Cell-free Production of Plasmids for Genetic Medicines

Stanford scientists have developed a cell-free method for producing high-purity plasmid DNA without the use of bacterial hosts. This approach eliminates contamination risks, reduces production time, and streamlines manufacturing for gene and cell therapies.

Cell and gene therapies require GMP-compliant production of plasmid DNA, which traditionally relies on production in E. coli. This bacterial approach faces several critical limitations, including risk of endotoxin contamination, inconsistent yields, complex purification processes, and lengthy production timelines. Moreover, certain therapeutically relevant sequences, such as human mitochondrial DNA, cannot be cloned in bacterial hosts at all. While alternative cell-free methods exist, they require insertion of specific DNA sequences that remain in the final product, compromising plasmid purity and functionality. A scalable method for producing scarless circular plasmid DNA without bacterial hosts could address these manufacturing challenges and unlock new therapeutic possibilities.

The cell-free plasmid production method uses rolling circle amplification and strategic enzymatic processing to generate high-purity circular DNA without bacterial hosts. Importantly, this approach is sequence-independent and produces scarless plasmids without any residual carrier sequences in the final product. The method has successfully produced functional human mitochondrial DNA, demonstrating its ability to generate sequences that are unclonable by conventional bacterial methods. Consequently, cell-free plasmid production has the potential to streamline gene therapy manufacturing, accelerate production timelines, and enable development of previously inaccessible therapeutic targets.

Stage of Development

• Proof of concept
• Continued research: Method optimization to increase yield and scale