Genome editing-based therapies typically aim to treat disease by correcting underlying genetic mutations in patient’s cells. However, most genetic disorders are caused by dozens or even thousands of unique mutations spread across a gene; this diversity results in challenges of scale when it comes to developing customized therapies for many individuals. A more universal strategy could involve the precise insertion of an entire copy of a healthy gene into a specific location in the genome, which could, in principle, provide a single, widely applicable treatment for all patients irrespective of their mutation within that gene.
Taking a step towards developing genetic therapies at scale, researchers from Mass General Brigham and colleagues describe a novel, non-toxic approach for inserting gene-sized DNA in a study published in Nature .
“Overall, this study demonstrates that large-scale genome writing is now possible without triggering dangerous immune responses and can be done independently of viral vectors,” said senior author Benjamin P. Kleinstiver, PhD , Kayden-Lambert MGH Research Scholar associate investigator in the Center for Genomic Medicine at Mass General Brigham. “With this approach, we may be able to move beyond the treatment of single mutations at a time. Our goal is to improve the systems we’ve developed in ways that can broaden their applicability to genetic treatments.”
Most approaches to insert large corrective DNA sequences into the genome utilize recombinase enzymes that rely on double-stranded DNA (dsDNA) molecules carrying the intended DNA cargo, which can trigger strong immune reactions that cause toxicity and ultimately limits how much treatment can be given, especially when therapies are delivered directly into the body. Although viruses can act as vectors to shuttle the DNA cargo into the nucleus, their use is subject to several caveats in terms of cost, safety, and other factors. Developing a virus-free targeted DNA integration method has therefore been a long-standing goal for the genome editing field.
The team recognized that circles comprised of only single DNA strands were largely able to evade immune detection. However, this apparent solution resulted in a different problem: recombinase enzymes evolved to recognize DNA molecules with two strands, making them incompatible with single-stranded DNA.
The team then investigated ways that natural systems had overcome similar problems, and realized that bacteria and bacteriophages had developed tricks to insert single-stranded DNA into a dsDNA host genome using recombinases.
“This was an exciting realization,” said lead author Connor Tou, PhD, a postdoctoral fellow in the Kleinstiver lab. “We then wondered whether these mechanisms could be recapitulated in human cells, hoping to solve the innate immunity challenge that has impeded efficient gene insertion.”
The team designed a different configuration using a DNA circle comprised largely of only a single strand that could remain ‘stealth’ to innate immune sensors, and a short region of two DNA strands to enable compatibility with recombinases. This dsDNA region could be designed to be long enough to allow the recombinase to function, but short enough to avoid immune detection.
The team then demonstrated that their new approach, called INSTALL, could enable non-toxic DNA integration in multiple human cell types. Furthermore, when performing in vivo experiments in mice by delivering the DNA via lipid nanoparticles (LNPs), INSTALL could successfully and safely insert large genetic payloads in the livers of mice, compared to fatal immune reactions observed when conventional dsDNA molecules were used.
"One of the most exciting moments of this study was seeing the stark differences in how well the mice responded to INSTALL compared with the toxicity of dsDNA," Tou noted. "When the INSTALL-treated mice looked similar to untreated controls, we knew this could be a game changer."
The authors are optimistic that continued efforts to improve the DNA cargo, alongside recombinase enzyme engineering, will result in substantial advances in large sequence insertion technologies in the near future.
Authorship: This study was a large collaborative effort, with key intellectual and experimental contributions from Howard Wu and Keqiang Xie, of Full Circles Therapeutics, a company that manufactures and commercializes circular single stranded DNA, as well as developing DNA based non-viral genomic medicines. The laboratory of Patricia Musolino of the Mass General Brigham Center for Genomic Medicine helped to lead DNA integration and immunology studies in mice. The laboratory of Natalie Artzi at the Mass General Brigham Gene and Cell Therapy Institute and the Wyss Institute for Biologically Inspired Engineering led the design and formulation of LNPs for delivery of mRNA and DNA cargos and contributed to the development and execution of the immune profiling strategy for the platform. Additional authors include Joana Ferreira da Silva, Pazhanichamy Kalailingam, Eliz Amar-Lewis, David Rufino-Ramos, William Sawyer, Madeline L. Eller, Jakob Starzy, Ishita Majumdar, Jiao Wang, Danna Lee, Shaobo Yang, Ronald J. Meis, Gary A. Dahl, Jiahe Li, and Richard Shan.
Paper cited: Tou C et al. “Immune evasive DNA donors and recombinases license kilobase-scale writing” Nature DOI: 10.1038/s41586-026-10241-z
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Mass General Brigham is an integrated academic health care system, uniting great minds to solve the hardest problems in medicine for our communities and the world. Mass General Brigham connects a full continuum of care across a system of academic medical centers, community and specialty hospitals, a health insurance plan, physician networks, community health centers, home care, and long-term care services. Mass General Brigham is a nonprofit organization committed to patient care, research, teaching, and service to the community. In addition, Mass General Brigham is one of the nation’s leading biomedical research organizations with several Harvard Medical School teaching hospitals. For more information, please visit massgeneralbrigham.org.
Nature
Experimental study
Cells
Immune evasive DNA donors and recombinases license kilobase-scale writing
11-Mar-2026
Tou and Kleinstiver are listed as inventors on a patent application filed by Mass General Brigham (MGB) that describes INSTALL. Tou, Ferreira da Silva, and Kleinstiver are listed as inventors on additional patents or patent applications filed by MGB that describe genome engineering technologies related to the current study. Xie, Shan, and Wu are listed as inventors on patent applications filed by Full Circles Therapeutics that describe the production and use of circular single stranded DNA (cssDNA). Xie, Starzy, Majumdar, Wang, Lee, Shan, and Wu are current or former employees of Full Circles Therapeutics. Musolino holds scientific research agreements with Angea Biotherapeutics and Minoryx and is a consultant for Ionis, Biogen, Vertex, Bluebird, Atlas Ventures and Inozyme. Patricia Musolino reports grants to Massachusetts General Hospital from the NIH for research on ALD. Kleinstiver is a consultant for Novartis Venture Fund, Generation Bio and Jumble Therapeutics, and is on the scientific advisory boards of Life Edit Therapeutics and Prime Medicine. Kleinstiver has a financial interest in Prime Medicine, a company developing therapeutic CRISPR–Cas technologies for gene editing. Kleinstiver’s interests were reviewed and are managed by MGH and MGB in accordance with their conflict-of-interest policies. The other authors declare no competing interests.