A new method for safely inserting large chunks of DNA into genomes has now measured up in mice, potentially paving the way for the next generation of gene editing medicines.
The approach, which is described in a Nature paper published March 11, uses circular, single-stranded DNA rather than the typical double-stranded version of the hereditary molecule. Dubbed “integration through nucleus-synthesized template addition of large lengths”—INSTALL for short—the technique was pioneered by a team led by Connor Tou, Ph.D., and Benjamin Kleinstiver, Ph.D., at Mass General Brigham.
With INSTALL proving capable of inserting DNA into the genomes of mice and lab-grown human cells, the team is now working to use it in new medicines for liver diseases, blood disorders and metabolic conditions, Kleinstiver told Fierce Biotech in an interview.
“This is beautiful work that addresses in an elegant way one of the big challenges with controlled DNA insertion into the genome,” Kiran Musunuru, M.D., Ph.D., co-founder of Verve Therapeutics and a scientist at the Children’s Hospital of Philadelphia and the University of Pennsylvania, told Fierce. Musunuru co-led development of the first custom CRISPR gene editing therapy and was not involved with the new study.
The key issue INSTALL addresses is the immune response to large chunks of double-stranded DNA, which aren’t normally found floating freely inside of cells.
DNA in the cytoplasm of the cell “usually means something is wrong,” such as a viral infection, Kleinstiver explained. When the cell detects DNA outside of its proper home of the nucleus, the cell launches an immune response that causes “downstream toxicity.”
To sneak DNA through the cytoplasm and into the nucleus so it can be edited into the genome, Tou and Kleinstiver were inspired by certain bacteria and viruses that can naturally insert circular, single-stranded DNA into double-stranded genomes.
“This was an exciting realization,” Tou said in a March 11 release. “We then wondered whether these mechanisms could be recapitulated in human cells.”
The researchers teamed up with genome engineering specialist Full Circles Therapeutics to fashion large chunks of DNA into this spherical shape and found they could slip through the cell undetected.
Because gene editing enzymes need double-stranded DNA to latch onto, they welded snippets of double-stranded nucleotides—too small to be flagged by the immune system—onto the single-stranded circles.
The technique allows for large DNA strands to be inserted into the genome more precisely than is possible with viral vectors, Kleinstiver told Fierce. INSTALL uses a lipid nanoparticle for delivery instead of the viruses commonly used in gene therapy.
“The potential and upside of these tools is really high, because you could develop one editor that could treat lots [of] or all patients,” Kleinstiver said.
Techniques like INSTALL could be especially handy for diseases that arise when large segments of a gene are missing, Musunuru explained.
“That's where gene replacement, as forecast by the work in this paper, will have a critical role to play,” he said. While INSTALL “has a long way to go before it might be useful for human therapeutics,” Musunuru described it as “a promising step in the right direction.”
The gene editing field got a big regulatory boost at the end of February with the official unveiling of the FDA's plausible mechanism pathway, a new route for bespoke therapies designed for individual patients to garner agency approval.
Kleinstiver’s team, which provided the gene editing enzyme used in baby KJ’s groundbreaking custom treatment, is pursuing the new pathway to develop personalized gene editing therapies for patients with an ultrarare genetic disease called multisystemic smooth muscle dysfunction syndrome.
“We've developed a therapy for the most common mutation, but leveraging some of these [plausible mechanism] principles might allow us to go and pursue therapies for some of the other mutations as well,” Kleinstiver said.
In cases where INSTALL or a similar tool scores regulatory approval, should that ever happen, it would render parts of the plausible mechanism pathway moot, Kleinstiver said. That's because in diseases where large-scale gene editing succeeds, treatments wouldn't need to be tweaked to match each individual.
“We wouldn't need 100 tools to treat 100 mutations,” Kleinstiver said. “We would have one tool to treat those 100 mutations.”
But both Kleinstiver and Musunuru said such a time would be far off, while Musunuru stressed that it would never be the case that every genetic disease could one day have a universal therapy.
“There isn't going to be a one-size-fits-all solution to all genetic diseases,” he added.