DNA-origami-based biomimetic interfaces for T-cell activation

DNA Origami Nanoparticles, also referred to as DNA-NPs, are nanostructures that take advantage of the Watson-Crick complementary base pairing rules to create precise DNA nanostructures with precise and customizable size and shape within the 10-nanometer to 100-nanometer size range. DNA-NPs provide a highly programmable interface for function as nanocarriers of proteins and are highly biocompatible, allowing them to be administered in vivo as they can be broken down by natural enzymes within the body (such as DNAse). DNA-NPs have also shown significant promise in the realm of T-cell activation, acting as nanocarriers for conjugated tumor antigens and antibodies, allowing for the activation of T-cells and recognition of target antigens (such as those overexpressed in tumor cells). Here, we aim to determine favorable nanoscale organizations of simple DNA-NP motifs—specifically ligand stoichiometry, protein orientation, and position—to determine the efficacy of different T-cell signaling pathways in CD8+ T-cell expansion and proliferation. Building on previous work done under previous URSPs, the previous wheel motif will be utilized in order to move forward with assembling such interfaces. Various protein conjugation methods will be tested with motif—mainly copper free click chemistry techniques, such as DBCO-azide, maleimide-thiol, and NHS/NH2 —and conjugation efficiency will be tested through the use of surface plasmon resonance experiments. Motifs will then be tested with fluorophores/mock proteins (i.e., streptavidin) to verify the nanoscale control of antigen presentation using the DNA Origami technique, most likely through the use of atomic force microscopy (AFM) and fluorescence resonance energy transfer (FRET) experiments. After validating optimal conjugation methods with the motifs, interfaces will be produced with attached cytokines and tested by our collaborator in vitro with CD8+ T-cells.

Unknown Speaker 0:01
Ben, hi everyone. My name is Ben Safa, and my project is DNA origami based biomedic interfaces for T cell activation.

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So a key problem in adoptive cell therapy, which is a form of cancer immunotherapy,

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is basically the immunosuppressive environment of the tumor microenvironment and the related cell exhaustion adopted. U cell therapy is a form of cancer where we extract white blood cells from patients, modify them, and then re administer them. And these white blood cells are susceptible to a variety of things, but the main problem that we’re focusing on is cell exhaustion, which causes limited responses across cancers.

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The need for a memory type is integral to solving this problem of cell exhaustion, and

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there have been different types of platforms created for this, but DNA origami is something that may provide a very efficient, scalable platform and very precise platform to solving this problem. DNA origami is basically

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DNA nanostructures that are created through a long, single strand scaffold and several short staple strands that help fold the structure into any arbitrary shape.

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This research thus attempts to create such interfaces,

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namely 2d

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structures to enrich memory like qualities in T cells, in the context of improving adoptive cell therapy and fusion products.

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So the methods we employed for this were one design and two synthesis. We started with a target geometry, in this case a wheel, and used the software predicts to generate scaffold and staple sequences, and we used we ordered those Safa scaffold and scaffold sequences from

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a manufacturer and added different types of

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synthesized the scaffold, first using DNA polymerase and different types of materials such as DNTPs, and added the staple strands to get our final structure.

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Then after that, what we hope to do is attach any type of protein you want. Likely this will be different types of stimulatory ligands, such as 41 BB. This is common

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stimulatory ligand for memory for T cells. We also could attach stuff like fluorophores to track our interfaces and different types of

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beads to characterize these structures,

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so the results that we experienced

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are detailed. So at the top here we have our scaffold synthesis. So we started with a template, primers and additives. So these are DNTPs, which allow us to make the scaffold, and we have a faint Ben at the bottom, which is our single strand DNA, which you want to isolate, as well as our double strand at the top,

Unknown Speaker 3:27
the single strand DNA here is has been purified through centrifugation, and after that, we folded it with our structures, with Our staple strands, and did a variety of different characterization on this at the bottom here, you’ll see a dynamic light scattering graph on the left, then a atomic force microscopy graph image, and then a gel electrophoresis image on the left. Our DLs graph shows the diameter of our particles, and we measured it to be around 58 nanometers, and we got something around that value. So that means it’s consistent. Our AFM images showed that there were some defects in our structure, so that would require us to go back to the staple sequence design, which is what we did and what we’re currently working on. And our gel electrophoresis showed that we were able to successfully purify our structures.

Unknown Speaker 4:27
So

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over the course of the spring, basically what we’ve done is verify the folding of our structures and modify our staple sequences such that we are able to get a structure that does not have the defects that you see in these AFM images. We’ve also proceeded to add overhangs, and we are going to be trying conjugation protocols over this coming summer. Thank you. Applause.