Tailor-Made DNA Plasmids For Viral-Vector Manufacturing

The viral vectors used for gene delivery are generally produced via transfection of several plasmid DNA molecules, one of which contains the gene of interest. The actual number depends on the type of viral vector being produced. Adeno-associated viral (AAV) vectors, which are widely used for in vivo gene therapies, typically require three plasmids, while lentiviral (LV) vectors, which are most often used for the production of CAR T-cell therapies, require four.

The specific requirements of each plasmid DNA depend on the type of viral vector and the gene of interest to be delivered. Indeed, the structure and properties of each plasmid DNA can have a direct impact on not only the transfection yield, but also on the quality of the viral particles produced. 

The conventional approach to plasmid DNA design and construction, however, is not amenable to fine-tuning the structure and properties of these molecules. A regular restriction ligation is inserted into a pre-existing plasmid with a backbone that cannot be easily modified. Often an optimum molecule is not obtained, and the entire process must be repeated to see if insertion at a different location may yield a plasmid with better properties.

To overcome these significant limitations, a new method has been developed for the design and construction of plasmid DNA from scratch. The sequence for the gene of interest is combined with the various required functions of the plasmid, and all of these components are assembled together in one step.