Jill Makin, Ph.D., chief scientific officer, Touchlight highlights how emerging DNA formats are shaping the future of biopharma.
Touchlight
Plasmid DNA (pDNA) has been the foundational starting material for genetic medicines for more than 20 years. It has powered the rise of viral vectors, underpinned the first wave of gene therapies, and supported the rapid expansion of mRNA and engineered cell therapy platforms. But as genetic medicines become more complex, potent, and are employed in more regulatory-sensitive modalities, the limitations of plasmid-based manufacturing are becoming increasingly apparent.
At the same time, a new generation of cell-free DNA formats, including enzymatically produced double-stranded and single-stranded DNA constructs, has matured into credible and scalable alternatives. These emerging formats represent a fundamental shift in how the industry sources, designs, and deploys DNA for advanced therapeutics.
Together, these trends point to a future in which plasmids are no longer the default choice, but one option among many, and increasingly, they may not be the most suitable one.
The plasmid problem?
Plasmid DNA has served the industry well, but its biological origins impose constraints that are becoming more pronounced as therapeutic demands evolve. Firstly, plasmids must be propagated in E. coli, which introduces sequence-dependent challenges. Large constructs, repetitive elements, inverted repeats, and GC-rich regions can destabilise plasmids, leading to heterogeneous populations, reduced yields, or complete batch failure. As genetic medicines increasingly rely on larger and more complex payloads, these issues become more acute.
Even under optimised conditions, fermentation introduces variability in plasmid copy number, host strain performance, metabolic burden, and growth phase dynamics. This variability can translate into inconsistent yields and unpredictable timelines, an unwelcome reality for programmes operating under compressed development schedules.
Plasmid production generates a wide range of host-derived impurities, including endotoxins, host cell DNA and proteins, RNA, and residual genomic fragments, including antibiotic resistance genes. Removing these to meet regulatory expectations requires multi-step purification trains that increase cost, time, and batch-to-batch variability.
Additionally, fermentation does not scale linearly. Oxygen transfer, metabolic stress, and bioreactor performance can significantly influence both yield and quality. As dose requirements rise, particularly for in vivo gene therapies, these scale-up risks become harder to absorb.
These constraints make plasmid manufacturing increasingly misaligned with the needs of modern genetic medicines.
A cell-free solution
One way to overcome the limitations of E. coli–based production is to remove cells from the equation entirely. Enzymatic, cell-free DNA contains no bacterial backbone sequences and is manufactured using recombinant enzymes to amplify DNA in a defined, linearly scalable process that avoids the biological variability of fermentation. Since no cells are used, enzymatic DNA contains no endotoxins, no host cell DNA or proteins, no antibiotic resistance genes, and no bacterial backbone sequences. This dramatically simplifies purification and reduces the risk of residual contaminants. This is becoming an increasingly important consideration as the field gains a deeper understanding of how DNA-related impurities influence both the safety and potency of cell and gene therapies.
Unlike plasmids, enzymatic DNA is not constrained by bacterial propagation. GC content, repeats, and structural motifs have minimal impact on yield. Developers can design constructs that would be unstable or impossible to produce in E. coli, enabling new architectures for gene editing, non-viral delivery, and next-generation RNA therapeutics.
Enzymatic reactions scale proportionally with reaction volume and input materials. This allows seamless progression from milligram-scale R&D batches to multi-gram GMP production using the same core process, which is an advantage that fermentation cannot match.
Beyond double‑stranded DNA: The rise of new DNA formats
The shift away from plasmids is not only about replacing fermentation with enzymatic amplification. A deeper transformation is underway as developers begin to rethink the architecture of DNA itself. Modern genetic medicines increasingly require formats that are structurally flexible, biologically compatible, and purpose built for specific therapeutic mechanisms. As a result, the field is moving beyond the traditional choice between plasmids and linear double-stranded DNA toward a broader portfolio of engineered structures designed to meet the demands of advanced therapies.
This evolution is driven by the growing complexity of therapeutic design. The rise of various gene editing technologies, in vivo delivery, and multiplexed engineering all place demands on DNA that plasmids were never intended to meet. Developers now need formats that minimise extraneous sequences, avoid innate immune activation, support large or intricate inserts, and perform reliably in sensitive primary cells. They also need the ability to prototype multiple structural variants quickly, something that is difficult to achieve when relying on bacterial propagation.
One of the most significant advances in this space is Touchlight’s Megabulb DNA (mbDNA), a circular single-stranded DNA construct engineered specifically for homology-directed repair (HDR). Departing entirely from plasmid architecture, it is free from bacterial sequences, and produced through cell-free methods. Its circular structure enhances stability, while its single-stranded nature reduces the innate immune responses that often accompany double-stranded DNA delivery. What distinguishes it further is its dual-domain design: a single-stranded “bulb” containing the gene of interest and homology arms, paired with a short double-stranded stem that includes the nuclease target site. This configuration allows the nuclease to bind and cleave the stem, remain associated with the DNA, and carry it into the nucleus via its own localisation signals. Once inside, the nuclease releases a clean single-stranded HDR template precisely aligned for genomic insertion.
The performance advantages of this engineered construct are striking. In primary T-cell editing, it routinely achieves homology-directed repair (HDR) efficiencies above 50 percent, far exceeding what is typically observed with plasmid donors. It also supports faster cell recovery and expansion, leading to substantially higher yields of edited cells. Plasmid delivery, by contrast, introduces both early electroporation-mediated toxicity and later innate immune activation, which together suppress cell proliferation and dramatically reduce knock-in efficiency. As gene editing moves into more sensitive cell types and more ambitious engineering strategies, these biological differences become increasingly consequential.
A turning point for DNA manufacturing
Taken together, these developments signal a broader shift in how the industry thinks about DNA. Instead of defaulting to formats based on historical convenience or manufacturing familiarity, developers are now choosing DNA based on biological performance and therapeutic fit. High-dose in vivo delivery benefits from utilisation of single-stranded DNA free of bacterial sequences to minimise immunogenicity. Precision editing requires donors that minimise toxicity and maximise HDR. Complex constructs demand architectures that cannot be stably propagated in E. coli. And rapid design–build–test cycles favour cell-free systems that can generate multiple variants without the delays of fermentation.
In this context, plasmids represent a legacy format optimised for an earlier era of genetic medicine. The future is moving toward DNA architectures engineered for purpose: cleaner, more adaptable, and better aligned with the needs of next-generation therapies.