Synthetic biology has the potential to upend existing paradigms of adeno-associated virus (AAV) production, helping to reduce the high costs of gene therapy and thus make it more accessible, according to a recent paper.

AAVs are an important vector for gene therapy, but AAV manufacturing is complex and expensive. Furthermore, first author Logan Thrasher Collins, a PhD candidate at Washington University in Saint Louis, tells GEN. “Many current industry approaches to enhancing AAV yields involve incremental process optimization. Synthetic biology has the potential to offer more radical improvements, yet is relatively underappreciated in the context of AAV production.”

Large-scale production poses challenges not typically found during preclinical stages, such as batch-to-batch variations in plasmid yield and purity, and poor yields from producer cells, the research team notes. Likewise, downstream processing challenges also are present, such as AAV aggregation, chemical lysis, and filtration complications. The rational approach to AAV design offered by synthetic biology, however, enables scientists to programmably design systems that assemble complex macromolecular structures and to avoid—or at least minimize—many of those challenges.

While several synthetic biology methods are available, Collins says tetracycline-enabled self-silencing adenovirus (TESSA) is among the most promising. “It uses a helper adenovirus equipped with a clever genetic circuit to prevent helper virus contamination while producing numerous high-potency AAVs. TESSA can produce 30-fold more AAVs than triple transfection methods.”

Doggybone DNA

Another up-and-coming technology is doggybone DNA (dbDNA). “It consists of linear double-stranded DNA that is covalently closed at its ends,” the researchers write. It can produce gram-scale quantities of GMP-grade DNA for AAV manufacturing, making it a contender to replace plasmid approaches.

The convergence of synthetic biology and AAV production is in its early days, but appears to offer multiple, diverse commercialization opportunities. For example, it could result in cellular chassis circuits that give host cells new functions; ways to induce formation of large multinucleate cellular syncytia as high-volume/high-density AAV nanofactories; or yield rational protein engineering strategies for scalable column-based purification of AAVs regardless of serotype.

“Cultivating innovative, high-risk/high-reward projects in this field will generate more opportunities for industry to acquire and scale up transformative technologies,” Collins says. “While we cannot predict the future, we think a system that produces AAVs a thousand times more efficiently than can be achieved today is a reasonable possibility through synthetic biology.”

First, however, “Greater communication and collaboration is vital between industry and academia,” Collins says, “to help academics gain a more nuanced understanding of existing AAV manufacturing pipelines and pain points,” and thus better address the challenges facing the AAV manufacturing sector.

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