Minicircles

Minicircles are small circular DNA molecules derived from plasmids that lack the bacterial backbone used in traditional plasmid vectors. They primarily carry a transgene expression cassette, including a promoter and regulatory elements, while the bacterial origin of replication and antibiotic resistance genes are removed to create a compact, largely nonbacterial vector.

Production typically uses a two-plasmid system in which a parental plasmid containing the expression cassette is designed with recombination sites flanking the bacterial sequences, and a helper plasmid expresses a site-specific recombinase. Upon induction, the bacterial backbone is excised, and the resulting minicircle DNA is isolated. Minicircles are usually supercoiled and range in size from about 2 to 6 kilobases, though larger constructs are used in some cases.

Advantages include reduced CpG motifs and bacterial sequences, which can lessen innate immune activation and transcriptional silencing. The compact size can improve transfection efficiency and in vivo transgene expression, yielding higher and more durable expression than conventional plasmids in many settings.

Applications span gene therapy research and vaccine development, where minicircles are used to deliver therapeutic genes or DNA-encoded antigens with the aim of achieving sustained expression and lower inflammatory responses. They have been explored for liver and muscle delivery, ocular applications, and cancer immunotherapy.

Challenges include scalable production and purification, ensuring complete removal of backbone sequences, and regulatory considerations tied to the recombination steps and residual sequences. Ongoing optimization of production workflows, delivery methods, and safety assessments continues to shape their development as research and therapeutic tools.