, PhD, while highlighting the contributions of other luminaries including Daniel Stanton, PhD, Mark Akeson, PhD, and George Church, PhD. Marks reveals that the nanopore proof-of-concept study, co-authored by Deamer and Daniel Branton, PhD, published in PNAS in 1996, was— like many profound papers before and since—rejected by both Nature and Science.

As Marks points out, “ONT’s nanopore sequencing tools are now being used in over 100 countries by researchers in human, plant, animal, microbiological or environmental genetics.” It has been used for experiments on the International Space Station and come into its own as a portable tool for monitoring viral outbreaks (Zika, Ebola, and so on) in places like Africa and South America. It is currently seeing new applications in monitoring COVID-19 variants, using a diagnostic test called lamPORE.

Despite the technology’s progress, some big hurdles remain. Nanopore sequencing “challenges the prevailing culture of how sequencing is done,” Marks writes. “Many researchers have so far been hesitant about using the technology because they prefer to continue with PCR with which they have more familiarity.” ONT encountered similar resistance from the UK government when discussing COVID-19 testing operations.

The Marks article vividly demonstrates just how far nanopore sequencing has come from its humble beginnings. ONT’s upward journey will be one to watch for the next decade. It brings to mind another classic line from Monty Python:

“Kilimanjaro is a pretty tricky climb you know, most of it is up until you reach the very, very top—and then it tends to slope away rather sharply.”

The post Nanopore Sequencing: The Long and Winding Road appeared first on GEN - Genetic Engineering and Biotechnology News.

Stephen J. Russell, MD, PhD, moved from the United Kingdom to the Mayo Clinic more than 20 years ago and has never looked back. He is a Professor of Medicine at Mayo and also President and CEO of Vyriad. His passion remains oncolytic virotherapy. Some remarkable successes in the clinic give Russell—and his commercial and pharmaceutical partners, including Regeneron—the belief that this can become an increasingly important aspect of cancer therapy.

Kevin Davies recently spoke to Russell – who is also serving as the 2021 president of the American Society of Gene & Cell Therapy — about his passage to America, his research and clinical successes, and his hopes for the future of oncolytic virotherapy. (This interview has been lightly edited for length and clarity.)

GEN Edge: When did you come over to the United States? Clearly you have resisted any temptation to go back.

Stephen Russell: I graduated from Edinburgh University back in 1982. I decided while I was at medical school that I wanted to use viruses to treat cancer—that has been a lifelong passion… After I got my PhD, I moved to Cambridge into Greg Winter’s laboratory and completed my hematology training there. I became a consultant hematologist and got a laboratory of my own in the Center for Protein Engineering. I was hell bent on staying in the United Kingdom and building my career at Cambridge…

Then I got a call from Mayo Clinic saying we have this big new program in molecular medicine that we would like to build. We have seven faculty positions to fill, 10,000 square feet of laboratory space negotiated by previous (candidates) who decided not to go. I think they were scraping the barrel, but anyway, they asked me, do you want to come and direct the program? Given that I wanted to do oncolytic viruses in a translational mode, they were throwing down the gauntlet. I absolutely had to come. It was a phenomenal opportunity, a wonderful move [in 1998].

GEN Edge: You mentioned oncolytic viruses. What got you switched on to knowing that that was what you wanted to do?

Russell: It was in medical school. I had just been told after my microbiology final examination in the third year that I was coming back for a distinction oral—and the following day I got a phone call to say to my sister had died in a house fire. I went home on the train from Edinburgh to the south coast and I just lost myself in virology because I needed something to take my mind off what had happened. I realized, just reading about viruses, that it was the last untapped bioresource. We should use them to destroy something we need to get rid of. I decided I am going to do viruses for cancer. Once you realize the destructive power of viruses can potentially be harnessed, it is quite addictive.

The whole idea of oncolytic virotherapy is that viruses destroy different tissues. For HIV, it is the immune system; hepatitis, the liver; encephalitis, the brain. What is the basis of that tissue tropism and can you engineer viruses to make them highly specific for tumors? It turns out you can. The targeting strategies that you can use are not just at the level of entry, which is targeting viruses by antibody display on surface proteins (engineered to destroy their natural binding), where we have had a lot of success. They can also be targeted inside the infected cells, where they are good biosensors because, once the viral genome is inside the tumor cell, it can register whether appropriate transcription factors are available, or other parameters needed to complete its life cycle.

We also developed a micro-RNA targeting strategy—if you have a virus that damages normal tissue, and you want to remove that tropism, then you can stitch into the viral genome a micro-RNA target, such that the genome is destroyed in the tissue you do not want to damage. We did it first for a picornavirus, with a muscle tropism that we wanted to get rid of, and there are plenty of muscle-specific micro-RNAs. So targeting is absolutely central to the whole oncolytic virotherapy field.

GEN Edge: What are one or two of the most successful examples of oncolytic virotherapy?

Russell: The biggest event in my life as an oncolytic virotherapy person was a patient named Stacy Erholtz, whom we treated at the Mayo Clinic 7 years ago with a measles virus. She had multiple myeloma, she had had two stem cell transplants, and she was resistant to all available myeloma therapy. She had rapidly relapsing disease after a second transplant and a large tumor on her forehead. Her PET-CT (positron emission computed tomography) scan showed four other tumors and she had diffuse infiltration of her bone marrow.

We gave her a single infusion of this oncolytic measles virus at the top dose level—it was a very high dose 1×10¹¹. Within a few days, she noticed that the tumor on her forehead was disappearing. She went into complete remission and remains in complete remission today, seven and a half years out. That illustrated that with oncolytic virotherapy, you can hit disseminated disease targets by intravenous administration of a virus. And you can get rid of diffuse infiltrating disease in the bone marrow and it can be durable.

We were very excited when we achieved that and thought “Aha, we’ve unlocked the secret to this.” We have subsequently discovered that it is not so easy — there were some special things about Stacy that led to her responding. One factor was she had no anti-measles antibodies in her bloodstream… She did, however, have anti-measles T cells, and so the virus could get to the tumor and the anti-measles T cells could then enhance the immune response to those infected tumor cells. Also, when we compared her with other myeloma patients, she had a higher mutational burden than the vast majority of myeloma patients that were in the public databases.

We think that high neo-antigen burden was probably contributory. When you give oncolytics, it is a two-stage therapy. Stage one is the virus infects and kills tumor cells—inflammatory killing. Step two is that you get long-term immune control because you have amplified the tumor-specific cytotoxic T lymphocytes in the context of that reaction against the virus.

GEN Edge: The case study that you just mentioned, was that published?

Russell: We put it in the Mayo Clinic Proceedings.¹ We submitted (the article), while we had two patients who responded, to the New England Journal of Medicine. They came back saying, “we want a third patient.” Given I was working at Mayo Clinic, I thought, let us put this in Mayo Clinic Proceedings. It was probably one of the biggest media events that Mayo Clinic had had. The attention that it attracted was phenomenal, which made it all the more dismaying that we could not just repeat it…

I think systemic oncolytic virotherapy is where we need to get to. Because of the anti-measles antibodies we moved to the vesicular stomatitis virus (VSV). It is a virus that causes a blistering illness in hoofed animals. It is endemic in Central America and the southern United States. Most people have no pre-existing exposure or antibody and you can easily grow it to high titer. We shifted our attention to that virus and we have been running it in intravenous clinical trials. It is starting to look very promising. We have unpublished data—we have a complete remission and a very deep partial remission in some patients.

GEN Edge: Have there been other successful clinical treatments during the past 5 or 10 years?

Russell: There is very little on systemic—I am not aware of other comparable cases. As I said, last year we had a patient that we treated with VSV who had extensive treatment refractory disease, who went into complete remission and remains in complete remission almost a year out from therapy. We think we are back there, but we had to take a deep dive and develop the VSV as a therapy and then go through the dose escalation. I have not seen comparable results from others. I think most of the success has been with intra-tumoral delivery and in the context of melanoma where it is an immunogenic tumor.

GEN Edge: Does oncolytic virotherapy have the chance to become a bigger and bigger part of the oncologist’s armory?

Russell: There is massive interest in it from big pharma at the moment. The hunt is on for something that can be used fairly generically in combination with checkpoint inhibitor antibodies. If you think about all the different approaches that have been taken in combination therapies, many of them are intratumoral delivery of something that inflames the tumor. The idea is, if you can inflame tumors then the checkpoint inhibitor antibodies are going to have something to work on, because you will have a lot of T cells.

So what is the best innate agonist or vaccine approach? If you think about oncolytic virotherapy it can come in destroy tumor cells and provoke a response against unknown tumor antigens. So it is an antigen-agnostic vaccine that can be used in conjunction with checkpoint inhibitor antibodies. That is really where everybody is going—all the pharma companies that have bought or entered into collaboration with oncolytic virotherapy companies, it is because of that.

At Vyriad, we have a major partnership with Regeneron Pharmaceuticals and a multicenter multi-indication phase II clinical trial combining intravenous VSV coding for interferon beta and NIS, in combination with the anti-PD1 antibody. So far, so good—it is looking pretty promising. The combinations are synergistic between the virus and the checkpoints preclinically. That study got underway fairly recently. We have 10–12 patients accrued so far out of a total expected of 140.

GEN Edge: You are President and CEO of Vyriad. Why did you launch the company?

Russell: I’ve spent my life in academic medicine until the launch of Vyriad. I have been committed to developing oncolytic virotherapy, no matter what it takes. At Mayo Clinic in the academic environment it could go so far—we could make lots of different viral constructs, test in mouse models, take through manufacturing on a small scale, and start local clinical trials. But accrual would always be slow because it is a single center.

Mayo was never going to become a pharmaceutical company and start developing product. We knew we needed a company. Mayo changed the rules back in 2013 about faculty getting involved in companies, starting an employee entrepreneurship program. As soon as they fired that starting gun, I was on it, because I have been desperate to get involved in that way.

We were able to put the technologies that we were developing at Mayo Clinic into the Vyriad and start thinking about multicenter clinical trials and moving things more aggressively. For me it has been a real eye opener to understand how things really work. In academic research you are often working on something to get it into the clinic. But you do not really think beyond that. There is so much more that has to happen for a product to finally get approved.

GEN Edge: Is Regeneron your biggest partnership to date?

Russell: Yes. It is a substantial partnership—we finally signed the Regeneron contract in October 2019, after a 2-year negotiation. It is a very strong collaboration with two components to it—this multicenter clinical trial that combines their antibody with our virus. There are frequent meetings to discuss how that is going, whether to add new arms, what indications we are chasing down.

Then there is a preclinical component in which we are engineering new configurations of VSV. It is so much bigger than anything I ever did in academia, because they have huge scientific firepower at their headquarters. They have a lot of their scientists working on this, we have 15 scientists working on it on our end. It is a very different approach to the development of the new oncolytics. By having the scale, you march through everything, nothing is left to chance. Any question that anyone raises leads to another animal experiment, defining things mechanistically. I have been impressed with the next-level science that it is taking me into.

This interview is adapted from Human Gene Therapy (February 2021).

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It is rare that a breakthrough study performed as a graduate student sets the course for the rest of a scientist’s distinguished career. But Richard Jude Samulski’s work in the early 1980s on an emerging viral vector—adeno-associated virus (AAV)—played a profound role in shaping the evolution of the gene therapy field. Samulski and colleagues stuck to their beliefs in the clinical potential of AAV, riding out some difficult times for the field to emerge emboldened.

Until 2016, Samulski was the head of the Gene Therapy Center at the University of North Carolina School of Medicine. In October 2020, the German big pharma Bayer agreed to acquire Samulski’s company AskBio for an impressive $4 billion.

Kevin Davies recently interviewed Samulski to learn about his early research career highlights, the launch and evolution of AskBio, and his hopes for the future of AAV therapy. (This interview has been lightly edited for clarity and length.)

Kevin Davies: Jude, how did you first get interested in gene therapy?

Jude Samulski: You’re really going back in time! In 1978, I was a graduate student at University of Florida. I was the first student for a new professor named Nicholas Muzyczka. If you could spell his name, you can join his lab..! About a year or two into the project, it became clear that SV40 was very unstable due to recombination. So we started looking for a new virus as a gene delivery system.

That’s when we came across adeno-associated virus (AAV) as a unique virus. One, it infected humans. Two, it was non-pathogenic. And three, it looked like it had a unique life cycle: in one phase, it would go into the cell and be latent, which would be ideal for delivering genes. In the other phase, it required a helper virus to propagate itself. The key at that point was to see if we could segregate the production component from the latent component and manipulate it—produce virus in one setting and then have it deliver genes in another setting.

My job was to clone AAV into a bacterial plasmid and test whether the recombinant plasmid, when introduced into human cells, would mimic the latent phase, where it would be persistent until adenovirus co-infection. Then, when adenovirus came into the cell, it would activate AAV to go into the lytic phase and make copies of itself. We were able to do that in 1978—we published that in PNAS in 1982…¹

I went on to do a postdoc at Princeton in Tom Shenk’s lab. We generated one of the vector substrates commonly used in every lab around the world for cloning genes into AAV. Our production system became very popular… We stripped it down—96% of the viral genome was removed. That made the packaging capacity of AAV about 5 kb that everybody uses today.

Davies: How excited were you about the clinical potential of AAV at this time?

Samulski: You have to appreciate, we were thinking about putting genes into cells, not people. The target was: can you get something back into the cell and validate it works? It was cells first and then animals next…

We realize[d] that by physical techniques, we could get the virus exposed to cells that it never saw in a normal infection. This opened up a plethora of target tissues simply based on AAV receptors—put it in the bloodstream it’ll go into the liver; put it into the hippocampus, it will go into neurons; AAV into the heart will go into cardiomyocytes. Everybody started studying diseases that were either tropic for ocular, liver, muscle, neuronal, and so forth. It really exploded.

The paper that really launched a thousand labs to jump from retroviruses and adenovirus to AAV was the one that Xiao Xiao and we published in 1996.² Realize this: everyone who put a gene into an animal’s muscle—whether it was adenovirus, retrovirus, lentivirus, plasmid, naked DNA or liposomes—they all had an immune response to the gene product and it was cleared from the tissue within two weeks to a month.

Xiao’s paper was the first example where we put in a gene and sacrificed the animal at two weeks, four weeks, etc.—each time he did it, he showed me the results using beta galactosidase… He went out over a year-and-a-half until they died of old age and said, “Jude, the gene is still on.” At that point, we knew this vector was special. This was that eureka moment… The promise of gene therapy was officially launched.

Davies: Where was that paper published?

Samulski: In the Journal of Virology in 1996.² We could not get that paper published for two years! People wouldn’t believe it and each time we presented it, they kept saying there’s something wrong, you are staining the tissue wrong, etc. We went to a number of journals until we finally got some virologists that looked at it from a virology perspective. “Oh, it’s set up latency. That’s what one would expect.”

Davies: You founded AskBio back in 2001. How did you manage to launch a gene therapy company when the field was in such turmoil back then?

Samulski: Good question. Not only that, the day we launched it was 9/11. Talk about all the cards stacked against you! We did struggle. It was such a bad time with respect to gene therapy. People couldn’t segregate the difference between adenovirus and AAV after the death of Jesse Gelsinger. We immediately got thrown into that group of the “bad virus.”

We actually came up with a different name—we went around saying, “We don’t do gene therapy. We’re working on biological nanoparticles.” Nanoparticles were big back then; anything that was 20 nanometers or smaller was a nanoparticle. AAV was 20 nanometers and we published this in 2008.³

We said it delivers genes and they were all excited about it. It was a phase for a while until people became educated and realized that viruses were still the golden chariot as far as getting things to work with respect to efficient gene delivery. Most of our funding came from parents, foundations and SBIRs. Thank goodness for President Obama, because when the crash of 2009 happened, they were putting out money for small businesses and we were able to get some to keep our efforts going.

Davies: Your company is named AskBio for short, but tell us about the full name.

Samulski: It refers to Asklepios—the god of healing incurable diseases. In fact, the two snakes you see wrapped around the staff for the medical symbol [Caduceus]? That’s because one day, someone cut off the head of a snake and Asklepios supposedly put it back together and healed it…

In classic Greek mythology, Zeus was told by the god of death that [Asklepios] is taking his business away and he wants this guy taken out. Zeus decided, you can’t kill him. He’s doing something remarkable. Why don’t we make him into a god? So they turned him into the god of incurable diseases. We felt that was appropriate because we were working on incurable diseases. But we couldn’t say it or spell it, so we shortened it!

Davies: At some point you created subsidiaries—Bamboo, Chatham and NanoCor. I presume this was an important way, not only to specialize in certain therapeutic areas but also, once acquired, a great source of revenue?

Samulski: It was done for two reasons. I was part of the first wave of the gene therapy companies, but more as a consultant. I was part of a famous company that many people don’t remember called Somatix. Somatix had Inder Verma from the Salk Institute, Richard Mulligan from Harvard, Tom Shenk from Princeton, and they invited me to be part of that. They had retrovirus and adenovirus and naked DNA…

We failed miserably because we were ahead of our time. We were trying to develop these delivery systems and nobody was willing to fund because they were waiting for one delivery system to actually prove itself. When I saw that the whole company went towards cancer and then it was using adenovirus, and none of the technology we developed was ever going to be utilized, I backed off and said, I will never let that happen again.

The template we used was that AskBio would be the mothership. Rather than have a company take a clinical trial forward and, if it failed, the whole platform failed, we dropped it down into special purpose entities (SPEs) that went forward as Chatham Therapeutics or Bamboo or NanoCor. That way that structure could partner with someone and if it succeeded, it would go forward. If it failed, it didn’t kill the platform.

Davies: Did you consider taking AskBio public?

Samulski: We were getting ready to go public, but another variable that was hard to weigh in was being in the middle of a pandemic with ten million people out of work. How long is this going to last? What’s the market going to do? There’s a presidential election. We might go public and be dead in two months because all the money dries up.

When a number of large pharmas started knocking on the door saying, “we’re interested,” we saw that as an opportunity for our funding entity. If they leave us alone, we get everything we wanted as if we went public. It turned out to be more conducive for us being independent and not having to flow with the markets to see what would happen.

Davies: Bayer’s deal was reportedly for $4 billion, but you’re being left as a fairly autonomous unit within the pharma?

Samulski: We only did it if we were autonomous. They were very insightful saying, “We want to leave you alone because you’re innovators and we need you to keep doing that. We don’t want to burden you with our bureaucracy and processes.” That was a tremendous opportunity to finally get enough gas in the engine that you could drive the whole track without having to stop.

Davies: What do you have in the pipeline and what does this infusion of funds allow you to do that perhaps you couldn’t execute before?

Samulski: Almost everything we did before was [funded by] a foundation or family or a grant. That meant you did one project at a time and try to get it in the clinic. Now we have Pompe disease—we’ve treated six patients. The heart trial has treated three patients. We have a Parkinson’s trial by Krys Bankiewicz that’s treated 16 patients, and a trial lead by him for amino acid decarboxylase [deficiency] that has treated around 23 children.

Let me make this clear: We wanted to make sure that our technology, which we’re hoping will address many patients’ needs, didn’t turn into a technology for only those that have money, or only for diseases that are prevalent enough to be marketable. So we started a foundation called the Columbus Children Foundation. We treat diseases with prevalence of under 100 patients total in the foundation with the same technology that we’re developing for the larger mass markets.

Right now, amino acid decarboxylase deficiency has about 90 patients in the world. This is a form of juvenile Parkinson’s—these kids are born frozen. Krys Bankiewicz is the primary person doing all these trials. We’ve now treated 26 children in Warsaw, Poland, and in the U.S. We hope that this is the first disease that gets eradicated by gene therapy. The Foundation’s goal is to systematically knock these diseases with smaller prevalence out one by one. We see the Foundation as a mechanism to treat ultra-rare diseases that, while we’re developing through AskBio drugs for larger markets like Pompe or hemophilia. Both use the same technology, just under different structures. If you try to develop gene therapy drugs for ultra-rare diseases with the goal of making money, you’re never going to be successful. So we’re funding the development of ultra-rare diseases by a different mechanism. Bayer was tremendously behind it and said keep going.

Davies: AAV has an excellent safety profile, but last summer came news about adverse events in clinical trials. Is there more work to be done to ensure that AAV is as safe as it can possibly be in a clinical setting?

Samulski: You need to be aware of the following data that led to where we are today. The Avigen trial done by Kathy High showed that in a human, if you put in 10¹² AAV particles per kilo for hemophilia indication, they saw liver toxicity. Everybody backed up saying, “this concern was not obvious in the animal models at any level. What’s going on here?”

While people were trying to figure out that immunology and suppressing it with steroids, we developed the self-complementary vectors which would allow you to drop down in dose and achieve the same therapeutic level with a much lower particle number.

It was Brian Kasper and Jerry Mendell at Ohio State that broke the sound barrier of the gene therapy field when they went into SMA (spinal muscular atrophy) children with 10¹⁴ AAV particles per kilo. We were scared to death that it would be an all-out toxicity problem. When they were able to manage that and still be able to cure these children, everybody decided the new ceiling went from 10¹² to 10¹⁴.

We have two logs of toxicity that we’re playing around with. We know if we stay below that we’re safe. Anything above that, you’re running a risk.

The full version of this interview was originally published in Human Gene Therapy (January 2021).

References
1. Samulski RJ, Berns KI, Tan M & Muzyczka N. Cloning of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells. PNAS 1982;79:2077-2081.
2. Xiao X, Li J, Samulski RJ. Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J Virology 1996;70:8098-8108.
3. Li W, Asokan A, Wu Z et al. Engineering and selection of shuffled AAV genomes: a new strategy for producing targeted biological nanoparticles. Molecular Therapy 2008;16: 2052-2060. doi: 10.1038/mt.2008.100.Epub

The post AAV Jude: A Conversation with AskBio’s Jude Samulski appeared first on GEN - Genetic Engineering and Biotechnology News.

In the new article, Liu’s group, in collaboration with NIH director Francis Collins, MD, PhD, have successfully treated the genetic mutation in a mouse model of progeria, a form of premature aging. GEN previously reported the highlights of this work in early 2020, after Liu presented his team’s progress at a scientific conference.

The new work seeks to treat a mouse model of Hutchinson-Gilford progeria syndrome (HGPS), a Mendelian disorder that results in premature aging. Most HGPS patients do not live beyond 15 years of age. Although a rare dominantly inherited disorder, most HGPS patients possess the same mutation—a C-to-T mutation in the nuclear lamin A gene. This sets up the disease as a candidate for treatment using adenine base editors (ABEs), developed by Nicole Gaudelli, PhD, and colleagues in Liu’s group a few years ago. Base editors are capable of engineering precise base substitutions to repair a genetic defect.

Grad student Luke Koblan, Michael Erdos, PhD, and their colleagues used an ABE to correct the pathogenic mutation in a mouse model of HGPS as well as cultured cells of HGPS patients. The authors used lentivirus vectors to deliver the ABE into patient fibroblasts, generating about 90% correction, reduced production of the aberrant progerin protein, and no detectable off-target effects.

In the mouse model, the researchers used an adeno-associated virus to deliver the ABE. Levels of mutation correction were 20–60% in various organs; the investigators also recorded lowering of progerin levels and restoration of normal vascular pathology. Strikingly, a single injection of ABE into mice two weeks after birth not only enhanced animal activity but also more than doubled the animals’ lifespan, from 215 days to 510 days.

“The toll of this devastating illness on affected children and their families cannot be overstated,” Collins said in a press release. “The fact that a single specific mutation causes the disease in nearly all affected children made us realize that we might have tools to fix the root cause.” He added that the crucial base editing technology was only developed thanks to long-term government investment in basic genomics research.

“It’s incredibly exciting to think that an idea you’ve been working on in the laboratory might actually have therapeutic benefit,” said Jonathan Brown, MD, a study co-author and assistant professor of medicine in the division of cardiovascular medicine at Vanderbilt University Medical Center. “Ultimately our goal will be to try to develop this for humans, but there are additional key questions that we need to first address in these model systems.”

The new results are encouraging in themselves, but the authors highlight further reasons for optimism. Since the genesis of the Nature report, Liu and coworkers have developed a suite of advanced ABE reagents with much greater activity. And progress is being made in other forms of drug therapy for progeria, which could complement the ABE approach, including the approval of a new drug in late 2020.

In an accompanying News & Views article, Wilbert Vermeij, PhD, and Jan Hoeijmakers, PhD, strike a positive note. “If base editing is to be used to treat human disease, the safety of such an intervention must be ensured. If it can be, and if this method successfully repairs the progeria-causing alteration in the crucial tissues, such an approach holds tremendous promise as a way of prolonging health, extending lifespan, and improving the quality of life of those who have this mutation.”

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Susan Catalano, founder and chief science officer of Cognition, trained in neurobiology, working on synaptic plasticity and mechanisms of learning and memory. She did her doctorate at UC Irvine, and a postdoc with renowned neuroscientist Carla Schatz PhD at UC Berkeley. She moved to Caltech and worked with another famous neuroscientist, Mary Kennedy, PhD, “before going to the dark side” and starting at Roche Palo Alto.

After a stint working outside the area of neurobiology, she joined Acumen Pharmaceuticals and then co-founded Cognition in 2007 in the Bay Area before relocating the company to Pittsburgh. The company is still small, with 22 employees, but has attracted significant government funding and is pushing forward in the clinic with a promising small-molecule drug for Alzheimer’s disease. Cognition is also making important strides in understanding the genetics of the disease, information that reinforces and refines its therapeutic rationale.

Kevin Davies asked Catalano to talk about Cognition’s clinical progress and the underlying rationale for its Alzheimer’s drug program. (This interview has been lightly edited for length and clarity.)

GEN Edge: What was behind your move to Pittsburgh?

Catalano: The Bay Area is certainly a hotbed. But we’re operating a company for about a quarter of the price. Pittsburgh is a great neuroscience town. They have a historically very high focus of neuroscience faculty at the university so we’ve been able to build a company, staff it, and grow it.

The economic development agencies in Pennsylvania, which include the Ben Franklin system: our local chapter here in Pittsburgh is called Innovation Works and Rich Lunak and the team were instrumental in rolling out the red carpet to us. We also had the support of John Manzetti and the Pittsburgh Life Sciences Greenhouse. So starting Cognition here and incubating it was really seamless.

GEN Edge: Is Alzheimer’s disease your sole focus?

Catalano: It’s part of a wider area of therapeutic interest. The company has been focused on Alzheimer’s disease for a while, but we’ve recently added additional indications that leverage what we’ve learned about our target to other disease areas where there’s a lot of unmet medical need.

GEN Edge: What is currently in the clinic or heading that way?

Catalano: Our lead drug candidate is CT1812, currently in four phase II clinical trials with a fifth about to start. These are studies where we are looking at the clinical pharmacology to understand the drug’s mechanism of action and its impact on disease. These trials are all being done in patients with mild-to-moderate Alzheimer’s disease, with the exception of the one that’s going to start [in 2021].

We announced the results of the first set of 24 patients to be dosed with the drug for six months [in the SHINE study] last July at the Alzheimer’s Association conference (AAIC).

GEN Edge: Is the hope for this drug to slow the course of the disease or to actually reverse the pathogenesis of the disease?

Catalano: We believe this is a disease-modifying therapeutic that will have a very broad and profound impact on Alzheimer’s disease progression. It works by reducing Aβ [amyloid beta] oligomer binding affinity. CT1812 is literally the only drug candidate in clinical development that can destabilize the binding pocket where Aβ oligomers bind on synapses on neurons and create all of the downstream damage and memory failure that we see in Alzheimer’s disease. The drug binds to sigma-2 receptors receptors, allosterically modulates them, which in turn modulates the receptors that oligomers bind to, causing an increase in the off rate of oligomers as a result of this destabilized binding pocket.

GEN Edge: You are firm believers in the amyloid hypothesis then?

Catalano: We are indeed. We think there’s pretty overwhelming evidence that supports that. Not only the very well-known risk genes that result in early onset inherited disease but also in sporadic disease and of course with this very profound protective mutation (in the amyloid precursor protein APP gene] that was discovered back in 2012.

GEN Edge: You wouldn’t have been able to advance this drug into Phase II without some pretty impressive preclinical data. What was the most impressive animal model data that got the drug into the clinic?

Catalano: We’ve been able to use three separate methods to actually demonstrate that the drug increases the off rate of the Aβ oligomer—this pathological ligand that’s only there in the disease. We looked with mature primary neurons in the dish. We looked with Alzheimer’s patient post-mortem tissue sections. We also looked in the brains of living mice. In all cases, we observed an increase in the off rate of Aβ oligomers, in some cases after a single dose of drug.

In the case of the patient tissue sections, this is what we classically refer to in the industry as an ex vivo binding experiment. You’re not adding anything to the tissue sections, you’re just putting solution on top of them as they sit on the slide that contain identical volumes with ascending concentrations of drug.  Then you look to see what’s left in the tissue and what’s extracted out of the tissue in the supernatant. We saw less oligomer in the tissue section around the plaques in AD patient brains and an increase in oligomers in the supernatant that was dose-dependent.

GEN Edge: Are you doing this by yourself or have you partnered with anybody?

Catalano: We’ve been fortunate to have enormous amounts of support from the National Institute on Aging (NIA). I think they recognize the necessity for very novel approaches even to traditional targets in Alzheimer’s disease. We are the only sigma 2 allosteric antagonist in the clinic for any indication, and we’re the only one pursuing this Aβ oligomer displacement mechanism in Alzheimer’s disease.

NIA has supported the entire clinical development of this drug—close to $120 million in grant funding, the majority of that being clinical. Last June, we announced that we had received a $75.8 million award from NIA to support a 540-patient efficacy study that we’re about to launch in collaboration with the ACTC. That was the largest grant they’ve ever given to a single drug company.

GEN Edge: Have you settled on the dosage? How is the drug administered?

Catalano: This is a once daily oral medication. We’ll be testing two doses versus placebo. All of our clinical trials have been double-blind placebo controlled.

GEN Edge: Let’s talk about the genetics of Alzheimer’s. How important is it to understand the genetics and how much of an influence is this understanding in terms of guiding your therapeutic course?

Catalano: We think that genetics is the guiding light as you might imagine. What gets lost in the rather dramatic sets of clinical failures that we’ve had in Alzheimer’s disease is the genetics.

In Alzheimer’s disease, when you simply look at the risk genes or the causative genes, all roads lead to Aβ. The vast majority of mutations that cause the early-onset form lead to a single phenotype—an increase in the concentration or the ratio of this longer, more aggregation-prone form of the Aβ protein. When we finally discovered the first robust protective mutation that has strong functional evidence, as well as epidemiological evidence of its impact, it was also a mutation in the Aβ sequence.

In every other neurodegenerative disease that you would look at, there are 2, 3, sometimes multiple genes mutations that could lead to an early onset form the disease that’s indistinguishable from the sporadic form. That’s not the case in Alzheimer’s disease. Alzheimer’s is unique. All roads really did lead to Aβ.

GEN Edge: You’ve got a new study in the Journal of Neurochemistry about a mutation called A673T first discovered in the Icelandic population. It’s arguably the strongest protective AD mutation discovered to date. What is the significance of this new finding?

Catalano: Collaborators at Genentech and Iceland’s DeCode Genetics discovered this mutation [in the APP gene] by genotyping everybody. It’s also position 2 in Aβ. This mutation is actually quite rare—in the first publication there were only about 41 folks who had it, and all but three were heterozygotes. Since then it’s been found in other Scandinavian populations at low frequency, but the folks who carry it — even the folks who are heterozygous for it—have a four-fold lower incidence of Alzheimer’s disease and age-related cognitive decline. So, it’s profoundly protective in a way that a lot of the other putative protective genes that have been identified really can’t demonstrate.

Many people have studied this mutation since it was discovered in 2012. Scientists have looked at the rate of fibrilization: the morphology of those fibrils. We wanted to look at the oligomeric form of that protein. Aβ is an intrinsically disordered protein. It takes a variety of shapes—it’s never a nice well-folded globular form. So we have the monomer; an infinite polymer known as a fibril that looks like a zipper; and we have a globular intermediate known as an oligomer—probably the best analogy of this is a Medusa’s head—with a hydrophobic central core that self assembles and then these floppy N termini around the outside of that central core.

The oligomers are the focus of most AD research for the past decade for one simple reason—the oligomers are the most toxic form of the protein. The reason for this toxicity is they behave like ligands. They bind to a single receptor site on neuronal synapses. When they’re bound, they trigger a variety of downstream changes that include memory failure—the inability to encode new memories. This is a very prominent feature of Alzheimer’s disease.

It’s this pathological ligand that everyone’s been studying for the past decade. It’s challenging to study because it’s intrinsically disordered, you can isolate it from patient brain or you can make it in the dish by taking peptide and allowing it to self-assemble and using experimental systems to measure toxicity.

That’s what we did. We chose a method of making these synthetic oligomers that is most physiologically relevant, a method pioneered by Bill Klein years ago. Using this method to make the oligomers we looked at wild-type and mutation containing oligomer side by side. We found that mutation-containing oligomers are less likely to form in the first place (by about half).

What was really surprising was when we took the wild-type oligomers and the protective mutation-containing oligomers and we put them on neurons and looked at the binding, (which is how the toxicity is actually happening), we found that the mutant oligomers bound with fourfold lower affinity. We had no idea that we would see this profound fourfold lower affinity.

When you add all this up, the predominant effect of this protective mutation is to lower the binding affinity of this pathological ligand. That was unexpected, and to us it was significant because our drug reduces affinity—lowers the ability of the oligomers to bind to this receptor at synapses in the brain.

GEN Edge: This reinforces what Decode and others had observed from the genetics, you’ve now got solid experimental proof that really backs up the genetic findings. Does that have any direct bearing on your ongoing clinical trials?

Catalano: This is a very important genetic validation of the approach that we’re taking. It was truly unexpected. It also points us in directions for looking at target engagement within those clinical trials in a fresh way. It’s going to be important to look at the movement of these oligomers — to look at the total equilibrium of Aβ in all three of its structural forms.

GEN Edge: Your lead drug candidate is in phase II trials. Might there be ways to further improve the specificity, reduce the toxicity of this small molecule?

Catalano: Like any drug discovery company, we do look for more effective forms of the molecule. We think CT1812 will be an effective therapeutic, but there’s always strategies that you can engage in to pursue follow-on molecules in the pipeline.

GEN Edge: What does the future hold for Cognition?

Catalano: We recently announced the results from the first group of mild-to-moderate Alzheimer’s patients that were treated with the drug once daily for six months, the SHINE trial, reported back in July. These 24 patients showed a trend for improvement in their cognitive scores compared to placebo-treated patients, meaning they had a slower decline than placebos were exhibiting.

In terms of biomarker evidence, we observed significantly reduced Aβ in drug-treated patient’s CSF [cerebrospinal fluid] compared to the placebo patients.

We have evidence of slowed cognitive decline and evidence of an impact on the Aβ biology in Alzheimer’s as well. This was something that the secretase inhibitors were hoping to achieve. We know that it’s very preliminary—this is a very small number of patients; it is not adequately powered for cognitive outcomes—but it’s encouraging and supportive of further development.

Next year, we’re going to get the results from two additional cohorts of patients where we’re planning to look at the cognitive score as well as the protein expression in these patients and a lot of imaging parameters, including the first ever clinical trial (SPARC) to look at synaptic density using a very exciting new PET tracer UCB-J that measures the synaptic protein SV2A. This is the world’s first trial to look at how synaptic density measured with this PET tracer changes longitudinally in Alzheimer’s patients.

This PET tracer was developed by Richard Carson and Chris van Dyck at Yale, who reported a hypo-intensity in the hippocampus using this PET (positron emission tomography) tracer in living Alzheimer’s patients—very different from what they saw in normal patients.

A tracer that allows us to measure synaptic density is groundbreaking and it would impact all of neurology. We’re excited to see the outcome from this trial. The first half of next year is going to be a very big year for Cognition.

GEN Edge: It’s my impression that several big pharma companies have pulled out of the neuroscience field because it’s just too difficult. Have you seen competitors drop out because they found trying to try to drug a disease like Alzheimer’s is just too complicated?

Catalano: No question about it. There have been a lot of failures and several large pharma companies discontinued their programs. Ironically and unfortunately this presents an opportunity for the therapeutic approaches that remain. Science marches on and our understanding of the disease progresses and we remain hopeful that that science will be reflected in therapeutics that are going to be more effective. We think that CT1812 will be one of them.

Ultimately, we expect Alzheimer’s to be treated like other chronic diseases with polypharmacology—therapeutics that are effective anti-inflammatories, therapeutics that effectively address tau build up in a patient brains, are combined with anti-amyloid approaches. But you’re going to have to address the Aβ oligomer pathology that sets the whole thing in motion and keeps it in motion. Otherwise, you’re, you’re not going to be able to fundamentally halt disease progression.

GEN Edge: So CT1812 Is perhaps part of a cocktail down the road where different drugs are tackling different elements of the pathology?

Catalano: That’s exactly right, Kevin. These poor patients and their families really need a breakthrough.

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Benchling is a new breed of Laboratory Information Management System (LIMS) platform helping academic research groups, biopharmas and other life science companies manage their data in the cloud. The Silicon Valley company was founded in 2012 by Saji Wickramasekara and Ashu Singhal. Both had backgrounds in software engineering and computer science, along with wet lab experience. Singhal had applied to MD PhD programs but decided that building tools for the industry was actually his calling. Similarly, Wickramasekara felt he could make a bigger contribution applying his software engineering skills and leaving the bench, so to speak.

In this interview, GEN’s Kevin Davies spoke to Benchling CEO Saji Wickramasekara and head of product marketing, Michael Schwartz. The discussion explores the client needs that Benchling strives to fill and the company’s mission and future plans. (The interview has been lightly edited for length and clarity.)

GEN EDGE: Saji, how did you launch Benchling?

Saji Wickramasekara: I’m actually a software engineer by training, not a scientist per se, but I spent a few years in molecular biology wet labs doing basic academic research. I was very interested in the design and development of medicines and thought I was going to pursue a career in biotech. I was ready to go to grad school for my PhD. Ultimately, I didn’t do that…

In software, it’s much easier to work with other people. You have really great tools for collaboration around fairly complex work. If I compared that to the life sciences, everything was running on paper, email, and spreadsheets! It was very challenging to work with others on increasingly complex work.

I founded Benchling to scratch that itch and to build tools that I would have liked to use as a lab scientist—build to the point where I’d be excited to do scientific research again! We initially built this product for academic scientists as more of a productivity tool, but as we took it to industry, we found there was a greater need there. That was facilitated by the shift from chemistry to biology and the complexity of the work.

A couple of things happened: There were major transformations in science. Today, eight of the top 10 drugs are biologics, and the first generation of CAR-T cell therapies have been introduced. We have CRISPR! The complexity of the work is very different. From a data management perspective, that’s a totally different data model and different set of processes.

All of a sudden, you have this exponential growth and complexity of work that scientists are trying to do, but the tools are still paper and spreadsheets! They just had not evolved. There was a huge need in industry as well and innovation really grew from there.

GEN Edge: Michael, how did you arrive at the company?

Michael Schwartz: I’ve been at Benchling for a year leading product marketing. I started off as a bioengineer, doing medical devices. I co-founded a company that was doing cellular analyses – I even wrote an article for GEN about nine years ago!

As a bio-engineer, I was looking to have the greatest impact on the world. Life sciences was great, but I got turned on to this world of software. We’d observed these massive transformations in other industries, living in Silicon Valley and San Francisco.

I moved into software-as-a-service about five years ago, leading the life science vertical at Salesforce. Saji really impressed me, not just for the company but with the mission of the company. I’d seen software transform the commercial and medical sides of the pharmaceutical industry, even clinical development. But R&D was this final frontier. If you think about where great medicines are discovered and developed, it’s upstream, but the software landscape here was the most fragmented.

GEN Edge: Saji did you have any previous company experience or was this literally going from academia to CEO?

Wickramasekara: Yes, it was. But in the beginning, you’re just the CEO of you and another person, and it’s not too big a change. Initially I got into this to scratch a personal itch. The company part came later. As a software engineer, I wanted to build tools that I thought should exist. It turned out there was a market for those tools and here we are.

GEN Edge: Has your main focus evolved away from academia?

Wickramasekara: It wasn’t necessarily just to serve the academic market so much that it was a great place to start, and to make sure that what you were building was loved by scientists, doing cutting-edge biology, working with CRISPR.

Today, we’re a business, we have a pretty ambitious set of products to build and the majority of our paying customers are in industry—cutting-edge biotech upstarts like Beam Therapeutics, Prime Medicine, Verve Therapeutics, Mammoth Biosciences, Editas Medicine. And big pharma companies as well, such as Gilead and Regeneron. I spend a lot of time in Westchester County!

GEN Edge: How is Benchling different than any other LIMS — Lab Information Management System—company. Can I even bracket you in that category?

Wickramasekara: The first generation of those electronic lab notebook [ELN] and LIMS companies was focused on the paper-to-glass transformation. The science wasn’t as complex back then, so you didn’t need as flexible a system. Those workflows were fairly rigid and so we transplanted that over and said let’s make it digital.

When that first evolution happened, 10–15–20 years ago, patents were awarded “first to discover,” not “first to file”. So there was also a compliance tailwind, not a scientific complexity tailwind that we’re experiencing now. But today, R&D teams are much more sophisticated and thinking very holistically. They want their data and processes completely connected through their applications.

They want the data structured and searchable, running large distributed organizations with highly specialized teams and lots of handoffs. The stakes are just so much higher. We’re the next generation of those ELN and LIMS companies of the past.

GEN Edge: Where are you seeing the most activity and growth? Is it in biotech and pharma or are you moving into other areas?

Wickramasekara: We’ve seen dramatic adoption throughout the industry both in the development of medicines, but also outside medicine. We have customers in industrial biotechnology, agricultural biotechnology, and food science—cultured meats and dairy replacements and other industrial applications. They have similar needs to biopharma with many shared R&D functions between them.

GEN Edge: What are some of the new features on the platform that you’ve added in the last year, or that are coming out in 2021?

Schwartz: Yes, a lot. We’re quite proud of the product and engineering teams because this was the model that Saji and Ashu set up. It’s not just SaaS, but it’s a continual innovation model. We’re able to pump new features, literally every week, whereas a lot of the legacy systems got stuck in these annual updates that were a hassle really for customers.

Wickramasekara: Frankly, the science moves too fast to do an annual update anymore. Take CRISPR—there’s a new base editor like every week!

Schwartz: Some of the bigger things we’ve introduced in the last few months include a lab automation element. Now almost all our customers have analytical instruments and liquid handlers that power the throughput of their labs. These are more like out of the box integrations to bake those right into the workflow.

We added an application called Insights—embedded intelligence right in the application. Previously, most of our customers would get a bunch of data and pump it out into Excel or a business intelligence tool. That still serves a purpose in some instances. But having online insights that contextualize all the way back to the sequence or the cell line is the preferred way for most of the biotechnology industry.

GEN Edge: What are your top success stories? What organizational problems have you really been able to help solve?

Wickramasekara: There are hundreds of organizations listed on our website. Obviously, we work with household pharma companies—Regeneron, Gilead, etc.—they have many medicines in the market. Many smaller biotech companies grew up with us from the beginning and have gone from five people to ten scientists to hundreds of scientists and now have their first medicines in clinical trials. All credit to them for building the medicines, but we were the enabling tool and foundational infrastructure for them. That’s something we’re really proud of.

GEN Edge: What problems has COVID-19 caused for your customers and how have you and they adapted?

Wickramasekara: Two things to discuss. First, we’re seeing a dramatic acceleration in the business, especially from large research organizations who need to adopt much more bleeding edge collaborative tools. Two or three years of digital transformation initiatives are being pulled into three months, sometimes things are moving at breakneck speed which is not something we’ve experienced in the past. The old on-premise tools that aren’t built for collaboration just aren’t cutting it when you have scientists taking shifts in the lab and having to work from home. We’re seeing an acceleration there.

Benchling is software that scientists already use all day. But we’re still seeing in some companies, especially those working on the front lines of COVID, usage jumping about 30%. They’re doing nights and weekends and we’re having to be on call 24/7 for some of these companies working on COVID antibodies and vaccines, because nothing can go wrong for them. It’s been awesome to be part of that. Again, they’re the ones doing the hard work, we’re not trying to take credit here. But we’re really happy to support those efforts.

In addition, we’ve found a new growth market in clinical testing. It’s not something we’d really been a part of before, but we wanted to do what we could to help with testing capacity, which is a general constraint in many countries. We’re working with more than a dozen labs in different countries, different states. We’re processing hundreds of thousands of tests per month because our software is a transformative tool to have when you’re spinning up a new lab or repurposing an old research lab. Corteva Agriscience is one of the largest agricultural companies in the world. They have a partnership with their local health system Mercy One, and we’re the software powering that.

You still need to ingest samples, barcode, track them through the process, run assays, capture the data from instruments, and report the results back. If you need to get that up and running in a week, there aren’t a lot of answers except something like Benchling. Normally it takes six months to roll out a system like that.

GEN Edge: Data security and reproducibility is an ongoing concern. Does Benchling help groups in academia and industry establish the provenance and the authenticity of their primary data?

Wickramasekara: Yes, absolutely. That’s one of the whole premises of a modern cloud solution like Benchling—you know who did what, you’ve done your best to not only standardize data but automate the capture of it. So you’re even removing humans from the equation where there’s normally a surface area for manual errors to be made.

This is something I’m very sensitive to, coming from academia. Data integrity and the duplication of work are serious problems in science. In industry, you often hear about a drug being put on hold because in some animal study there was an error copying the data. When you have electronic systems instead of paper, you’re eliminating most of those errors.

Back when I was doing research at Duke University, the lab next door to mine had a huge reproducibility crisis, papers were getting retracted because clinical trials were starting off that [research]. So I’ve seen firsthand how damaging that can be. The solution is to build tools so good that scientists want to use them. If they’re using those digital tools, you can solve these reproducibility and data integrity issues.

GEN Edge: You mentioned a few bellwether CRISPR companies among your clients. What is it about CRISPR that Benchling is proving so helpful?

Wickramasekara: There are a couple of reasons. At the macro level, these companies are doing really complex science that’s never been done before. They need some way to track all the different cell lines, proteins, plasmids, CRISPR guides and link all them together to have full experimental context so they can keep moving quickly.

Those organizations are early adopters of tools like ours—we’ve been working with Jennifer Doudna, Feng Zhang’s lab, George Church, David Liu, and others, they were some of our first adopters. They were the voices in our ear telling us, “you need to build these capabilities”. CRISPR was by design—it’s a hard computational problem doing a full genome search and scoring these guides very rapidly. Prior to Benchling, the solution was a freeware tool, run by a grad student, which would take you 8–10 hours to get your results back. We do it in a couple of seconds.

GEN Edge: What are you most excited for over the next 12 months, besides the pandemic winding down, hopefully?

Schwartz: I’m excited for our push downstream to connect the dots across a wider swath of R&D. Our customers are building platforms, the centers of excellence, which continues all the way through the R&D process. We have some exciting announcements coming up next year that will directly support that well.

Wickramasekara: We’re becoming a global company. That’s really exciting to me. We’re making a big push into Europe right now. Many of our customers are multinationals, with sites in the U.S. and Europe, Asia as well. Becoming a global company is the next big milestone.

Schwartz: Saji might be too modest to talk about this, but at Benchling, the mission is focused on helping companies build better therapies in the world. He’s also built a company that’s trying to do good in the world. Our people are helping support STEM causes. We’re also trying to empower scientists to do better in the academic world and volunteering in the communities we live in. I know that’s something that makes me enjoy coming virtually to work every day.

Wickramasekara: Yes, all the COVID testing work is pro bono. We’re giving away the testing platform that is helping all these labs and communities do their COVID testing. That’s really important to us.

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The Chief Scientific Officer of Spark Therapeutics, Federico Mingozzi, surveys the hopes and challenges facing the human gene therapy community.

After a distinguished career in academic medicine, including stints at the Children’s Hospital of Philadelphia, INSERM and Genethon in France, Federico Mingozzi joined Spark Therapeutics three years ago as chief scientific officer.

Mingozzi aims to build on the company’s landmark success with Luxturna, the approved therapy for certain retinal diseases such as the type 2 version of Leber’s congenital amaurosis (LCA). In this recent interview with Kevin Davies, Mingozzi discusses his career highlights and surveys the hopes and challenges facing the human gene therapy community. (The text has been lightly edited for length and clarity.)

GEN Edge: Did you always want to be a gene therapist, or was there a “spark” when you decided that you had to move into this field?

Dr. Federico Mingozzi: I started early, when gene therapy was not that popular! My love for everything that is molecular biology and recombinant DNA technology started quite early. I studied biology in Italy at the University of Ferrara, and my PhD mentor, Professor Francesco Bernardi, was the person who introduced me to lab research.

Francesco made the link with Katherine High, and that is how I got involved with gene therapy. That was back—I am going to date myself—in 2000, when I moved from Italy to the Children’s Hospital of Philadelphia (CHOP) for a postdoc. I started working on liver gene therapy, adeno-associated virus vectors (AAV), and immunology. That was the thread of my career for the next 20 years—immunology and gene therapy.

I joined the laboratory of Roland Herzog, who is now professor at the University of Indiana. We worked together in defining the mechanisms responsible for the induction of immunological tolerance with gene transfer with AAV. Then I started working directly with Kathy High, and got involved in the early studies of hemophilia gene therapy and also the early work in ocular gene transfer in collaboration with Jean Bennett at the University of Pennsylvania—the work that today is Luxturna.

GEN Edge: When you first got into gene therapy, AAVs had all the appearances of becoming an important class of vector, correct?

Mingozzi: Yes and no, meaning that I started in gene therapy in probably the dark years of gene therapy, when the field was emerging from a number of important setbacks. There were problems with the early adenoviral platform and the tragic death of Jesse Gelsinger. There were all the issues that emerged with the AAV platform and immunogenicity in the early hemophilia trials. There were also issues in the ex vivo field, with the leukemia in the X-linked severe combined immunodeficiency trial and other trials.

That is when I entered gene therapy, which was the moment when academia really picked up the work. There were a number of companies back in the late ’90s/early 2000s, working in gene therapy, and today they are pretty much all gone, right? So when I entered gene therapy, there was no industry interest in this therapeutic modality. But there was investment from academic centers to try to address the limitations of the technologies and then bring it to patients. It was great, because I think without that the field would not exist as it is today.

The opportunity after my postdoc to join Kathy’s team was great because, at the time, the CEO of CHOP, Steven Altschuler, and CHOP decided to invest in the Center for Cellular and Molecular Therapeutics, a translational medicine center, to bring gene medicine from bench to the bedside.

That was, for a biologist, the wildest dream becoming true, bringing a therapy to patients. There are moments I will never forget the excitement of those days, for example, when we enrolled the first participant in the phase I trial of gene therapy for RPE65 deficiency. It was amazing!

The work that we did in hemophilia was more challenging, but perhaps the most important work I have done in terms of contributing to the understanding of the immune responses to AAV vectors in humans. When we initially approached gene therapy trials, we thought viral vectors were not immunogenic, but then we understood better what was happening, and that allowed for significant progress.

One example of the progress made was the hemophilia B trial led by Amit Nathwani and Andy Davidoff, who were able to modulate the immunogenicity of AAV vectors with corticosteroids, and then they were able to show that that gene therapy can potentially deliver a cure for a disease. We showed that also with RPE65 with Luxturna in the early days. I think these early successes were really the pivotal moment when the investors and the industry started to say, “Wow, there is something there. The technology can really deliver something that is transformational.”

There was also important progress made in other fields of gene therapy, including the work of the colleagues at the San Raffaele Hospital in Milan, Italy, on the ex vivo gene transfer platform, French and U.S. teams as well, in evolving the lentiviral vector platform to become safer. It was a nice parallel of in vivo and ex vivo gene therapy, and then clinical success started to become more real.

GEN Edge: How has the gene therapy field been able to recover from those setbacks? What were some of the biggest issues that the field as a whole has had to really work on and understand better to where we are today?

Mingozzi: I think a bit of everything. I will put it in two main categories. One is the platform, the other is understanding the biology. We started to have better gene delivery tools—there was the first-generation AAV, then more efficient AAV vectors came to fruition, to be used in the clinic. The same for ex vivo, the first platform was based on gamma-retroviral vectors, and then the next generation of retroviral vectors came along, which was a more amenable platform for clinical translation.

Then the biology piece—better understanding the immunogenicity of the vectors we use for gene therapy for in vivo, and for ex vivo—as you can imagine, a more in-depth understanding of the drivers of the risk associated with gene integration.

GEN Edge: What proportion of trials does AAV make up?

Mingozzi: It is a big proportion of the trials. The flavors of AAV that are now reaching the clinic span a broader range than in the past, starting from natural serotypes, which were of course the first ones adopted, to a growing number of engineered serotypes. The appetite for new serotypes is nearly infinite, and, if you look at the landscape, both in academia and in industry, you find more and more groups and companies working on engineering new AAV serotypes.

GEN Edge: What was the genesis of your decision to take a break from academia and join Spark Therapeutics about 3 years ago?

Mingozzi: The funny thing is that I left CHOP before Spark Therapeutics was funded, and I went to France to work for the French Muscular Dystrophy Association and Généthon. It was really a fantastic journey in gene therapy, and I think it was fantastic. A little more than 3 years ago, I started talking to Spark Therapeutics about one of the programs that was developed in my laboratory—a gene therapy for Pompe disease, which is now in the Spark Therapeutics pipeline. Finally, I was offered the position of chief scientific officer, which was a really humbling moment for me.

These days, if you work on gene therapy it is likely that you receive many offers for jobs in the industry every week. However in the case of Spark Therapeutics, there was an emotional attachment to the programs, but also the recognition that it was a great company with great science and a fantastic culture. So I decided to join the company. I was happy in France and had a tenured position with INSERM. It was a difficult decision, but at the end I decided to move back to Philadelphia and join Spark Therapeutics.

GEN Edge: Spark is best known for historic success with Luxturna. How has that drug fared since its approval? And how has the company benefitted from that experience? How does that experience provide momentum for the other clinical programs that are now coming through the pipeline?

Mingozzi: It is a great question. First of all, I feel lucky that I have been involved with Luxturna from early on. When I joined Spark Therapeutics is about when the drug was approved by the FDA.

There are many learnings. The first one is that gene therapy is a therapeutic modality that can go all the way to become a drug—importantly, all the regulatory hurdles can be overcome. That is very important for the field, and also to drive the interest of industry in the specific technology.

There are additional learnings related to doing gene therapy in the eye. We know more that, for example, the route of administration to the eye can present important complexities, and so as we think about larger indications, we need to consider that carefully.

Every time I hear about Luxturna, I am happy, because it is a very positive and successful story. It is a drug that is really changing the life of patients, and this is what matters the most. There are also additional learnings. Luxturna is a treatment for an ultrarare disease. There was no molecular diagnostics, for the most part, for retinal disease associated with biallelic mutations in the RPE65 gene. That means that, for example, we had to identify the eligible patient population, and the actual commercial development was complicated and brought a lot of learnings for Spark Therapeutics and required a lot of innovation.

GEN Edge: Luxturna is injected subretinally. There is a lot of work in developing new, evolved AAV vectors that could in principle provide a less invasive injection that might deliver the same therapeutic benefit. Would Spark Therapeutics employ this approach, if not for a new and improved version of Luxturna, maybe for other blindness conditions?

Mingozzi: Certainly, for example, there is a lot of interest around using AAV capsids that can transduce the retina through direct intravitreal injection in the eye. Then essentially you are simplifying the administration procedure. You do not need the operating room, you can do it theoretically in the outpatient setting.

At the moment, there are a couple of trials wherein this paradigm is being tested. The jury is still out. Definitely there is interest also on our end, how to improve the delivery to the eye. Of course, as we think about bigger indications, it would be very hard to serve large patient populations when your administration procedure is very cumbersome, it requires a lot of operating room time.

GEN Edge: As Spark Therapeutics’s chief science officer, how does the pipeline look to you? What are you most excited about that we will be hopefully seeing moving toward approval in the coming months and years?

Mingozzi: I am excited about each and every asset in our pipeline. And I am also excited about the lines that we are not going to tell you about!

The emphasis and engagement of Spark Therapeutics in hemophilia remain very very important. Hemophilia B is now in phase III with Pfizer, and for hemopnilia A, the program is advancing well. We recently presented at the International Society of Thrombosis and Hemostasis, where we showed very important evidence of durability in the patients enrolled to date. So that is very exciting.

I am also excited about what is coming up next in the liver. We have an open trial for Pompe disease. We have ocular programs that are advancing well, and last, but not least, there is the central nervous system—we have Huntington and other programs wherein I think there is a lot of interest, and a huge unmet medical need. Gene therapy there has also the potential to deliver transformational results.

GEN Edge: What is your approach in Huntington’s disease, which is of course a dominantly inherited disorder?

Mingozzi: The approach is a silencing approach, the idea is to knock down the expression of the Huntingtin (HTT) gene. It is similar to other approaches of gene therapy that are in development. It is not allele specific. The idea is that as long as you knock down the mutant HTT allele sufficiently, and it does not matter whether you knock down also the allele that is not detrimental, then you would have a therapeutic impact.

GEN Edge: There have been three reported fatalities in an AAV trial that happened earlier this year. What has been your reaction, or perhaps the reaction of your peers, to this news? Is this a wake-up call for the gene therapy field as a whole?

Mingozzi: Definitely the field is watching closely. It is too early to know exactly what happened. I think clearly it is an unfortunate chain of events, and based on what it takes to bring our gene therapy to the clinic, the assumption is that this was not predicted in preclinical studies.

Now what we need to do is to understand what happened. The myotubular myopathy results in the gene therapy trial are quite impressive in terms of what can be done for this disease, which does not currently have a treatment. Now we need to understand what happened, and then move forward.

This is a very specific situation wherein very high doses of vector were used. I do not think we have the same safety concern when lower doses of vector are used. But to tackle certain diseases, we need more vector infused, and we will have to do our best in the field, to understand what the drivers of the observed toxicities are.

GEN Edge: Are you interested in nonviral delivery methods? Are you seeing promising data using nanoparticles or liposomes that might solve some of these immunogenicity problems?

Mingozzi: I will say clearly Spark Therapeutics is interested in gene transfer as a whole, and we are keeping ourselves up to speed with all the technologies, including nonviral (delivery). There is a saying: mice lie and monkeys do not tell the truth… Until we have got clinical data, it will be hard to decide whether these technologies are really up to the promise. I think it is exciting, potentially.

GEN Edge: Would you consider using CRISPR or genome editing for a particular therapeutic indication if traditional gene therapy or gene silencing could not work? Or is genome editing really the specialized arena for companies that are set up to do gene editing? Do you feel you can use genome editing down the road if the situation calls for it?

Mingozzi: What we are seeing is that all lines are blurred, because the lentiviral vectors are primarily being used ex vivo, and now there are initiatives to use them in vivo. AAV vectors are a platform for in vivo. And AAVs are used as a donor template for ex vivo in gene editing, right? So the line is not clear cut. Gene editing is relying on some delivery platform. You need to deliver the gene editing tools in one way or the other.

I think gene editing has many applications and huge potential, and ex vivo gene editing is already in the clinic. Ex vivo is the ideal setting, because you modify a cell, and you do it often times in a physiological way by correcting a gene defect. And then you implant it back, and you even overcome potential issues with immunogenicity of these gene editing tools.

For in vivo gene editing, I think there is promising data, and potential applications, and there is even nuclease-free gene editing, that has been proposed, based on technologies based on AAV that integrate in the genome. Again, things will have to be tested in the clinic to see how safe and efficent they are.

But then there are definitely areas where they could be very useful. Let us think about gene editing in a developing liver, where AAV would not have long-term expression. Then gene editing could be the way. The alternative would be to be able to readminister AAV.

GEN Edge: Spark Therapeutics’s founder Kathy High has now retired. How are you and the rest of the Spark Therapeutics team going to fill her shoes?

Mingozzi: First of all, I do not think Kathy will ever retire. She is too smart and has too much energy. You are right, she left a big void, we are doing our best to fill the void! Spark Therapeutics has many very smart people, very engaged and motivated people.

We are recruiting amazing people. We just recruited a new Chief Medical Officer, Dr. Gallia Levy, who joined Spark Therapeutics from Genentech, which like Spark Therapeutics is a member of the Roche group. It is a new exciting era for the company. Kathy represents the legacy of Spark Therapeutics, and we are very excited about the future ahead of us.

Human Gene Therapy, published by Mary Ann Liebert, Inc., is the premier, multidisciplinary journal covering all aspects of gene therapy. The Journal publishes in-depth coverage of DNA, RNA, and cell therapies by delivering the latest breakthroughs in research and technologies. The above article was first published in the October 2020 issue of Human Gene Therapy with the title “The Italian Job: An Interview with Federico Mingozzi”. The views expressed here are those of the authors and are not necessarily those of Human Gene Therapy, Mary Ann Liebert, Inc., publishers, or their affiliates. No endorsement of any entity or technology is implied.

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The award was announced today by the Nobel Prize Committee in Stockholm.

Doudna, a structural biologist at the University of California Berkeley and the Howard Hughes Medical Institute (HHMI), was reportedly woken from a deep sleep in the middle of the night by a journalist.

A stunned Charpentier, shortly after the news broke, reflected on the historical significance of the award, the first time two women have shared the Nobel Prize.

“I think it’s very important for women to see a clear path,” she said. “I think the fact that Jennifer Doudna and I were awarded this prize today can provide a very strong message for young girls.”

“Congratulations to Jennifer and Emmanuelle, now even more uniquely distinguished CRISPR pioneers,” said Rodolphe Barrangou, PhD, chief editor of The CRISPR Journal [a GEN sister journal]. “This is a momentous time to celebrate them and the field, and a great opportunity to engage the public to further the acceptance of this revolutionary technology. They are up to the task and have also been leaders in science advocacy, so I am hopeful they will propel the field to new heights.”

Thelma and Louise

Doudna and Charpentier traveled in very different circles before their paths converged in early 2011, meeting for the first time at a microbiology conference in Puerto Rico. Doudna had enjoyed a highly successful scientific career, training with two Nobel laureates and joining the ranks of HHMI investigators in 1997. Charpentier, a French microbiologist, had kept a lower profile before her own breakthrough paper on one of the key components of CRISPR, called tracrRNA, was published in Nature in 2010.

In Puerto Rico, the two women agreed to forge a collaboration to study the mechanism of Cas9, the DNA-cutting nuclease at the heart of the one of the model CRISPR bacterial immune systems. Leading the charge was Martin Jínek, PhD, a Czech postdoctoral fellow in Doudna’s lab, and Krzysztof Chylinski, a Polish grad student in Charpentier’s group. The team made a key discovery, fusing the CRISPR (cr) RNA and tracrRNA into a “single-guide” RNA that could be programmed to target and cleave any sequence of DNA. Although genome editing was a powerful technology using other molecular systems such as zinc finger nucleases, CRISPR transformed scientists’ ability to rewrite the genetic code—in any organism.

Jínek reflected on the key steps in developing the sgRNA a CRISPR Journal interview published earlier this year:

“We then came up with the idea that if [crRNA and tracrRNA] are part of a duplex, then presumably the 3′ and the 5′ ends must not be too distant from each other. Then you can stitch them together with a loop. This was the kind of thinking one had to do in the field of structural biology of RNA… It was still a leap, but we were primed to have these ideas.”

The Doudna/Charpentier paper was published in Science in June 2012, with both women credited as corresponding authors. Leading commentators, including University of Utah biochemist Dana Carroll, PhD, and microbiologist Rodolphe Barrangou (North Carolina State University), the chief editor of The CRISPR Journal, wrote contemporaneous commentaries discussing the implications of their breakthrough for genome editing in humans and other higher organisms.

“Only the future will tell whether this programmable molecular scalpel can outcompete ZFN and TALEN DNA scissors for precise genomic surgery,” Barrangou wrote in Nature Biotechnology. Carroll agreed. “Whether the CRISPR system will provide the next-next generation of targetable cleavage reagents remains to be seen, but it is clearly well worth a try,” he wrote in Molecular Therapy. “Stay tuned.”

But by this time, the Broad Institute’s Feng Zhang, PhD, Harvard Medical School geneticist George Church, PhD, had successfully performed CRISPR gene editing in human cells. Both of those studies were published in Science in January 2013. Several other groups, including Doudna and Jínek, demonstrated the same thing.

Despite the bitter ongoing patent dispute over the legal invention of CRISPR genome editing, Charpentier and Doudna’s discovery has already won numerous prestigious scientific awards, including the “Nobel Prizes” of Japan, Canada, Israel, and other countries. In 2018, they shared the Kavli Prize in nanoscience with Virginijus Šikšnys, a Lithuanian biochemist. The French newspaper Le Monde dubbed the duo the “Thelma and Louise” of modern biology.

Nobel speculation

Predictions over who would win the Nobel Prize for CRISPR have raged for several years. While some speculated that the award might come from the Physiology or Medicine committee, CRISPR-based therapeutics have only just entered the clinic. It will be many years before we see significant medical impact from the technology. That put the spotlight on Chemistry.

There was little doubt that, should a Nobel Prize for CRISPR be awarded in Chemistry, Doudna and Charpentier would claim two of the possible three slots. There has been much speculation about who a third recipient might be. Francisco Mojica, PhD, a Spanish microbiologist, first described the viral sequence homology of the “spacers” in the CRISPR arrays. Barrangou and Philippe Horvath, PhD, led a landmark 2007 study that proved experimentally the anti-viral function of CRISPR.

Šikšnys actually submitted a similar study to the Doudna/Charpentier work in early 2012, in which he coined the term “DNA surgery.” But his manuscript, which did not feature a single-guide RNA, was rejected by Cell. By the time it was published in PNAS, the story had moved on.

And of course there is Zhang, who published the first demonstration that CRISPR could edit the DNA of mammalian cells, in a collaboration with Rockefeller University microbiologist Luciano Marraffini, PhD, alongside a similar study from George Church’s group.

Church commented on the “credit” question in an interview last year with The CRISPR Journal. Doudna and Charpentier “had not shown editing, and [Zhang’s paper] was side by side with ours… I did not want anybody to be left out… I felt that Martin Jínek had been left out of the story, and Prashant Mali and Luhan Yang and Le Cong.”

While debate over the Nobel selections might simmer in some quarters, Doudna’s former student (and co-author of A Crack in Creation), Samuel Sternberg, PhD, reflected on the award on Twitter.

“Nobels are also bittersweet, because [so] many more folks deserve the award than can receive it. Michael ‘Michi’ Hauer was the first to purify Cas9 in Jennifer’s lab, while working with Martin Jínek. He died last year, but would have been ecstatic to hear the news today.”

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Few medical non-profit organizations have had the sort of impact in developing genuine treatments as the Cystic Fibrosis (CF) Foundation. Based in Bethesda, Maryland, the CF Foundation has championed cystic fibrosis researchers for decades, dating back well before the CF gene was identified in 1989. Since then, research has focused on better understanding the underlying pathophysiology of the genetic disease, and researching novel therapies, including gene therapies and small-molecule approaches. The Foundation’s support has played a key role in the successful development of multiple CF drugs by Vertex Pharmaceuticals, which have significantly improved the life expectancy of many CF patients.

Physician-scientist Michael Boyle, MD, took over as president and CEO of the CF Foundation at the beginning of 2020. Here, in an exclusive interview with Kevin Davies, Boyle looks back at the role of the Foundation, with particular emphasis on its support of gene therapy. Boyle also explains how interest in gene therapy and genome editing is growing, as the Foundation strives to leave no CF patient behind in its search for a cure. (This interview is lightly edited from the original, published in Human Gene Therapy.)

Kevin Davies: Michael, please start by telling us a bit about your background and what you did before you came to the CF Foundation?

MICHAEL BOYLE: My background is as a physician-scientist. I started my medical training at Johns Hopkins as a medical student in 1986 and stayed at Hopkins for 29 years until I was a full professor in pulmonary and critical care medicine.

An interesting bit of background: as a pulmonary fellow, my research was in CF gene therapy. Much of my equipment had your chief editor, Terry Flotte’s name on it, including my ice bucket, which I used every day! So I saw Terry’s name regularly, because he’d been at Hopkins and left shortly before I arrived in that lab.

I started and directed the Johns Hopkins adult CF program for 15 years and was the principal investigator for several of the Vertex CFTR modulator trials. I assumed I’d stay at Johns Hopkins for the rest of my career but had the opportunity to lead clinical research at the Foundation about five years ago—probably the one job that I was willing to leave for. I initially oversaw the CF Foundation’s clinical research and therapeutic development network of 92 academic centers, and then started as president and CEO in January 2020. And what a year it’s been!

Davies: Give us a snapshot of the CF Foundation. What do you spend on research over the course of the year?

BOYLE: We have about 700 employees. We’re based in Bethesda, Maryland, and we have local chapters supporting the CF community in almost every state in the U.S. Our science really comes from two places—the Bethesda office, where we review the science, issue grants, and form collaborations with companies, and our lab in Boston, which has about 45 scientists. The lab focuses on assisting both companies and researchers in CF, particularly in genetic technologies. Overall we spend nearly $200 million each year on CF research.

Our goal is to advance therapies for people with CF, to support the care of patients, and help address the other challenges that come with having CF. We also support a care center network and play a role in connecting and advocating for patients. But our primary goal is to advance the treatments and the cure of CF.

Davies: What’s the current state of play with the Vertex drugs that are now on the market and what proportion of CF patients are they able to treat? What difference have these drugs made in terms of the prognosis and lifespan of CF patients?

BOYLE: It’s been an amazing story! For the CF Foundation, a big focus early on was identifying the gene in 1989. All along, the intention was to use that information to develop therapies. As understanding advanced on the protein product of the gene, a chloride channel in lung epithelia, it was recognized there was an opportunity to directly address the protein misfolding that occurred in the most common types of CF.

There are about 1,700 different mutations that can cause CF and while those have a wide variety of effects on the protein, many cause misfolding. CFTR modulators—this new class of drug that has revolutionized the treatment of CF—help to specifically address that misfolding. This started with work that the Foundation supported with Aurora Biosciences, identifying early leads for compounds that could refold the protein and open the channel. Vertex Pharmaceuticals took over that program and has done a remarkable job of rapidly advancing these transformative therapies.

Over the past decade, the percentage of patients able to be treated by CFTR modulators has steadily increased. Initially, it was a small percentage, maybe 5%, with a drug called ivacaftor, which addressed rare gating mutations by increasing opening time of the channel. Over time, a two-drug combination of lumacaftor/ivacaftor allowed initial treatment of the most common folding mutation—F508del.

The big breakthrough however, about one year ago, was the approval of a three-drug combination, which dramatically restores CFTR function for anybody with CF that has a single F508del mutation. The early treatments for F508del required patients to be homozygous, because there was a gene dose effect.

This new three-drug combination of elexacaftor, tezacaftor and ivacaftor called Trikafta, is strong enough to restore CFTR function to approximately 50% of its wild-type levels, which is sufficient to make a dramatic clinical improvement in anybody with CF and a single F508del mutation. Approximately 90% of people with CF have this mutation and therefore have the potential to significantly benefit from this three-drug combination. That’s why it’s been such an amazing story!

The specific clinical benefits of this treatment include a large increase in lung function, return of their sweat chloride, which is the diagnostic test, into a non-diagnostic range, weight gain, and fewer problems with infection. Current median predicted survival is around 45 years and we fully expect that number will increase significantly in years to come.

We always say however that benefitting 90% of people with CF is not good enough. Despite CFTR modulators, many people with CF still suffer from challenging complications, and the role of the CF Foundation is to make sure we treat—and eventually cure—100% of people with CF. That last 10% will require other approaches, because a significant portion of that group has mutations that do not make protein. This group is going to require genetic approaches—including approaches such as read-through agents of premature truncation codons (PTCs) to restore CFTR function, and RNA or DNA replacement.

Davies: Let’s talk about gene therapy. The CF Foundation was, I believe, quite heavily involved and there was a lot of excitement about gene therapy as an approach in the early 90s. What’s your current thinking about gene therapy for CF?

BOYLE: I think you’ve identified something that’s a key point. After the discovery of the CF gene, there was an initial wave of excitement that we’re going to be able to quickly solve this. There was a somewhat infamous prediction that within five years, we would likely see a cure for CF through gene therapy. Looking back, it taught us we have to be cautious about such statements because it’s always more difficult and more complicated than you initially think. There were numerous gene therapy projects early on, including multiple clinical trials. Unfortunately, we experienced a little dose of reality.

Initial work with adenovirus and AAV all ran into obstacles—inflammation, limited levels and duration of expression. People realized that it wasn’t going to be as easy as we thought. There was a shift to other therapeutic approaches including CFTR modulators. Overall there have been over 20 trials of gene therapy in CF, all of which have showed perhaps modest expression but no clear clinical benefit and insufficient duration of effect. There has also been recognition that there are challenges that are specific for lung gene therapy and CF, including cell turnover of lung epithelia and tenacious CF mucus.

The flip side, however, is that there’s been continued clear progress in gene therapy in other diseases. These advances and proof of concept from other diseases, as well as the requirement in CF to develop genetic approaches to assure we can treat every person with CF—no matter what their mutation—has  led to a renewed focus on CF gene therapy.

This past November, at our North American CF conference, the CF Foundation simultaneously announced the data on the three-drug modulator combination, and the launch of our Path to a Cure initiative. This is a $500-million program over the next five years focused on advancing genetic technologies, which will help us to develop therapies for 100% of people, no matter what their mutation. Right at the heart of this is gene therapy.

Davies: Let’s talk a bit more about the gene therapy and even the gene editing approaches. Now you you’re using the AAV vector. Is the plan simply to deliver the full protein? It’s a big protein—how would that work?

BOYLE: Let’s talk about gene transfer first. We know there has been success with AAV in other organ systems including retina and muscle. There’s an opportunity in CF if we’re able to address some of the unique challenges that the lungs face.

AAV is our main approach currently. We recently made large awards to two AAV companies, Spirovant and 4D Molecular for a total of up to $20 million to advance CF AAV therapy. There are some challenges however. AAV allows a cargo capacity of about 4.8 kb. CFTR cDNA is 4.5 kb. So when you add the other required regulatory elements, it’s actually too big. Work has included use of a mini-gene approach—a truncated functional DNA.

There’s also been some work in non-viral approaches—lipid nanoparticles. There was a large CF gene therapy trial completed a couple of years ago in the U.K. utilizing lipid nanoparticles. There’s also interest in lentivirus, as it may help address the duration of expression challenge.

Davies: What about gene editing? We’re seeing some very exciting work clinically and gene editing fronts using CRISPR-Cas9 and maybe base editing coming into the portfolio as well. Are there other approaches using either of those technologies that could work for CF?

BOYLE: We think that gene editing has the potential to be the best long-term solution in CF. There has already been successful editing of mutant CFTR in cell lines. The challenges are well-known—first, the safety issues. Then we’re going to have to deliver to stem cells. Right now, we are supporting work to identify those key progenitor cells in the airway, because that’s going to be a requirement. Another challenge is going to be in the delivery of CRISPR-Cas9 or whatever platform we end up using. The components may be big and require using dual vectors with AAV. I think there is a lot of promise in gene editing, but it is going to take significant work.

There’s one other potential challenge in CF, and that’s the variety of 1,700 different mutations. We’re not going to be able to do this one mutation at a time. Options for editing to reach a large number of CF patients include inserting a super exon or targeting the most common mutations.

Davies: Last year, there was a rather protracted saga where Vertex was trying to reach an agreement with the British National Health Service, in order to bring these transformative medicines to British CF patients. Did the CF Foundation play any role in resolving that impasse? I’m sure you’re not involved in telling Vertex how to price their drugs—you want to see them be rewarded for the pioneering R&D that they’ve done. But at the same time to see a large pool of CF patients potentially denied or delayed access to the drugs that you’ve helped fund must be a conflicted feeling?

BOYLE:  We would love every person with CF to be able to benefit from these transformative drugs. And you’re right, we do not have a say in the price of the drugs. We’ve always tried to say there is a middle ground, which will allow people to have access to these drugs while still providing incentive for companies bringing forward transformational science.

Our goal is to cure CF. As exciting as these modulator drugs are, there are additional drugs which are going to be even better. We want there to be an incentive to bring forward new drugs and novel gene therapy and gene editing which will treat everybody with CF and eventually lead to that cure.

Davies: Given you’re now funding $200 million a year, have a $500-million Path to a Cure program in place, have you been able to help other societies who are working to find cures for other Mendelian diseases? They may not have your assets they may have a much smaller patient population, but have you established some best practices to help other non-profits?

BOYLE: We frequently meet with other nonprofits. A couple of things come to mind.

First, we know that some of the science that we’re helping to support, particularly related to nonsense mutations including PTC read-through therapy, has potential to benefit a large number of genetic diseases. There are thousands of diseases which are caused by nonsense mutations and may directly benefit from the science we are supporting. That’s going to help not just people with CF, but genetic illnesses everywhere.

Second is more specific for other foundations. What we say is learn from our model. There are a couple of things in the model to highlight. One of these is supporting basic science to make sure that the understanding needed to drive new therapies is present. This harkens back to our work to allow the initial discovery of the CF gene.

Another key is developing a collaborative relationship with industry. We have an incredible history of such collaboration through venture philanthropy. We’ve taken our funding and used it to support the best science available in small biotech or drug companies. We have incentivized them to focus on CF with funding, with expertise, through the CF clinical trial network, and with information from our patient registry. When we meet with other foundations, we highlight these components.

Davies: Finally, you’re really open to new ideas to continue to elevate the basic science and develop new gene therapy or gene editing approaches. How should people get in touch with you?

BOYLE: We support any research that we think could end up making a difference for people with CF. That includes academic research through traditional RFAs and grant cycles where any researcher can apply. We recently had a Path to a Cure RFA specifically focused on supporting all aspects of genetic technologies, from basic research to delivery issues.

At the same time, we have an entirely separate program intended for industry called therapeutic development agreements (TDAs). This is for companies that have technology or therapeutic programs that may benefit people with CF. We don’t have TDA deadlines. There is opportunity for millions of dollars of support, depending on the quality and stage of the research, and the potential for making a difference for people with CF.

Most recently, we have started a new incubator fund in collaboration with Longwood Fund, a biotech-focused venture capital firm, for companies with genetic technologies who are just being formed to incentivize them to focus on CF.

For the Path to the Cure program, we have a couple of key research areas that we’re interested in. For gene therapy, we’re particularly interested in expression and delivery issues. We need to have sufficient expression and efficiency of delivery to the airway epithelium. We need tropism for the tissues most affected by CF. And we need to address long-term expression issues, including immune response and neutralizing antibodies. For gene editing, we are very interested in identifying airway progenitor cells.  This is going to be a requirement for successful lung gene editing in the future.

For all of these research programs, our website at www.cff.org is the place to start.

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