BIOPHYSICS MEETS GENE THERAPY: HOW EXPLORING SUPERCOILING-DEPENDENT STRUCTURAL CHANGES IN DNA LED TO THE DEVELOPMENT...

Supercoiling affects every aspect of DNA function (replication, transcription, repair, recombination, etc.), yet the vast majority of studies on DNA and crystal structures of the molecule utilize short linear duplex DNA, which cannot be supercoiled. To study how supercoiling drives DNA biology, we developed and patented methods to make milligram quantities of tiny supercoiled circles of DNA called minicircles. We used a collaborative and multidisciplinary approach, including computational simulations (both atomistic and coarse-grained), biochemical experimentation, and biophysical methods to study these minicircles. By determining the three-dimensional conformations of individual supercoiled DNA minicircles, we revealed the structural diversity of supercoiled DNA and its highly dynamic nature. We uncovered profound structural changes, including sequence-specific base-flipping (where the DNA base flips out into the solvent), bending, and denaturing in negatively supercoiled minicircles. Counterintuitively, exposed DNA bases emerged in the positively supercoiled minicircles, which may result from inside-out DNA (Pauling-like, or "P-DNA"). These structural changes strongly influence how enzymes interact with or act on DNA. We hypothesized that, because of their small size and lack of bacterial sequences, these small supercoiled DNA circles may be efficient at delivering DNA into cells for gene therapy applications. "Minivectors," as we named them for this application, have proven to have therapeutic potential. We discovered that minivectors efficiently transfect a wide range of cell types, including many clinically important cell lines that are refractory to transfection with conventional plasmid vectors. Minivectors can be aerosolized for delivery to lungs and transfect human cells in culture to express RNA or genes. Importantly, minivectors demonstrate no obvious vector-associated toxicity. Minivectors can be repeatedly delivered and are long-lasting without integrating into the genome. Requests from colleagues around the world for minicircle and minivector DNA revealed a demand for our invention. We successfully obtained start-up funding for Twister Biotech, Inc. to help fulfill this demand, providing DNA for those who needed it, with a long-term goal of developing human therapeutics. In summary, what started as a tool for studying DNA structure has taken us in new and unanticipated directions.