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 with conventional 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 . 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.

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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]

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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 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 a function of Performing the duties of unknotting, unlinking, plasmid supercoiling in E. coli (x-axis). I had deter- and regulating supercoiling requires the topoisom- mined the efficiency of a site-specific recombination erases to pass DNA helices through each other. reaction (shown as % on the y-axis) as a function of Although the reactions catalyzed by topoisomer- plasmid supercoiling in E. coli (x-axis) (Figure 1B). ases are essential, there is inherent danger involved in breaking and rejoining DNA (7). A long-standing conundrum in the field is how these enzymes recog- nize when and where to act (2), a question that also occupies us to this day.

A B

Figure 1. Correlation of negative supercoiling and DNA activity in E. coli. (A) Effect of DNA supercoiling (σ) on decatenation. Similarly to (A), were isolated from E. coli after recombination and split into two portions. One portion was nicked with DNase I and catenanes were separated by high resolution electrophoresis on agarose gels and quantified (11). The other portion was used to measure DNA supercoiling levels using agarose gel electrophoresis run in the presence of chloroquine. (B) Correlation of DNA supercoiling (σ) and λ Integrase recombination. Plasmids were isolated from E. coli following λ Integrase recombination and divided into two parts. One part was subjected to assays that quantify the amount of recombination. In the other part, supercoiling levels were quantified using agarose gel electrophoresis run in the presence of chloroquine (reprinted with permission from Genes & Development, copyright 1997 (9)). The line was drawn for ease of visualization. 430

These graphs hinted at an extremely strong depen- (13). These minicircles were typically made by cir- dence of two different DNA activities on the degree cularizing a linear fragment by ligating the ends (14). that DNA is negatively supercoiled in vivo. For both Such an approach results in extremely limited yield, recombinations, there was a distinct threshold of generating microgram quantities at best. If I were to supercoiling, below which the reaction did not take make enough minicircles for the experiments I was place. These results were foremost on my mind at envisioning, a different approach would be required. the FASEB meeting. It was subsequently determined that FokI requires One of the most exciting talks at the conference, at two copies of its cognate DNA site (each bound by a least in my opinion, was by Dr. Aneel K. Aggarwal. He FokI molecule), stimulating dimerization of the cleav- had succeeded in crystalizing Fok1 nuclease bound age domain, allowing full activation of cleavage (15). to DNA. FokI consists of two functionally distinct The effect of supercoiling on FokI remains undeter- domains, a DNA recognition domain that binds to mined, and I never actually tested my hypothesis. a specific DNA sequence and a separate cleavage Nevertheless, it served as the intellectual catalyst for domain that cleaves a short distance away from the the idea of using supercoiled minicircles for many cognate sequence. The modular nature of the enzyme other interesting questions. facilitated the engineering of artificial enzymes with novel sequence specificities by swapping out the rec- REDUCTION OF THE IDEA TO PRACTICE ognition domain. Zinc-finger nucleases and TALENS To determine (9) which topoisomerase unlinked exploit this feature of FokI and became a promi- DNA, DNA gyrase or the newly discovered topoisom- nent tool in genome editing although they have been erase IV (16), I utilized a clever system (17) engineered superseded in recent years by the development of by Dr. James B. Bliska when he was a trainee with my CRISPR/Cas9. postdoctoral advisor, Dr. Nick Cozzarelli. In this sys- As I gazed upon the co-crystal structure (12), I first tem (Figure 2), λ-integrase is induced in E. coli and noticed that the cleavage domain was sequestered catalyzes recombination between two sites on a plas- away from the DNA, indicating that the enzyme must mid. The product of this recombination is catenated undergo a conformational change to swing the cleav- DNA. I furthermore used this system to uncover age domain over to the DNA for activity to occur. key differences between in vitro (17) and in vivo Dr. Aggarwal’s structure provided an unprecedented recombination (9). Although I observed the cate- insight into the mechanism of this enzyme despite nated intermediates by blocking topoisomerase IV that fact that it depicted an inactive conformation. activity, I never directly observed the smaller of the Various mechanisms for activating the enzyme were two unlinked rings because it was only ~600 bp in postulated, but little thought was given to the DNA length, and the assays to observe and quantify the cat- substrate itself. From my own research, I knew that enanes were designed for analyzing plasmid-length DNA supercoiling could dramatically affect enzyme DNA (>3,500 bp). Despite several challenges, this activity. Looking at the short duplex of relaxed DNA recombination system has the inherent advantage in the structure, I thought to myself, “if we could that the recombination occurs in bacteria. In effect, connect the ends of the DNA and supercoil the mole- the bacteria would “do the work,” rather than us hav- cule, maybe we could determine the active structure.” ing to deal with multiple isolated components (18). Now lost in my own thoughts as Dr. Aggarwal fin- Bacterial cultures can be scaled up almost indefinitely, ished his talk and fielded questions, I worked out giving the potential to make milligram quantities in my mind how to make large quantities (such as of supercoiled DNA. Achieving that goal, however, might be needed for any subsequent crystallogra- would take many years of difficult and often tedious phy attempts) of tiny supercoiled DNA circles. In work. Fortunately, I was able to find collaborators, fact, for the rest of the meeting, I could think only most especially Dr. Jonathan M. Fogg, who were con- about getting back to the laboratory to get started. vinced of the potential utility of minicircles despite Some earlier pioneering work by others had already skepticism we encountered. shown the utility of supercoiled DNA minicircles BIOPHYSICS MEETS GENE THERAPY 431

Figure 2. Generation of minivector DNA. Minivector DNA encoding a desired DNA sequence (red) is generated using the λ-inte- grase (Int) site-specific recombination system (17,19). Site-specific recombination between the attB and attP sites, directly orientated on the same plasmid, exchanges the DNA sequences denoted by the arrowheads to form two catenated DNA circles. In E. coli, topoisom- erase IV (Topo IV) unlinks these to generate a minicircle and a large ring containing the rest of the original parent plasmid DNA.

I met crystallographer Dr. John J. Perona at the structure of the nucleosome core particle in 1997 (23). 1996 FASEB meeting, and he was the first to hear With 146 bp wrapped into a superhelix by the bind- the idea. He had recently started his laboratory at ing of histone proteins, it was in a similar size range the University of California at Santa Barbara (UCSB) to the minicircles we wanted to study. Because of the and was particularly interested in the role of DNA inherent rigidity of DNA, we hypothesized that the bending in the induced fit mechanism employed by minicircles would be constrained into a limited num- the restriction enzyme EcoRV to help recognize spe- ber of conformations, thus favoring crystallization. cific DNA sequences (20–22). EcoRV is amenable to We discovered many years later that the minicircles, crystallization, and Dr. Perona’s group has solved the and supercoiled DNA in general, are far more con- structure of the enzyme in numerous different crys- formationally dynamic than previously realized (24). tal lattices, both with and without DNA. We had the Despite our eagerness to get started on the project, idea that the binding of EcoRV, and the concomitant it had to go on the backburner as I wrapped up pub- bending of the DNA by the enzyme, might lock the lications and started my assistant professor position supercoiled minicircle DNA into a particular con- at Baylor College of Medicine in 1997. Dr. Perona formation and allow crystallization. Fortunately, Dr. remained committed to the project, even coming to Perona thought the idea had potential and was worth Houston in 2001 for a short sabbatical, during which a try despite there being no enabling data at the time. he cloned the plasmids used to make the first mini- Others had expressed doubt that small DNA circles circles. Dr. Fogg, who joined Dr. Perona’s group near could be crystallized because of their relatively large the end of 2001, was intrigued enough by the idea to size. We were encouraged, however, by the crystal begin working on the problem. Isolating minicircle 432

DNA in large quantities proved challenging, how- much too soon after that conversation. ever, especially given that the project was largely Many researchers were reaching the same conclu- unfunded. Although the idea of crystallizing super- sion as we had—that supercoiled minicircles were coiled minicircle with EcoRV remained a goal, we useful to study DNA acting enzymes. For about the could not continue the project without generating next two years, we found ourselves spending most some tangible results to justify continuing. of our time making minicircles for others. One day, Supercoiled minicircles are excellent substrates when I was writing up the progress report for one for the study of topoisomerases (14), my research of our grants, I realized that because we were spend- field. Dr. Fogg decided to redirect his efforts toward ing so much time making minicircles for others, our topoisomerases, eventually moving to my labora- own work and our own ideas had nearly ground to a tory, where he has remained ever since. He is now a halt. That is when the idea to form a company came senior staff scientist and has been involved in every to mind. With start-up funds from Baylor College aspect of the development of the project. of Medicine Technologies, Twister Biotech, Inc. was Along the pathway to successfully making these founded to take the burden of making minicircles for supercoiled circles, we learned much about DNA others off my laboratory. supercoiling (2,3,19,24,25). These discoveries forced us to stop and rethink what we thought we under- THE INITIAL GOAL: UNDERSTANDING stood about active DNA. DNA is far more dynamic SUPERCOILED DNA and far more interesting than previously recognized. The study of DNA supercoiling and proteins that The topic of how DNA supercoiling impacts DNA interact with and manipulate supercoiled DNA has activity remains an active area of research in our lab- been hampered by the lack of a biophysically trac- oratory, proving to us that our time spent figuring table DNA substrate. Short duplex , although out how to make and isolate supercoiled minicircles easy to make and manipulate, cannot be supercoiled. was well worth it. Plasmid DNA, although supercoiled, is too large for many biochemical and biophysical assays, par- DEMAND FOR MINICIRCLES NECESSITATED ticularly the ones that we were interested in, which THE FORMATION OF TWISTER BIOTECH, INC. involved subtle changes. These likely subtle struc- As we were generating surprising results about tural effects of supercoiling get averaged out over DNA supercoiling using minicircles, other research- such long molecules. ers studying DNA processes began to be interested. After experimenting with various different-length Shortly before he died in 2006, Dr. Nick Cozzarelli minicircles, we settled on 339 bp (and later 336 bp called me. Whereas other researchers had thought and 333 bp) minicircles for the majority of our stud- making supercoiled minicircles in vivo was an ies. Minicircles of this length can be isolated in high interesting idea, most doubted that we could obtain yield and are small enough to allow isolation of mul- sufficient quantities using this method and felt that tiple different individual topoisomers (minicircles a biochemical approach would be the only way to with the same size and sequence but different levels make it work. Like John Perona’s, Nick’s encourage- of supercoiling). Each purified topoisomer, then, has ment from the start was invaluable to us. When Nick a precisely defined level of supercoiling. (In contrast, phoned that day, he was excited to be back at work supercoiled plasmids are typically purified as a large following a successful bone marrow transplant and mixture of topoisomers; isolating individual topoiso- had taken on two new graduate students in the lab- mers is technically challenging with very low yield.) oratory. During our conversation, I told him that we We also developed methods to manipulate the super- had successfully generated supercoiled minicircles in coiling, allowing a wide range of supercoiling to be high yield. His immediate reply was, “Great! Send me studied, from hyper-negatively to positively super- some!” So, the first person we made supercoiled mini- coiled. From the combination of 333 bp, 336 bp, and circles for was my old boss. Sadly, one of his postdocs 339 bp minicircles, we are able to generate and iso- returned them, unused, at Nick’s memorial service, late 29 unique topoisomers, providing us exquisite BIOPHYSICS MEETS GENE THERAPY 433 control over the supercoiling level. open and some very compact. Atomic force microscopy (AFM) provided our We have been continuously surprised by super- first look at the conformations of individual super- coiled minicircle data, first with AFM and then with coiled minicircles in collaboration with Dr. Helen cryoEM. Many of the conformations observed, and Hansma at UCSB (19). In this technique, the DNA the majority of conformations observed for the most is adsorbed onto a flat surface. A sharp tip is scanned negatively or the most positively supercoiled minicir- over the surface, and displacement of the tip provides cles, should be energetically unfavorable because they a high-resolution read-out of the structure. We were contain regions of very sharp bending, and DNA is excited to see the shapes of individual supercoiled an intrinsically rigid molecule. To better understand DNA minicircles, the path of the DNA backbone, what is happening at the base-pair level, we turned to and how the DNA helices juxtaposed (19). computational simulations and have been fortunate AFM is undoubtedly powerful; however, the DNA to collaborate with talented computational scientists, may distort as it absorbs onto the flat mica surface. including Dr. B. Montgomery Pettitt, formerly at the At Baylor College of Medicine, we have world-class University of Houston and now at the University of facilities and researchers in electron cryomicroscopy Texas Medical Branch in Galveston, Texas, and Dr. (cryoEM), led for many years by our collaborator Dr. Sarah A. Harris at the University of Leeds in the UK. Wah Chiu and now by our collaborators Dr. Steven Dr. Graham L. Randall, then a graduate student in Ludtke and Dr. Zhao Wang. our group co-mentored by Dr. Pettitt, used molec- We had been working to obtain a complete struc- ular dynamics simulations to investigate the effects ture of the full-length topoisomerase IIα by cryoEM. of underwinding and overwinding on short DNA The protein contains intrinsically disordered regions duplexes that were restrained to prevent any coiling that were missing from our preliminary single-par- of the helices in 3-D space, which is known as writhe ticle reconstruction of the structure. Similar to our (26,27). One of the major findings of the study was hypothesis with FokI, we postulated that these disor- that even with slight underwinding, localized devi- dered regions would become ordered upon binding to ations from the canonical B-form helical structure supercoiled DNA. Before starting work on the struc- occur (28). Slight underwinding causes sequence-de- ture of the enzyme-DNA complex, however, we had to pendent base-flipping; increased underwinding causes study the “control” structure of the minicircles in the multiple base-flipping events in succession, which absence of topoisomerase. The results were so much results in denaturation (28). This base-flipping and more interesting than we anticipated! Supercoiled denaturation relieves the torsional strain, allowing DNA adopts a surprisingly wide and varied distri- the remainder of the molecule to revert back to the bution of three-dimensional conformations (24). B-form double helix. Overwinding beyond a criti- Minicircles migrate through a polyacrylamide cal threshold induced the formation of an inside-out gel at rates depending upon the degree of super- conformation called Pauling-DNA (P-DNA) with coiling. Generally, the more negatively or positively the DNA backbone on the inside and bases on the supercoiled the DNA is, the faster the DNA migrates outside exposed to solvent, as had been previously through the gel (19,24). Because different topoisomers postulated to explain the results from single-mole- are all the same size, the faster migrating topoisomers cule manipulation experiments (29). presumably adopt more compact conformations to At about the same time, Dr. Sarah Harris was per- allow them to pass more rapidly through the pores in forming molecular dynamic simulations of DNA the gel matrix. Although each topoisomer migrates minicircles up to 178 bp in length and obtaining as a discrete band on a polyacrylamide gel, when we some intriguing results (30). Excited by the possi- cut out an individual band from the gel, isolate the bilities brought forth by the results from Dr. Harris, DNA from the band, and visualize it by cryoEM, we initiated a collaboration. The computing power each individual topoisomer adopts multiple confor- required for molecular dynamics simulations mations (24). From each purified topoisomer band, increases exponentially with the number of atoms, we observed a variety of conformations—some very especially when including solvent, as was needed for 434 our goals. Fortunately, computing power has contin- Could the exposed bases be because of the bending ued to increase, and Dr. Harris is able to simulate strain in the highly-writhed minicircles (31,32)? Or increasingly larger DNA circles. As we were studying could the exposed bases indicate Pauling-DNA (29), them by cryoEM, Dr. Harris was, in parallel, sim- the unusual inside-out conformation observed in our ulating our 336 bp minicircles (24). She observed earlier simulations of overwound DNA (28)? that, during the simulations, the minicircles fluctu- In collaboration with Dr. Pettitt, we used computa- ated among the conformations we observed. Being tional simulations to investigate the effect of placing able to atomistically simulate the minicircles that we sequence-dependent hyperflexible sites at different observe experimentally has proven to be extremely locations on the minicircle (25). The simulations sug- beneficial; simulations provide atomistic detail to gest that it is possible to modify the conformational low-resolution cryoEM structures. In contrast, plas- distribution of supercoiled minicircles by altering mids are too large for all-atom molecular dynamics the sequence. This finding allows us to potentially simulations. Any experiments investigating plasmid design novel DNA nanoparticle shapes, a tool that structure will be subject to interpretation and depen- may have utility for gene therapy. dent on the coarse-grained model used to fit the data. Relatively little is known about positively super- A SECOND PURPOSE: GENE THERAPY coiled DNA despite its biological importance. As Soon after having the idea for how to make milli- mentioned above, positive supercoils are generated gram quantities of pure supercoiled minicircles, we ahead of the replication and transcription machin- recognized that they might be good ery, and several topoisomerases preferentially relax vectors because of their small size. We decided to positively supercoiled DNA (2). These topoisomer- call these minimal supercoiled DNAs for gene ther- ases are the target of important antibacterial (DNA apy applications “minivectors” to distinguish them gyrase and topoisomerase IV) and anticancer (human from other non-viral vectors and from the minicir- topoisomerase IIα) drugs (2). To better understand cles used for biophysical studies. In the late 1990s and how these drugs act, we need to better understand early 2000s, the emerging field of gene therapy was the DNA structures they affect. Our cryoEM (24) and undergoing a crisis. After initial encouraging results molecular dynamics studies (24,28) revealed numer- that progressed gene therapy to clinical trials, the field ous differences between the structures of negatively suffered a number of setbacks that almost completely and positively supercoiled DNA; we are currently obliterated the early confidence. Most notable was the investigating how topoisomerases and the drugs that tragic death of Jesse Gelsinger, who suffered from a affect topoisomerases affect or are affected by the fatal reaction to the adenovirus used to deliver the structures. gene (33). In European clinical trials, several children Localized base-flipping and denaturation play an who were successfully treated for severe combined important role in modifying the structure of super- immunodeficiency using gene-therapy later went coiled DNA. When a base-pair is broken during on to develop leukemia caused by erroneous inte- base-flipping, the helix becomes much more flexible gration of the viral vector into an oncogene; one (24,25). Using Bal-31, an enzyme that detects exposed child died (34). bases in DNA, we detected and mapped these local- The safety concerns with viral vectors made ized denaturations (24). They occur, for extremely non-viral vectors a promising and safer alternative. negatively supercoiled minicircles, at regions 180° Supercoiled plasmids, the most commonly used apart from each other, perhaps at each apical end of non-viral vector, are much less efficient at deliv- rod-shaped minicircles (24). ering their payloads than viruses. Many clinically Somewhat surprisingly, we also observed exposed important cell types are refractory to transfection bases in positively supercoiled DNA, albeit at higher with plasmids. We thought surely minicircles would supercoiling levels than required for negatively super- transfect cells better than large supercoiled plasmids coiled DNA (24). The reason for the exposed bases in because they are so much smaller (and more nega- positively supercoiled DNA remains undetermined. tively supercoiled), but using them to deliver a long BIOPHYSICS MEETS GENE THERAPY 435 gene would remove the size advantage. The discovery Minivectors are not limited to delivery of shRNA. of RNA interference in 1998 (35) provided a potential Any therapeutic sequence may be encoded on a min- means to regulate genes by encoding a short hairpin ivector, including genes. In principle, any length of RNA (shRNA). The sequence encoding shRNA, sequence can be delivered. To take advantage of the coupled with a minimal-length promoter (e.g., H1), small size, however, the optimal therapeutic sequence requires as little as ~150 bp of DNA sequence to is typically less than 2,000 bp. Although it would seem encode. like this sequence length would rule out using min- Years later (specifically, in 2006), a brave graduate ivectors to deliver long genes, this is not the case. student, Dr. Michelle C. Swick, who had experience Larger genes may be split into multiple smaller frag- with cell culture, emboldened us to meet with a gen- ments (37), each encoded on a different minivector. erous colleague then in our department, Dr. Richard When expressed in vivo, the protein fragments com- Sutton, who quickly agreed to help us test whether bine to reconstitute a functional protein (37). the idea could work. The idea was to test whether Meanwhile, another colleague, Dr. Brian E. Gilbert, minivectors encoding shRNA against the gene encod- had been thwarted in his attempts to aerosolize plas- ing the receptor, CCR5, would knock down gene mid DNA for the treatment of lung diseases. Plasmid expression of this receptor. CCR5 is normally used DNA is extensively degraded during aerosolization by the human immunodeficiency virus (HIV) to gain because of hydrodynamic shear, the force exerted entry into specific CCR5-expressing cells. Knocking on the DNA by the rapid flow of liquid in the nebu- down CCR5 was postulated to make cells resistant lizer. It was already known that shear force survival to HIV and was therefore a potential therapy. To our was inversely correlated with DNA length, but even delight, a minivector of 385 bp efficiently knocked the smallest plasmids were degraded by aerosoliza- down expression of CCR5. These early results were tion. Attempts to protect the DNA by using chemical very encouraging, but further progress was hindered agents to condense the DNA help to alleviate shear- when Dr. Sutton was recruited to Yale University ing; the vehicles used, however, are highly cytotoxic in 2008. We found ourselves with a promising tool and induce inflammation (38–40). Plasmid shear- but unsure what disease to use it for. The possibili- ing not only reduces the effective dose, but there was ties were seemingly endless, and we decided to take also concern that the degraded DNA fragments could advantage of the additional expertise in the TMC. induce DNA repair pathways that may even trigger The promising early results led us to join the Baylor apoptosis (41). These fragments could also potentially College of Medicine Center for Cell and Gene Therapy recombine into novel and potentially toxic sequences (CAGT) (we are grateful to Dr. Malcolm Brenner, or may integrate into the genome (41). then head of the CAGT, for his support and encour- Dr. Gilbert wanted to test whether minivectors agement). We gave a talk to the CAGT members could survive nebulization. We therefore delineated and met Dr. Youli Zu. With him, we published our the dramatic effect of DNA vector length on aero- first gene therapy paper demonstrating that minivec- solization survival (41). Minivectors less than 2,000 tors were as good as small interfering RNA (siRNA), bp completely survived aerosolization, even with- synthetic RNA duplexes, equivalent to shRNA, that out any vehicle. feed into the RNA interference pathway directly, and The gene therapy field is undergoing a renais- much, much better than conventional plasmid vec- sance, with confidence and excitement returning after tors in knocking down anaplastic lymphoma kinase the earlier failures. In 2017, Luxturna, from Spark and blocking growth of human lymphoma cells (36). Therapeutics, became the first gene therapy treatment Unlike siRNA, which must be repeatedly readminis- approved by the U.S. Food and Drug Administration. tered because it is destroyed during the knockdown With a price tag of $850,000, the treatment for a rare process, minivectors continuously express shRNA cause of blindness will likely only be used in a limited to produce a prolonged response. Therefore, mini- number of cases. Nevertheless, it heralded the arrival vectors had potential to be used therapeutically and of a new host of possible gene therapy treatments. not just in cell culture. Billions of dollars are currently being invested in the 436 gene-therapy field. Adeno-associated viral (AAV) remnant of the recombination process used to gener- vectors are currently in favor because of their effi- ate the minivectors. Minivectors have all the benefits cacy and low incidence of inserting into the genome. that arise from removing bacterial sequences but Although considered safer than the viruses used in with additional unique advantages. Minivectors can earlier trials, there remains a strong risk of toxicity be made much shorter than other vectors, as small and immunogenicity (42). AAV vectors cannot be as 250 bp. The small size improves transfection effi- delivered repeatedly to the same patient because the ciency (50). For cell types that are easy to transfect, patient will rapidly develop immunity to the virus such as HeLa, minivectors provide only a modest (43,44). The high cost of AAV vectors also makes improvement over plasmids. There are many cell them prohibitively expensive. types, however, that cannot be easily be transfected For the reasons above, and others discussed in with plasmids, and this is where minivectors provide our recent review (45), there is enormous potential the biggest transfection benefit. These difficult-to- for non-viral gene therapies. They are particularly transfect cell types include many clinically important attractive for diseases that will require repeated treat- cell lines, such as the aforementioned lymphoma cells, ment over time. Their low toxicity, long shelf life, and therefore minivectors provide a much-needed and relatively low production costs make them a tool for transfecting these cells. logical choice. One advantage of short length becomes evident The limitations of plasmid-based vectors have during production. Minivectors are much smaller motivated the development of alternative non-viral than their parent plasmids. They can be, therefore, vectors (reviewed in (45)). Plasmids require bacterial readily separated from their parent plasmids and sequences for propagation. One particular concern is from the other by-products of recombination. Other the use of antibiotics and resistance genes for selec- vectors include up to 5% contamination from parent tion, which is strongly discouraged by regulatory plasmid (51,52). This contaminating plasmid may agencies because of the risk of transferring these cause immunotoxicity. Minivectors can be isolated genes into the microbiome of the patient or into the with high purity, devoid of any contaminating plas- environment, thus promoting the spread of antibi- mid, because of the huge size difference between the otic resistance. minivector and parent. Another advantage of small Bacterial sequences can also lead to silencing of length is that more copies are delivered per mass, the (46–48). “Minicircle” vectors (histori- suggesting that the same therapeutic effect could be cally the term minicircle, as we used above, describes achieved with less vector. With less mass of DNA to circular DNAs of less than ~500 bp or the circles cat- deliver, less vehicle would be required, thereby reduc- enated to maxicircles that make up DNA ing transfection vehicle-mediated toxicity. in trypanosomes, but in this use describes circular We are currently exploring a number of poten- DNAs—of any length—lacking bacterial genes used tial therapeutic avenues to exploit the advantages for propagation) exhibit ten-fold or higher levels of of minivectors. After demonstrating the potential sustained gene expression over plasmids (46). Also of minivectors, we are hoping to move these toward of concern are CpG motifs, cytosine-phosphate-gua- clinical uses as quickly as possible. This change in nine dinucleotides, which can be recognized by the research emphasis required a team crash course in immune system. CpG motifs are approximately gene therapy. Fortunately, we continue to be assisted four times more prevalent in bacterial DNA than in this journey by a number of excellent trainees and eukaryotic DNA. Removing CpG motifs reduces the collaborators. The outlook is promising. immunogenicity of a vector (49). Conversely, a vec- tor may be loaded with CpG motifs if the goal is to SUMMARY stimulate the immune system. Our basic work on DNA structure and function Minivectors allow control over the sequence that led to a new tool in the gene therapy toolbox: mini- is delivered. The only required sequence is a short vectors (Figure 3). For both applications, these small 106 bp (attL) or 183 bp (attR) sequence that is the DNA circles are proving to have incredible utility. BIOPHYSICS MEETS GENE THERAPY 437

Supercoiled minicircles have provided an unprec- grant RO1 GM115501. The authors hold numer- edented insight into the structure and diversity of ous patents on the generation and purification of supercoiled DNA and will continue to be used to minicircle and minivector DNA as well as the use answer fundamental questions related to DNA func- of minivector DNA for gene therapy. Both authors tion. As small as ~250 bp, minivectors can be made have equity stake in Twister Biotech, Inc. at any length and still have the major advantages of being devoid of undesirable and potentially toxic REFERENCES bacterial sequences and being more negatively super- 1. Deibler RW, Mann JK, Sumners DWL, Zechiedrich coiled than other DNA vectors. Minivectors only L. Hin-mediated DNA knotting and recombin- encode the DNA sequence of interest and can be ing promote dysfunction and mutation. made to encode shRNA, miRNA, RNAi, hormones, BMC Mol Biol. 2007;8:44. peptides, antibody fragments, protein fragments, 2. Fogg JM, Catanese Jr DJ, Randall GL, Swick MC, protein binding sites, or a DNA template for repair. Zechiedrich L. Differences between positively and negatively supercoiled DNA that topoisom- erases may distinguish. In: Benham CJ, Harvey S, Olson WK, Sumners DWL, Swigon D, editors. Mathematics of DNA structure, function and interactions. Vol. 150. New York (NY): Springer; 2009. pp. 73-121. 3. Fogg JM, Randall GL, Pettitt BM, Sumners DWL, Harris SA, Zechiedrich L. Bullied no more: when and how DNA shoves proteins around. Q Rev Biophys. 2012;45(3):257–299. 4. Crisona NJ, Strick TR, Bensimon D, Croquette V, Cozzarelli NR. Preferential relaxation of posi- tively supercoiled DNA by E. coli topoisomerase Figure 3. Minivectors are 10 to 50 times smaller than plasmids. IV in single-molecule and ensemble measure- To the left is schematized plasmid containing the bacterial origin ments. Genes Dev. 2000;14(22):2881–2892. of replication (ori) that could transfect normal cells to replicate 5. McClendon AK, Rodriguez AC, Osheroff N. antibiotic resistance. Minivectors (mv) encode only the therapeu- tic sequence. To the right are atomic force micrographs of each. Human topoisomerase IIalpha rapidly relaxes positively supercoiled DNA: implications for ACKNOWLEDGMENTS enzyme action ahead of replication forks. J Biol We are indebted to a number of people for encour- Chem. 2005;280(47):39337–39345. agement, collaboration, advice, and reagents. 6. Bizard AH, Yang X, Débat H, Fogg JM, Foremost is the late Dr. Nicholas R. Cozzarelli, who Zechiedrich L, Strick TR, Garnier F, Nadal M. thought “it just might work!” thus kicking off the TopA, the Sulfolobus solfataricus topoisom- endeavor. Additionally, Dr. James B. Bliska, Dr. erase III, is a decatenase. Nucleic Acids Res. Malcolm K. Brenner, Dr. Daniel J. Catanese, Jr., Dr. 2018;46(2):861–872. Wah Chiu, Dr. Brian E. Gilbert, Dr. Helen G. Hansma, 7. Bates AD, Berger JM, Maxwell A. The ancestral Dr. Sarah A. Harris, Dr. Steven J. Ludtke, Dr. Neil role of ATP hydrolysis in type II topoisomerases: Osheroff, Dr. John J. Perona, Dr. B. Montgomery prevention of DNA double-strand breaks. Nucleic Pettitt, Dr. Graham L. Randall, Dr. Buck S. Samuel, Acids Res. 2011;39(15):6327–6339. Dr. Richard E. Sutton, Dr. Michelle C. Swick, and 8. Zechiedrich EL, Cozzarelli NR. Roles of topoisom- Dr. Youli Zu. Others, including all the former and erase IV and DNA gyrase in DNA unlinking current members of the Zechiedrich laboratory, and during replication in Escherichia coli. Genes Dev. anyone else we accidentally left off this list. This work 1995;9(22):2859–2869. was supported by National Institutes of Health (NIH) 9. Zechiedrich EL, Khodursky AB, Cozzarelli NR. 438

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