The work, which is published online today in Nature in the paper “In situ targeted base editing of bacteria in the mouse gut,” was a team effort by the scientists at Eligo, led jointly by Jesus Fernandez-Rodriguez, PhD, (Eligo’s VP of Technology), David Bikard, PhD, (Eligo co-founder and head of the Synthetic Biology Group at Institute Pasteur), and Xavier Duportet, PhD, (Eligo’s CEO and chairman).

Before this report, notes Bikard, “it was still an outstanding question whether it would even be possible to genetically modify a whole target bacterial population in an animal. There could have been fundamental barriers that would’ve made this impossible. But here we show that we can do it!”

Eligo’s advance, which combined research in both vector engineering and payload modification, is exciting on two fronts: it could open the door to new microbiome genome-editing therapeutic modalities. And it launches a microbiome-editing toolbox that has been previously unavailable.

This work is “ushering in a new era of microbiome engineering,” notes Rodolphe Barrangou, PhD, professor at North Carolina State University and editor-in-chief of The CRISPR Journal. “This proof-of-concept study is just not for E. coli or the mouse gut microbiome; it can be used much more widely, for all kinds of things, and can be deployed at scale.”

Brady Cress, PhD, principal investigator of microbiome editing technologies at the Innovative Genomics Institute at the University of California, Berkeley, agrees. Cress told GEN that this is “a massive step forward that opens the door to rewriting our microbiomes for optimal health.”

Tweaking, not altering

Duportet co-founded Eligo with Bikard a decade ago; the two friends were still in training when they had the idea for the company—Duportet was a graduate student at MIT, Bikard a postdoctoral fellow in Luciano Marraffini PhD’s lab at the Rockefeller University. (Marraffini and Tim Lu, MD, PhD, are Eligo’s other scientific co-founders.) Today, Duportet and Bikard are a dynamic duo—with Duportet at the helm of the company and Bikard a scientific advisor, who remain close friends while collaborating scientifically.

Current microbiome approaches are typically based on altering the compositions of the bacteria. The idea is to introduce bacterial species to change the balance (like probiotics) or to remove others. Eligo’s focus here was different. The idea is not to kill the bacteria but rather, as Duportet explains, to “inactivate its pathogenic potential and leave the bacteria in place.”

“If you are trying to target bacteria that has a niche,” notes Bikard, “completely removing it from the niche might be very challenging. Unless there is something else there to take its place, it will just grow back. So, it is a better strategy to disarm it, rather than kill it.”

Two-fold

Eligo’s new data are not the first to demonstrate editing of the microbiome in vivo, however. In May 2023, research from the Danish company SNIPR Biome was published in Nature Biotechnology in a paper entitled, “Engineered phage with antibacterial CRISPR–Cas selectively reduce E. coli burden in mice.” In it, the researchers identified eight phages (after screening a library of 162 phages) that delivered a CRISPR-based gene-editing payload that resulted in a reduction of E. coli in the mouse gut. In the SNIPR Biome study, the E. coli were killed by CRISPR.

It has been known for a decade—since Bikard and Marrafini’s Nature Biotechnology paper in 2014—that cutting the bacterial chromosome with CRISPR-Cas kills bacteria efficiently. But the first generation of gene-editing tools were not very efficient tools, notes Bikard. They could be used in a lab setting to make modifications but most of the bacteria would be killed in the process. Therefore, if the goal is to make modifications in vivo, and maintain the bacterial population, the first generation of CRISPR tools for bacteria “would not cut it.”

The game changed when base editing—a more precise form of genome editing developed in the Broad Institute lab of David Liu, PhD—entered the toolbox. Eligo worked to bring together their knowledge in genome editing and delivery to allow for efficient editing of bacteria without changing the composition of the microbial population.

When targeting E. coli strains colonizing the mouse gut, Eligo’s technology modified the target gene in more than 90% of the bacteria, reaching up to 99.7% in some cases. These modifications remained stable for at least 42 days.

Barrangou notes that the penetrance of the edits showed remarkable efficiency. “They are setting the bar,” he told GEN. “Being able to do it is one thing. But being able to do it with that kind of longevity and efficiency is practically important and sets the stage for new opportunities in the field.”

For Bikard, working out the gene editing was not as big a hurdle as the delivery. Eligo modified the site fibers on a phage chassis (from phage lambda) to target specific bacterial strains. The phage vector can be modified to target different bacterial strains or species with additional engineering.

Cress thinks of it as “a reprogrammable platform” for targeting different bacteria. That said, while this research provides an impressive blueprint using the most well-studied phage-bacteria pair, Cress notes that expanding it to other microbes will necessitate developing efficient genetic tools for non-model bacteria and a deeper understanding of the genetics and biology of less well-studied phages.

Another advance, Duportet notes, is that his team was able to demonstrate the same efficiency of editing using a non-replicative plasmid in the target bacteria. This is an additional benefit because they don’t maintain a transgene in the microbiome of the animals.

Long road ahead

The long-term goal at Eligo is to develop therapeutics—not necessarily for infectious diseases. The interest extends to those that would change the genetic content of the microbiome that alter a factor of host–focused diseases.

One example where this could be applicable is the delivery of a base editor to commensal intestinal E. coli that express the toxin colibactin, to inactive its mutagenic potential, therefore preventing the progression of human colorectal tumors.

But there is a long road ahead and challenges remain. One, notes Cress, is that this approach uses short-lived delivery of gene editing machinery to make gene disruptions, but other types of edits like gene insertions take longer to write (e.g. CRISPR-associated transposases) and thus will likely require different delivery approaches. Another point Cress considers is that “the type of edits made in this study could potentially revert through natural mutation, making gene removal a more durable solution than gene disruption.”

This study also raises new questions. Teasing apart the genetic network of the microbiome is in its infancy. Do researchers have enough knowledge to use this new tool?

“It’s important to have the genome editing tools,” notes Barrangou. But in the end, “what really matters is knowing what to target. Knowing what to target and what edit you want is part of the secret sauce.”

But Bikard reckons that this work will help answer some of those questions. This will be an extraordinary tool for researchers, he says, because it offers the possibility to probe gene function directly in the animal. He is excited to use it in his academic lab on the other side of Paris from Eligo’s base.

Duportet hopes that scientists will use the method and is happy to issue “a call for collaboration.” “We cannot work on everything, and we cannot find all the targets to edit,” he notes. “But we have the knowledge to design the vectors and the payloads to make it happen.”

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The research, published today in Science in a report entitled “A molecular glue degrader of the WIZ transcription factor for fetal hemoglobin induction,” was led by Pamela Ting, PhD, associate director of hematology at Novartis Biomedical Research. Ting leads a team of 40 co-authors, including Jay Bradner, MD, PhD, the former president of Novartis Institutes for BioMedical Research, who left the company in 2022 and joined Amgen late last year. Developing small-molecule drugs for SCD has been a long-standing interest of Bradner’s, dating back almost 15 years to research he performed on histone deacetylase inhibitors in collaboration with Stuart Schreiber, PhD, and colleagues at the Broad Institute.  

Over the past decade or more, predating her own arrival at the company in 2015, Ting estimates that Novartis has screened some two million compounds in the search for a promising compound to take to the clinic. Along the way, the company briefly pursued its own CRISPR gene editing strategy, following a similar path to the approach that Vertex Pharmaceuticals took in developing Casgevy by boosting HbF production. (Although Novartis and collaborators published promising initial clinical data last year, the company decided to shelve the gene editing program.) 

Despite the remarkable clinical success of the CRISPR ex vivo approach—the first American patient in the Vertex trial, Victoria Gray, celebrated the fifth anniversary of her stem cell transfusion earlier this week—it is clear that this demanding (and expensive) ex vivo strategy will not help the millions of patients in Africa and beyond—a point that Ting and colleagues hit home in their report in Science.  

“Regrettably,” Ting and her co-authors write, “substantial challenges exist for [hematopoietic stem and progenitor cell] therapies to reach most SCD patients, who live in medically underserved communities and low and middle-income countries. Safe, efficacious, and globally accessible HbF-inducing medicines therefore remain an important unmet need.” 

Fyodor Urnov, PhD, director of technology and translation at the Innovative Genomics Institute, told GEN that the 2023 approval of Casgevy provided definitive clinical evidence that the strategy of upregulating HbF is “a safe and effective approach to resolve major symptoms of sickle cell disease.” The new work builds on the theme of modulating a transcription factor to upregulate HbF, but this time using a small molecule. “Even people who live and breathe CRISPR will agree that, on balance, such a small molecule could have a wider global reach than gene editing,” Urnov said. 

“There is a pressing need for oral agents that can more effectively induce fetal hemoglobin,” agreed hematologist Vijay Sankaran, MD, PhD, professor of pediatrics at Harvard Medical School. (Sankaran played a key role in cementing the HbF upregulation strategy to treat SCD 15 years ago.) He called the new Novartis report impressive and “exciting work and identifies a promising new target for fetal hemoglobin induction.” 

Screen time 

The Novartis study falls into a category known as “molecular glue pharmacology.” The researchers recognized that very small modifications to the chemical structure of a class of drug compounds that mediate targeted protein degradation (TPD) could profoundly affect the choice of protein for degradation. (A good example was work published by Novartis colleagues last year on the discovery of selective glue degraders for cancer immunotherapy).

“This was a big conceptual leap,” Ting explained. “You could do just a little bit of work around a fundamental structure and then broadly look for changes to the substrate selectivity.”

She added: “We decided, if this [chemical] library is capable of drugging a new class of transcription factors, then we should think about how we can apply it to an age-old problem that, at the root, is really a question of how do we regulate genes? And can we target transcription factors to regulate gene expression?” 

Ting’s team embarked on a screen of a library of almost 3,000 compounds that target cereblon, a component of the E3 ubiquitin ligase complex that is involved in targeted protein degradation. Using a cellular assay that detected the upregulation of HbF) in erythroblasts, Novartis researchers identified scores of candidate molecules before whittling down the list and focusing on “compound C” for further study.   

After mass spectrometry experiments showed that this chemical targeted the WIZ transcription factor, Ting’s group renamed the drug candidate dWIZ-1. The WIZ target was emphatically confirmed by CRISPR gene knock-out experiments. All told, these results suggested that WIZ was a previously unrecognized repressor of HbF expression. dWIZ-1 could recruit WIZ to the cereblon-ubiquitin ligase complex to trigger targeted protein degradation. The Novartis researchers continued experiments with a modified molecule termed dWIZ-2. 

The Science report is packed with multiple types of experiments—molecular assays, mouse and non-human primate models, protein modeling using AlphaFold, and X-ray crystallography. Part of the rationale, Ting says, “was to convince ourselves that by every measurement that we could think of, we could see a reproducible effect.”  

Some of the most important data in the report are results administering dWIZ-2 in a trio of cynomolgus monkeys. This in vivo study monitored the animals for 28 days and showed robust up-regulation of the γ-globin gene and HbF production in virtually all peripheral reticulocytes. “We saw almost pan-cellular HbF expression, which is very promising,” Ting said, and no signs of cytotoxicity. Curiously, one of the three animals was termed a non-responder. Ting calls it “the coolest monkey, because I feel like it must be telling us something and I don’t know what it is!” 

The Novartis team found evidence for WIZ binding directly at the β-globin locus, which was unexpected. “Seeing WIZ for the first time is quite surprising for such a deeply studied locus,” Ting said. “For whatever reason, the globin locus seems to be highly sensitive to the loss of WIZ.” What is striking, she adds, is that “there is relatively strong overlap with binding sites for BCL11A,” the transcription factor that Vertex successfully targets in its Casgevy cell therapy.

“That’s where we leave it in the paper, as a bit of a teaser, something that we hope that we can further study in the future and that others will be inspired to study.”  

Dealbreaker 

As Ting readily acknowledges, there are still many unanswered questions, including the specificity of the inhibition. WIZ naturally binds at many spots in the genome, including a dozen or more in the β-globin gene cluster. “For whatever reason, when we modulate WIZ, the effects are fairly moderate and fairly mild,” Ting said. And the results in the non-human primates bode well. “We hope with these new chemical probes that this is something that we can continue to study and understand in the long run,” Ting says.  

“I think the [Novartis] approach is promising, but with the current data it’s difficult to know what degree of HbF induction a human would experience,” said Vivien Sheehan, MD, PhD, associate professor and director of Sickle Cell Translational Research at Emory University School of Medicine in Atlanta. Sheehan, who first heard these results presented at the American Society of Hematology conference late last year, would like to see additional quantification data including HbF measurements using high-performance liquid chromatography. She also observes that “the lack of myelosuppression is promising for a potential combined therapy with hydroxyurea.”  

Sankaran cautions that the non-specific effects of targeting WIZ lead to expression changes of hundreds of genes in erythroblasts. “More studies are needed before and during human clinical studies,” he said. A rigorous assessment of the safety of systemic WIZ degradation will be needed. “That could be a dealbreaker,” Sheehan says. 

Understandably, Ting would not be drawn on a timeline for progress into the clinic, but her closing comments underlined her team’s belief in the program. “What we’re really focused on now is making sure that we’ve found a molecule that can really go all the way to patients,” she said. “We are highly committed to sickle cell patients around the world. We see the unmet need,” she said. “We’re progressing as quickly as possible, and we really have our eye on a medicine that will reach patients where they are.” 

Nevertheless, while a small-molecule drug should prove both more affordable and accessible in the countries where most SCD live, IGI’s Urnov cautions that access to drug therapies outside the U.S. is by no means guaranteed. For example, a decade ago, Gilead slashed the price of its drug Solvadi by almost 99 percent in order to provide meaningful access to hepatitis patients in Egypt. The Novartis study, Urnov says, offers “an important opportunity to think ahead to how to solve this challenge when and if a medicine based on this work is approved in the U.S.” 

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The riboswitch technology is just part of MeiraGTx’s work in gene therapy. The company has technologies for the optimization of adeno-associated virus (AAV) vectors and for the design of promoter sequences. Also, the company has an internally developed manufacturing platform process and several production facilities. Finally, the company has several gene therapy programs in late-stage clinical trials.

In recent months, MeiraGTx has reported several business successes. For example, the company entered into a $415 million asset purchase agreement with Janssen Pharmaceuticals related to botaretigene sparoparvovec (bota-vec) for the treatment of X-linked retinitis pigmentosa. MeiraGTx has yet to turn a profit, but the company has a cash runway that should support operations well into 2026.

Capsid/promoter optimization

The degree of vector optimization at MeiraGTx differentiates it from other gene therapy developers. Its gene therapy platform, which optimizes capsids and promoters, has resulted in extensive, proprietary libraries that contain capsids with increased tropism to different tissues, and that enable insights into promoters and how specific genes are expressed in particular cells.

By making minute changes in capsids, the company can affect gene delivery efficiency. Likewise, by optimizing promoters, the company’s scientists can affect expression levels. For example, they can limit expression to certain cell types.

AI-driven in silico cloning, which the company initiated a few years ago, enhances those results by helping to identify “small, strong, and specific promoters and control elements,” says Alexandria “Zandy” Forbes, PhD, MeiraGTx’s CEO.

Forbes asserts that the resulting libraries enable MeiraGTx to optimize any viral vector, making it up to three logs better than the original. The company makes a point of testing its capsids and promoters in organoids derived from human stem cells to ensure that the vectors are optimized for human potency.

A broad pipeline

MeiraGTx developed its pipeline through partnerships and in-licensing rights from third parties “in an indication-agnostic fashion,” Forbes relates. Initially, the company focused on products for the eye, collaborating with Moorfields Eye Hospital in London and University College London (UCL). “UCL has a very strong retinal organoid group,” Forbes observes. Technology developed by a UCL spinout company, Athena Vision, has become part of MeiraGTx’s ocular program.

“The resulting gene therapy for X-linked retinitis pigmentosa was the one of the first products we tested in human retinal organoids made from stem cells of actual patients who are blind,” Forbes says. “It made the cells function as if the mutation wasn’t there.” It recently completed Phase III trials.

MeiraGTx has also focused on developing immunogenic therapies that needn’t be administered in massive, systemic doses. The company believes that local delivery could allow very small doses to change tissue function or, in the case of Parkinson’s disease, rewire the brain, thus minimizing safety concerns and lowering the cost of goods.

Late-stage clinical programs at MeiraGTx include a gene therapy for Parkinson’s disease, another for a salivary gland condition, and the Janssen program. Multiple programs for inherited retinal diseases are in Phase II trials, and there are candidates for additional indications at the IND stage.

Regulation of gene expression

“When we started the company,” Forbes recalls, “we saw a really big gap in the technology of gene therapy.” This gap was the inability to easily control gene expression after a gene was delivered to a patient. To fill it, the company “designed a platform technology based on RNA structure,” Forbes says. “[It] allowed us to completely and precisely control—with oral small molecules—the delivery of any peptide or protein in the body at any time.”

The platform technology, called Riboswitch, has enabled the delivery of multiple antibodies, peptides, and hormones, including epoetin, parathyroid hormone, growth hormone, glucan-like peptide-1 (GLP-1), gastric inhibitory polypeptide (GIP), and multiple gut peptides and combinations.

“Our ability to deliver any peptide, hormone, or antibody with a pill,” Forbes declares, “solves many, if not all, of the issues we’re seeing with the extensive use of the gut peptides for metabolism, such as efficacy, tolerability, muscle loss, and fat regain, as well as the manufacturing burden of producing large amounts of peptides outside the body.”

Manufacturing capabilities

Manufacturing is, in many ways, at the heart of the company. Recognizing the lack of broadly available and highly effective manufacturing processes for gene therapy, MeiraGTx built its own capacity in-house from the beginning. “We now have a proprietary process that can, within two to three months, take an AAV vector and fit it to a GMP process,” Forbes notes. This time frame, she predicts, will be reduced to a few weeks.

The company boasts two flexible and scalable viral vector facilities to provide appropriate product volumes throughout development, from Phase I trials through commercialization, using the same cells and same processes.

“Because we are the commercial manufacturer for Janssen, we also built our own quality control facility, so we can perform release and stability testing for our batches,” Forbes says. “We did that not because we wanted to, but because it wasn’t possible to get rapid, high-quality testing from the current CDMOs.”

This means the company can go from making IND quantities to commercial quantities without making large changes in the process. Additionally, she says that during dialogs with the FDA before MeiraGTx launched its first controlled study for xerostomia, “We were able to answer the questions about the assay within three weeks.”

“Having these internal capabilities,” Forbes points out, “saves two to three or maybe even four years in the development timeline of any one of our products.”

Future plans

MeiraGTx’s Riboswitch technology goes beyond gene therapy to newly emerging applications in which short-acting agonists act as drugs. “This is an ‘aha!’ moment we didn’t expect,” Forbes admits. “Most pharmaceuticals are inhibitors,” but there is an enormous world of activators—agonists such as GLP-1 and GIP, for example—that are short-acting. “Only a few of them have been turned into drugs.”

Forbes suggests that a better alternative to blasting the body constantly with a gene product would be the delivery of short-lived agonists, an approach that “appears to massively increase efficacy and tolerability.” For example, as Forbes says, switching on the chimeric antigen receptor (CAR) in a CAR T cell with a pill increases efficacy fourfold compared to the approved anti-CD-19 drug “and has important implications for safety and durability.”

Metabolism is an area of keen interest as the company moves forward. Forbes says it may be possible to inject a patient with the DNA template for GLP-1 or GIP, and then administer a daily pill to precisely control the timing and level of the peptide’s production. She adds that MeiraGTx has developed a peptide that is the target of myostatin inhibition and “actively enhances muscle.” (Muscle loss is a prominent downside to currently approved GLP-1 therapies.) The company is also working to address the regain of fat.

Forbes notes that MeiraGTx’s big goals are accompanied by big challenges—none greater, she stresses, than the need to “find a workforce that’s as good as the one we currently have.” 

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Unleashing potential with CRISPR technology

In the dynamic landscape of biological research, CRISPR technology has revolutionized our approach to genetic disorders and the future of personalized medicine. Of these advances, cell and gene therapy is front and center as an example of where CRISPR has been forging inroads into these novel therapeutic possibilities.

Established as a pioneer in genome engineering, EditCo Bio spun out from the CRISPR heavy weight, Synthego, in March of 2024 with a mission to redefine how CRISPR tools are used. Leveraging data from over 500,000 edits, EditCo’s automated gene editing design and application platform has handled a variety of complex genomic challenges, offering streamlined solutions from optimized gene knockout kits or custom-edited cells to high-throughput projects and advanced data analysis.

High-throughput screening in iPSCs for cell therapies

This novel gene editing platform has opened the door to a wide range of challenging projects and partnerships with companies whose research is moving the world of cell and gene therapy forward quickly. An example highlighting how EditCo Bio’s CRISPR platform has solved gene editing challenges is through a recent partnership with bit.bio, a synthetic biology company with a platform to program human induced pluripotent stem cells (iPSCs) into a wide range of cell types for discovery research and cell therapy.

Combining EditCo’s ability to perform high-throughput edits in many medically relevant cell types, including human iPSCs, with full automation integration, and bit.bio’s expertise in leveraging iPSCs for cell therapies, the team performed a high-throughput screen of hundreds of guides across 25 genomic safe harbor (GSH) sites. Additionally, EditCo’s ability to analyze many biological and technical replicates with the use of an integrated robotic workcell for next-generation sequencing (NGS) allowed the generation of a highly reproducible dataset by sequencing more than 4,600 NGS libraries with a success rate ranging from 88% to 98%.

Through meticulous optimization of editing conditions, careful validation through NGS, and automated high-throughput capacity, the project showcased the precision and flexibility made possible through EditCo’s gene editing platform. This allowed for rapid identification of hot spots for high knock-in editing levels with the most effective guides in genomic regions not typically targeted for gene editing. Ultimately, this research has the aim of maximizing safety and efficacy of CRISPR-Cas editing across the 25 genomic loci.

Addressing Alzheimer’s disease with advanced CRISPR applications

Beyond large-scale editing optimization, EditCo’s genome editing process has been important for providing a unique solution for CRISPR screening in clonal cells. In collaboration with the National Institute of Health (NIH), the Inducible Pluripotent Stem Cell Neurodegeneration Initiative (iNDI) project by EditCo Bio targeted 22 genes, generating 12 variants per gene, which resulted in 264 CRISPR iPSC clones all in less than five months.

This massive screening of 8,108 clones and the execution of 6,138 sequencing reactions highlighted the platform’s capability to handle large-scale, high-throughput operations efficiently. This project is particularly crucial for advancing understanding and treatments of complex diseases like Alzheimer’s, showcasing EditCo’s pivotal role in cutting-edge genetic research.

Shaping the future of healthcare

By marrying the precision of CRISPR with the expertise and innovation within EditCo Bio, significant progress continues to be made to elucidate and mitigate risks associated with genetic diseases. Opening up new avenues for enhancing human health is proving that EditCo’s technology will continue to be a powerful solution within the field of cell and gene therapy and healthcare overall. And, as CRISPR technology continues to evolve, EditCo Bio plans to remain at the forefront, committed to partnering with researchers and leading the charge toward transformative health solutions.

References
1. Published with permission from bit.bio
2. Figure created with BioRender.com

To learn more visit editco.bio

Contact us partner@editco.bio

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In March 2022—less than three years after his initial diagnosis—Pirovolakis received an AAV-based gene therapy. He is the only patient in a clinical trial being run at the Hospital for Sick Children (SickKids), affiliated with the University of Toronto.

Now, findings from the clinical trial (NCT06069687) are published in Nature Medicine in the paper, “AAV gene therapy for hereditary spastic paraplegia type 50: a phase 1 trial in a single patient.” The authors report that, after treatment, the course of Pirovolakis’ condition was altered significantly. In addition, they present a road map for individualized treatment of an ultra-rare disease.

“When we heard that Michael had been diagnosed with this terrible disease, our world fell apart. We were lost and broken as a family,” said Pirovolakis’ parents, Terry and Georgia. “Thankfully, we had an amazing team at SickKids and a supportive community that lifted us up and gave us the confidence to raise millions of dollars and create a therapy, not only for Michael, but for other children affected by this disease for generations to come.”

In Pirovolakis’ case, SPG50 is caused by biallelic pathogenic variants in the AP4M1 gene, encoding a subunit of the adaptor protein complex 4 (AP-4). SPG50, the authors noted, is “an ideal candidate disease for gene therapy. The coding sequence is small (1,359 base pairs) and fits within a self-complementary adeno-associated virus (scAAV) vector.”

Led by Jim Dowling, MD, PhD, staff clinician in the division of neurology and senior scientist in the genetics and genome biology program at SickKids, the clinical research team delivered the healthy AP4M1 gene into Michael’s spinal fluid. More specifically, AAV9-AP4M1 was administered at 1 × 10¹⁵ vector genomes through intrathecal delivery. This is, the authors noted, “among the largest doses of AAV9-based gene therapy ever administered into the cerebrospinal fluid.”

In the trial, the primary endpoints were safety and tolerability, and the secondary endpoints evaluated efficacy. In the 12 months after he received the treatment, Pirovolakis experienced no serious side effects and, contrary to the hallmark of neurodegenerative conditions like SPG50, his condition does not seem to be progressing further. Preliminary efficacy measures suggest a stabilization of the disease course.

He also began to show potential signs of improvement. For the first time, Pirovolakis was able to stand with his heels on the ground. He also experienced improvements in some aspects of his neurodevelopment.

The clinical research team continues to follow Pirovolakis’ progress, but the trial provides important initial evidence of the safety and efficacy of gene therapy to reduce or halt the progression of SPG50.

“While these ultra-rare diseases are unique, our workflow provides a road map for gene therapies that could help many of the thousands of children in Canada with rare genetic conditions,” said Dowling.

Importantly, the results also highlight how gene therapy can be developed quickly and personalized for individual patients with rare genetic conditions. They hope that this approach can be used for other conditions in the future to help achieve Precision Child Health, a movement at SickKids to deliver individualized care for every patient.

“There are over 10,000 individual rare diseases and most are without therapy,” said Dowling. “We are providing a blueprint that, with adequate funding and support, has the potential to change the lives of patients with rare diseases and a future where every child can benefit from precision medicine.”

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The collaboration between researchers at the Whitehead and Broad Institutes has been published in Science in the paper titled, “Brain-wide silencing of prion protein by AAV-mediated delivery of an engineered compact epigenetic editor.”

“The spirit of the collaboration since the beginning has been that there was no waiting on formality,” said Sonia Vallabh, PhD, senior group leader at the Broad Institute. “As soon as we realized our mutual excitement to do this, everything was off to the races.”

The joint team, led by Vallabh and Jonathan Weissman, PhD, a core member at the Whitehead Institute, combined their labs’ expertise to develop a novel way to target prion diseases. While mad cow disease is a widely known infectious prion disease, there are also inherited prion diseases. In both cases, the misfolded prion proteins trigger a cascade of misfolding events in the brain resulting in neurodegeneration and neuron death. In genetic prion disease, that Vallabh herself is currently asymptomatic for, misfolding of the PrPs leads to profound dementia and inability to sleep, culminating in the death of the affected individual (human or otherwise).

Current treatments target the proteins themselves, but Vallabh, Weissman, and their team endeavored to take a step back, both in their strategy and in the cellular production process of proteins to target protein precursors. CHARM works on an epigenetic level to silence the PrP gene so that the protein that may eventually become a prion will not be produced in the first place.

The project was led by a Whitehead Institute graduate student, Edwin Neumann and Tessa Bertozzi, PhD, a postdoc in the Weissman lab. The lab had previously developed the technology to silence specific genes using the tool CRISPRoff. CRISPRoff functions by adding methyl groups to specific target genes to prevent transcription. The gene itself remains unedited, but its ability to function is inhibited.

The next hurdle to clear was delivery of the CRISPRoff into the brain. CRISPRoff was initially designed to use Cas9, however the use of adeno-associated viruses (AAV) is limited by the size of Cas9. The team replaced Cas9 with a smaller zinc finger protein (ZFP) to target the correct genes. An added bonus is that in humans, ZFPs are less likely to cause an immune response compared to bacterially derived Cas9 and alleviate off target effects seen with the previous Cas9 system.

Another innovation to the system was the use of the cells’ own methylation mechanism. Originally the technique involved the inclusion of part of a methyltransferase, however the team found that they could reduce cellular toxicity and further reduce the content load of AAVs by using using the cells’ own enzyme, DNMT3A.

“From the perspectives of both toxicity and size, it made sense to recruit the machinery that the cell already has; it was a much simpler, more elegant solution,” Neumann said. “Cells are already using methyltransferases all of the time, and we’re essentially just tricking them into turning off a gene that they would normally leave turned on.”

Using mouse models, they tested ZFP-guided CHARMs, finding that over 80% of the prions in the brain were eradicated. Previous research has indicated that a much smaller reduction in prions, approximately 21%, will improve symptoms in patients. The reduction in PrPs prior to misfolding is also not an overt concern. The current research indicates that these proteins are not essential for healthy adults and their removal therefore should not cause adverse effects in those without symptoms. On the contrary, removing the prions may halt symptom progression or prevent development of symptoms in asymptomatic genetic carriers.

With CHARM, there is a potential for a safe, efficient, and effective treatment and preventative therapy for treating prion diseases. Within a short time, this collaboration resulted in an adaptable tool that, with appropriate tests and scalability trials, may move toward the clinic as a viable treatment option. Bertozzi commented, “It’s been a privilege to be part of this; it’s pretty rare to go from basic research to therapeutic application in such a short amount of time.”

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Physician-scientist Matthew Porteus, MD, PhD, has been a mainstay in the genome editing field for more than two decades. He trained at Stanford University Medical School before completing his residency and hematology/oncology fellowship at Boston Children’s Hospital/Dana Farber Cancer Institute. During his postdoctoral research at Caltech with Nobel laureate David Baltimore, Porteus began his journey in gene targeting, in particular homologous recombination, as a means to repair disease genes. He has been on the Stanford faculty since 2010, treating patients with sickle cell disease and other hematological disorders at the Lucille Packard Children’s Hospital. 

Porteus is a scientific co-founder of CRISPR Therapeutics, the company that launched the exa-cel CRISPR trial that culminated in the approval of Casgevy in December 2023. With the latest companies he has co-founded—first Graphite Bio, now Kamau Therapeutics— Porteus remains steadfast in translating the promise of homologous recombination to the benefit of patients with sickle cell disease. 

In this wide-ranging interview with GEN editorial director Kevin Davies, PhD, Porteus candidly discusses some of the scientific milestones along his 25-year genome editing journey, including an update on the clinical translation of homologous recombination to treat patients with sickle cell disease. 

(This interview has been edited for length and clarity. A longer version has been published in the June 2024 issue of The CRISPR Journal.) 

Kevin Davies: Matt, what’s your impression of the current state of gene therapy and gene editing? 

Matthew Porteus: The approvals in December 2023 of Casgevy [Vertex Pharmaceuticals/CRISPR Therapeutics] and Lyfgenia [Bluebird Bio] are super exciting to see. Within a decade, a discovery made in a test tube translated to an approved therapy for a disease that has a large unmet medical need. I’m proud to have made my contributions to that process.  

That said, this is a first step but we’re nowhere close to where we need to go on multiple axes. From an efficacy standpoint, I’d argue that it’s not as good as a bone marrow transplant. If I could do an allogeneic bone marrow transplant as easily as doing an autologous transplant, the results are still better. Then there’s the issue of cost and accessibility. Certainly, an autologous transplant (using the patient’s own cells) is going to be more accessible because it’s a much less complicated procedure. 

The manufacturing, of course, is more complicated. If you had a patient and they had a perfect allogeneic transplant match, that’s the direction you would probably go. If you have a matched sibling donor—a perfect HLA match donor without sickle cell disease or even with sickle cell trait—the trials exclude you from getting a gene therapy or gene-edited product because the results there are so spectacular. It’s when you don’t have that donor that you look for alternative sources. That is 85% or more of patients who don’t have that gold-standard matched sibling donor.  

Davies: You have a long history in the genome editing space, going back to the early 2000s. Can you summarize some of those early landmark studies?  

Porteus: I started thinking about this during my MD/PhD training. During my PhD work, I would argue the work that led to the first Nobel Prize in genome editing was performed. Mario Capecchi and Oliver Smithies showed that you could get targeted integration at measurable frequencies in mouse embryonic stem cells. Maybe we should start calling that the first Nobel in gene editing—they called it ‘gene targeting’ but the reality is that’s what a lot of us are doing.  

The second thing was taking care of patients with sickle cell disease during medical school and recognizing the gulf between the contributions this disease had made to our understanding. of almost everything—blood development, genomic regulation, genomic structure, molecular pathophysiology—and that we did nothing that was driven by that understanding. We had this tremendous genetic and pathophysiological understanding— but it wasn’t being translated.  

At the beginning of my career, I thought I could go into lentiviral gene therapy. But I wanted to develop homologous recombination—the Smithies-Capecchi approach to this disease—because the basis is a single nucleotide change in a single gene. We know the cell that you need to fix because allogeneic transplant shows us—as Guido Lucarelli called it ‘allogeneic gene therapy.’ John Tisdale (NIH) called it ‘whole genome therapy.’ We knew fundamentally what needed to be done. 

Luckily, I found a postdoctoral mentor—David Baltimore—who was willing to say go at it. He admits he treats his trainees with ‘benign neglect’ in his words! But that was what I needed. Maria Jasin and others had shown that if you make a DNA break, the homologous recombination pathway is stimulated.¹ I was part of a group showing how to take an engineered nuclease at the time—zinc-finger nuclease (ZFN)—and create a genome-specific break.² Other than Sangamo, no one can really make good ZFNs. There was a real barrier because it was so difficult to engineer them. Many of us spent a decade doing the best we could with them.  

Casgevy is not based on gene correction or homologous recombination. And the recognition that DNA breaks can lead to site-specific indels (insertions/deletions), we need to really give Dana Carroll credit. He published a paper using ZFNs showing that you could make mutations in specific genes.³ And since that was a relatively simpler process, it really has exploded. I thought TALENs were going to be really transformative but just as they were getting off the ground, we have the 2012–2013 discoveries of CRISPR.  

Davies: You were a co-author on the famous Nature paper in 2005 that introduced us to the term ‘genome editing.’ How did your collaboration with Sangamo come about? 

Porteus: I was talking to Srinivasan Chandrasegaran, who biochemically had said ‘I can link a ZFN to the Fok I-chimeric nuclease domain.’ He had collaborated with Carroll. We needed to engineer these systems into mammalian cells and had shown how to use nucleases in a mammalian cell to get high frequencies of gene targeting. I could fix the GFP gene. And I was finishing up my postdoctoral work with David who said, ‘you need to talk to Sangamo…’ I remember visiting Casey Case, the CSO of Sangamo at the time…That prompted a collaboration as I was transitioning to my independent faculty position and their enterprise on engineering ZF proteins and then coupling them to the nuclease domain, which led to the Nature paper.⁴ It was the first demonstration that you could engineer a nuclease to target an endogenous sequence in a genome. In all the prior experiments, you inserted your target site first, and then retargeted it.  

Davies: Not long after the CRISPR gene editing papers in 2012–13, you became a scientific co-founder of CRISPR Therapeutics. How did you get involved with them?  

Porteus: I make the distinction that gene editing is a process and CRISPR is a tool to start the process. But any protein that makes a DNA break initiates the gene editing process. CRISPR just happened to be this amazingly powerful, easy-to-use specific tool. There was a clear excitement—it caught the attention of investors who wanted to start biotech companies. Investors were looking to people who knew about gene editing and some came knocking on my door. I started talking to some of the VCs and through those conversations became a scientific founder of CRISPR Therapeutics, got to know [co-founders] Sean Foy, Rodger Novak, and Emmanuelle Charpentier. They clearly had a sense that they had a hold of something. One of the things I think I can take credit for is pointing them in the direction that sickle cell disease and β-thalassemia were the perfect first indication.  

Sickle cell disease needed and deserved this attention. It was the right [patient] size, it had a large unmet medical need, the biology was well understood, and there were a lot of approaches that one could take. My lab has continued to work on directly correcting the mutation that causes the disease by homologous recombination because that’s where I got started.  

But the biology of understanding that fetal hemoglobin (HbF) could counteract the effects of sickle hemoglobin and that there were pathways that controlled the levels of HbF, and that there were people in the world who had genetic changes causing hereditary persistence of fetal hemoglobin, pointed people to the idea that perhaps a faster approach was homologous recombination—to harness the indel-based approach that Dana Carroll had [published]. I helped CRISPR Therapeutics work through potential targets with CRISPR-Cas9. 

They ended up building on the biology that came from Daniel Bauer and Stuart Orkin on BCL11A.⁵ The first approaches in fact were by Sangamo to target BC11A itself. We quickly learned that if you inactivate the protein, you disrupt stem cell function. When Dan identified this key enhancer—we helped them a bit with some TALENs [to show] that you could inactivate BC11A being turned on in the red cell lineage without inactivating all of its key functions in hematopoietic stem cells and B cells. 

They built on that target, but if you look at some of the subsequent programs that are targeting HbF, they’re targeting other pathways in BCL11A, the binding sites. They’re making changes that match what has been found in people. We often go back to the Orah Platt [Boston Children’s Hospital] paper showing that 8–9 is the level of HbF that starts to differentiate between less severe disease.⁶ And of course, hydroxyurea is a small molecule showing that if you upregulated HbF, you decrease the severity of the disease…  

The second part that I contributed to was that CRISPR-Cas, as originally discovered, simply didn’t work in hematopoietic stem cells (HSCs). Delivering the system as a plasmid, as DNA expression molecules, simply didn’t work. We at Stanford, in collaboration with Agilent, showed that you could deliver it as a Cas9 protein complexed to a synthetic guide molecule, with the guide molecule having modifications at the end. This probably serves a couple of purposes, one of which is to protect it from degradation, the other maybe is to shelter it from being recognized as a foreign molecule. I suspect that was a highly effective way of getting genome editing to occur in a range of primary cell types and keeping those cells healthy. You could deliver plasmids to CD34 cells and they would just die like a dog because cytoplasmic DNA is sensed as a viral infection and you get this robust type 1 interferon response. This was a way to deliver that nuclease in a highly efficient fashion to get that break where you wanted and maintain the quality of the cells. It’s that approach using the CRISPR-RNP complex that’s being used to manufacture Casgevy. 

CRISPR Therapeutics-Vertex found a good guide RNA, you have your target, you treat a patient, you see Victoria Gray doing fantastically well.⁷ The pace of their trial enrollment was appropriately slow, then you see this huge acceleration and the number of patients they enrolled. It is super exciting to see the results in Victoria basically generalize [to others] and the approval in December 2023. 

Davies: Let’s talk about your former company, Graphite Bio. What was the origin of the name? 

Porteus: We went through several names as most companies do, but we picked the name because the very first structure that Rosalind Franklin published was of graphite! So it was a call out to her contributions to our understanding of the structure of DNA that leads to sickle cell disease as a genetic disease. 

Davies: Do you still believe that direct correction of the pathogenic mutation will work better than upregulating HbF? 

Porteus: I certainly think it will be, it’s an approach that still is going to be the gold standard. Casgevy is still not a treatment of the root cause. This still has not been done for any disease—to take the pathologic variant and change it to the non-pathologic variant. That has not been done yet. [The HbF approach] is great but it’s a workaround, making mutations to compensate for the pathologic variant.…  

Even as we were supporting and advising CRISPR Therapeutics on their program, in my academic lab, we were building the technology to get high frequencies of gene correction in the endogenous beta-globin gene. We developed it with CIRM support from Stanford, generous philanthropic support from some Bay Area supporters, and got to the place where we could submit an IND and then raise capital to run the clinical trial. We made the decision to start Graphite Bio to run that clinical trial, and then maybe to more broadly develop the HDR [homology directed repair] platform. The company got started when times were good in the biotech investing world. Raising capital is always challenging but they were able to raise capital.  

Davies: The story of Graphite didn’t end the way you had planned it. There was an adverse event after you dosed your first patient? 

Porteus: The patient received her own cells in August 2022. When you get chemotherapy following a bone marrow transplant, there’s always a period where you wait for the cells to engraft and start making new red blood cells and platelets. Her period lasted longer than we expected but she was discharged from the hospital, she was doing fine, she felt great. But the treating team decided to stimulate her bone marrow by putting her on a growth factor for stem cells—a thrombopoietin agonist—which is being used in other bone marrow failure settings. But it wasn’t written into the protocol. And because we started an off-protocol medicine for a finding that was related to the drug product—slow engraftment— that triggered the SAE [serious adverse event] reporting. She was home! There were some [erroneous] reports, one even reported that she had died. She called her treating physician and said, ‘I’m not dead!’ Nonetheless, it was clear that the process that had come out of Stanford needed improvement….

Davies: So Graphite was at a crossroads. One could imagine a scenario where the company regrouped and decided to carry on—but you didn’t do that?  

Porteus: The company had appropriately decided to pause it on its own. After that [the patient’s] bone marrow started to recover. It was reported at ASH 2023 [American Society of Hematology] that she’s now transfusion independent, she’s off all her growth factors and clinically she’s doing great. There are some laboratory findings that we’ll continue to study and report in the future. 

It was clear it was going to take another 12–18 months to take the improvements had been identified both academically and within Graphite to treat the next patient. It was clear that the manufacturing process was too hard on the cells, but it was going to take another 12–18 months to get the manufacturing down and reboot the clinical trial. Graphite, the board and the investors thought that it was not in the best interest for that company to continue the program. 

So it came back to me because that makes sense. I got a lot of advice, thought long and hard. They were willing to do whatever I wanted to do. Some people said, ‘You should take it back to your Stanford lab and reboot it there.’ But I don’t think that works because we were ready to go. So instead, I started another company called Kamau Therapeutics…. 

Basically, the entire genome editing program has been transferred from Graphite into Kamau. We’re in the process of closing our seed funding. The trial was never closed, so we’re just reopening it under different names, the same general sites, with this markedly improved manufacturing process, some tweaks to the clinical protocol around cell dose and utilization of growth factors. We hope to treat the next patient with Nula-cel in 2025. 

Davies: We now have two approved therapies, priced at $2.2 and $3.1 million. Do you think those are fair prices?  

Porteus: One part of me agrees with what Julia Kanter (University of Alabama) has publicly said, which is that these patients and these therapies deserve that price: the benefit to patients absolutely justifies the lifelong savings and the indirect costs. [These prices] are not out of line for the prices for similar therapies. Lenmeldy, which just got approved for metachromatic leukodystrophy, got listed at $4.25 million! Why should sickle cell drugs be priced any less than any of the other peer drugs? 

That said, the price is clearly going to be a barrier to patients getting access. Where is the price coming from? Well, it’s coming from companies that are driven by shareholder value that need to make a return, and they develop the drugs and that’s the system we work in. It’s also driven by the fact that these autologous ex vivo manufactured therapies are expensive to make. Once you have a small molecule drug, you can make kilograms. If it’s an antibody and they do a 25,000-liter bioreactor run, one lot gives you doses for thousands, if not 10,000 patients. Every [gene editing] dose has to be manufactured specifically. So right now, there’s just a cost of goods that’s high.  

Some people said that’s why we need to figure out how to give an in vivo gene editing drug, but I’d argue right now the in vivo gene therapies are just as expensive and just as inaccessible as ex vivo therapies. So yes, it could get you there, but AAV gene therapies are also $3 million. There’s not a big difference. In terms of treating patients in Africa and India where most patients are, ex vivo therapy is pretty much [considered] a non-starter. But I don’t believe that.  

I think the other challenge with in vivo is the HSC is a pretty special cell. So in vivo editing and the Intellia programs are super exciting and the Verve Therapeutics programs are exciting in some senses. But delivery is challenging, even in the liver. The liver is designed to take up things. Macrophages are designed to take up things. HSCs are not designed to take up anything! They’re designed to be sheltered and protected. I think we’ve got a lot of biologic work to be done to find the right way to deliver an editor to that cell. 

In contrast, I think ex vivo autologous engineering is now, to me, an engineering problem. How do you stick process engineers on this and make the cost of goods cheaper, make the process cheaper and scalable? Instead of having five people make one product, can you have one person make ten products? I think engineering is something we are really good at and there are solutions out there and solutions being developed. The story of monoclonal antibodies is the analogy I turn to: once considered impossible to be a scalable drug and now a platform that is considered standard. I hope to be part of the story where autologous genetically engineered cell therapies go through a similar developmental process. 

I think we need competition. I’m going to put my effort in making ex vivo therapy as cheap as possible. Let’s set a bar that in vivo has to get to.  

Matthew Porteus, MD, PhD, is a physician-scientist at Stanford University School of Medicine, Palo Alto, CA. Kevin Davies, PhD, is Editorial Director of GEN.

A longer version of this interview has been published in the June 2024 issue of The CRISPR Journal. 

References

1. Urnov F. Genome Editing B.C. (Before CRISPR): Lasting lessons from the “Old Testament. Crispr J 2018;1(1):34–46.
2. Porteus MH, Baltimore D. Chimeric nucleases stimulate gene targeting in human cells. Science 2003;300(5620):763.
3. Carroll D. Genome engineering with zinc-finger nucleases. Genetics 2011;188(4):773–782.
4. Urnov FD, Miller JC, Lee Y-L, et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 2005;435(7042):646–651.
5. Orkin SH, Bauer DE. Emerging genetic therapy for sickle cell disease. Annu Rev Med 2019;70:257–271.
6. Platt OS, Brambilla DJ, Rosse WF, et al. Mortality in sickle cell disease—Life expectancy and risk factors for early death. New Engl J Med 1994;330:1639–1644.
7. Gray V, Thomas U, Davies K. Warrior spirit: An interview with Victoria Gray, sickle cell pioneer. Crispr J 2024;7(1):5–11.

The post A Story of Perseverance: An Interview with Matthew Porteus appeared first on GEN - Genetic Engineering and Biotechnology News.

Modified viruses have long served as valuable tools in molecular biology, providing researchers with a reliable way to deliver genetic material (DNA or RNA) to target cells. Naturally, viruses have become foundational elements in many CGT development pipelines. But, accumulating evidence suggests the use of viral vectors can come at a cost.^1–3

Inherent safety risks and complexities associated with these vectors drive up manufacturing costs while also increasing the potential for serious adverse events among patients. The FDA underscored this point in January 2024 by issuing new warning and guidance documents related to the oncogenic potential of common viruses used for cell therapy manufacturing.^4,5

While such risks may be tolerable when treating advanced stage cancers, emerging applications for CGTs extend well beyond terminal conditions.^6,7 It is increasingly necessary for developers to explore alternative, non-viral approaches that can enhance the safety and efficacy of these groundbreaking therapies.

Role for electroporation

Electroporation is well positioned to fill this need. Under optimized conditions, cells exposed to electrical pulses may become temporarily porous, enabling therapeutic components to enter the cell, and negating the need for a delivery vehicle (e.g., a virus). When used with clinical Good Manufacturing Processes, a clinically validated electroporation platform can be a viable way to circumvent the risks and complications associated with viral vectors, ultimately enabling simpler, safer, and more cost-effective therapeutic development.

CGTs refer to a wide spectrum of treatments that leverage cellular and genetic materials to either prevent or treat diseases, with examples ranging from mRNA vaccines to engineered tissues. The diversity of therapeutics in this field has grown in recent years, thanks to tools from synthetic biology that enable researchers to engineer novel proteins, edit genomes with precision, and transform ordinary cells into potent therapeutic agents.

T cells fortified with chimeric antigen receptors (CARs), for example, have emerged as powerful anti- cancer agents. T cells are naturally potent regulators of adaptive immunity, functioning as sentinels that recognize pathological antigens and may trigger either inflammatory or cytotoxic outcomes when activated. Such a response can be weaponized against malignant cells by modifying T cells with DNA or RNA coding for a synthetic CAR protein, one designed to both recognize tumor-associated antigens and subsequently activate a T cell-mediated immune response.⁸

The success of CAR-T cell therapy in acute lymphoblastic leukemia (ALL), large B cell lymphoma (LBCL), and other such cancers has inspired hope that this approach may be useful beyond cancer.^6,7 In fact, CAR-T therapy is already showing promise for treating autoimmune diseases.⁹

Although these therapies may be useful for a wide range of conditions, the use of CAR-T and other cell therapies is complicated by their considerable manufacturing costs and inherent safety risks.

Challenges of engineering cell therapies

Cell therapy manufacturing typically begins with the collection and isolation of immune cells, stem cells or other types of primary cells ex vivo. From there, cells can be transformed into therapeutic agents by exposing them to modified viruses that are capable of introducing custom DNA or RNA cargo into human cells. This genetic payload will contain genes and regulatory elements that enable the expression of the therapeutic construct, be it CAR or gene editing components. Altered cells are then infused into the patient for therapeutic effect.

There is a significant safety concern with viral vectors stemming from their origins as human pathogens In particular, vectors derived from retroviruses, lentiviruses, and adeno-associated viruses require complex engineering and manufacturing strategies to minimize chances that replication competent viruses might be generated during vector production. These strategies often involve dividing viral components among multiple plasmids, which must be co-transfected into producer cells, complicating manufacturing and reducing production efficiency.

Moreover, most regulatory agencies require that vector manufacturing lots intended for use in humans must be tested for replication competent viruses, which adds an additional complication to the manufacturing workflow.¹⁰ Additionally, viral vectors come with inherent safety risks related to both immunogenicity and insertional mutagenesis.

The majority of CGTs are produced using lentivirus (LV), murine γ-retrovirus, adenovirus (AD), and adeno-associated virus (AAV).¹¹ Each differs from the other in many crucial ways, including their ability to transduce various cell types, genomic complexity, the size of genetic cargo they can carry, and the ultimate fate of that cargo.¹⁰ LV, for example, is designed to integrate its payload with the host’s genome where it can endure through successive rounds of cellular division. By using a virus that embeds in the host’s genome, researchers gain long term, stable expression of therapeutic genes and the ability to expand the population of engineered cells.^{10, 12} 

It has also recently been reported that persistence of viral proteins in transduced cells may result in immunogenic responses with the potential to cause cytokine release syndrome—a life threatening adverse reaction.¹⁴ Therefore, use of any integrating vector comes with the added risk of potential insertional mutagenesis. The risk is great enough that the FDA saw fit to release a guidance document related to cell therapy manufacturing and integrating vectors, calling on researchers to consider alternatives when possible.^3,4

Non-viral alternatives

Electroporation stands out as a validated non-viral method for delivering genetic material into cells that can support the growth of CGT development. Where viral vectors transfer genetic material into a cell by binding to surface proteins, electroporation allows naked nucleic acids to permeate the cell by creating temporary perturbations in the cell membrane with electrical pulses.¹⁵ By avoiding the use of viruses, electroporation offers several distinct advantages for therapeutic development.

One such advantage is the lack of packaging limitations. With viral vectors, genes must be packaged inside the finite space of a capsid which limits researchers to approximately 4–10 kb of transgenic material, with diminishing packaging efficiency as they approach the upper limits for each virus.^16,17 This is a substantial constraint for CRISPR-based therapies that may require the delivery of a Cas9 enzyme, sgRNA, regulatory elements, and potential donor templates.¹⁸

To achieve this, multiple transductions may be necessary and the overall efficiency of the workflow decreases. Electroporation has a much larger size limit, and it allows simultaneous delivery of multiple and diverse loading agents. Therefore, researchers can readily deliver complex payloads when developing advanced therapies.

Such is the case with Vertex’s Exagamglogene autotemcel (trade name Casgevy), the world’s first FDA-approved CRISPR gene therapy. To correct transfusion-dependent beta thalassemia or sickle cell disease, an enhancer controlling the BCL11A gene is CRISPR modified in patient bone marrow stem cells, ultimately driving an increase in fetal hemoglobin production and reduced anemia or red blood cell sickling.

A CRISPR ribonucleoprotein complex—consisting of Cas9 protein and a sgRNA—are delivered to the stem cells ex vivo using electroporation.¹⁹ Not only does this allow for the efficient delivery of large CRISPR components, but it eliminates concern of virus induced immunogenicity and insertional mutagenesis. None of the 31 patients treated with Casgevy in clinical trials developed malignant side effects—a stark contrast to a similar vector-based CRISPR therapeutic for sickle disease which has received a black-box warning due to the risk of hematological malignancy.²⁰

As Casgevy demonstrates, electroporation enables researchers to deliver a wide range of payloads, from proteins to RNA, transposons, and reporters. This flexibility is key for improving the safety of cell therapies as it allows for innovation. Electroporation does not inherently eliminate the risk of insertional mutagenesis, but rather it allows researchers to use payloads and alternative strategies that avoid the issue altogether (as in the case of Casgevy) or greatly reduce the odds of oncogenic insertion.^15,21

Lastly, the use of electroporation in place of viral vectors requires less stringent biosafety precautions because it eliminates concerns about producing replication competent viruses. This significantly simplifies and reduces the cost of product manufacturing.

Viral vectors have historically been preferred because they allow cell engineering on a much larger scale. However, the development of flow electroporation technologies like those offered by Maxcyte have made it possible to perform automated, aseptic electroporation across a wide range of scales (5 × 10⁵ cells to 2 × 10¹⁰ cells and beyond).²² Accordingly, Maxcyte’s flow electroporation platform is used for the manufacturing of Casgevy and several other CGTs currently in development.

Electroporation has proven to be a robust, versatile, and safer alternative to viral vectors for CGT development. Its ability to efficiently deliver large and complex genetic payloads, combined with a reduced need for stringent biosafety measures, lower immunogenic risks, and flexibility in payload type, positions electroporation as a key technology in the future development of successful and safe cell and gene therapies.

James Brady, PhD, is senior vice president, technical applications and customer support, MaxCyte.

References

1. Goyal, Sunita, et al. “Acute Myeloid Leukemia Case after Gene Therapy for Sickle Cell Disease.” New England Journal of Medicine, vol. 386, no. 2, 13 Jan. 2022, pp. 138–147, https://doi.org/10.1056/nejmoa2109167.
2. Ghilardi, Guido, et al. “T-Cell Lymphoma and Secondary Primary Malignancy Risk after Commercial CAR T-Cell Therapy.” Nature Medicine, 24 Jan. 2024, pp. 1–1, www.nature.com/articles/s41591-024-02826-w, https://doi.org/10.1038/s41591-024-02826-w.
3. Magdi Elsallab, et al. “Second Primary Malignancies after Commercial CAR T-Cell Therapy: Analysis of the FDA Adverse Events Reporting System.” Blood, vol. 143, no. 20, 16 May 2024, pp. 2099–2105, https://doi.org/10.1182/blood.2024024166.
4. Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products Draft Guidance for Industry. 2022. https://www.fda.gov/media/156896/download
5. Research, Center for Biologics Evaluation and. “FDA Investigating Serious Risk of T-Cell Malignancy Following BCMA-Directed or CD19-Directed Autologous Chimeric Antigen Receptor (CAR) T Cell Immunotherapies.” FDA, 28 Nov. 2023, www.fda.gov/vaccines-blood-biologics/safety-availability-biologics/fda-investigating-serious-risk-t-cell-malignancy-following-bcma-directed-or-cd19-directed-autologous.
6. Chancellor, Daniel, et al. “The State of Cell and Gene Therapy in 2023.” Molecular Therapy, 1 Nov. 2023, https://doi.org/10.1016/j.ymthe.2023.11.001.
7. Bashor, Caleb J., et al. “Engineering the next Generation of Cell-Based Therapeutics.” Nature Reviews Drug Discovery, vol. 21, no. 9, 1 Sept. 2022, pp. 655–675, https://doi.org/10.1038/s41573-022-00476-6.
8. De Marco, Rodrigo C., et al. “CAR T Cell Therapy: A Versatile Living Drug.” International Journal of Molecular Sciences, vol. 24, no. 7, 1 Jan. 2023, p. 6300, www.mdpi.com/1422-0067/24/7/6300, https://doi.org/10.3390/ijms24076300.
9. Mackensen, Andreas, et al. “Anti-CD19 CAR T Cell Therapy for Refractory Systemic Lupus Erythematosus.” Nature Medicine, vol. 28, 15 Sept. 2022, pp. 1–9, www.nature.com/articles/s41591-022-02017-5, https://doi.org/10.1038/s41591-022-02017-5.
10. Bulcha, Jote T., et al. “Viral Vector Platforms within the Gene Therapy Landscape.” Signal Transduction and Targeted Therapy, vol. 6, no. 1, 8 Feb. 2021, www.nature.com/articles/s41392-021-00487-6, https://doi.org/10.1038/s41392-021-00487-6.
11. Shirley, Jamie L., et al. “Immune Responses to Viral Gene Therapy Vectors.” Molecular Therapy, vol. 28, no. 3, Mar. 2020, pp. 709–722, https://doi.org/10.1016/j.ymthe.2020.01.001. 
12. Lukjanov, Viktor, et al. “CAR T-Cell Production Using Nonviral Approaches.” Journal of Immunology Research, vol. 2021, 27 Mar. 2021, p. 6644685, www.ncbi.nlm.nih.gov/pmc/articles/PMC8019376/,https://doi.org/10.1155/2021/6644685.
13. Yan, Koon-Kiu, et al. “Integrome Signatures of Lentiviral Gene Therapy for SCID-X1 Patients.” Science Advances, vol. 9, no. 40, 6 Oct. 2023, https://doi.org/10.1126/sciadv.adg9959.
14. Jamali, Arezoo, et al. “Early Induction of Cytokine Release Syndrome by Rapidly Generated CAR T Cells in Preclinical Models.” EMBO Molecular Medicine, vol. 16, no. 4, 21 Mar. 2024, pp. 784–804, https://doi.org/10.1038/s44321-024-00055-9.
15. Moretti, Alex, et al. “The Past, Present, and Future of Non-Viral CAR T Cells.” Frontiers in Immunology, vol. 13, 9 June 2022, https://doi.org/10.3389/fimmu.2022.867013.
16. Dong, Wendy, and Boris Kantor. “Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives.” Viruses, vol. 13, no. 7, 1 July 2021, p. 1288, https://doi.org/10.3390/v13071288.
17. Lanigan, Thomas M., et al. “Principles of Genetic Engineering.” Genes, vol. 11, no. 3, 10 Mar. 2020, p. 291, www.mdpi.com/2073-4425/11/3/291/htm, https://doi.org/10.3390/genes11030291.
18. Uchida, Naoya, et al. “Cas9 Protein Delivery Non-Integrating Lentiviral Vectors for Gene Correction in Sickle Cell Disease.” Molecular Therapy – Methods & Clinical Development, vol. 21, June 2021, pp. 121–132, https://doi.org/10.1016/j.omtm.2021.02.022.
19. Frangoul, Haydar, et al. “CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia.” New England Journal of Medicine, vol. 384, no. 3, 5 Dec. 2020, https://doi.org/10.1056/nejmoa2031054.
20. Parums, Dinah V. “Editorial: First Regulatory Approvals for CRISPR-Cas9 Therapeutic Gene Editing for Sickle Cell Disease and Transfusion-Dependent β-Thalassemia.” Medical Science Monitor: International Medical Journal of Experimental and Clinical Research, vol. 30, 1 Mar. 2024, p. e944204, pubmed.ncbi.nlm.nih.gov/38425279/, https://doi.org/10.12659/MSM.944204.
21. Bozza, Matthias, et al. “A Nonviral, Nonintegrating DNA Nanovector Platform for the Safe, Rapid, and Persistent Manufacture of Recombinant T Cells.” Science Advances, vol. 7, no. 16, 16 Apr. 2021, https://doi.org/10.1126/sciadv.abf1333
22. Li, Linhong, et al. “Large Volume Flow Electroporation of MRNA: Clinical Scale Process.” Methods in Molecular Biology, 8 Nov. 2012, pp. 127–138, https://doi.org/10.1007/978-1-62703-260-5_9.

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Uduak Thomas | Senior Editor

Katalin Karikó was thrust into the limelight during the COVID-19 pandemic for her pioneering work that led to key advances in mRNA vaccination—work that was awarded the Nobel Prize in Physiology or Medicine last year. During that time, the world learned some of Karikó’s story, marked by challenges, determination, and a love of science. Now, Uduak is eager to read Karikó’s story told in her own words. Breaking Through is “a testament to one woman’s commitment to laboring intensely in obscurity—knowing she might never be recognized in a culture that is more driven by prestige, power, and privilege—because she believed her work would save lives.”

Christina Jackson | Associate Editor

A new book can be seen on Christina’s desk, in her bag, or in her hand, on a weekly basis. When asked what book she plans to pick up this summer, she pointed to The Gene by Siddhartha Mukherjee. The Gene is described as offering “a definitive account of the fundamental unit of heredity—and a vision of both humanity’s past and future.” With a history marked by a cast of characters including Darwin, Mendel, Crick, Watson, and Franklin, Mukherjee also injects the story of his own family and their mental illness. Undoubtedly, we’ll see the 600-page book on Christina’s desk for another week or so, before she devours it and moves onto something else. 

Corinna Singleman, PhD | Managing Editor 

Ben Goldfarb, an environmental journalist, traveled throughout the United States and the world to analyze how roads have transformed our planet. Corinna used to study the impacts of human contamination on fish, and maintains her passion for learning about ecology and conservation in her post-research life. In Crossings, she is eager to read about the impacts of human roadways on animal commuting strategies and how we can address these issues. The book has been described as an “eye-opening account of the global ecological transformations wrought by roads” and was a New York Times Notable Book of 2023 and an Editors’ Choice. 

John Sterling | Editor in Chief 

As a journalist and biotech expert, John loves nothing more than a good story about science. For that reason, he is looking forward to digging into Kira Peikoff’s latest book, Baby X, a fictional thriller set in a world where a person can have a baby with anyone else—using just a biological sample. In it, a black market operation (called The Vault) steals cells from people (imagine a used napkin or fork) to convert them into sperm and egg. The “thought-provoking look into the near future” raises questions about the progress of technology and the impacts it has on the world.

James Lambo | Art Director 

If you ever wanted to know about how CRISPR works from a layperson’s perspective, James says “The Code Breaker is the book for you.” James (who got a jump start on his summer reading and is already deep into the book) says that biography veteran Walter Isaacson weaves a fascinating tale about how—over billions of years—bacteria have outwitted viruses. The story describes how modern-day researchers have seized this new technology to overcome modern-day diseases, including cancer and SARS-CoV-2. Doudna’s story, Isaacson writes, “is a thrilling detective tale that involves the world’s most profound mysteries, from the origins of life to the future of our species.”

Katherine Vuksanaj | Online Editorial Manager

Women in Science Now shares stories and insights of “women from a range of backgrounds working in various disciplines, illustrating the journeys that brought them to the sciences, the challenges they faced along the way, and the important contributions they have made to their fields.” This is a fitting choice for Kathy who found her own way into science publishing and makes important contributions at GEN every day. In her book, Munoz combines stories with data to illuminate the challenges women scientists face, while “highlighting research-based solutions to help overcome these obstacles.”

Alex Philippidis | Senior Business Editor

It comes as no surprise that Alex chose one of the leading books on the COVID-19 pandemic for his summer read. For two years, Alex led COVID-19 coverage for GEN, covering the latest drugs, vaccines, and other SARS-CoV-2 research on a daily basis throughout 2020 and 2021. Gottlieb’s book, which was released in the fall of 2021, is “an intense ride through the pandemic with chilling details of what really happened. It is also sprinkled with notes of true wisdom that may help all of us better prepare for the future,” notes Sanjay Gupta, MD, CNN chief medical correspondent.  

Kevin Mayer | Senior Editor

While some of us are delving into modern questions about COVID-19 and CRISPR, Kevin chose to read about questions that have been around a bit longer. Kevin plans to read “Sky In A Bottle” this summer. In his book, Pesic introduces us to chemistry, optics, and atomic physics and describes the polarization of light, Rayleigh scattering, and connections between the appearance of the sky and Avogadro’s number. He discusses changing representations of the sky in art, from new styles of painting to new pigments that created new colors for paint.

Julianna LeMieux, PhD | Deputy Editor in Chief 

As a microbiologist, I have always been a fan of Dr. Fauci’s research on HIV and his work as NIAID director to lead the nation through several viral outbreaks including Ebola, SARS, West Nile, and anthrax. I cannot wait to dive into his new memoir that “reaches back to his boyhood in Brooklyn, NY” and “carries through decades of caring for critically ill patients, navigating the whirlpools of Washington politics, and behind-the-scenes advising and negotiating with seven presidents on key issues from global AIDS relief to infectious disease preparedness at home.”

Rob Reis | Production Editor

Not all science summer reads need to be about science, per se. When GEN‘s production editor, Rob, first started reading the medical thrillers written by doctor and author Robin Cook more than 40 years ago, the main appeal was the interesting characters and compelling stories, but as Cook continues to keep abreast of the changes affecting the world of medicine, Rob increasingly sees connections between the topics covered in GEN and the stories crafted by Cook. He will not only re-read Cook’s 2023 novel, Manner of Death, this summer, but he also looks forward to Cook’s 2024 novel, Bellevue, when it gets published in December. 

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Unquestionably, we will emerge from this revolutionary period with modified views of components of cells and how they operate, but only, however, to await the emergence of the next revolutionary phase that again will bring startling changes in concepts. —Barbara McClintock, Nobel laureate (1983)

 “Everything in the last 14 years of genome editing has been based on CRISPR. We have been whipping this horse for a decade and a half, but we need more programmable functions with complexity beyond the molecular scissors that cut RNA and DNA.” So says Patrick Hsu, PhD, co-founder of the Arc Institute, in whose lab the next revolutionary phase of genome engineering may have just been unearthed, even as the CRISPR revolution has barely begun.  

In January of this year, researchers in Hsu’s laboratory (he is also assistant professor of bioengineering and Deb Faculty Fellow at the University of California, Berkeley) posted a preprint on bioRxiv in which they claim to have discovered a new class of natural single-effector RNA-guided systems.¹ That story, now peer reviewed, is published online today in Nature.  

These systems retain the key property of programmability from RNAi and CRISPR, while enabling large-scale genome design beyond RNA and DNA cleavage. These modular, bi-specific bridge RNAs can be reprogrammed to enable sequence-specific fundamental DNA rearrangements, potentially accelerating the advancement of genome design (Fig 1).² 

“This is a much more complex molecular machine,” Hsu continues. “We’re excited about the potential of this for eventually achieving chromosome-scale genome engineering, where you can do long-range insertions, deletions, and genome translocations.” 

Complex genome assembly 

Hsu is a self-described technologist—he creates the genome engineering and biological design tools of tomorrow. As a graduate student at Harvard University, he worked with his mentor Feng Zhang, PhD, building some of the foundational components of CRISPR as a genome engineering tool.³ But Hsu came to realize that manipulating RNA might be a more flexible technique than making permanent, and sometimes unintended, changes to the genetic code. In 2018, Hsu and his team, which included Arc Institute co-founder Silvana Konermann, PhD, developed CasRx as a programmable RNA-binding module for efficient targeting of cellular RNA, enabling a general platform for transcriptome engineering and future therapeutic development with RNA targeting CRISPR.⁴  

Hsu believes that one of the greatest challenges today is the manipulation of eukaryotic genomes, particularly the integration and manipulation of large, multi-kilobase (kb) DNA sequences, which limits the rapidly growing fields of synthetic biology, cell engineering, and gene therapy. According to Hsu, who is quite the science historian, the prevailing modern genome editing method has been stuck on a road paved with milestones that began with the initial work of Mario Capecchi, PhD, in 1980 on the insertion of DNA into mammalian cells,⁵ Martin Evans PhD’s work in 1986 on chimeric mice,⁶ and then work from both Oliver Smithies, PhD, and Capecchi to successfully create specific modifications in the genomes of mice.^7,8   

“That was generally an attempt to do an insertion reaction, which is only one way to do genome editing,” Hsu told me. “Funnily enough, what happens today with base editing or prime editing—while exciting—is arguably even smaller in scope. You’re making single-nucleotide polymorphism changes or tens of bases, rather than the multigene-sized cassettes that we originally [envisioned]… Generally, all of this has been small-scale single-locus changes.” 

Hsu argues that CRISPR-Cas molecular scissors require a complex multistep process to make an edit that, in chemistry terms at least, has very low purity. “You mostly get indels, and only a small amount of time you get the edit, which then leads to chromosomal translocations and large lesions,” said Hsu.  

“You’re trying to create this chemical reaction that’s just not very predictable. Certainly, there are highly optimized cases like ex vivo T-cell engineering, where billions of dollars have been invested into this problem to get it to work right. [Scientists] will hang their hat on that and say that CRISPR is high efficiency and specificity. But generalizing this success to other cell types or in vivo has been really hard—it’s a fundamental mechanistic limitation.” 

Hsu is not alone in this view. In 2019, Harvard Medical School geneticist George Church, PhD, memorably referred to CRISPR as a “blunt axe” that performs “genome vandalism.” Instead of looking to take a major leap forward, Hsu said the genome engineering field is in a battle over bragging rights that centers around the size, toxicity, specificity, and efficiency of their nucleases.  

Some recently developed gene editing assemblies, such as PASTE and PASSIGE (both developed by former colleagues of Hsu’s at the Broad Institute), are showing considerable promise in their own right, although they require several distinct components to be delivered to a given cell.⁹   

By contrast, Hsu is trying to reframe the conversation completely. Hsu, who was already working on a solution to evolve the genome engineering field, did what many talented scientists have done in the past—he turned to nature for inspiration. For example, during conception, there’s a large amount of recombination and genomic rearrangements from crossover events at chromosomal scale between genes from the mother and father that results in the unique individuals that we are.  

Another example is Deinococcus radiodurans, a bacterium that is extremely resistant to radiation and other environmental stresses. When the organism is being battered by radioactivity, their genomes can shatter, but they always find a way to reassemble a complete genome. And then there are mobile genetic elements (MGEs), which brings us to the great Barbara McClintock, PhD. 

About 80 years after Gregor Mendel worked with peas in an abbey garden in (what is now) the Czech Republic to describe the transmission of genetic traits (before anyone knew genes existed), McClintock began experiments on maize kernels at the Cold Spring Harbor Laboratory that would lead to her profound discoveries on transposable elements.^10,11 McClintock observed that genetic elements can change position on a chromosome, causing nearby genes to become active or inactive, and that this correlated with the redistribution of genetic traits in maize as well as other organisms.¹² 

In her 1983 Nobel Prize lecture, McClintock spoke about genetic shocks created by MGEs that create selective pressures and, thus, new gene functions—even the origin of new species.¹³ “[McClintock] was one of the original people who discovered these elements, one of the first people that realized that they’re actually really powerful driving forces for genetic diversification in new functions,” said Hsu. “We’ve just been fascinated by these transposons and genomic elements and what else could be out there.” 

That framework got Hsu to begin examining large serine recombinases (LSRs). “The reason we did our LSR research in the first place, was not really to find recombinases per se—it was to solve this core technological problem of modern gene editing.” 

The bridge RNA recombinase mechanism 

A few years ago, two members of the Hsu lab, graduate student Nicholas Perry and computational biologist Matt Durrant, PhD, were working on LSRs (Fig 2). One of the perks of working in the non-profit Arc Institute is that it affords scientists no-strings-attached, multi-year funding.¹⁴ Perry and Durrant decided to sift through a huge genomic and metagenomic database that Durrant had been compiling for MGEs (Box 1).  

Upon investigating a family of cut-and-paste MGEs called IS110s, which all had a gene encoding a RuvC-like domain—one of the key nuclease domains in the Cas9 nuclease—the Arc team wondered if it might encode a whole RNA-guided transposase system.^15,16 This made sense to Durrant, as IS110 elements scarlessly excise themselves from the genome and generate a circular form as part of their transposition mechanism (Fig. 3). 

“Patrick threw around the idea that maybe there’s some kind of RNA-guided transposase out there,” Durrant recalled. “At that point, I had just been staring at sequences for so long that I felt like I had built up an intuition for what would be worth pursuing.” 

Hsu’s and Durrant’s hypothesis sent Perry on a quest to discover whether there could be some sort of RNA that existed, a pre-requisite for any sort of RNA-guided transposase system. Their first clue came from secondary structure analysis of a particular IS110 sequence, called IS621, which was predicted to contain an RNA with a 5′ stem loop and two large internal loops. Perry set out to demonstrate that an RNA was expressed from this sequence. 

Meanwhile, Durrant circled back to an idea that Hsu had pushed him to implement called covariation analysis to predict base-pairing interactions between the non-coding RNA (ncRNA) and the target or donor DNA.¹⁷ This comparative analysis method asks whether pairs of nucleotides change in tandem at specific positions of aligned DNA and RNA, which would indicate evolutionary pressure to conserved base-pairing interactions between ncRNA positions and target or donor positions. Projecting this covariation pattern onto the canonical IS621 sequence and ncRNA secondary structure, Durrant saw that the first internal loop may base-pair with the target DNA.  

“It was a really messy initial analysis… but we got lucky enough that it worked,” said Durrant. “We saw this signal of bits of the non-coding ends that co-vary with the target. It was the most exciting moment of my whole career, when it became clear that these are probably programmable.” 

Durrant immediately messaged Perry, who was at a CRISPR conference in Boston, saying they had to talk immediately. Perry hurriedly left the meeting and covertly called Durrant, wary of any potential eavesdroppers who might pick up on their findings about RNA-guided transposases. Although the team gave some thought to publishing the computational results alone, Durrant said there was “some sliding scale of confidence and doubt… We tried to deeply understand how everything worked.” 

For the next few months, Perry was buried in experiments to try to assemble pieces of the puzzle that had been predicted by Durrant’s computational analysis. On a Sunday morning, which happened to be his birthday, Perry was looking through data when he got confirmation for an experiment showing that a promoter on the circular form of IS621 expressed an encoded ncRNA that formed a functional complex with a transposase. 

“I spent probably six months before getting any conclusive positive data about the expression of a non-coding RNA, observing them transpose and showing that they worked in an orthogonal system, trying to engineer the system,” said Perry. “If we couldn’t even get these to function in the cell type that they’re native in, then how could we ever learn anything more about them? You can wish that they’re programmable, but if there’s no observable function, does it really matter?” 

Perry was able to confirm a mechanism for a programmable target loop, and a short time later, they repeated the whole process for identifying and confirming that the second loop was a programmable donor loop. This suggested to Perry and Durrant that the RNA acts as a “bridge” between target and donor DNA to enable recombination by the IS621 recombinase (Box 2). 

Then came the eureka moment, where the entire picture of their computational and experimental data came together. Perry and Durrant had discovered a single-effector recombinase system that uses a bridge RNA with two distinct binding loops that can be independently reprogrammed to bind and recombine diverse DNA sequences (Fig. 4). “We realized it was programmable on both [the donor and target] ends, which was completely unprecedented,” said Perry. 

The team went on to demonstrate that the modularity of each loop of the bridge RNA can facilitate the recombinase system to execute sequence-specific insertions, inversions, and excisions. This meant that the bridge RNA-guided single effector system provided “a unified, programmable, and modular mechanism for the fundamental DNA rearrangements required for genome design,” says Hsu. “We discovered a conceptually distinct mechanism of RNA-guided self recognition for a mobile genetic element and capitalized upon this mechanistic feature to enable a new method of genetic engineering.” 

A GUI for genome engineering 

Since the early days of bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs), assembling large sections of DNA has been technically incredibly challenging. As the field has moved towards making synthetic minimal genomes, the field has been limited to relatively short DNA synthesis and assembly techniques. With the concept of genome design in mind, Hsu explains the significance of these bridge RNAs in computer terms—they’re basically like a program that allows the user to install and uninstall packages. 

“The Xerox Alto was the first computer ever sold with a mouse and a graphical user interface (GUI),” said Hsu. “It was invented just off the street from where Arc [Institute] is today at Xerox PARC. It gave humans for the first time a simple and intuitive way to interact with information. Guide RNAs act like that mouse cursor to interact with nucleic acids in a large genome. What we’ve been doing so far is basically punching individual nucleotides and changing them, like punch-card programming. We want something that can operate at a much higher level of abstraction to design genomes. That’s where all of this is going.” 

Nature has invented it, and there are probably many more programmable systems. And Hsu will keep on looking to see what other biotechnology doors he can unlock. 

References 

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