Backbone-free synthetic DNA supports safer and more compliant AAV production
Adeno-associated virus (AAV) has become a leading vector for gene delivery and has been employed in eight approved gene therapies (1). Recent promising results in clinical trials for Huntington’s disease have once again reignited excitement for this technology and further underscore the potential of AAV-based treatments. However, just a few months before, patient deaths in Sarepta Therapeutics’ muscular dystrophy trials highlighted the critical need to address safety, placing it at the forefront of development and regulatory considerations (2). Of particular concern is the encapsidation of contaminants and the lack of standardization for the full characterization of final virus batches. In addition, addressing challenges related to scalability, rising manufacturing costs and complex purification processes, will be key to ensuring these therapies achieve widespread clinical impact (3).
AAVs are usually generated in producer cell lines through transfection, typically with three separate plasmid DNA (pDNA) constructs that encode the essential components for virus production. Traditionally, pDNA is produced via recombinant E. coli fermentation and contains backbone sequences, including antibiotic resistance genes and origin of replication to allow their propagation.This process can introduce variability in yield and purity and carries inherent challenges of endotoxin contamination and residual bacterial genomic DNA, all of which require extensive purification and testing. Further, essential sequences such as inverted terminal repeats are prone to recombination in bacterial hosts.
While it is expected that only the full-length genome sequence, i.e. the expression cassette flanked by ITR, should be encapsidated, recent advances in sequencing technologies have revealed that sequences derived from the plasmid backbone, can be aberrantly incorporated into the rAAV particles and account for up to 3-4% of the total reads aligning to encapsidated DNA. (4) Not only does this pose potential safety risks to patients, but the lack of standardized tests for their quantification is a key regulatory concern. Emphasizing the importance of identifying DNA impurities, a recent study correlated cross-packaged material with inflammation of the brain in non-human primates. (5)
But what if DNA could be produced at scale without bacterial backbone sequences? This is precisely what’s offered by synthetic, rolling-circle amplification-based DNA platforms. Using an entirely enzymatic cell-free approach, 4basebio amplifies DNA at scale (50mg to 10g at GMP-grade) from circular backbone-free templates, meaning the final product contains only the sequence of interest with limited non-coding sequences.Importantly, nanopore sequencing of rAAV produced from 4bb synthetic DNA has demonstrated that less than 0.2% of total reads aligned to bacterial backbone sequences, compared to 4.4% for the rAAV produced from pDNA. This was confirmed with a qPCR method where backbone sequences could not be detected at all in rAAV produced from hpDNA templates. Simply removing the possibility for the incorporation of unwanted sequences in this way should lead to safer therapies for patients and facilitate their pathway through regulatory approval.
Safety isn’t the only way synthetic DNA can enable the progression of AAV vectors to clinic. Manufacturing viral vectors is not only technically complex but also expensive, with high cost-of-goods driven in part by pDNA. GMP-grade pDNA production can exceed $500,000 for a single 500 L batch, reflecting both the scale of material required and the resource-intensive nature of bacterial fermentation (6). By contrast, enzymatic synthesis methods are less labor-intensive, have reduced process volumes and waste streams and do not require master cell banking, all directly translating to a lower cost of goods in AAV manufacture. Further, the absence of bacterial backbone enables less DNA and transfection reagent to achieve comparable viral titers to pDNA (6), which is approximately a 30% reduction in mass, depending on sequence length.
Ensuring the safety of AAV vectors is critical to the continued progress and acceptance of gene therapy. While pDNA has served as a cornerstone of vector manufacturing, its bacterial origins introduce risks that are increasingly harder to overlook with modern safety and regulatory expectations. Synthetic DNA provides a robust alternative, free from bacterial sequences and associated contaminants, delivering a higher level of purity and consistency.
References:
Zhao, Q., Peng, H., Ma, Y., Yuan, H., & Jiang, H. (2025). In vivo applications and toxicities of AAV-based gene therapies in rare diseases. Orphanet Journal of Rare Diseases, 20(1). https://doi.org/10.1186/s13023-025-03893-z
Bilodeau, K. (2025, March 31). A recent gene therapy death shines a light on Aav Safety. PharmaVoice. https://www.pharmavoice.com/news/sarepta-elevidys-gene-therapy-duchenne-death-aav-safety/743824/
Srivastava, A., Mallela, K. M. G., Deorkar, N., & Brophy, G. (2021). Manufacturing challenges and rational formulation development for AAV viral vectors. Journal of Pharmaceutical Sciences, 110(7), 2609–2624. https://doi.org/10.1016/j.xphs.2021.03.024
Jen, H.I., Wilkinson, P., Lu, X., & Zhang, W. (2025). Residual DNA impurities in AAV vectors-nature and transcription. Molecular Therapy Methods & Clinical Development, 33(3), 101503. https://doi.org/10.1016/j.omtm.2025.101503
Keiser, M. S., Ranum, P. T., Yrigollen, C. M., Carrell, E. M., Smith, G. R., Muehlmatt, A. L., Chen, Y. H., Stein, J. M., Wolf, R. L., Radaelli, E., Lucas, T. J., Gonzalez-Alegre, P., & Davidson, B. L. (2021a). Toxicity after AAV delivery of rnai expression constructs into Nonhuman Primate Brain. Nature Medicine, 27(11), 1982–1989. https://doi.org/10.1038/s41591-021-01522-3
D’Costa, S. (2025, September 2). Transformative Advances in Viral Vector Manufacturing: Unlocking Commercial Scalability, Consistency, and Cost-Effectiveness. Pharma’ Almanac. https://www.pharmasalmanac.com/articles/transformative-advances-in-viral-vector-manufacturing-unlocking-commercial-scalability-consistency-and-cost-effectiveness?utm_content=346931216&utm_medium=social&utm_source=linkedin&hss_channel=lcp-80288380