DNA nanostructures have been evolving fast in the past few decades and have found various new applications in biomedicine. The biocompatibility, binding-specificity and programmability of DNA nanostructures make them ideal drug carriers [1]. Currently, most functional DNA nanostructures are assembled in vitro, using chemically synthesized custom oligonucleotides. More efficient mass production of functional DNA nanostructures is crucial, not only to accelerate research progress in this promising field, but also to expand the application of such versatile molecules in biomedicine. In this project, we design new therapeutic DNA nanostructures, explore the potential of mass producing them in bacteria and endeavor to establish an in vivo library of functional DNA nanostructures.
Functional DNA nanostructures
Design principles: safety, efficient cell entry, flexibility, stability
In this project, 2 DNA nanostructures, namely Nano Drug Carrier (NDC) and Nano Drug Carrier-AS1411 (NDC-AS) for breast cancer therapy were designed and tested. Each nanostructure is made up of 5 single-stranded DNA (ssDNA) synthesized using 5 different BioBricks. The 2 nanostructures have 2 componant strands in common, so a total of 8 BioBricks were made and submitted.
Our Nano Drug Carriers are composed entirely of DNA, which is non-toxic and degradable inside human cells. The Nano Drug Carriers are designed as tetrahedrons to facilitate cell entry. Previous studies have shown that three-dimensional DNA nanostructure enter mammalian cells more efficiently than two-dimensional AS1411 or linear structures [2]. Tetrahedron is chosen because we consider it the simplest three-dimensional structure, which can be easily assembled from just a few DNA strands. Building the Nano Drug Carrier with separate DNA strands means that much flexibility is allowed for functional modifications. Functional DNA sequences can be conveniently added to the 4 vertices of the tetrahedron to achieve oligonucleotide delivery or cell antigen binding, enhancing the effect of the drug. Three-dimensional structures composed of double stranded DNA have been shown to be stable in extracellular compartment, making them ideal as drug carriers [3].
Bacterial nanostructure production
Our project stems from a previously described method of DNA nanostructures genetic encoding and self-assembly in living bacteria [4]. Despite its demonstration of successful in-vivo nanostructure production, the nanostructure produced does not have any function. We designed new biobricks to be operated in a DNA nanostructure production system named ETHERNO (E. coli-synthesized Therapeutic Nanostructures).
Each of the biobrick we submitted encodes a single-stranded DNA of specific sequence. These ssDNA synthesized can then be extracted for assembly of our originally designed therapeutic nanostructure. This way our therapeutic devices can be biologically produced in large scale. The components of all our devices are genetically stored and can be produced upon induction. Establishment of such a bacterial library allows convenient storage, production and extraction of DNA nanostructures, promoting DNA nanostructure research and application.
For more details on the operation of this system, please refer to Part Overview.
Therapeutics
According to WHO [5], breast cancer is the most frequent cancer among woman impacting 2.1 millions each year, and at the same time the greatest number of cancer related death. [6] As a result, it has been a field with significant researches and it has been shown that early breast cancer treatment usually result in high survival rate. However, there have been increasing reviews in the recent years indicating that there are high resistance against endocrine therapy and immunotherapy. [7] Furthermore, patients that are with metastatic traits such as lymph node presentation has a high rate of recurrence and therefore need a specific targeted drugs such that the metastatic breast cancer can be recognized and eliminated. According to a researched in 2016 “Doxorubicin resistance in breast cancer cells is mediated by extracellular matrix proteins” it was mentioned that the extracellular matrix proteins and interaction with the cells could possibly play a role in doxorubicin resistance as demonstrated in MDA-MB-231 cells. This trigger our HKU iGEM team to develop a specific drug carrier such that can carry the doxorubicin directly into the cell such that it can directly interact with the DNA and therefore inhibit the topoisomerase II directly. [8] [9] We hope that the DNA tetrahedral will play a significant role acting as a drug carrier and open up possibilities of new way of treatment, and potentially in the future not limited to breast cancer.
References
Jiang, D., England, C.G. & Can W. (2016, October 10). DNA Nanomaterials for Preclinical Imaging and Drug Delivery. J Control Release. 239, 27–38.
Xia, Z., Wang, P., Liu, X., Liu, T., Yan, Y., Yan, J....He, D. (2016, March 8). Tumor-penetrating peptide-modified DNA tetrahedron for targeting drug delivery. Biochemistry, 55(9),1326-1331.
Kumar, V., Palazzolo, S., Bayda, S., Corona, G., Toffoli, G. & Rizzolio F. (2016). DNA Nanotechnology for Cancer Therapy. Theranostics, 6(5), 710-725.
Elbaz, J., Yin, P. & Voigt, C.A. (2016, April 19). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nat Commun. 7, 11179.
"Breast Cancer." World Health Organization. September 12, 2018. Accessed October 17, 2018. http://www.who.int/cancer/prevention/diagnosis-screening/breast-cancer/en/.
Lancet, The. "Breast Cancer Targeted Therapy: Successes and Challenges." The Lancet389, no. 10087 (2017): 2350. doi:10.1016/s0140-6736(17)31662-8.
Sledge GW, Mamounas EP, Hortobagyi GN, Burstein HJ, Goodwin PJ, Wolff AC. Past, Present, and Future Challenges in Breast Cancer Treatment. Journal of Clinical Oncology. 2014;32(19):1979-1986. doi:10.1200/JCO.2014.55.4139.
Lovitt, Carrie J., Todd B. Shelper, and Vicky M. Avery. "Doxorubicin Resistance in Breast Cancer Cells Is Mediated by Extracellular Matrix Proteins." BMC Cancer18, no. 1 (2018). doi:10.1186/s12885-017-3953-6.
Thorn CF, Oshiro C, Marsh S, et al. Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenetics and Genomics. 2011;21(7):440-446. doi:10.1097/FPC.0b013e32833ffb56.
Patel AG, Kaufmann SH. How does doxorubicin work? eLife. 2012;1:e00387. doi:10.7554/eLife.00387.
