Acute myeloid leukemia (AML) is the most common and lethal leukemia in adults. AML consists of many genetic subtypes, which limits broad applicability of targeted therapy. We discovered that the hematopoiesis-restricted tetraspanin CD37 is expressed on the majority of primary AML blasts and thus may represent a common therapeutic target for AML regardless of subtype. We demonstrate that the internalization properties of CD37 are distinct in AML blasts when compared with normal blood cells, and that CD37 rapidly accumulates inside AML blasts via dynamin-dependent endocytosis. Our work revealed that the clinically relevant anti-CD37 antibody-drug conjugate (ADC) Debio 1562 (alpha CD37DM1) is highly cytotoxic to AML blasts, but not normal hematopoietic stem cells. We found that alpha CD37-DM1 improved clinical outcomes and overall survival in multiple in vivo models of AML. Together, these data demonstrate that targeting CD37 with an ADC such as alpha CD37DM1 is a feasible and promising therapeutic option for the treatment of AML.

Abstract Gene therapy is a cornerstone of modern medicine's quest for precision treatment to cure human disease including cancer. The current approach to gene delivery technology employs viral vector delivery systems. While effective, these vectors present significant challenges including immunogenicity, safety concerns due to off-target non-tissue specific delivery, payload limitations, and significant cost ultimately diluting their therapeutic potential. Thus, novel approaches to gene delivery are necessary. One such approach to address these critical limitations involves DNA Nanobots, a DNA-based nanotechnology that allows for a tissue-/target-cell specific gene delivery system. Utilizing DNA origami molecular assembly techniques, our DNA Nanobots offer a customizable and highly precise delivery platform, free from viral vector constraints. This innovative approach has the potential to transform the landscape of gene therapy by providing a safer, more efficient, and targeted alternative. Key technical advancements with our DNA Nanobots Gene Delivery Platform include: •CRISPR Cas-9 Integration: By incorporating CRISPR Cas-9 functional proteins, our nanobots were shown to enhance gene editing precision in primary human T cells, broadening therapeutic applications across various genetic conditions. •Targeted mRNA Delivery: Our nanobots revealed promising results in the targeted delivery of mRNA, vital for vaccine development and personalized treatments. •Efficient antisense/siRNA Delivery: Prior studies have overcome traditional challenges including endosomal escape and cytoplasmic entry, demonstrating effective siRNA delivery critical for gene silencing therapeutic development. In summary, DNA Nanobots mark a paradigm shift in gene therapy. Their ability to deliver various nucleic acids with high specificity and efficiency addresses many of the current methodological limitations especially off-target safety concerns and high cost. This advancement not only enhances the therapeutic index of existing treatments but also opens avenues for novel therapeutic strategies, potentially reshaping the future of molecular medicine and gene therapy. Citation Format: Melika Shahhoessini, Jeffrey R. Spitzner, Christopher R. Lucas, Carlos E. Castro, Patrick D. Halley. Exploring the potential of targeted DNA Nanobot delivery systems for gene delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7238.

Abstract Acute Myeloid Leukemia (AML) treatment faces considerable challenges, in particular the delivery of therapeutics with sufficient potency and specificity. Traditional Antibody-Drug Conjugates (ADCs) offer a strategic approach to cancer therapy by combining the targeting capabilities of monoclonal antibodies with the cell-killing effect of cytotoxic drugs. However, the limitations in drug-to-antibody ratio (DAR) and the challenges in controlling the release kinetics have often hindered their therapeutic window suggesting novel approaches are necessary. Our approach integrates DNA nanobot technology to facilitate targeted delivery of anthracycline agents directly to CD37-expressing leukemic cells. CD37, a tetraspanin superfamily antigen, has shown promise as a target for AML due to its selective expression on immune cells and favorable internalization properties. By harnessing DNA origami assembly techniques, we developed a customizable platform capable of accommodating a significantly higher payload of chemotherapeutic agents than traditional ADCs. This increased payload potential, paired with the precision targeting of CD37, presents a potent therapeutic strategy with the possibility of reduced systemic toxicity. Here we present preclinical evidence demonstrating targeted delivery of our DNA nanobot drug delivery device and anthracycline payload to CD37+ AML target cells in vitro. In addition, we show targeted efficacy in vitro and in vivo of our anthracycline-loaded DNA nanobot-enabled delivery system, which significantly outperformed free anthracycline in AML target cells. Collectively, our findings suggest an improvement in the therapeutic index of anthracycline and antimitotic agents as well as the ability to combine multiple drug pathways in a single delivery device. The implications of this work extend beyond AML, offering a versatile platform that could revolutionize ADC chemistry and targeted drug delivery. We propose that our DNA nanobot delivery system signifies a pivotal advancement in the field of targeted cancer therapy, with the potential to overcome longstanding barriers in the treatment of AML and potentially other malignancies. Citation Format: Nicholas Vantangoli, Patrick D. Halley, Jeffrey R. Spitzner, John C. Byrd, Karilyn T. Larkin, Carlos E. Castro, Christopher R. Lucas. Enhanced payload delivery for acute myeloid leukemia treatment through CD37-targeting DNA nanobots [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 479.

ABSTRACT DNA origami is a rapidly emerging nanotechnology that enables researchers to create nanostructures with unprecedented geometric precision that have tremendous potential to advance a variety of fields, including molecular sensing, robotics, and nanomedicine. Hence, many students could benefit from exposure to basic knowledge of DNA origami nanotechnology. However, due to the complexity of design, cost of materials, and cost of equipment, experiments with DNA origami have been limited mainly to research institutions in graduate-level laboratories with significant prior expertise and well-equipped laboratories. This work focuses on overcoming critical barriers to translating DNA origami methods to educational laboratory settings. In particular, we present a streamlined protocol for fabrication and analysis of DNA origami nanostructures that can be carried out within a 2-h laboratory course using low-cost equipment, much of which is readily available in educational laboratories and science classrooms. We focus this educational experiment module on a DNA origami nanorod structure that was previously developed for drug delivery applications. In addition to fabricating nanostructures, we demonstrate a protocol for students to analyze structures via gel electrophoresis using classroom-ready gel equipment. These results establish a basis to expose students to DNA origami nanotechnology and can enable or reinforce valuable learning milestones in fields such as biomaterials, biological engineering, and nanomedicine. Furthermore, introducing students to DNA nanotechnology and related fields can also have the potential to increase interest and future involvement by young students.

Abstract DNA origami is a platform nanotechnology allows for the generation of nanostructures with precisely defined geometric shape that may be easily functionalized with a variety of payloads for therapeutic delivery. These payloads include nucleic acids (miRNA, siRNA, gene sequences), associated Cas protein molecules, therapeutic small molecules drugs, functional peptides (adjuvants or cell penetrating peptides), and incorporated targeting and/or therapeutic antibodies. Hence, DNA origami is a highly promising vehicle to deliver targeted therapeutic payloads to cancer cells or other diseases. Previous studies by our laboratory and others revealed DNA origami mediated drug delivery efficiently delivered daunorubicin or doxorubicin to cancer cells while outperforming free drug in both solid and liquid tumor model systems in vitro and in vivo. Our recent findings showed significant survival advantage by doxorubicin-loaded DNA origami over delivery of free doxorubicin in an aggressive, acute monocytic leukemia model. We also showed antibody-directed drug delivery by DNA origami may be directly targeted to monocytic leukemia cells expressing the target surface antigen, and superior efficacy in vitro against acute myeloid leukemia cells when loaded with daunorubicin relative to free drug at low concentrations. Thus, antibody targeted drug loaded DNA origami represent a promising novel precision medicine approach. Additionally, recent findings by our laboratory in vivo determined that DNA origami nanostructures alone administered at high dose (12.0mg/kg) distribute well, are non-toxic, and illicit mild immunogenicity, making them an attractive candidate for further development towards therapeutic applications. We recently developed a DNA origami structure functionalized at a high density with adjuvant (CpG) and antigenic peptides (OVA) to stimulate a directed antigen-specific immune response. We have shown this DNA origami vaccine platform displays significant improvement in efficacy by an OVA-pulsed antigen-killing assay in vivo relative to alum and CFA standard adjuvant formulations. Thus, our DNA origami vaccine platform represents a promising novel approach to optimize an anti-cancer immune response, a finding also shown by other laboratories.Collectively, DNA Nanobot technology offers 1) a promising a cancer drug delivery system that can deliver a high number of therapeutic drugs (100s of drug molecules per targeted delivery system) utilizing existing ADC conjugation methods; 2) An optimal vaccine delivery method to enhance efficacy; and 3) cell specific targeted delivery of therapeutic nucleic acids and genes to modify oncogene and/or tumor suppressor gene expression. Citation Format: Patrick Halley, Niksa Roki, Nicholas Vantangoli, Tom Zupancic, Jeff Spitzner, Meixiao Long, Karilyn Karilyn Larkin, John Byrd, Carlos Castro, Chris R. Lucas. DNA origami nanostructures as a targeted payload delivery system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 828.

DNA origami (DO) nanotechnology enables the construction of precise nanostructures capable of functionalization with small molecule drugs, nucleic acids, and proteins, suggesting a promising platform for biomedical applications. Despite the potential for drug and vaccine delivery, the impact of DO vehicles on immunogenicity in vivo is not well understood. Here, two DO vehicles, a flat triangle and a nanorod, at varying concentrations are evaluated in vitro and with a repeated dosing regimen administered at a high dose in vivo to study early and late immunogenicity. The studies show normal CD11b+ myeloid cell populations preferentially internalize DO in vitro. DO structures distribute well systemically in vivo, elicit a modest pro-inflammatory immune response that diminishes over time and are nontoxic as shown by weight, histopathology, lack of cytokine storm, and a complete biochemistry panel at the day 10 end point. The results take critical steps to characterize the biological response to DO and suggest that DO vehicles represent a promising platform for drug delivery and vaccine development where immunogenicity should be a key consideration.

Single molecule force spectroscopy is a powerful approach to probe the structure, conformational changes, and kinetic properties of biological and synthetic macromolecules. However, common approaches to apply forces to biomolecules require expensive and cumbersome equipment and relatively large probes such as beads or cantilevers, which limits their use for many environments and makes integrating with other methods challenging. Furthermore, existing methods have key limitations such as an inability to apply compressive forces on single molecules. We report a nanoscale DNA force spectrometer (nDFS), which is based on a DNA origami hinge with tunable mechanical and dynamic properties. The angular free energy landscape of the nDFS can be engineered across a wide range through substitution of less than 5% of the strand components. We further incorporate a removable strut that enables reversible toggling of the nDFS between open and closed states to allow for actuated application of tensile and compressive forces. We demonstrate the ability to apply compressive forces by inducing a large bend in a 249bp DNA molecule, and tensile forces by inducing DNA unwrapping of a nucleosome sample. These results establish a versatile tool for force spectroscopy and robust methods for designing nanoscale mechanical devices with tunable force application.

Despite demonstrating exciting potential for applications such as drug delivery and biosensing, the development of nanodevices for practical applications and broader use in research and education are still hindered by the time, effort, and cost associated with DNA origami fabrication. Simple and robust methods to perform and scale the DNA origami self-assembly process are critical to facilitate broader use and translation to industrial or clinical applications. We report a simple approach to fold DNA origami nanostructures that is fast, robust, and scalable. We demonstrate fabrication at scales approximately 100–1,500-fold higher than typical scales. We further demonstrate an approach we termed low-cost efficient annealing (LEAN) self-assembly involving initial heating at 65 °C for 10 min, then annealing at 51 °C for 2 h, followed by brief quenching at 4 °C that leads to effective assembly of a range of DNA origami structures tested. In contrast to other methods for scaling DNA origami assembly, this approach can be carried out using cheap and widely available equipment (e.g., hot plates, water baths, and laboratory burners) and uses standard recipes and materials so is readily applied to any existing or new DNA origami designs. We envision these methods can facilitate device development for commercial applications and facilitate broader use of DNA origami in research and education.

SmallVolume 12, Issue 3 p. 307-307 FrontispieceFree Access DNA Origami: Daunorubicin-Loaded DNA Origami Nanostructures Circumvent Drug-Resistance Mechanisms in a Leukemia Model (Small 3/2016) Patrick D. Halley, Patrick D. Halley Department of Mechanical and Aerospace Engineering, College of Engineering, The Ohio State University, Columbus, OH, 43210 USA Department of Chemical and Biomolecular Engineering, College of Engineering, The Ohio State University, Columbus, OH, 43210 USASearch for more papers by this authorChristopher R. Lucas, Christopher R. Lucas Department of Mechanical and Aerospace Engineering, College of Engineering, The Ohio State University, Columbus, OH, 43210 USASearch for more papers by this authorEmily M. McWilliams, Emily M. McWilliams Biomedical Sciences Graduate Program, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH, 43210 USASearch for more papers by this authorMatthew J. Webber, Matthew J. Webber Biophysics Graduate Program, The Ohio State University, Columbus, OH, 43210 USASearch for more papers by this authorRandy A. Patton, Randy A. Patton Department of Mechanical and Aerospace Engineering, College of Engineering, The Ohio State University, Columbus, OH, 43210 USASearch for more papers by this authorComert. Kural, Comert. Kural Biophysics Graduate Program, Department of Physics, The Ohio State University, Columbus, OH, 43210 USASearch for more papers by this authorDavid M. Lucas, David M. Lucas Department of Internal Medicine, College of Medicine, College of Pharmacy, The Ohio State University, Columbus, OH, 43210 USASearch for more papers by this authorJohn C. Byrd, John C. Byrd Department of Internal Medicine, College of Medicine, College of Pharmacy, The Ohio State University, Columbus, OH, 43210 USASearch for more papers by this authorCarlos E. Castro, Corresponding Author Carlos E. Castro Department of Mechanical and Aerospace Engineering, College of Engineering, Biophysics Graduate Program, The Ohio State University, Columbus, OH, 43210 USASearch for more papers by this author Patrick D. Halley, Patrick D. Halley Department of Mechanical and Aerospace Engineering, College of Engineering, The Ohio State University, Columbus, OH, 43210 USA Department of Chemical and Biomolecular Engineering, College of Engineering, The Ohio State University, Columbus, OH, 43210 USASearch for more papers by this authorChristopher R. Lucas, Christopher R. Lucas Department of Mechanical and Aerospace Engineering, College of Engineering, The Ohio State University, Columbus, OH, 43210 USASearch for more papers by this authorEmily M. McWilliams, Emily M. McWilliams Biomedical Sciences Graduate Program, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH, 43210 USASearch for more papers by this authorMatthew J. Webber, Matthew J. Webber Biophysics Graduate Program, The Ohio State University, Columbus, OH, 43210 USASearch for more papers by this authorRandy A. Patton, Randy A. Patton Department of Mechanical and Aerospace Engineering, College of Engineering, The Ohio State University, Columbus, OH, 43210 USASearch for more papers by this authorComert. Kural, Comert. Kural Biophysics Graduate Program, Department of Physics, The Ohio State University, Columbus, OH, 43210 USASearch for more papers by this authorDavid M. Lucas, David M. Lucas Department of Internal Medicine, College of Medicine, College of Pharmacy, The Ohio State University, Columbus, OH, 43210 USASearch for more papers by this authorJohn C. Byrd, John C. Byrd Department of Internal Medicine, College of Medicine, College of Pharmacy, The Ohio State University, Columbus, OH, 43210 USASearch for more papers by this authorCarlos E. Castro, Corresponding Author Carlos E. Castro Department of Mechanical and Aerospace Engineering, College of Engineering, Biophysics Graduate Program, The Ohio State University, Columbus, OH, 43210 USASearch for more papers by this author First published: 14 January 2016 https://doi.org/10.1002/smll.201670014Citations: 5AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract DNA origami "Trojan horse" nanostructures provide an effective delivery approach to circumvent drug resistance in leukemia cells. These 100 nanometer delivery vehicles, described on page 308 by C. E. Castro and co-workers, are easily fabricated in 10 min and can be precisely loaded with commonly used anthracycline chemotherapeutic drugs. Drug-loaded DNA origami nanostructures enter cells via endocytosis, allowing the drug to bypass defenses in the cell membrane that are effective against free drug. This approach leads to larger amounts of drug in cells, which is later released due to decreasing pH and enzymatic degradation, ultimately improving drug efficacy. Citing Literature Volume12, Issue3January 20, 2016Pages 307-307 RelatedInformation

Many cancers show primary or acquired drug resistance due to the overexpression of efflux pumps. A novel mechanism to circumvent this is to integrate drugs, such as anthracycline antibiotics, with nanoparticle delivery vehicles that can bypass intrinsic tumor drug-resistance mechanisms. DNA nanoparticles serve as an efficient binding platform for intercalating drugs (e.g., anthracyclines doxorubicin and daunorubicin, which are widely used to treat acute leukemias) and enable precise structure design and chemical modifications, for example, for incorporating targeting capabilities. Here, DNA nanostructures are utilized to circumvent daunorubicin drug resistance at clinically relevant doses in a leukemia cell line model. The fabrication of a rod-like DNA origami drug carrier is reported that can be controllably loaded with daunorubicin. It is further directly verified that nanostructure-mediated daunorubicin delivery leads to increased drug entry and retention in cells relative to free daunorubicin at equal concentrations, which yields significantly enhanced drug efficacy. Our results indicate that DNA origami nanostructures can circumvent efflux-pump-mediated drug resistance in leukemia cells at clinically relevant drug concentrations and provide a robust DNA nanostructure design that could be implemented in a wide range of cellular applications due to its remarkably fast self-assembly (≈5 min) and excellent stability in cell culture conditions.