DNA Nanobots for Gene Delivery

Mar 22, 2023 | Gene Delivery, Gene Therapy, Uncategorized | 0 comments

By: Christopher R. Lucas, PhD


The ability to deliver genes to the nucleus of diseased patient cells, either to replace mutated genes or add missing ones to ultimately restore healthy, normal cell function has the potential to revolutionize genetic disease therapy. Currently (as of this writing, 3/2023) there are 27 FDA approved cell and gene therapies in the United States1 impacting genetic diseases including rare disease (spinal muscular atrophy, -thalassemia, Leber’s congenital amaurosis, Lipoprotein lipase deficiency, and severe combined immunodeficiency) and cancer (leukemia/lymphoma and melanoma)2. Although extremely promising, with numerous treatments currently under clinical development (153 trials currently recruiting; clinicaltrials.gov), a host of limitations exist including production costs and price3, as well as multiple risks associated with viral delivery vectors including: undesired immune system reaction, targeting the wrong cells, infections caused by the virus, and the possibility of causing a tumor4 suggesting that new non-viral gene delivery approaches must be developed to limit cost and significantly reduce patient side effects.

            One such approach is through DNA Nanotechnology, namely the DNA origami technique pioneered by Paul Rothemund in 2006 to create highly ordered nanostructures (nanobots) with precisely controlled geometric shape5–7, easy to functionalize6, and recently demonstrated the ability to deliver genetic material to cell nuclei8–10. The first report by Lin-Shiao and colleagues (which included Alexander Marson, DNA origami world leader Carlos Castro, and Nobel Laureate Jennifer Doudna) showed that genes themselves (a human gene and a reporter gene including CRISPR-Cas9 ribonucleoprotein binding sites) may be folded into DNA nanobots and efficiently delivered to nuclei and incorporated into the genome of cell lines (HEK293T) and primary human T cells via transfection and electroporation via CRISPR-mediated homology directed repair (HDR) method8. Of critical importance, the authors went on to show that the DNA nanobot gene delivery particles could be effectively packaged into viral like particles for efficient nuclear delivery and genomic integration enabling gene delivery by DNA nanobots in both a non-viral and non-transfection/electroporation manner8.

The second report by Kretzmann et. al. out of Hendrik Ditez’s lab explored the applicability of the scaffolded DNA origami approach to deliver and co-express genes in a cell line (HEK293T) transfection model9. By including and tuning functional sequences and DNA origami (nanobot) structures from custom designed scaffolds, including virus inspired inverted-terminal repeat-like (ITR) motifs, the authors effectively demonstrated enhanced gene expression efficiency when genes were incorporated directly into a brick-like DNA origami nanobot after transfection9. Furthermore, the report confirmed the ability to load and deliver multiple genes in a single DNA origami nanobot, as well as and express multiple genes in the same cell in a stoichiometrically controlled manner upon transfection9. The third report by Liedl and colleagues from Hendrik Ditez’s lab explored scaffold DNA origami nanobot mediated gene delivery in the context of active nuclear transport by developing a construct that included a custom ssDNA scaffold, a mammalian-cell expressible reporter gene (mCherry), and multiple Simian virus 40 (SV40) derived DNA nuclear targeting sequences (DTS)10. The authors showed that inclusion of the DTS after transfection allowed for gene expression rescue in arrested cells suggesting active nuclear transport10, further validating the DNA origami approach for gene delivery at the proof-of-concept level. Collectively, these findings provide compelling in vitro evidence to support applicability of the DNA origami nanobot platform as a gene delivery vehicle, warranting pre-clinical validation in vivo in relevant disease models.

Indeed, the highly customizable DNA origami nanobot gene delivery platform includes multiple advantages over the current standard adenovirus or adendo-associated virus (AAV) gene delivery vehicle including low cost11, a safe, non-toxic, biocompatible vehicle12–16, moderate immunogenicity15,16, and allows for targeted delivery17–21. In addition, there is remarkable control over the ability to decorate and functionalize DNA origami nanobots with genes9,10, cell penetrating peptides22, nuclear localization sequences10, viral-like particles encapsulating a gene DNA nanobot itself8, and antibody molecules17,23 to target DNA nanobot gene delivery devices only to intended diseased cells in vivo. Furthermore, the cost to produce DNA origami nanobots is nominal and scalability potential of the platform is high11 compared to costly production of viral gene delivery vectors, where the average cost of goods and services (COGs) of a single gene therapy is an estimated $500,000-$1M24. In stark contrast, the DNA nanobot gene delivery platform alone is expected to cost $0.18/mg of raw material, and since the cost of custom oligonucleotide production of a gene begins around $0.09/base pair25, there is a remarkable economic advantage to using the non-viral DNA nanobot gene delivery platform over the conventional viral gene delivery vectors. In conclusion, there are numerous disadvantages to viral gene delivery vectors including extreme cost and harmful side effects to patients. The time is right to explore novel, non-viral approaches such as the customizable, targeted DNA nanobot gene delivery vehicle.


1.         The TransMission: Current FDA Approved Gene and Cell Therapies. https://www.mirusbio.com/blog/fda-approved-gene-cell-therapies.

2.         Papanikolaou, E. & Bosio, A. The Promise and the Hope of Gene Therapy. Frontiers in Genome Editing 3, (2021).

3.         High Price for Gene Therapy ― But Cost-Effective in Sickle Cell. Medscape https://www.medscape.com/viewarticle/985411.

4.         Gene therapy – Mayo Clinic. https://www.mayoclinic.org/tests-procedures/gene-therapy/about/pac-20384619.

5.         Rothemund, P. W. K. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 (2006).

6.         Kearney, C. J., Lucas, C. R., O’Brien, F. J. & Castro, C. E. DNA Origami: Folded DNA-Nanodevices That Can Direct and Interpret Cell Behavior. Advanced Materials 28, 5509–5524 (2016).

7.         Castro, C. E. et al. A primer to scaffolded DNA origami. Nat Methods 8, 221–229 (2011).

8.         Lin-Shiao, E. et al. CRISPR-Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells. Nucleic Acids Res 50, 1256–1268 (2022).

9.         Kretzmann, J. A. et al. Gene-encoding DNA origami for mammalian cell expression. Nat Commun 14, 1017 (2023).

10.       Liedl, A., Grießing, J., Kretzmann, J. A. & Dietz, H. Active Nuclear Import of Mammalian Cell-Expressible DNA Origami. J. Am. Chem. Soc. 145, 4946–4950 (2023).

11.       Halley, P. D., Patton, R. A., Chowdhury, A., Byrd, J. C. & Castro, C. E. Low-cost, simple, and scalable self-assembly of DNA origami nanostructures. Nano Res. 12, 1207–1215 (2019).

12.       Zhang, Q. et al. DNA Origami as an In Vivo Drug Delivery Vehicle for Cancer Therapy. ACS Nano 8, 6633–6643 (2014).

13.       Li, S. et al. A DNA nanorobot functions as a cancer therapeutic in response to a molecular trigger in vivo. Nat Biotechnol 36, 258–264 (2018).

14.       Jiang, D. et al. DNA origami nanostructures can exhibit preferential renal uptake and alleviate acute kidney injury. Nat Biomed Eng 2, 865–877 (2018).

15.       Palazzolo, S. et al. An Effective Multi-Stage Liposomal DNA Origami Nanosystem for In Vivo Cancer Therapy. Cancers 11, 1997 (2019).

16.       Lucas, C. R. et al. DNA Origami Nanostructures Elicit Dose-Dependent Immunogenicity and Are Nontoxic up to High Doses In Vivo. Small 18, 2108063 (2022).

17.       Douglas, S. M., Bachelet, I. & Church, G. M. A Logic-Gated Nanorobot for Targeted Transport of Molecular Payloads. Science 335, 831–834 (2012).

18.       Pal, S. & Rakshit, T. Folate-Functionalized DNA Origami for Targeted Delivery of Doxorubicin to Triple-Negative Breast Cancer. Frontiers in Chemistry 9, (2021).

19.       Weiden, J. & Bastings, M. M. C. DNA origami nanostructures for controlled therapeutic drug delivery. Current Opinion in Colloid & Interface Science 52, 101411 (2021).

20.       Balakrishnan, D., Wilkens, G. D. & Heddle, J. G. Delivering DNA origami to cells. Nanomedicine 14, 911–925 (2019).

21.       Fan, Q., He, Z., Xiong, J. & Chao, J. Smart Drug Delivery Systems Based on DNA Nanotechnology. ChemPlusChem 87, e202100548 (2022).

22.       Yan, J. et al. Growth and Origami Folding of DNA on Nanoparticles for High-Efficiency Molecular Transport in Cellular Imaging and Drug Delivery. Angewandte Chemie International Edition 54, 2431–2435 (2015).

23.       Shaw, A., Benson, E. & Högberg, B. Purification of Functionalized DNA Origami Nanostructures. ACS Nano 9, 4968–4975 (2015).

24.       Macdonald, G. J. Vector Production Processes Play Key Role in High Gene Therapy Prices. GEN – Genetic Engineering and Biotechnology News https://www.genengnews.com/topics/bioprocessing/vector-production-processes-play-key-role-in-high-gene-therapy-prices/ (2022).

25.       High Quality Gene Synthesis – Twist Bioscience. https://www.twistbioscience.com/products/genes?tab%27overview=&adgroup=114820676383&creative=491174669905&device=c&matchtype=p&location=9014872.

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