The bacteriophage PY54 Tel/pal recombinase system has been exploited as a novel platform for the efficient production of linear covalently closed (LCC) DNA minivectors, termed DNA ministrings, to serve as safe and effective DNA delivery vectors. This work aims to construct efficient and scalable processes to generate DNA vectors in vivo as simplified, scalable and optimized strategies for vector production. In characterizing these DNA vectors, we compared them in safety and effectiveness to conventional DNA delivery vectors. This work has culminated in the development of the most efficient in vivo LCC DNA vector production system to date, and we continue to optimize the efficiency and scalability of the production system [Nafissi et al. 2014. Mol Ther Nucleic Acids 3: e165]. Our modified and optimized DNA ministrings have proven to be the most effective vectors for transgene delivery, demonstrating superior cellular trafficking, an efficiency of transfection that rivals adenoviral vector transduction and gene expression. In addition, our vector offers a superior safety and bioavailability profile relative to conventional vectors due to its minimal cistron composition that removes unwanted immunostimulatory prokaryotic sequences, and unlike its circular counterpart, protects against potentially malignant vector integration events. The patent describing this technology has a filing date of April 24, 2014.

 Figure 1. Ministring Production System: Under thermal repression (30°C) The engineered Eschericia Coli (W3NN) processes the parent plasmid (pNN9) into DNA ministrings. At induced conditions (42°C) the engineered E.coli cells lead to the production of linear covalently closed (LCC) DNA ministrings by Tel protelomerase activity on its target site, encoded within two Super Sequence (SS) sites on pNN9.

 Figure 1. Ministring Production System: Under thermal repression (30°C) The engineered Eschericia Coli (W3NN) processes the parent plasmid (pNN9) into DNA ministrings. At induced conditions (42°C) the engineered E.coli cells lead to the production of linear covalently closed (LCC) DNA ministrings by Tel protelomerase activity on its target site, encoded within two Super Sequence (SS) sites on pNN9.

Learn more about how to employ DNA ministrings for a variety of gene delivery and therapeutic applications.