Putting DNA in stealth mode: an INSTALL approach for non-toxic gene delivery
A new approach for delivering DNA into cells has demonstrated non-toxic delivery and integration, bypassing innate immune detection.
A research team from Mass General Brigham (MA, USA) has unveiled a novel approach for inserting gene-sized DNA into cells, addressing a key challenge in DNA delivery and integration for gene editing-based therapies. By harnessing the natural mechanism of integrative prokaryotic viruses, the study demonstrated the non-toxic, enhanced delivery and integration of DNA into cells using their “INSTALL” approach. Could this innovative approach pave the way for developing effective, non-viral delivery methods for gene editing-based therapies?
Gene editing is a promising avenue for addressing or correcting the genetic mutations underlying many disorders. However, scaling gene editing-based therapies can be challenging – among the key challenges is the diversity of mutations responsible for these conditions, for there may be dozens or even thousands of unique mutations causing a disorder.
Designing new gene delivery systems
A project has been launched to engineer and test libraries of optimized gene therapy vectors designed to improve delivery of large DNA payloads.
Most gene therapy approaches rely on various modes of DNA delivery. One method involves utilizing recombinase enzymes to insert large, corrective DNA sequences into a patient’s cells. This method typically uses double-stranded DNA molecules to deliver the corrective sequences; however, as effective as this mode of delivery is, there is a risk of triggering strong immune reactions that cause toxicity and can limit dosages. Another method uses viral vectors to deliver DNA corrections into the nucleus, but these methods have high development and manufacturing costs, as well as safety concerns related to potential toxigenicity.
These limitations highlight the need for a virus-free method of DNA integration – one that is safer and more cost-effective at scale. But, if foreign double-stranded DNA causes an immune response within the body and viral vectors also have a risk of unwanted toxigenicity, what is the next approach?
The team recognized that circular, single-stranded DNA molecules were largely able to evade immune detection. Integrative prokaryotic viruses use a similar mechanism, having developed tricks to insert single-stranded DNA into a double-stranded DNA host genome using recombinases. The team “wondered whether these mechanisms could be recapitulated in human cells, hoping to solve the innate immunity challenge that has impeded efficient gene insertion,” according to lead author Connor Tou.
The team designed DNA circles primarily composed of a single strand of DNA that could remain “stealthy”, avoiding innate immune detection. Within this DNA circle, they added a small region of two DNA strands, an essential factor for compatibility with recombinases. The team ensured that the double-stranded DNA was long enough for recombinase function, but short enough to avoid innate immune detection. They called this approach INSTALL.
The team demonstrated that the INSTALL approach could enable non-toxic integration in multiple human cell types. Additionally, in in vivo experiments, the researchers used lipid nanoparticles to deliver the DNA circles designed with the INSTALL method into the livers of mice. They found that the approach successfully and safely inserted large genetic payloads – the opposite is usually observed when conventional double-stranded DNA molecules are used.
“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,” explained senior author Benjamin P Kleinstiver. “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.”
Looking ahead, the team plans to focus on enhancing the DNA cargo and advancing recombinase engineering, paving the way for significant progress in large sequence insertion technology.
