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Zika and Dengue Infections Differentiated Using Epitope Surrogate Technology

Antibodies for Zika virus, a mosquito-borne virus that spread to the Americas in 2015 and still causes sporadic disease, can be confused in many diagnostic tests with antibodies for dengue virus, making it difficult to tell if someone who tests positive previously had dengue, Zika, or both. It is especially important for reproductive-age women to know if they already had Zika—and likely have immunity—because infection during pregnancy can cause birth defects.

“If you go to places like Brazil, nearly everybody is going to have some immunity to dengue, and also to Zika,” said first author Priscila Castanha, PhD, an assistant professor in the department of infectious disease and microbiology at Pitt Public Health. “This makes it very difficult to test new treatments or determine how widespread an emerging disease may be in areas endemic for flaviviruses, which cause a high burden of illness globally.”

Zika and Dengue
Thomas Kodadek, PhD [Scott Wiseman, The Wertheim UF Scripps Institute]
Now, a newly discovered Zika virus-specific synthetic molecule is capable of differentiating Zika-immune patient samples from samples of patients previously infected with the related dengue virus. The technology may lead to the development of better diagnostics and vaccine candidates.

The research team used an approach pioneered by Thomas Kodadek, PhD, professor of chemistry at the Wertheim University of Florida Scripps Institute to screen half a million “peptide-inspired conformationally constrained oligomers,” or PICCOs, against blood samples from people infected with dengue or Zika virus. PICCOs, which are attached to microscopic plastic beads, mimic epitopes of a pathogen.

This research was published in PNAS in the paper, “Identification and characterization of a nonbiological small-molecular mimic of a Zika virus conformational neutralizing epitope.

The researchers identified 40 PICCOs that engaged Zika virus antibodies. After screening against dengue-positive blood, one PICCO, dubbed CZV1-1, was specific to Zika antibodies. This single CZV1-1 PICCO synthetic molecule correctly identified people previously infected with Zika virus 85.3% of the time and generated false positives in 1.6% of tests.

The study is the first to apply an innovative “epitope surrogate” technology to Zika. Until now, researchers and clinicians have lacked diagnostic tools to easily differentiate between prior infections with different flaviviruses, a family of mostly mosquito- and tick-borne viruses that include Zika and dengue. This has posed challenges for clinical-epidemiologic studies, viral diagnostics, and vaccine development.

Zika and Dengue
Priscila Castanha, PhD [Amro Nasser, University of Pittsburgh]
“The technology is amazing. You don’t need to know the sequence, or the structure, or even the pathogen,” said Donald Burke, MD, Pitt Public Health dean emeritus. “As long as you have chosen the right sets of patient blood samples to compare, you can tease out the important antibodies that differ between the patient sets, along with the corresponding synthetic molecule biomarkers.”

This research team has been studying Zika virus since it emerged in the Americas in 2015. Prior to its emergence in Brazil, they were studying dengue virus. “For every sample we tested during the 2015 outbreak, we had to do 10 different blood tests to confirm Zika,” said Castanha. “These tests are technically difficult and time-consuming, making them impractical for providing clinical guidance. I think if we had this molecule back then, it would’ve been fantastic.”

Importantly, the PICCO screening technology used to identify the Zika-specific molecule doesn’t require refrigeration and could also be used for other outbreaks. “The molecule is a molecular mimic that cannot unfold,” Kodadek said. “That means there’s no cold chain required, making it particularly useful for outbreaks occurring in remote or low-resourced areas.”

Could Single Technologies’ 3D Sequencing Deliver the $10 Genome?

Johan Strömqvist, PhD, has always been interested in “seeing” molecules, particularly in three dimensions (3D). Strömqvist worked with optical single-molecule imaging for his PhD and then founded a company in 2010 to look at protein-protein interactions to see how different compounds modulated those interactions to improve the attrition rate of drug candidates in clinical testing. Later on, Strömqvist began working with fiberoptics innovator Bengt Sahlgren—who was instrumental in the rise of Hexatronic, the largest Nordic fiberoptic company—to take confocal imaging to the next level.  

Around 2014, in the basement of a suburban home in Stockholm, Strömqvist and Sahlgren began working together on an optical technology for proteomics and spatial biology. After ditching a multiplex method for looking at multiple genes using barcodes, like a spatial gene-expression platform, the duo switched their attention to in situ next-generation sequencing (NGS). That’s when they realized they could use their optical system to sequence using a 3D matrix for hugely parallel reactions in three dimensions.  

“We realized that the technology scales with a cube, which is a game changer because all NGS technologies, including long-read applications, scale with a square,” Strömqvist told GEN Edge 

“What Illumina did was scale reads with a square, which is why it took over Sanger sequencing—they went from one dimension to two dimensions. Now we’re going to three dimensions.”

Too good to be true?  

A few key innovations combine to make Single Technologies’ 3D DNA sequencing a potential game changer in NGS technology.  

The first innovation is the development of a high-speed confocal scanner that enables imaging of large areas in three dimensions with single fluorescence molecule sensitivity. This is an approach that has been explored by the likes of Illumina but was abandoned because of speed issues leading to the use of line scanning. 

The second innovation concerns ditching the concept of a single, two-surface flow cell patterned with billions of nanowells for a 3D matrix. Strömqvist explained that their matrix works a bit like a stack of Post-it notes containing DNA information. According to Strömqvist, this format enables 3D DNA sequencing in situ.

The size of Single Technologies' 3D DNA sequencing matrix
The size of Single Technologies’ 3D DNA sequencing matrix (left) compared to Illumina’s S4 flow cell (right).

“If you take all the Post-it notes and put them on the table, you could fill a huge table,” said Strömqvist. “So, if you have a mouse brain, you could either chop it up into small 10-micron slices and distribute it over a huge area, or you can basically keep it pretty much like it is.” 

Strömqvist and Sahlgren have been working out the kinks to turn their technology, which they call “3D DNA sequencing,” from a basement DIY project into a commercial solution with their startup, Single Technologies. According to Strömqvist, Single Technologies has generated data over the past two years using a prototype of their fully automated 3D DNA sequencer Theta and is currently developing two commercial systems that should be ready by the start of 2025. 

However, not everyone is convinced that the revolution is happening at Single Technologies. In a breakdown of Single Technologies’ patents, sequencing technologist Nava Whiteford, PhD, said that Single Technologies doesn’t describe much innovation in their intellectual property (IP) outside of the optical system. Whiteford said the patterned flow cell—which seems to be (in addition to the imager) the core innovation for Single Technologies—is not described. 

Strömqvist also claimed that Single Technologies’ 3D DNA sequencing can be applied to almost any fluorescent-based sequencing chemistry. Whiteford is dubious, noting that the company lacks employees with the backgrounds required to develop a new sequencing chemistry.  

According to Whiteford, the best bet for Single Technologies may be to partner with a company with existing sequencing chemistry or to be acquired by an existing sequencing company, a situation in which Single Technologies could provide a throughput advantage with their improved optical system.   

Keith Robison, a well-known NGS expert and founder of the “Omics! Omics!” blog, is also skeptical, telling GEN Edge that he hasn’t seen anything “solid” from Single Technologies. Single Technologies has only raised €16 million ($17 million) so far, through a mixture of traditional fundraises and equity financing over the past decade.  

Citizen Kain

However, Single Technologies does have its converts, perhaps most notably Bob Kain, who gained fame for his role in the development of Illumina’s Hi-Seq, which brought the sequencing cost of a human genome down to around $1,000. Since leaving Illumina in 2014, Kain has been advising several NGS companies. Kain told GEN Edge that, besides what he’s seen from Single Technologies, he hasn’t been particularly impressed.  

“While many companies out there have interesting improvements in sequencing, they all have certain drawbacks. I don’t think they’re compelling enough to get around the barriers to entry that Illumina has put up into switching costs,” said Kain.  

“The reality behind sequencing with Hi-Seq and Nova-Seq is really all about how many bases per reagent dollar and bases per day you can get at the appropriate accuracy in the long run. That’s what Illumina wins with.”  

But Kain also doesn’t think that Illumina has made any major improvements to NGS or innovated much beyond the Hi-Seq.  

“The Hi-Seq was a series of exponential technologies that went into one instrument, and as those technologies individually moved forward, the instrument itself could get higher performance without major reinvention,” said Kain. “Even the Nova-Seq is more of a reinvention based on the user interface, things like that, and how you handle reagents. It’s not a reinvention of the main components.”  

When Kain first heard about Single Technologies’ 3D DNA sequencing, he thought it was just an interesting, very early-stage technology that wasn’t far enough to get him to buy in.  

“Interesting technologies often mean problems to solve,” said Kain. “But over months, I realized that they have been quietly solving these problems for a while, and the hurdles I might have thought of when I understood their technology were already addressed. I was really impressed by that.”  

Searching for a spark 

Last December, Kain flew to Stockholm to visit Single Technologies. He was impressed by how well the technology was developing and the state of their instrument. In April 2024, Kain joined Single Technologies as an advisor to support the commercialization of Theta.  

“When I saw this technology, I thought this optical architecture had the potential to bring the cost per human genome down to $10 and below,” said Kain. “It’s a new exponential curve. Maybe it’s a little dramatic, but it’s like going from tubes to transistors and transistors to microcircuits…Each time, you get on a new exponential curve.”  

But this technology that Kain touts as being transformational to NGS hasn’t materialized. The only live wire coming from Single Technologies pertains to a project called “Regenerar,” which aims to use epigenetic manipulation to reprogram glial cells in the brain into neurons. Single Technologies is not playing a leading role in the project, which has received €3 million ($3.2 million) from the European Commission’s European Innovation Council Pathfinder program, but rather is providing its 3D DNA sequencing technology. 

Though Kain and Strömqvist want to capitalize on the purported agnosticism of their platform and stand alone at the top of the NGS pyramid, the solution to summiting may require partnering with a company with a proven sequencing chemistry. The best way for Single Technologies to show the exponential cost-saving and experimental value of “digitizing” samples is to prove it, and to do so they may not need to reinvent the sequencing chemistry wheel.  

It is unclear if Single Technologies will be able to survive financially through the four years of the Regenerar project. Perhaps Kain’s endorsement will trigger a chain reaction of excitement and investment, taking Single Technologies from patents and prototypes to the pinnacle of an already crowded NGS market

But without more cash, Single Technologies might struggle to make it out of the basement. 

Stem Cell Model Illuminates Genetic Drivers of Neuroblastoma

Neuroblastoma is the most common childhood tumor occurring outside the brain. Until now, studying genetic changes and their role in neuroblastoma initiation has been challenging due to the lack of suitable laboratory methods. A new model developed by researchers at the University of Sheffield, in collaboration with the St. Anna Children’s Cancer Research Institute in Vienna, replicates the emergence of early neuroblastoma cancer-like cells, providing insight into the genetic pathway of the disease.

Their findings are published in Nature Communications in an article titled, “A human neural crest model reveals the developmental impact of neuroblastoma-associated chromosomal aberrations.”

“Early childhood tumors arise from transformed embryonic cells, which often carry large copy number alterations (CNA),” the researchers wrote. “However, it remains unclear how CNAs contribute to embryonic tumorigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumor neuroblastoma (NB).”

The international research team found that specific mutations in chromosomes 17 and 1, combined with overactivation of the MYCN gene, play a pivotal role in the development of aggressive neuroblastoma tumors.

Childhood cancer is often diagnosed and detected late, leaving researchers with very little idea of the conditions that led to tumor initiation. Models that recreate the conditions that lead to the appearance of a tumor is needed in order to understand tumor initiation.

The formation of neuroblastoma usually starts in the womb when a group of normal embryonic cells called “trunk neural crest (NC)” become mutated and cancerous.

In an interdisciplinary effort spearheaded by stem cell expert Ingrid Saldana, PhD, from the University of Sheffield’s School of Biosciences and computational biologist Luis Montano, PhD, from the St. Anna Children’s Cancer Research Institute in Vienna, the new study found a way in which to use human stem cells to grow trunk NC cells in a petri dish.

These cells carried genetic changes often seen in aggressive neuroblastoma tumours. Using genomics analysis and advanced imaging techniques, the researchers found that the altered cells started behaving like cancer cells and looked very similar to the neuroblastoma cells found in sick children.

The findings offer new hope for the creation of tailored treatments that specifically target the cancer while minimizing the adverse effects experienced by patients from existing therapies.

Anestis Tsakiridis, PhD, from the University of Sheffield’s School of Biosciences and lead author of the study, said: “Our stem cell-based model mimics the early stages of aggressive neuroblastoma formation, providing invaluable insights into the genetic drivers of this devastating childhood cancer. By recreating the conditions that lead to tumor initiation, we will be able to understand better the mechanisms underpinning this process and thus design improved treatment strategies in the longer term.

“This is very important as survival rates for children with aggressive neuroblastoma are poor and most survivors suffer from side effects linked to the harsh treatments currently used, which include potential hearing, fertility, and lung problems.”

Florian Halbritter, PhD, from St. Anna Children’s Cancer Research Institute and second lead author of the study, added: “This was an impressive team effort, breaching geographic and disciplinary boundaries to enable new discoveries in childhood cancer research.”

Multiply Labs and Retro Biosciences Partner on Cell Therapy Manufacturing for Age-Related Diseases

Multiply Labs, a robotics firm, and Retro Biosciences, which is developing therapies to address age-related diseases, signed an agreement valued at up to $85 million to automate Retro’s approach to target the cellular drivers of aging.

With this partnership, Multiply Labs marks the first commercial sale of its robotic system in support of Retro’s efforts.

By enabling Retro to manufacture at a cost-efficient scale, Multiply Labs empowers them to expedite the transition from process development to clinical trials and commercialization, ultimately expediting the development and delivery of life-changing therapies to patients worldwide, according to Multiply Labs.

“The accelerating rate of change in cell therapies motivated us to look for a manufacturing platform with flexibility at its core,” said Joe Betts-LaCroix, CEO of Retro. “The modular structure of the Multiply Labs robots will help enable Retro to bring its unique cell therapies to patients.”

“This collaboration signifies a profound step forward in our efforts to harness cutting-edge robotic technology that helps patients access life-saving therapies,” added Fred Parietti, PhD, co-founder and CEO of Multiply Labs. “We are thrilled to enter this agreement with Retro, a like-minded organization with a shared commitment to extending human longevity and saving lives.”

While robotics and automation are common approaches to reducing labor costs in other industries, it is more challenging in the biotech field as changing instruments or processes in FDA-approved therapeutics manufacturing requires regulatory resubmissions and lengthy comparability studies. This is why Multiply Labs’ approach, in collaboration with a partnership ecosystem (including companies such as Akron Bio, Charles River, Fedegari, and GenScript) focuses on robotic systems that can operate market-leading GMP instruments from different vendors which are already extensively deployed for cell and gene therapy manufacturing, pointed out Parietti, adding that this technology is designed to enable plug-and-play-like capabilities, faster automation timelines, and fewer regulatory barriers.

Platelet Pathway More Traveled with Age, Leads to Excessive Clotting

As people age they become more prone to blood clotting diseases, which can be caused when blood platelets clump together. This clumping can lead to strokes and cardiovascular disease. For decades, scientists have studied why older people’s blood cells behave in this way.

Studies in mice by researchers at the University of California, Santa Cruz (UCSC), and collaborators have now uncovered a distinct, secondary population of platelets that appears with aging. The researchers traced this population of platelets to its stem cell origins, and identified what they describe as the first-ever-discovered age-specific development pathway from a stem cell to a distinct mature platelet cell. The team found that these types of platelets display hyperreactive behavior and unique molecular properties, which could make them easier to target pharmacologically.

“The question for decades and decades has been: why are aging people at such high risk for excessive blood clotting, stroke, and cardiovascular disease?” said research lead Camilla Forsberg, PhD, UC Santa Cruz professor of biomolecular engineering. “We have this discovery of a whole new pathway that progressively appears with aging—troublemakers! That was never part of the discussion.”

The investigators reported on their studies in Cell, in a paper titled “An age-progressive platelet differentiation path from hematopoietic stem cells causes exacerbated thrombosis,” in which they concluded, “… our findings may profoundly impact the millions of elderly people at risk of experiencing adverse thrombotic events.”

Red cells, white cells, and platelets (Plts) are the three types of blood cell produced by the body, the authors explained. Millions of platelets float around in the blood at all times, and their job, when an injury occurs either internally or externally, is to clot together to form a natural, living band aid. Platelet dysregulation, which is known to increase with age, occurs when these cells are either hyperreactive and form clots too often, or are under performing. Hyperreactivity is the much more widely seen problem, but both cases result in the body being unable to effectively manage bleeding and clotting. “Tipping the homeostatic balance toward inadequate Plt production is associated with higher risk for bleeding disorders, whereas Plt overproduction and hyper-reactivity lead to pathologic clot formation in thrombotic diseases such as deep vein thrombosis and ischemic stroke,” the authors stated.

All blood cells begin as a special type of stem cell known as hematopoietic stem cells (HSCs), and then mature through a series of intermediary steps that take them down a differentiation pathway that determines their fate as either platelets, red blood cells, or white blood cells. It’s been known for decades that hematopoietic stem cells decline with age, but that then presents scientists with a contradiction. If these hematopoietic cells are less healthy, why are the platelets they create hyperreactive? Moreover, the authors commented, “Although drastic increases in Plt dysregulation and adverse thrombotic events in aging populations have been clear for decades, distinct differentiation paths and cell populations have not been envisioned as underlying mechanisms of disease susceptibility upon aging.”

As stem cell biologists, the researchers at UC Santa Cruz traced the lineages of these stem cells in mouse models. They discovered that some platelets in aged animals did not follow the expected differentiation pathway. Instead, these platelets took what the UCSC researchers dubbed a “shortcut” pathway, skipping over the intermediary steps and immediately becoming megakaryocyte progenitors (MkPs), the blood cell stage immediately before platelet production. To the researchers’ knowledge, this is the first age-specific stem cell pathway ever discovered.

“… we employed the FlkSwitch model as a powerful tool for tracking hematopoietic differentiation pathways to demonstrate an unexpected cellular mechanism for the etiology of Plt-related disorders upon aging,” they stated. “The age-induced megakaryocyte progenitors have a profoundly enhanced capacity to engraft, expand, restore, and reconstitute platelets in situ and upon transplantation and produce an additional platelet population in old mice.”

Upon vascular injury, platelets in young mice form much smaller blood clots (left) than platelets in old mice (right), where the clot almost occludes the blood vessel. Fluorescence microscopy image from Poscablo et al, CELL, 2024.
Upon vascular injury, platelets in young mice form much smaller blood clots (left) than platelets in old mice (right), where the clot almost occludes the blood vessel. Fluorescence microscopy image from Poscablo et al, CELL, 2024. [Poscablo et al]
The reported studies found that production of hyperreactive secondary platelets start around midlife for the mice, with the population of this type of platelet growing progressively with aging. And while the population of platelets produced from the shortcut pathway were hyperreactive, the platelets produced from the primary differentiation pathway continued to behave like the platelets in a young person, the team found. “People think of [platelets and red blood cells] as one lineage that shares regulation and intermediate stages until the very end,” Forsberg said. “To see that [the secondary platelet population] were completely separated all the way from the stem cell level, only in aged mice, was really surprising.” Added first author Donna Poscablo, PhD, Forsberg’s former PhD student, and now a postdoctoral scholar at Stanford University, “The gradual differentiation cascade maintains a youthful property, and I feel like that is also surprising within itself.”

 

The studies did not identify a trigger that might set off activation of this secondary pathway, but the investigators the results did indicate that it was not triggered by the aging environment itself. The scientists experiments showed that transferring a young hematopoietic stem cell into an aged environment did not trigger the shortcut pathway. And when an aged hematopoietic stem cell was put into young environment, the old stem cells continued to behave as old stem cells.

“That was surprising, the age resilience of the other pathway,” Forsberg said. “One of the platelet populations is not affected at all [by aging], whereas the one we have discovered is—so the whole phenomenon is not primarily induced by the environment, but by the differentiation path.”

Knowing that this secondary population of platelets exists will help scientists identify new ways to target and regulate these problematic cells via their stem cells. Researchers have not to date tried to target these upstream cells. “The emergence of Plt subpopulations during aging provides evidence that Plt heterogeneity is a determinant of age-related Plt diseases,” the scientists pointed out. “The production of mature cells via distinct differentiation paths offers a paradigm of stem cell aging that is currently unexplored.”

Poscablo said, “From our expertise, we can ask the questions of how to target the hematopoietic stem cell and now the megakaryocyte progenitor, which has never really been highlighted before as a place to target.”

Targeting these cells may not require the development of new drugs, but more simply inform the prescription of existing blood thinners, such as aspirin, which treat different patients to varying degrees even if they present with similar clotting-related symptoms. The researchers aim to use their mouse models to help identify which of the two populations of stem cells are more sensitive to aspirin and to other platelet drugs on the market. In the mouse models, they will also continue to study how to manipulate and control the shortcut pathway, with funding from the National Institutes of Health (NIH).

In parallel, and with the support of a grant from the California Institute for Regenerative Medicine (CIRM), the UCSC researchers are also working on finding this secondary population of platelets in humans. They concluded, “Our identification of the cellular origins and mechanisms of aging-enriched Plts provides compelling therapeutic opportunities for targeting HSCs and MkPs to control both Plt generation and functional reactivity throughout life.”

The State of CRISPR and Gene Editing 2024

Broadcast Date: 
  • Time: 

Following the landmark approval of the first CRISPR-based cell therapy in December 2023, the CRISPR community is looking ahead to the next wave of commercial successes, fueled by continued innovation in the development of new gene editing and delivery tools and technologies. Equally exciting advances are occurring in livestock editing, xenotransplantation, and many other specialties.

In The State of CRISPR and Gene Editing virtual summit, GEN proudly gathers a tantalizing line-up of luminaries from academia and industry to discuss the latest research developments, innovations, and advanced technologies that are expanding the CRISPR toolbox, delivering new therapies to patients and safeguarding our food supply.

Highlights of this summit include:

  • An opening keynote from David Liu, PhD (Broad Institute) on the state of base and prime editing.
  • A keynote conversation with Laura Sepp-Lorenzino, PhD (CSO Intellia) as the biotech advances a pair of landmark in vivo therapies in the clinic.
  • Talks on two cutting-edge areas of CRISPR toolbox research—transposases from Sam Sternberg, PhD (Columbia) and anti-CRISPRs with Joe Bondy-Denomy, PhD (UCSF).
  • The state of livestock gene editing—a panel discussion with Elena Rice, PhD (CSO, Genus plc) and Alison Van Eenennaam, PhD (UC Davis).
  • A fireside chat with the co-founders of Tome Biosciences, Omar Abudayyeh, PhD and Jonathan Gootenberg, PhD (Harvard Medical School) on programmable genome engineering.
  • A fireside chat with two leading genome engineers and co-founders of the non-profit Arc Institute—Patrick Hsu, PhD and Silvana Konermann, PhD.

Registration is free.

Produced with support from:

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Guest Speakers Include

The State of CRISPR and Gene Editing 2024 speakers

Bye, Bye, Baltimore: GEN’s Takeaways from ASGCT

At the conclusion of the ASGCT meeting, Julianna LeMieux, PhD, GEN’s deputy editor in chief, and Corinna Singleman, PhD, GEN’s managing editor, chat about how they spent their time at the conference and their unique takeaways from the research and news presented through the week.

They discussed general insights on how diverse the topics have been. Julianna discussed David Liu’s, PhD, keynote on Wednesday and Corinna shared about an interview she had with Frederic Revah, PhD, Genethon.

ASGCT 2024: Interview with Frédéric Revah, CEO of Généthon

A noticeable trend at last week’s American Society for Gene & Cell Therapy (ASGCT) conference was one of retrospection in the face of progress. In many cases, speakers expressed the need to reflect on the history of their field and the progress made over the past few decades to aid in decision-making for future endeavors.

Frederic RevahFrédéric Revah, PhD, is one such scientist who has consistently considered the history of biotechnology, research, and pharmacology. As the CEO of Généthon, Revah has reflected on the history of the non-for-profit’s origins to direct the next steps for the organization, and those organizations that have branched out of it.

Revah sat down with GEN’s managing editor, Corinna Singleman, PhD, to discuss the past, present, and future of Généthon and particularly its pioneering research in muscular dystrophy.

Généthon was created and funded by the French Muscular Dystrophy Association, AFM-Telethon in 1990. AFM-Telethon arose from the grass-roots efforts of nine families advocating for study into Duchenne muscular dystrophy (DMD) and hosting telethon events beginning in the 1950s. They began a national phenomenon in France to raise awareness and funding for muscular dystrophy research, which has expanded to study other rare genetic disorders.

Généthon was created with a primary focus on research in gene therapy. Initially, this work concentrated on better understanding the human genome as a whole, as researchers around the world were working on the Human Genome Project. With the successful sequencing of the human genome, Généthon’s goals refocused on gene therapies, culminating in its first gene therapy trial for severe combined immune deficiencies (SCID).

“Généthon progressively constructed the competencies, the know-how in designing those drugs, testing them, manufacturing them. We were involved in manufacturing as early as 2008… The idea is that, if we didn’t have manufacturing tools, we would go nowhere.”

Through strategic partnerships and collaborations, Généthon has continued to grow in its research abilities as it develops therapies, bringing many treatments to clinical trials.

This interview has been edited for length and clarity.

Singleman: How would you describe your contribution since you joined Généthon as CEO? Have you changed or modified the direction of where the company was going to where it is now?

Revah: I came in 2010 at a moment when there was no clinical trial ongoing, with the mandate to transform what was a very efficient research institute into some kind of not-for-profit biotech. And now, 14 years later, we have this pipeline and industrial partnerships. I had an initial academic background then moved to the pharma industry. And it’s really with that training, bringing my pharma expertise to the association, ready to have this shift from an R&D institute to a more biotech type of operation. That’s where we are today, trying to secure industrial financing and move progressively from discovering drugs, to using those drugs in the clinic, and possibly moving to the next step of taking some of those drugs to the market ourselves. This is a challenge and an endeavor because the number of resources that is needed is very high to achieve this transition in an environment that is not very easy for rare genetic diseases.

Gene therapy for rare diseases was not of interest for investors or for pharma. Things changed with the first initial “Wow!” results that were so impressive. Then pharma got interested and then realized that for rare genetic diseases, by definition, the market for the business model based on recouping the investment on one single injection for a small population was a challenge.

It does worry me. We’re seeing in the pharma industry, some of the players stepping back from their investment in rare disease. See what happened with Pfizer. And we see pharma company, biotech investors in particular, in this moment where access to capital is difficult, are still interested in gene therapy but for larger markets. Our challenge in the coming years will be to try to find the right balance to move those drugs forward, but under strained financial conditions.

Singleman: In some of the talks at ASGCT, there has been discussion about rare diseases through the lens of how to apply those therapies to more common diseases, like heart disease which is one of the top killers. What are your thoughts on that?

Revah: You should not neglect the fact that rare diseases are a public health issue. There are thousands of these diseases. Three hundred million people around the world are affected by them and there’s many of those individually that could benefit from gene therapy. Whatever we do for rare diseases also benefits frequent disorders and working on rare diseases allows us to develop technologies, develop approaches, develop science, and it’s really a springboard for more common diseases.

There are only a few kids per year in Europe, probably a similar number of kids in the United States, with DMD. The work to study and treat this disease is now applied to cancer with CAR T cells because CAR T cells are really the extension of the same type of technology, and now we can think about heart disease and diabetes. The research into rare diseases really feeds research for widespread diseases and rare disease research should not neglected.

There is a systemic question then about how do we finance development for these rare genetic diseases? The Bespoke Gene Therapy Consortium (BGTC) is an organization launched by the FDA and National Institutes of Health (NIH), trying to finance developments for some genetic diseases with no commercial models. Their symposium exemplified how the fight of parents has been important. But this is not sufficient. We have to find a systemic way in addressing the financing and it’s not only regulatory. Peter Marks (FDA) said that we have to be pragmatic and we have to adjust the regulatory hurdles throughout the season. That’s good but it doesn’t solve the cash problem.

Généthon is one of the pioneers in gene therapy. We’re probably one of the oldest organizations in the room working on gene therapy. It started in the ‘90s at a time when nobody was, and we were still one of the leaders in the sense of the breadth of our portfolio. We work not only on therapies, but on some technologies. We are convinced that bringing down the cost of production is key.

Singleman: How are you doing that?

Revah: We have a group of 40 people working on that, in close conjunction with the production side. We were pioneers, for instance, in suspension methods; we have been using cell suspension methods at a time when everybody was using a different method. The scaling up becomes much easier when the cells are in suspension. We’re trying to find new production systems, new production cells. We are also trying to improve the transfection step, which is very important. We’re trying to improve downstream methods and purification methods.

I would say incremental improvement of the process, but also trying to bring some breakthrough approaches in the way plasmids are designed and moving to plants for instance, which is something that we’re testing. It could be a breakthrough approach and the objective here is really to decrease the cost of production. Some of these productions could be as expensive, from a few hundred thousand [Euros] per dose. I’m just talking about cost of production, cost of goods, bringing that cost down is also an important element in making development affordable.

Singleman: If we’re going to have these treatments, there needs to be some equity in distribution. Is all this manufacturing bioprocessing work being done in-house or do you also work with corporate partners?

Revah: We strongly believe that improving bioprocessing is really a transdisciplinary approach because you have to understand how these viruses assemble in cells. We ask questions: What’s the transfection? How are these bioreactors working? It’s really a mix of virology, cell biology, instrumentation sensors, and more, because in order to understand what happens in vivo, you have to develop sensors and we use artificial intelligence to assess the data that comes out and try to improve. So basically, we’re not doing all that by ourselves, trying to create networks. We’re working with AI specialists, in virology and cell biology. We’re working with solution providers, getting people in other fields interested in the gene therapy arena. We believe that there’s technology out there that we should at least test. So yes, we’re not doing it on our own.

Singleman: Is there anything exciting or interesting coming up with either partnerships or any new trajectories in your research or processing that you are looking forward to?

Revah: We’re working on producing AAV in plants, also very systematic genetic engineering of production cells in order to be able to identify those genes that might modify and improve productivity. That’s something that we’re doing in a very systematic way. We’ve been working on AI with a very large conglomerate and the defense industry in France. The idea is really to create a numerical twin of our bioreactors. For the time being, we’re trying to segment the process and we have been concentrating on the downstream process. We’d like to be able to predict from the chromatography profile of our products, some very key parameters that we would not have to test in the analytical lab. We’re progressing on that in order to reduce the time and the cost of production.

Singleman: You said you have a lot of stepwise processes that you’re being very precise about. Have there been any pitfalls or areas that are very clearly not working and that need improvement?

Revah: I think the whole industry is facing the same questions. There is a ratio of full to empty to consider. This includes a capsid with the exact genome that we want to put it in, there are the capsids that are exactly the way we want them. But there are capsids that are empty, or capsids that are partially full. This has been a challenge for the whole industry trying to improve this ratio. After all, we’re forcing cells to assemble a virus with DNA, which is not the DNA that is supposed to be there. So how does this happen? And it takes us into both basic virology, and also design of the bioreactors. Without going into detail, we think we have interesting solutions there.

Singleman: You mentioned Pfizer earlier. Do you have any thoughts or comments about the situation with Pfizer’s DAYLIGHT project and the death of the young patient in the project? Généthon has its own clinical trial coming up. Will this sad news impact the trial?

Revah: Of course, we’re following this very closely. We shared information on some of our respective side effects with Sarepta, Pfizer, and Solid Biosciences. We presented what we encountered and the solution we came up with, which was different from the side effect that we’ve seen this week, which is cardiac.

It is another type of side effect that appears almost a year after treatment and so of course, there are some elements that we’re thinking about, having studied the disease for more than 20 years. We will definitely take that into account in our thinking and the way we treat patients.

Antiviral Protein Combinations Are Responsible for Lupus Symptoms

In a new study, researchers from Johns Hopkins School of Medicine report they have discovered insights as to why lupus symptoms and severity present differently in individuals with the autoimmune condition. The researchers believe their finding is a crucial step forward in understanding biological mechanisms behind lupus, and may also lead to shifts in how clinicians treat patients.

Their new study is published in Cell Reports Medicine in an article titled, “Uncoupling interferons and the interferon signature explains clinical and transcriptional subsets in SLE.”

“Systemic lupus erythematosus (SLE) displays a hallmark interferon (IFN) signature. Yet, clinical trials targeting type I IFN (IFN-I) have shown variable efficacy, and blocking IFN-II failed to treat SLE,” the researchers wrote. “Here, we show that IFN type levels in SLE vary significantly across clinical and transcriptional endotypes.”

Interferons normally help to fight infection or disease, but are overactive in lupus, causing widespread inflammation and damage.

“For years, we have accumulated knowledge that interferons play a role in lupus,” said corresponding author and rheumatologist Felipe Andrade, MD, PhD, associate professor of medicine at the Johns Hopkins School of Medicine. He says this research began with questions about why certain lupus treatments were ineffective for some patients. “We have seen instances where the patient surprisingly didn’t improve—we wondered if certain interferon groups were involved.”

Some lupus treatments are designed to suppress a specific group of interferons, known as interferon I. The researchers believed that two other interferon groups, interferon II and interferon III, may be to blame for these poor treatment responses.

To investigate, the researchers observed at how different combinations of interferon I, II, or III, and their overactivity, may present in people with lupus. They took 341 samples from 191 participants to determine the activity of the three interferon groups, and used human cell lines engineered to react to the presence of each specific interferon group to analyze the samples. Through this process, the researchers determined that the majority of participants fell into four categories: those only with increased interferon I; those with a combination of increased interferons I, II, and III; those with a combination of increased interferons II and III; or those with normal interferon levels.

The researchers were able to use these findings to also make several associations between these interferon combinations and lupus symptoms. In those with elevated interferon I, lupus was mainly associated with symptoms affecting the skin, such as rashes or sores. Participants with elevated levels of interferon I, II, and III exhibited the most severe presentations of lupus, often with significant damage to organ systems, such as the kidneys. However, not every symptom found in lupus was associated with elevated interferons.

The formation of blood clots and low platelet counts, which also affect clotting, did not have an association with increased levels of interferon groups I, II, or III. Researchers say this indicates that both interferon-dependent and other biological mechanisms are involved in this complex disease. The study also found that genetic testing of genes associated with these interferon groups, or the interferon signature, did not always indicate elevated interferon levels. They plan to investigate this in future studies.

“What we’ve seen in our study is that these interferon groups are not isolated; they work as a team in lupus and can give patients different presentations of the disease,” said rheumatologist Eduardo Gómez-Bañuelos, MD, PhD, assistant professor of medicine at the Johns Hopkins University School of Medicine and the study’s first and additional corresponding author. Evaluating a patient’s elevated interferon combinations allows for a better understanding of how they may react to treatments, and would allow clinicians to group them into clinical subtypes of lupus, Gómez-Bañuelos explained.

Recursion Completes Supercomputer for AI Drug Discovery

Artificial intelligence (AI)-based drug developer Recursion said it has completed the fastest supercomputer to be wholly owned and operated by any pharmaceutical company worldwide, using technology developed by collaboration partner Nvidia.

The supercomputer, BioHive-2, ranks No. 35 on the TOP500 list of the world’s most powerful supercomputers across all industries as of this month.

BioHive-2 operates four times faster than Recursion’s original supercomputer BioHive-1 in benchmark performance tests, according to the company.

BioHive-2 consists of an NVIDIA DGX SuperPOD AI supercomputer, powered by 63 DGX H100 systems with a total of 504 NVIDIA H100 Tensor Core GPUs interconnected by NVIDIA Quantum-2 InfiniBand networking.

Recursion has spent the last decade generating and aggregating one of the largest biological and chemical datasets in the world, purpose-built for training new AI models. With BioHive-2 now online, we have significantly more computational horsepower to accelerate our use of our ever-growing dataset, extending our ability to train larger and more generalizable foundation models and AI agents to industrialize our drug discovery efforts,” Ben Mabey, CTO, said in a statement.

Recursion has also developed new foundation models that include Phenom-1, a deep-learning model designed to extract biologically meaningful features from images of cells. The company says it has demonstrated a need for training larger models since the performance of its model has increased as the size of the training data and the number of model parameters have grown.

The experimentation and training to produce Phenom-1 required several months of computational time using BioHive-1. By developing BioHive-2, Recursion said, it can now execute multiple AI projects of similar or greater size in parallel in shorter timeframes, enabling teams from both Recursion and its Valence subsidiary to pursue advanced AI drug discovery and unlock additional value from Recursion’s data.

Valence is a pioneer in low-data small molecule drug design which Recursion bought for $47.5 million last year.

Investors signaled approval of Recursion’s supercomputing advance with a buying surge that sent the company’s stock rising 10% Monday, from $8.60 to $9.48. Nvidia was all but flat, rising 0.58% from $898.78 to $903.99.

A smaller model similar to Phenom-1, called Phenom-Beta, was released for external use earlier this year through NVIDIA BioNeMo, a generative AI cloud-based platform designed to enable faster discovery and design of drugs. Phenom-Beta is designed to flexibly process cellular microscopy images into general-purpose embeddings at any scale, from small projects to billions of images.

“Meaningful representations”

“Phenom-Beta can turn a series of image inputs into meaningful representations that are foundational to analyzing and understanding the underlying biology,” Mabey wrote on the company’s blog.

Phenom-Beta was trained using the RxRx3 dataset, a publicly available dataset Recursion released last year that contains approximately 2.2 million images of HUVEC cells across ~17,000 genetic knockouts and 1,674 known chemical entities.

The Phenom series is among several different models Recursion has developed to accelerate drug discovery using biological, chemical, and real-world patient data. Phenom-Beta is the first third-party model to be made available on BioNeMo.

Last year Recursion’s Nvidia-based platform enabled the AI drug developer to develop a large protein-ligand interaction prediction dataset. Recursion predicted the protein target or targets for approximately 36 billion chemical compounds on Enamine’s REAL Space chemical library, which consists of 48 billion make-on-demand molecules.

REAL Space offers the largest supply of commercially available compounds, according to Kyiv, Ukraine-based Enamine and Salt Lake City, UT-based Recursion. Last December, the companies launched a collaboration of undisclosed value to generate compound screening libraries with insights from Recursion’s protein-ligand interaction predictions spanning across Enamine’s massive library.

An NVIDIA DGX SuperPOD based system called Eos ranks No. 10 among supercomputers worldwide. Eos is based on the NVIDIA DGX H100 with Xeon Platinum 8480C processors, NVIDIA H100 accelerators, and Infiniband NDR400 and it achieves 121.4 Pflop/s.

“Accelerated computing, combined with the power of generative AI, is propelling the pharmaceutical industry into a new, advanced era of drug discovery,” said Rory Kelleher, global head of business development for life sciences at Nvidia. “BioHive-2, powered by NVIDIA DGX AI supercomputing, is poised to accelerate the development of additional industry-leading foundation models across biology, chemistry, and healthcare.”

Flaviviridae viruses, illustration.
Antibodies for Zika virus can be confused with antibodies for dengue virus, making it difficult to tell if someone who tests positive previously had dengue, Zika, or both. Now, a newly discovered Zika virus-specific synthetic molecule is capable of differentiating Zika-immune patient samples from samples of patients previously infected with the related dengue virus. The technology may lead to the development of better diagnostics and vaccine candidates.
Digital blue background with DNA double helix structure. Nucleic acid sequence. Genetic research. 3d illustration. Pixelated effect.
Around 2014, in the basement of a suburban home in Stockholm, Johan Strömqvist and Bengt Sahlgren began working together on an optical technology for proteomics and spatial biology. After ditching a multiplex method for looking at multiple genes using barcodes, the duo switched to in situ next-generation sequencing (NGS). That’s when they realized they could use their optical system to sequence using a 3D matrix for hugely parallel reactions in three dimensions.
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A new model developed by researchers at the University of Sheffield, in collaboration with the St. Anna Children's Cancer Research Institute in Vienna, replicates the emergence of early neuroblastoma cancer-like cells, providing insight into the genetic pathway of the disease.
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Robotics and automation are common approaches to reducing labor costs in many industries. It is more challenging in biotech as changing instruments or processes in FDA-approved therapeutics manufacturing requires regulatory resubmissions and comparability studies. Multiply Labs says it focuses on robotics that operate market-leading GMP instruments from vendors already extensively deployed for cell and gene therapy manufacturing with a technology designed to enable plug-and-play-like capabilities.
Formation of a blood clot
As people age they become more prone to blood clotting diseases, caused by abnormal blood platelet function. Mouse studies uncovered a specific population of platelets that appears with aging, produced by what the scientists say is the first discovered age-specific development pathway from a stem cell to a distinct mature platelet cell. Experiments showed that these “troublemaker” platelets display hyperreactive behavior and unique molecular properties, which could make them easier to target pharmacologically.
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In The State of CRISPR and Gene Editing virtual summit, GEN proudly gathers a tantalizing line-up of luminaries from academia and industry to discuss the latest research developments, innovations, and advanced technologies that are expanding the CRISPR toolbox, delivering new therapies to patients and safeguarding our food supply.
image of ASGCT banner outside of conference center
In this video, at the conclusion of the ASGCT meeting, GEN’s Julianna LeMieux, PhD, and Corinna Singleman, PhD, chat about how they spent their time at the conference and their unique takeaways from the research and news presented through the week.  
female researcher looking at cell culture
Frédéric. Revah, PhD, shares some history of Généthon’s creation and its future path under his guidance as CEO. Généthon is a not-for-profit biotherapy research center founded by the French Muscular Dystrophy Association, which has broadened its research scope to investigating other rare diseases. In this interview Revah speaks with GEN's Corinna Singleman, PhD.
Lupus erythematous
Researchers from Johns Hopkins Medicine report they have discovered insights as to why lupus symptoms and severity present differently in individuals with the autoimmune condition. The researchers believe their finding is a crucial step forward in understanding biological mechanisms behind lupus, and may also lead to shifts in how clinicians treat patients.
Group of Recursion executives and staffers who developed the BioHive-2 supercomputer
The supercomputer, BioHive-2, ranks No. 35 on the TOP500 list of the world’s most powerful supercomputers across all industries as of this month. BioHive-2 operates four times faster than Recursion’s original supercomputer BioHive-1 in benchmark performance tests, according to the company. BioHive-2 consists of an NVIDIA DGX SuperPOD AI supercomputer, powered by 63 DGX H100 systems with a total of 504 NVIDIA H100 Tensor Core GPUs interconnected by NVIDIA Quantum-2 InfiniBand networking.