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Technology and Innovation, Vol. 20, pp. 427-440, 2019 ISSN 1949-821 • E-ISSN 1949-825X http:// Printed in the USA. All rights reserved. dx.doi.org/10.21300/20.4.2019.427 Copyright © 2019 National Academy of Inventors. www.technologyandinnovation.org BIOPHYSICS MEETS GENE THERAPY: HOW EXPLORING SUPERCOILING-DEPENDENT STRUCTURAL CHANGES IN DNA LED TO THE DEVELOPMENT OF MINIVECTOR DNA Lynn Zechiedrich and Jonathan M. Fogg Department of Molecular Virology and Microbiology, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, and Department of Pharmacology and Chemical Biology, Baylor College of Medicine, Houston, TX, USA Supercoiling affects every aspect of DNA function (replication, transcription, repair, recom- bination, 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), biochem- ical 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 appli- cations. “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, includ- ing 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. Key words: Innovation; Inventor; Patents; Supercoiled DNA; Gene therapy; Structural biology; Minicircle DNA; Minivector DNA; Twister Biotech, Inc. _____________________ Accepted: November 1, 2018. Address correspondence to Lynn Zechiedrich, Ph.D., Department of Molecular Virology and Microbiology, One Baylor Plaza, Mail-stop: BCM-280, Baylor College of Medicine, Houston, TX 77030, USA. Tel: +1 (713) 798-5126. E-mail: [email protected] 427 428 The 2018 annual meeting of the National late Dr. Nicholas Cozzarelli, and I was beginning to Academy of Inventors (NAI) was titled Exploring plan and submit applications for the next stage of my the Intersections of Innovation. As a new NAI fellow academic career. I was invited by the meeting orga- inductee, I was invited to submit an abstract on the nizers to give a short talk describing my research in topic of dual-purpose inventions. I originally thought understanding the various essential cellular func- that everyone else had invented to fill a need and that tions carried out by DNA topoisomerases in E. coli. I was odd for inventing for one purpose but uncover- Topoisomerases are ubiquitous enzymes that per- ing another purpose in a different field. How exciting form the essential roles of unknotting, decatenating to know that I was not alone! From my abstract sub- (unlinking), and maintaining a specific degree of neg- mission (above), I was chosen to give a talk and to ative DNA supercoiling in cells. The combined length contribute this article covering the journey from a and compaction of DNA in cells lead to self-entan- quest to understand how supercoiling affects DNA glement (knotting). If not untied, these knots can function to developing a new gene therapy vector for inhibit DNA replication and transcription (1). Knots treatment of human diseases. The following article also become weak-points in the DNA, analogous to is an encapsulation of the presentation, providing a knots in a fishing line, because stress becomes con- recollection of how a seemingly “pie in the sky” idea centrated at the knot, leading to breakage of the DNA became, 20 years later, a main focus of my research. (1). As an aside, the method we would use to gen- As such, this article is not a research or review article erate supercoiled minicircles was the same method in the conventional sense but a personal perspective we used to generate DNA knots (1). in keeping with the theme of this special issue dedi- DNA supercoiling, the coiling of the DNA helix cated to the 2018 NAI annual meeting. Moreover, this about itself, modulates the properties of DNA and is article illustrates the sometimes unpredictable path of critical for cell survival (2,3). In organisms studied to research. In an age where the cost of doing research date, DNA is maintained in a partially underwound must be continuously justified, the following story (negatively supercoiled) state (relative to a completely serves as an example of how basic research can ulti- relaxed molecule). This underwinding reduces the mately lead to unexpected therapeutic applications. energy required for strand separation. Modulating I had the great fortune to develop the invention DNA supercoiling thereby provides a means to at Baylor College of Medicine in Houston’s Texas regulate gene expression (reviewed in (3) and refer- Medical Center (TMC) with many excellent collab- ences therein). Overwinding (positive supercoiling) orators. In describing the original inspiration for of the DNA occurs transiently during replication the idea, the narrative is from my own perspective. and transcription. If not promptly relaxed, positive The project quickly became a multi-person effort. As supercoiling inhibits these processes, which may be such, the narrative will switch to reflect the combined why some topoisomerases have evolved to prefer- effort. I am particularly indebted to Dr. Jonathan M. entially relax positively supercoiled DNA (2,4,5). Fogg, the co-author of this article, who moved the Understanding the differences between positively invention from idea to reality. Originally envisioned and negatively supercoiled DNA and how these are as a tool for the study of the structure of DNA and recognized by topoisomerases have been questions its interactions with proteins, the invention now has that have motivated our research (2). A more thor- the promise to treat human diseases. ough explanation of supercoiling and its biological consequences may be found in Fogg et al. (2,3). THE IDEA DNA catenanes are a normal consequence of DNA The idea for making minicircles of DNA that could transactions, including DNA replication. At the be supercoiled arose in the summer of 1996 at a end of replication, the two “daughter” replicons are Federation of American Societies for Experimental linked together (catenated). These catenanes must be Biology (FASEB) meeting, Enzymes that Act on unlinked by topoisomerases for chromosomal segre- Nucleic Acids, in Saxtons River, Vermont. At the time, gation to occur. Catenanes may also be generated by I was a postdoctoral fellow in the laboratory of the site-specific recombination between two sites. When BIOPHYSICS MEETS GENE THERAPY 429 two sites are engineered on a plasmid, the site-specific My talk at the FASEB meeting described work recombinase, for example λ-integrase, recombines the delineating the roles of the four Escherichia coli DNA to make catenanes, which are intermediates in topoisomerases. I reported that topoisomerase IV, the process we use to generate supercoiled minicir- not gyrase as previously thought, unlinked catenated cles. Prior to the development of the process to make DNA replication intermediates (8) and catenated supercoiled minicircles, I used the same λ-integrase recombination intermediates (later published as (9)). site-specific recombination system to generate cat- Additionally, I discussed a surprising DNA relax- enanes in bacterial cells and study their unlinking ation role for topoisomerase IV in countering the by the cellular topoisomerases (described below). supercoiling activity of DNA gyrase (eventually pub- Many years later, using the techniques developed to lished as (10)). I had just generated the data shown generate large quantities of minicircles, we success- in the graphs below (Figures 1A and B). In Figure fully isolated catenanes to study their unlinking by 1A, I had quantified the efficiency of topoisomer- topoisomerases in vitro (6). ase-mediated decatenation (y-axis) as

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