DNA nanotechnology, that encompasses the self-assembly of nucleic acids into nanostructures, has shown promise for various applications, including nanomachines, nanopores, drug delivery systems, and biosensors. Despite the potential, existing assembly techniques face challenges in scalability, consistency, and pharmaceutical compatibility.

A common strategy to assemble these types of nanostructures is the “scaffolded DNA-origami” where a long single-stranded DNA (ssDNA) molecule (scaffold) is folded with the assistance of short oligonucleotides (staples) into the target nanostructure. Computational algorithms guide the design of ssDNA and staples. Scaffolds are produced biologically, enzymatically, or through asymmetric PCR, while smaller staples are commercially available. Assembly occurs through thermal annealing with high magnesium concentrations, followed by purification to eliminate impurities using methods such as agarose gel extraction and PEG precipitation. DNA-origami nanostructures (DON) offer advantages over metal nanoparticles, including precision, addressability, lower biotoxicity, increased stability, and superior adaptability. Functionalization of DON introduces added functionality by conjugating proteins, polymers, metal nanoparticles, or quantum dots to nanostructures, enhancing their potential in drug delivery, biosensing and cell imaging.

However, the development of drug delivery and bio-imaging applications based on DNA-origami requires the use of large amounts of well-folded and impurity-free DNA nanostructures. Current methods for biologically-produced scaffold purification rely on agarose gel extraction or ethanol precipitation, which are not scalable or selective, preventing from delivery a product with therapeutic quality. For this reason, the primary goal of this project is to develop scalable processes for the biomanufacturing of scaffolds and DON. Moreover, the possibility of using functionalized DON for image-guided drug delivery will be evaluated though DON functionalization using etoposide (ETOP), a chemotherapeutic agent used in the treatment of prostate cancer, bombesin (BBN) and NODAGA for 67Ga radiolabeling will be evaluated. ETOP exhibits efficacy in preclinical prostate cancer models by inhibiting DNA topoisomerase II, inducing crucial errors in DNA synthesis, and triggering apoptosis. BBN conjugation with nanoparticles has shown promise in delivering imaging and therapeutic radionuclides to tumor cells overexpressing the gastrin-releasing peptide receptor (GRPR). NODAGA ensures stable coordination of metal ions, including 67Ga, suitable for single photon emission computed tomography (SPECT).

We will initially develop a scalable method for the downstream processing of DNA-origami scaffolds based on M13mp18 genome (Task 1), evaluating either the feasibility of go directly from a phage lysate to a chromatographic step or do a first step of concentration and diafiltration, resorting to centrifugal filters, followed by chromatography. Different chromatographic modalities will be explored. Then, to scale-up the production and purification biomanufacturing process of DNA-origami scaffolds based on M13mp18 genome batch and/or fed-batch bioreactors will be used to produce large titers of phage particles (Task 2). A downstream process based on tangential flow filtration and chromatography will be integrated to deliver a high-quality product. Host cell contaminants will be analyzed to ensure the compliance with Good Manufacturing Practices (GMP) and the delivery of a consistent and high-quality product, that can then be used to assemble DON suitable for therapeutic applications.
Our innovative proposal will also address the potential use of DON for prostate cancer treatment, since this type of cancer is the second-leading cause of cancer-related death in men. For this we will use the produced scaffolds to fold and functionalize the target DON with ETOP, BBN and NODAGA (Task 3). Finally, the functionalized structure will be radiolabeled with 67Ga and characterized in vitro in terms of toxicity, effectiveness, and specificity (Task 4), through assays that determine the binding affinity to GRPR, cellular uptake, programmed cell death, among others.
This cutting-edge project exploits the unique expertise of a multidisciplinary team with expertise in upstream and downstream processing, biomolecule characterization, chemical functionalization, radiolabeling, and in vitro analysis studies from iBB, CQE and C2TN. It will capitalize on the deep knowledge of the PI on biomolecules downstream processing. Our proponent team is also specialized in bioengineering, chemistry, and nuclear technology. This frontier project will provide a scalable biomanufacturing platform for ssDNA scaffold production for DON and show the use of these type of nanostructures for image-guided drug delivery.