As interest in nonviral gene delivery surges, new advances in lipid nanoparticle (LNP) and electroporation technologies are paving the way for more efficient and safer therapeutic solutions. While viral-vector-based gene therapies face challenges such as adverse immune reactions and limited re-dosing options, synthetic dbDNA from Touchlight offers a promising alternative. With its scalability, flexibility, and improved safety profile, dbDNA is poised to revolutionize the field of nonviral gene therapy, unlocking new therapeutic possibilities.
Renewed Interest in Nonviral Gene Delivery
Delivering DNA directly into cells has always been challenging, primarily because of its large size and strong negative charge, which makes it difficult for DNA constructs to cross cellular membranes. Early attempts at nonviral delivery, which relied on physical and chemical methods, faced significant limitations in terms of efficiency, durability, and safety.
However, recent breakthroughs, particularly with LNP technology, have renewed interest in nonviral gene delivery. LNPs were instrumental in the success of mRNA vaccines against COVID-19, demonstrating their potential for nucleic acid delivery, and they are now being explored for DNA-based therapies. Simultaneously, challenges associated with viral vectors like adeno-associated viruses (AAVs) have become more apparent as in vivo gene therapies move further through clinical development. Specifically, issues such as the inability to re-dose patients who develop antibodies to AAVs, risks of adverse immune reactions when high or multiple doses are required, and the high cost and complexity of producing large quantities of viral vectors are driving researchers to reconsider nonviral alternatives.
These converging factors have sparked a resurgence of interest in nonviral gene delivery platforms, which could offer safer and more flexible therapeutic options. While AAV vectors are highly efficient and known for their long-lasting effects due to the episomal stabilization and concatemerization of genomes, they face significant challenges with anti-vector immunity, limiting the ability to re-dose. In contrast, nonviral delivery systems may require repeated dosing to maintain therapeutic levels, but they provide the advantage of being re-dosable without triggering the same immune response. An ideal nonviral delivery system would efficiently transport genetic material into the nucleus, enable durable gene expression, and be well-tolerated across multiple doses, offering a potential solution to some of the key limitations faced by AAV-based therapies.
Both AAV developers and nonviral gene therapy companies are actively investigating these approaches. Beyond LNPs, technologies like electroporation are also being optimized, while a growing focus is being placed on alternatives to plasmid DNA (pDNA), which is typically produced via bacterial fermentation. This conventional pDNA production process has several drawbacks, including safety concerns linked to bacterial sequences and difficulties scaling up. Consequently, researchers are exploring every aspect of nonviral delivery — from improved formulations to advanced DNA constructs like synthetic dbDNA (doggybone™ DNA) — aiming to enhance the tolerability, safety, and functionality of these therapies.
Formulation Innovations Leading the Nonviral Revolution
Current efforts to advance nonviral gene delivery are predominantly focused on formulation-based strategies. LNPs have emerged as the leading delivery technology, having already secured regulatory approval for RNA-based vaccines and therapies. Interestingly, some LNPs were originally developed for DNA delivery before being adapted for RNA applications. However, many newer players in the field focused initially on RNA and are now adapting these LNPs for DNA delivery. Additionally, researchers are exploring other promising solutions, including polymeric nanoparticles, lipid–-polymer hybrids, and various encapsulation techniques designed to safely and efficiently deliver genetic material into target cells.
Early nonviral formulations often accumulate in the liver when delivered systemically, which is effective for certain therapeutic targets. However, there is growing interest in refining these systems to achieve more precise targeting of specific cells, tissues, or organs. The challenge lies in identifying the optimal formulations for different types of DNA and ensuring they reach their intended destinations effectively and consistently.
While formulation-based approaches remain the front-runner, alternative delivery technologies are also gaining attention. Notably, sonoporation, which uses ultrasound to temporarily disrupt cell membranes, and low-voltage electroporation, which minimizes cell damage while facilitating DNA entry, show considerable promise in improving nonviral gene delivery outcomes.
Why dbDNA is the Future of Scalable Genetic Medicine
The growing demand for genetic medicines has exposed a significant bottleneck: shortcomings associated with the supply of pDNA, which is produced through a time-consuming and costly bacterial fermentation process that faces serious scalability issues. Furthermore, most pDNA contains unwanted elements like antibiotic-resistance genes and bacterial sequences. These not only raise safety and quality concerns but also lead to larger, less efficient genetic constructs.
In contrast, Touchlight’s dbDNA is created synthetically through an enzymatic process that is highly optimized for both scalability and the production of complex sequences. dbDNA is a double-stranded, linear, covalently closed DNA construct, produced via rolling circle amplification from a circular template.1 Crucially, it contains no bacterial sequences or antibiotic-resistance genes, and can accommodate genes of interest up to 30 kilobases in size.
Another key advantage of dbDNA is its small manufacturing footprint. Processes conducted at scales below 5 liters can yield equivalent quantities to what would require fermentation batches exceeding 50 liters — sometimes even up to 300 liters — using conventional methods. The enzymatic amplification process scales linearly, reduces biological waste, and maintains high fidelity, even for complex sequences that would be difficult to produce through traditional bacterial fermentation.
dbDNA is available in a range of scales — Catalogue, Discovery, Research, Smart-GMP, and GMP grades — supporting everything from small-scale experimental work to large commercial programs. In 2022, Touchlight’s Drug Master File (DMF) was accepted by the U.S. Food and Drug Administration (FDA), and dbDNA is currently being used in investigational new drug (IND) applications and clinical trial applications (CTAs) across a variety of modalities in the United States and Europe, spanning phase I and II trials. Additionally, a pivotal trial is anticipated for 2025, further advancing the clinical validation of dbDNA.
Enhanced Therapeutic Windows with dbDNA in Nonviral Gene Therapy
Beyond its general benefits over pDNA, dbDNA has demonstrated substantial potential as an alternative specifically for nonviral gene therapy, whether it’s formulated into LNPs or delivered via low-voltage electroporation. Its smaller size — typically 2 to 3 kilobases smaller than pDNA — enables the creation of more compact, uniform LNPs without compromising encapsulation efficiency, a critical advantage over pDNA-based formulations
The structural simplicity of dbDNA, along with its lack of a bacterial backbone and CpG islands, supports the incorporation of larger and more complex genetic payloads, significantly expanding the therapeutic possibilities for nonviral gene therapies. This absence of immunogenic bacterial sequences contributes to improved safety and reduced risk of immune reactions, making dbDNA an attractive option for advancing nonviral delivery systems. dbDNA also exhibits dose-sparing capabilities, with higher potency and improved durability compared with matched plasmid constructs. This provides a better balance between tolerability and therapeutic effect, effectively broadening the therapeutic window — a vital consideration in nonviral delivery approaches.
Preclinical studies have consistently shown that dbDNA enables sustained gene expression, while initial observations suggest it may also reduce inflammatory cytokine production and liver injury markers, further reinforcing its potential clinical relevance in nonviral systems. These advantages make dbDNA an attractive solution for advancing the safety, efficiency, and durability of nonviral gene therapy applications.
Superior Gene Expression and Safety with dbDNA LNPs
Touchlight, in collaboration with several partners, has extensively investigated the performance of dbDNA formulated into LNPs and polymeric nanoparticles. In one internal study, two off-the-shelf dbDNA constructs were formulated as LNPs using a lipid composition originally optimized for pDNA, with only slight adjustments made to accommodate the linear structure of dbDNA. These were then compared to a client-supplied, optimised pDNA–LNP formulation.
The results were compelling: dbDNA formulated efficiently as LNPs, demonstrating comparable encapsulation efficiency to that of the optimized pDNA–LNP, despite the fact that minimal dbDNA-specific lipid optimization was performed. In addition, dbDNA LNPs were smaller and exhibited superior size uniformity compared with the pDNA–LNPs, even while encapsulating a larger DNA payload (Figure 1).
Figure 1. dbDNA formulates well in LNPs compared with plasmid. Physicochemical characterization of DNA–LNPs. dbDNA, parental plasmid DNA, and customer control circular DNA compared.
In vivo studies demonstrated the advantages of these differences versus formulated plasmid. Both dbDNA constructs, featuring different promoters, showed improved gene expression over the matched pDNA–LNP formulation (Figure 2). Notably, dbDNA constructs also induced lower levels of inflammatory cytokines and liver injury markers compared with pDNA–LNPs, a critical advantage given the known inflammatory potential of nucleic acid–LNP systems (Figure 3). The dose-sparing properties of dbDNA add additional value, reinforcing its potential for safer and more efficient nonviral gene delivery.
Figure 2. Improved in vivo expression of dbDNA. DNA-LNPs administered via tail vein (at 0.3 mg/kg and 0.1 mg/kg) to seven-week-old female CD-1-IGS mice. Luminescence measured periodically by IVIS.
Figure 3. dbDNA shows lower inflammatory cytokine & liver injury markers. Pro-inflammatory cytokines and liver injury markers assessed four hours post tail vein administration of DNA–LNPs.
Electroporation: Enhanced Delivery with dbDNA
Low-voltage electroporation has emerged as a promising method for nonviral gene delivery, allowing DNA to enter cells with minimal damage compared with traditional electroporation techniques. Touchlight conducted a study comparing the performance of dbDNA and pDNA delivered using a low-voltage electroporation device (MYOÔ electroporation device), with funding support from the Bill & Melinda Gates Foundation. Naked DNA constructs expressing an anti-flu haemagglutinin antibody were administered to mice and serum antibody levels monitored for eight weeks.
The results were supportive and similar to other data generated in non-viral gene therapy: dbDNA exhibited superior in vivo expression levels compared with pDNA, clearly underscoring the advantages of dbDNA over pDNA when using electroporation for delivery. dbDNA’s ability to achieve strong gene expression by multiple delivery methodologies highlights its potential for broader use in nonviral gene therapies (Figure 4).
Figure 4. Non-viral delivery using intramuscular injection (IM) / electroporation (EP) device. 25 µg of dbDNA or pDNA encoding the human anti-flu haemagglutinin 2-12C monoclonal antibody was administered intramuscularly using the MYOÔ device to SCID mice (MYOÔ, www.RenBio.com). “TL” architecture utilised a muscle-specific promoter; “RB” used a promoter active in most cell types. Serum antibody titres were evaluated periodically using ELISA. Disclaimer: This publication is based on research funded in part by the Bill & Melinda Gates Foundation. The findings and conclusions contained within are those of the authors and do not necessarily reflect positions or policies of the Bill & Melinda Gates Foundation.
Collaborating to Optimize dbDNA Delivery
To enhance the awareness and adoption of dbDNA across diverse applications, Touchlight has adopted a strategic approach — partnering with leading companies in the field of nonviral gene delivery. A key focus is on collaborations with prominent LNP manufacturers and manufacturers of physical delivery devices, such as low voltage electroporation. Current partnerships include well-established players, with more collaborations in the pipeline.
These partnerships are essential for advancing the understanding of critical process parameters required for optimizing LNPs that contain linear dbDNA, as compared with supercoiled pDNA. The primary variables under investigation include the choice of ionizable lipids, the ratio of the different lipid types, and, most importantly, the ratio of lipids to DNA. The objective is twofold: to generate robust data that can be shared with customers and to ensure that Touchlight can confidently recommend formulation partners who can help clients optimize their nonviral delivery systems.
Unlocking Additional Opportunities for Optimisation
As a relatively young field, nonviral gene delivery presents many opportunities for optimization. Significant improvements can be made not only in the development of more effective delivery methods and targeted therapies but also in customizing the DNA sequence. While this flexibility is also true for pDNA, dbDNA offers unique advantages, particularly in terms of copy number, performance, and durability. Thanks to the enzymatic process used to produce dbDNA, there is exceptional flexibility in modifying both the therapeutic transgene sequence and the regulatory elements that influence key performance factors, such as intracellular transport to the nucleus and overall expression efficiency. Touchlight’s extensive experience in this area enables us to guide clients through this customization process — whether they are already experts or require more tailored support.
Additionally, Touchlight continues to develop new technologies designed to improve properties, such as nuclear uptake and nucleotide modification, further enhancing the potential of Touchlight’s enzymatically synthesized DNA for applications in nonviral delivery systems.
To foster further innovation, Touchlight is also making research-grade dbDNA readily available at an affordable cost, offering small quantities suitable for screening experiments and candidate selection. This accessibility opens the door for further exploration of tailored sequences, enabling continued innovation in nonviral gene therapy.
Nonviral Gene Delivery is Coming of Age: What’s Next?
Nonviral gene delivery is still in its early stages, but the field is expanding rapidly with a range of innovative methods under development. These include non-targeted LNPs, LNPs formulated with proprietary lipids designed for targeted delivery, polymeric nanoparticles, lipid–polymer hybrids, and other chemical encapsulation techniques. Researchers are also exploring physical delivery methods and advanced targeting strategies that use peptide or monoclonal antibody (mAb) fragments to achieve higher specificity. Significant advances are anticipated, particularly in improving the tolerability of DNA–LNP formulations. These developments will move the industry beyond basic LNPs to more refined, targeted approaches, with further insights into optimal DNA topologies for specific tissues expected.
While viral-vector-based delivery will remain commonplace for in vitro cell modification and in vivo gene therapy, there has been a steady increase in the number of candidates using nonviral delivery entering clinical trials — particularly for larger transgenes, where conventional AAV vectors are too small, and the complex split-AAV approaches face significant challenges. Nonviral delivery has strong potential to address these limitations, and Touchlight values working with clients exploring these innovative solutions. Both large pharmaceutical companies and smaller biotech firms are heavily investing in this space, working to optimize all facets of nonviral DNA delivery. Tailored dbDNA, with its unique advantages, is at the forefront of these efforts.
With clinical trials underway and two nonviral gene therapies (neovasculgen and beperminogene perplasmid) already receiving regulatory approval, Touchlight anticipates that as more candidates successfully navigate the approval process, the floodgates for nonviral gene delivery will truly open. Touchlight is eager to collaborate with DNA therapy developers and nonviral delivery specialists to bring these groundbreaking therapies to patients as quickly as possible.
