Several secretive and well-funded biotechs are sharing the first glimpses of data from a new suite of gene editing tools that promise to overcome one of the field’s grand challenges: precisely inserting large sequences of DNA, or even whole genes, into the genome.
So far, most work with CRISPR gene editing has used the tool to turn off problematic genes. Newer versions of the technology, like base editing and prime editing, can make small changes to one or a few letters of genetic code, respectively. But replacing large swathes of errant code, or uploading brand new ones, has remained a challenge.
ReNAgade Therapeutics, SalioGen Therapeutics and Tome Biosciences have been quietly working on new gene insertion techniques. So has David Liu, a prominent gene editing scientist from the Broad Institute of MIT and Harvard. Those startups and Liu all disclosed new technologies this week at the American Society of Gene & Cell Therapy’s conference in Baltimore.
Jason Cole“This is our coming-out party, scientifically,” SalioGen CEO Jason Cole told Endpoints News.
His company on Tuesday presented data on its experimental therapy for an inherited form of vision loss called Stargardt disease. In a mouse experiment, the approach restored expression of the gene in 40% of photoreceptors and reduced a harmful byproduct believed to contribute to vision loss by a similar amount.
Stargardt disease has been tough to tackle with other approaches. The broken gene is too big to squeeze into the viral vectors commonly used in traditional gene therapies. And since the disease can be caused by a large number of different mutations in that gene, fixing those mutations with existing CRISPR tools is impractical.
Gene insertion offers a new solution. SalioGen uses a new lipid nanoparticle to deliver a fresh copy of the gene into the eye, which gets stitched into the genome with an enzyme called a transposase, which is familiar to biologists for moving so-called “jumping genes” throughout the genome.
SalioGen is working on a similar approach to treat cystic fibrosis in the lungs, which is also caused by several mutations in a large gene. And it hopes that’s just the start. “These integrating technologies can really open up the number of indications we can go after with genetic medicine,” Cole said.
A ‘final chapter in genomic medicine’
Gene insertion is the new vanguard of gene editing. The original three biotechs built around CRISPR gene editing — CRISPR Therapeutics, Editas Medicine and Intellia Therapeutics — have all begun early discovery work on gene insertion technologies. And many small startups are developing their own gene insertion tools.
Although adding or replacing entire genes isn’t always necessary to treat a disease, it feels like the last major technological hurdle for manipulating DNA for some scientists. Tome Biosciences, which launched in December with $213 million, even called its programmable genomic insertion approach “the final chapter in genomic medicine.”
On Friday, Tome presented glimpses of its first data, pairing CRISPR with other enzymes to drag and drop small and large stretches of DNA into cells. The company was able to edit roughly 40% of stem cells in a dish and up to 10% of liver cells in a monkey, numbers that it expects will rise with further iterations of the technology.
John Finn“There are a lot of different variables that we’re changing. We will not be getting into the specific details,” Tome chief scientific officer John Finn said in an advance interview. “We have a lot of knobs we can turn, and so we’re continuing to optimize. This is nowhere near a ceiling.”
This week, Tome disclosed its pipeline for the first time, too. Its two most advanced programs are a pair of off-the-shelf cell therapies for autoimmune kidney diseases, including lupus nephritis. The engineered natural killer cells are among the most heavily edited in the industry, with three genes knocked out and four to five genes added to improve safety and efficacy. The addition is a stretch of DNA measuring 12,000 letters long, much larger than what’s possible with widely used cell engineering techniques.
The company is also turning its gene insertion technology to several genetic liver diseases, with phenylketonuria up first, by aiming to completely replace the broken gene that leaves people unable to break down a toxic molecule. And it believes that inserting the gene into a seemingly modest 10% of liver cells is enough to sufficiently restore vital proteins.
“If we reach these levels in a human being, we’ve essentially already reached curative levels for a lot of indications, including PKU,” Finn said.
An increasingly crowded field
While prior iterations of CRISPR, such as base editing and prime editing, debuted in prestigious scientific papers, much of the gene insertion work is happening behind closed doors. The companies’ reluctance to divulge details has raised questions about how well each technology really works, and how similar or different they are from each other.
Many of these technologies pair a CRISPR enzyme, which can be directed to make edits at a specific location in the genome, with an increasingly complex cast of characters that are adept at lugging around big chunks of DNA.
Enzymes borrowed from microbes or dug up from the dark matter of the human genome — including integrases, recombinases and reverse transcriptases — feature prominently in the makeup of these new gene insertion methods.
ReNAgade Therapeutics, which launched with over $300 million last year, disclosed its own gene insertion technology on Tuesday for the first time. The company is combining CRISPR tools with bacterial retrons — composed of a reverse transcriptase and an RNA molecule — to make insertions dozens to hundreds of letters long.
Pete Smith“The idea of doing gene insertions is big white space for us,” ReNAgade chief scientific officer Pete Smith said. He thinks these medium-sized insertions could be “very valuable” for replacing segments of genes known as exons in conditions where many mutations are clustered in one region.
Prime editing, a version of CRISPR developed by David Liu at the Broad Institute, also centers around using a reverse transcriptase to write a DNA sequence from an RNA template. Prime Medicine, one of several companies that Liu has co-founded, has licensed that technology and recently got clearance for the first human tests.
Companies like Tome and Tessera Therapeutics, which has raised $580 million for its gene writing tools, have faced scrutiny for their techniques appearing too similar to prime editing. And while Tessera presented its most sweeping view of preclinical data yet at ASGCT this week, showcasing its ability to edit the blood stem cells, immune cells and the liver, the company still hasn’t disclosed the precise makeup of its DNA writers.
“We’ve focused on efficacy data, which we think are very remarkable,” Tessera CEO Michael Severino said. “We’re not getting into the specifics of the individual constructs at this point.”
A CRISPR pioneer’s latest inventions
David LiuAlthough prime editing is best at making small changes to DNA or adding sequences under 200 letters in length — much smaller than the average gene — Liu has previously shown that prime editing can be used to create a landing pad in the genome for another enzyme, such as recombinase, to install a full gene.
The method, called PASSIGE, worked modestly when Liu first revealed it in 2022, with only a few percent of cells typically having genes successfully integrated. At ASGCT on Thursday, Liu presented his lab’s unpublished work to improve the system. By evolving and engineering new recombinases better suited to the task, they bumped gene integration up to 20% to 35%.
Liu also showed data suggesting that his new engineered and evolved method, dubbed eePASSIGE, was significantly better at inserting genes than a similar method called PASTE, developed by MIT scientists Jonathan Gootenberg and Omar Abudayyeh and licensed to Tome.
Rahul KakkarLike PASSIGE, the version of PASTE that was first published in 2022 uses a CRISPR enzyme, a reverse transcriptase and a recombinase. But Tome CEO Rahul Kakkar said the company has improved and expanded upon the technology. Tome might be able to use another writing enzyme in place of the reverse transcriptase, or forgo that component altogether and just use a CRISPR enzyme and an integrase, he said.
Liu also disclosed a second project, in collaboration with Samuel Sternberg at Columbia University, to engineer and evolve a class of enzymes known as CRISPR-associated transposases, or CAST. They are remarkable at inserting genes in bacterial cells, but have “zero to very low activity” in mammalian cells, Liu said.
The team’s improved CAST enzymes got genes integrated in human cells about 10% to 30% of the time. “The integration efficiencies of eeCAST are already sufficient to offer potential benefit to patients suffering from a variety of loss-of-function genetic disorders,” Liu said.
Lei Lei Wu contributed to reporting.