After becoming the world’s first patient treated with a bespoke base editing therapy, baby KJ Muldoon is now healthy and free from the toxic ammonia buildup caused by his rare genetic metabolic disorder that initially presented a 50% mortality rate in infancy. While his story highlights the life-changing potential of gene editing, it also underscores a major challenge for the field: expanding these therapies to benefit broader patient populations.
KJ’s urea cycle disorder stemmed from a single disease-causing mutation that could be precisely targeted. However, many genetic disorders arise from numerous mutations scattered across a gene, making individualized corrections far too resource-intensive to scale.
Ben Kleinstiver, PhD, associate investigator at Massachusetts General Hospital (MGH) and co-author of the NEJM study describing KJ’s case, told GEN that insertion of large DNA sequences at programmable locations in the genome holds tremendous promise as a generalizable medicine that could treat patients regardless of their underlying disease-causing mutations. His team has recently taken one step closer to making large gene insertions safer for therapeutic applications.
In the new study published in Nature titled, “Immune evasive DNA donors and recombinase license kilobase-scale writing,” Kleinstiver and colleagues, in collaboration with Full Circles Therapeutics, have developed a circular single stranded DNA donor (ssDNA) that enables kilobase-scale integration while remaining non-toxic to cells.
The technology, named integration through nucleus-synthesized template addition of large lengths (INSTALL), provides an alternative to double-stranded DNA (dsDNA) donors that actively evoke harmful immune responses, yet are required for recognition by the diverse suite of genome editing enzymes, including recombinases. Notably, INSTALL maintains recombinase compatibility by attaching a short region of dsDNA, whose length can go undetected by the cytosolic DNA sensor and immune system activator, cGas.
INSTALL results showed successful and safe non-viral insertion of large genetic payloads in the livers of mice when delivered by lipid nanoparticles (LNPs). In contrast, mice experienced fatal immune reactions when receiving conventional dsDNA molecules.
When asked about the origins of the technology, Connor Tou, PhD, postdoctoral fellow in the Kleinstiver lab and lead author of the study, recalls observing how the hurdle of DNA innate immunity to the donor seemed to be less of a focus in efforts to scale gene insertion, despite the emergence of new recombinase and transposase technologies.
“We knew ssDNA could evade key immune sensors in the cytosol, but it was fundamentally incompatible with recombinases,” Tou told GEN. “The ‘aha’ moment came when we realized nature had already developed a potential solution: many phages and bacteria naturally use circular ssDNA for insertion. We just had to recreate those mechanisms in mammalian cells.”
Going non-viral
The reliance of recombinases on dsDNA donors has traditionally favored viral delivery systems, such as adeno-associated viruses (AAVs), which traffic directly to the nucleus and avoid cytosolic immune sensing. However, AAV vectors are limited by high manufacturing costs, restricted cargo capacity, and the inability to redose due to acquired vector immunity, driving interest in non-viral delivery approaches for large gene insertion.
INSTALL represents the first demonstration of non-viral and non-toxic large sequence insertion in primary human cells and mice using LNPs. However, less than 1% of mouse liver cells showed successful DNA integration. Although low levels of correction could be beneficial for some diseases, most disorders require single or double digit efficiencies to benefit patients
John Finn, PhD, CSO at Basecamp Research, affirms that the study is a “big advance” when addressing the key issue of dsDNA toxicity in genetic medicines. Yet, he does not anticipate AAVs to be overthrown in the near-term future. Downstream challenges, such as effective delivery to the nucleus of non-dividing cells, remain a bottleneck for translation.
“Almost every single cell we care about in our bodies, including brain, liver, and muscle cells, are mostly non-dividing,” Finn told GEN. “Until that’s been solved, I don’t see a big impact in the clinic.”
Basecamp Research has been an active player leveraging AI to augment gene editing technologies. Earlier this year, the company unveiled the EDEN family of AI models trained on 9.7 trillion tokens of evolutionary data to enable the design of programmable recombinases for large gene insertion.
Kleinstiver concurs that improving the efficiency rate is a key future direction of the work. While the study uses standard LNPs designed for delivering mRNA, the vehicle can be optimized for carrying the new circular ssDNA donor. He also highlights that an under-explored space is engineering the nucleic acid component of recombinases for new functionalities. Integration efficiency may be further improved by adding chemical modifications that assist delivery to the nucleus to the short oligo annealed to the circular single stranded DNA, among other approaches.
Tou acknowledges that the study provides an initial proof-of-concept with further work needed before the approach can reach the clinic.
“We’re optimistic that next-generation INSTALL systems, combined with continued engineering of recombinase proteins, will have translational utility for mutation-agnostic genome editing and other applications,” he says.
