Harnessing synthetic DNA for more efficient and safer AAV manufacturing

Adeno-associated viruses (AAVs) have emerged as one of the most promising vectors for gene therapy, offering efficient and targeted gene delivery with a favorable safety profile. However, traditional AAV manufacturing methods often face challenges related to production efficiency, safety, and regulatory compliance. One of the primary obstacles stems from the reliance on plasmid DNA (pDNA), which is essential to produce AAV vectors but comes with several drawbacks.

Challenges of Plasmid DNA for AAV Manufacturing 

In the early stages of development, when using pDNA for AAV manufacturing, the main challenge is the production of a master cell bank (MCB). It can often be extremely challenging and time consuming, as this step can impact yields and sequence integrity. Furthermore, pDNA manufacturing relies on bacterial fermentation, which often raises safety concerns in AAV manufacturing, due to the presence of a bacterial backbone. This can often lead to unwanted packaging of exogenous sequences, which has a major influence on the efficiency of the final AAV vector.  

The process of large-scale plasmid production is time-consuming and can lead to inconsistencies in yield and purity. As gene therapy developers transition to large scale AAV manufacturing, issues may arise around the quality and concentration needed to progress. Usually, three different plasmids are required for transfection, meaning the amount of pDNA required for AAV production is substantial, which creates further hurdles to overcome. These limitations not only slow down manufacturing but also pose potential safety and regulatory concerns for clinical applications.  

Introducing hpDNA: Ideal construct for AAV Manufacturing 

With the various challenges associated with pDNA manufacturing, there’s a growing need for reliable alternatives for pDNA manufacturing. 4basebio’s synthetic DNA is manufactured using a fully cell-free process, the resulting DNA is free of bacterial sequences. 4basebio’s synthetic DNA platform produces application-specific DNA constructs to suit a number of therapeutic applications. 

hpDNA is ideally suited for viral vector manufacturing, it is a double-stranded, linear construct, which is covalently closed with single strand hairpins at the 5’ and 3’ ends.

Why Synthetic DNA is a Game-Changer for AAV Manufacturing?

1. Reduced Lead Times 

One of the most significant advantages of using synthetic DNA for AAV manufacturing is the drastic reduction in lead times. Traditional plasmid-based approaches require time-consuming processes, which can extend production timelines. Synthetic DNA, on the other hand, is produced enzymatically, which eliminates bacterial fermentation processes. This accelerates the production process, allowing for faster turnaround times in gene therapy development and clinical applications. 

2. Enhanced Safety and Compliance 

Regulatory requirements for gene therapy products continue to evolve, with increasing emphasis on the purity and safety of AAV vectors. Synthetic DNA minimizes the risk of contamination from bacterial endotoxins and antibiotic resistance genes due to the lack of the bacterial backbone.  

3. Equivalent Titers  

Maintaining high and consistent viral titers is essential for the efficacy and scalability of gene therapies. AAV vectors produced using synthetic DNA achieve comparable titers to those produced via plasmid-based methods. Cost reduction is observed, since less DNA is required to achieve comparable titers, due to the linear construct lacking a bacterial backbone.  

4. No Risk of Reverse Packaging  

Reverse packaging, where undesired elements are mistakenly incorporated into the AAV capsid, poses a significant risk with using pDNA. Synthetic DNA eliminates this concern, as there is no bacterial backbone present, ensuring that no reverse packaging occurs.  

5. Enhanced Product Quality  

The integrity of inverted terminal repeats (ITRs) is crucial for maintaining AAV vector stability and function. Plasmid-based methods are prone to recombination events during bacterial fermentation, that can lead to deletions or mutations within the ITRs, negatively impacting vector efficacy. Synthetic DNA offers greater sequence stability and precision, eliminating the risk of recombination and ensuring a higher-quality AAV product.