Using Spherical Nucleic Acids to Track and Treat Disease

Thought LeadersDr. Chad MirkinDirectorInternational Institute for Nanotechnology

An interview with Dr. Chad Mirkin, Northwestern University, conducted by April Cashin-Garbutt, MA (Cantab)

What are spherical nucleic acids (SNAs)? What do they consist of and how do they differ from linear nucleic acids?

Spherical nucleic acids are structures that are made by taking a nanoparticle template and using chemistry to arrange short strands of DNA or RNA on the surface of those particles. The spherical core of the nanoparticle creates a spherical arrangement of DNA or RNA, similar to tiny little balls of nucleic acids.

Even though the sequences can be identical, the properties of spherical nucleic acids are very different from linear nucleic acids. For example, SNAs bind complementary DNA or RNA much more tightly than linear nucleic acids.

This means that in the context of detection and the use of SNAs as diagnostic probes, you can use a lower concentration of a nucleic acid target, for example, associated with a given disease. And so, these have become the basis for high sensitivity and also very high selectivity probes, in molecular diagnostic tools.

How can SNAs be used for the detection of infections?

There's a technology called the Verigene system that was commercialized by Nanosphere, a company I had started, which was then sold to Luminex. The Verigene system is used to sort signatures associated with disease, infectious disease in particular, and at very low concentrations, meaning at very early time points, to measure the presence of a particular infection. For example, in blood.

This is important because it can then be used, for example, to diagnose patients with sepsis, where being able to diagnose very early is really important because for every hour that a patient goes undiagnosed and untreated, the chance of mortality increases substantially.

Technology like this is changing the way molecular diagnostics is carried out. It is a very simple and rapid point-of-care medical diagnostic tool that allows for the detection of bacterial infections way before conventional tests. It is not necessary to go through the process of culturing the sample, which takes a long time and, therefore, increases patient risk.

So ultimately, you have a tool that is better for the patient because you get an accurate diagnosis earlier and better for the doctor, because the doctor is not needlessly prescribing a lot of unnecessary antibiotics, wasting money, and contributing to antibiotic resistance. Instead the tool can be used to figure out who has a bacterial infection and who doesn't;  the appropriate treatment with effective measures then can be taken.

What does SNA synthesis involve?

In the case of developing a biological label, a gold nanoparticle is used for the template, and the SNA is made by bringing the template in contact with short strands of DNA that can be chemically anchored to it. In the case of gold, the anchoring groups are thiols.

We have developed a process that allows you to load the DNA or RNA on the surface of a particle to very high extents. The reason that's important is that it forces the orientation and gives the architecture both its spherical shape and also the properties that I've been mentioning.

Can you please outline your upcoming talk at Pittcon 2017 on ‘Nano-Enabled In Vitro and In Vivo Diagnostic Tools for Tracking and Treating Disease’? What bioassays will you be focusing on?

At Pittcon I will be focusing on two different types of bioassays:

• Ones based upon the Verigene system
• A new technology that allows one to measure intra-cellular nucleic acid targets -- mRNA

Both technologies are based on SNAs, which are structures that can enter a live cell, bind to a particular target, in this case an mRNA target, and elicit or liberate a fluorophore signaling entity that lights up the cell.

This allows you to then measure for the first time, the genetic content of live cells. In addition to measuring the genetic content, cells can be differentiated based on mRNA expression levels. The location of the RNA within the cell can also be measured, which is especially exciting because nobody has ever been able to do that in live cells before now.

This is especially interesting because when coupled with a technology like flow cytometry, you are able to sort cells based upon genetic differences. Millipore is a company that has commercialized this technology and produced many variations of these types of architectures, so that researchers can begin to look, for example, for rare cell populations and pick out circulating tumor cells, in the presence of healthy cells.

This becomes a way of studying the cells and the number of them. It  also allows you to isolate them so that you can study them after the fact. You can pull them away from majority cell populations, culture them, and use them to understand the origins of genetic differences. For example, looking at how a cancer patient’s cells respond to different types of therapeutics.

This is a major step towards personalized medicine and increasing our capabilities with respect to probing cellular systems. It's also potentially useful for high throughput drug screening, where you can look at how different types of drug molecule activate or suppress different types of genes. You can get a visual readout in this case, based upon the use of this technology, we refer to as nano-flare technology. Millipore has commercialized a form of nanoflares they refer to as smart-flares.

What will be the focus of your second talk at Pittcon 2017, ‘Spherical Nucleic Acids as Potent Immunomodulation Agents for Cancer Therapy’?

SNA structures also represent the basis for an entire new class of nucleic acid therapeutics. There are three central arteries of drug development:

• Small molecules

Benefits are well known, aspirin being a great example.

• Biologics

Seven of the top ten drugs are based upon biologics; these are antibodies, protein-based architectures. They have a lot of advantages and capabilities that go beyond what small molecules offer.

• Nucleic acid medicines

Here short snippets of DNA or RNA are used to treat disease and attack it at its genetic roots.

Antisense drugs are based upon DNA and are used to soak up mRNA in cells and stop translation of that RNA and production of proteins that we associate with disease. The idea behind antisense is that you can regulate a person’s cells and convert an unhealthy cell into a healthy cell by knocking down the production of a specific type of protein.

Then came along siRNA technology – a similar concept in the sense that you're knocking down the production of specific types of proteins, but via different pathways. The idea of developing genetic medicine is really the concept of a type of digital medicine, where instead of every time you need a new drug you don't look for a new small molecule, you change the sequence of DNA or RNA being used based upon an understanding of biological pathways.

From a conceptual standpoint, these were really powerful technologies. They led to the development of many commercial approaches but have had limited success. The reason being, to truly realize digital medicine you need multiple things in play. One is you have to be able to synthesize DNA and RNA, and two, you have to be able to understand pathways.

These two issues have now been overcome; we know how to synthesize DNA and RNA, and thanks to the human genome project, we also know a lot about the pathways of disease and how to attack different types of pathways to treat disease. But the third, and perhaps most important requirement, is the ability to get the DNA or RNA to the site that matters. And that's where most attempts have fallen short.

This is where spherical nucleic acids are very important. SNA structures, which have no natural equivalent, can interact with natural systems completely differently from the native DNA and RNA from which they're derived. Almost every cell type in your body, other than mature red blood cells, recognize SNAs and rapidly internalize them without the need for transfection agents.

This is particularly interesting because, for example, putting normal DNA or RNA in creams and putting them on your skin won't make them go into your skin cells; but with spherical nucleic acids they'll rapidly take them up. This discovery therefore opens up the ability to create topical medicines, local medicines, that allow you to treat a lot of diseases.

And so we've been looking at this capability in terms of developing new types of treatments for skin disease.  There are over 200 diseases with a known genetic basis. One can begin to think about creating therapeutics for the eye, ear, lung, bladder, and colon via similar approaches.

The fundamental properties of SNAs make nucleic acids relevant for treating a wide range of medical conditions not addressable with conventional nucleic acids. The first SNA constructs are in human trials for treating psoriasis.

How could SNAs be used in cancer vaccines?

Another application we've been researching is the use of structures as potent regulators of the immune system. SNAs will enter immune cells, dendritic cells, and if the sequence is correct, they will activate toll-like receptors, so that you can take an animal, or a patient in principle, and selectively activate their immune system.

This allows for the creation of new forms of vaccines, for example, where you can train a person’s body to fight a specific type of cancer. This is what is happening right now, we have a whole series of drug candidates based upon this approach, and I will be talking primarily about prostate cancer at Pittcon.

In principle vaccines like this could be developed to treat many different types of cancers, including cancer of the brain, bladder, colon, and melanoma.

What stage of development are SNA cancer vaccines currently at and what hurdles still need to be overcome?

The cancer vaccine work is just about to go into human clinical trials this year. The technology has been extensively vetted in animals and proven to be safe, for example, in primates.

The human trials are extremely important. With a cancer vaccine, you are modulating a person's immune system and there is a risk of creating autoimmune responses.

What are the next steps in your research?

For me, it's all about understanding what makes these structures so special and continuing to understand how we can build different forms of spherical nucleic acids, and use the unique properties of them to solve major problems in medicine and other areas of research.

Do we currently know why the spherical nucleic acids are internalized or is further research needed to fully understand this?

At the moment, we believe that they are recognized by what are called scavenger receptors; these are structures common to many cell types, and they're used to move cargo in and out of cells.

They have also been shown to recognize and bind to spherical nucleic acids much more tightly than linear nucleic acids, and so effectively we have, in part by accident, discovered and designed an architecture that is recognized by natural biological machinery, scavenger receptors that lead to their internalization into a cell.

There are several papers that explore this for different cells types, and all of our research thus far is consistent with that conclusion.

What are you looking forward to at Pittcon 2017?

It's honestly a really exciting venue for anybody interested in analytical chemistry, new instrumentation, or new techniques associated with that instrumentation, and so, I particularly enjoy the frontier talks. But of course, I also enjoy the expo hall and seeing all the new technology on display.

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Where can readers find more information?

1. Cutler, J. I.; Auyeung, E.; Mirkin, C. A. “Spherical Nucleic Acids,” J. Am. Chem. Soc., 2012, 134, 1376–1391, doi: 10.1021/ja209351u.
2. Alhasan, A. H.; Kim, D. Y.; Daniel, W. L.; Watson, E.; Meeks, J. J.; Thaxton, C. S.; Mirkin, C. A. “Scanometric microRNA (Scano-miR) Array Profiling of Prostate Cancer Markers Using Spherical Nucleic Acid (SNA)-Gold Nanoparticle Conjugates,” Anal. Chem.,2012, 84, 4153-4160, doi: 10.1021/ac3004055, PMCID; PMC3357313.
3. Zheng, D.; Giljohann, D. A.; Chen, D. L.; Massich, M. D.; Wang, X.-Q; Iordanov, H.; Mirkin, C. A.; Paller, A. S. “Topical Delivery of siRNA-based Spherical Nucleic Acid Nanoparticle Conjugates for Gene Regulation,”  Proc. Natl. Aca. Sci. USA, 2012, 109, 11975-11980, doi: 10.1073/pnas.1118425109, PMCID: PMC3409786.
4. Young, K. L; Scott, A. W.; Hao, L.; Mirkin, S. E.; Liu, G.; Mirkin, C. A. “Hollow Spherical Nucleic Acids for Intracellular Gene Regulation Based Upon Biocompatible Silica Shells,” Nano Lett., 2012, 12, 3867–3871, doi: 10.1021/nl3020846, PMCID: PMC3397824.
5. Zhang, K.; Hao, L.; Hurst, S. J.; Mirkin, C. A. “Antibody-linked Spherical Nucleic Acids for Cellular Targeting,” J. Am. Chem. Soc., 2012, 134, 16488–16491, doi: 10.1021/ja306854d, PMCID: PMC3501255.
6. Choi, C. H. J.; Hao, L.; Narayan, S. P.; Auyeung, E.; Mirkin, C. A. “Mechanism for the Endocytosis of Spherical Nucleic Acid Nanoparticle Conjugates,” Proc. Natl. Aca. Sci., 2013, 110, 7625-7630, doi: 10.1073/pnas.1305804110, PMCID: PMC3651452.
7. Jensen, S. A.; Day, E. S.; Ko, C. H.; Hurley, L. A.; Luciano, J. P.; Kouri, F. M.; Merkel, T. J.; Luthi, A. J.; Patel, P. C.; Cutler, J. I.; Daniel, W. L.; Scott, A. W.; Rotz, M. W.; Meade, T. J.; Giljohann, D. A.; Mirkin, C. A.; Stegh, A. H. “Spherical Nucleic Acid Nanoparticle Conjugates as an RNAi-Based Therapy for Glioblastoma,” Science Trans. Med., 2013, 5, 209ra152, doi: 10.1126/scitranslmed.3006839, PMCID: PMC4017940.
8. Rink, J. S.; McMahon, K. M.; Zhang, X.; Chen, X.; Mirkin, C. A.; Thaxton, C. S.; Kaufman; D. B. “Knockdown of Intra-Islet IKKβ by Spherical Nucleic Acid Conjugates Prevents Cytokine-Induced Injury and Enhances Graft Survival,” Transplantation, 2013, 96, 877-884, doi: 10.1097/TR.0b013e3182a4190e, PMCID: PMC3839058.
9. Walter, S. R.; Young, K. L.; Holland, J. G.; Gieseck, R. L.; Mirkin, C. A.; Geiger, F. M. “Counting the Number of Magnesium Ions Bound to The Surface-immobilized Thymine Oligonucleotides That Comprise Spherical Nucleic Acids,” J. Am. Chem. Soc., 2013, 135, 17339-17348, doi: 10.1021/ja406551k.
10. Alhasan, A. H.; Patel, P.C.; Choi, C. H. J.; Mirkin, C.A. “Exosome Encased Spherical Nucleic Acid Gold Nanoparticle Conjugates as Potent MicroRNA Regulation Agents,” Small, 2014, 10, 186-192, doi: 10.1002/smll.201302143, PMCID: PMC3947239.
11. Mirkin, C. A.; Stegh, A. H. “Spherical Nucleic Acids for Precision Medicine,” Oncotarget, 2014, 5, 9-10, PMCID:  PMC3960185
12. Wu, X. A.; Choi, C. H. J.; Zhang, C.; Hao, L.; Mirkin, C. A. “Intracellular Fate of Spherical Nucleic Acid Nanoparticle Conjugates,” J. Am. Chem. Soc., 2014, 136, 7726-7733, doi: 10.1021/ja503010a, PMCID: PMC4046773.
13. Rouge, J. L.; Hao, L.; Wu, X. A.; Briley, W. E.; Mirkin, C. A. “Spherical Nucleic Acids as a Divergent Platform for Synthesizing RNA-Nanoparticle Conjugates Through Enzymatic Ligation,” ACS Nano, 2014, 8, 8837-8843, doi: 10.1021/nn503601s, PMCID: PMC4174098.
14. Banga, R. J.; Chernyak, N.; Narayan, S. P.; Nguyen, S. T.; Mirkin, C. A. “Liposomal Spherical Nucleic Acids,” J. Am. Chem. Soc., 2014, 136, 9866-9869, doi: 10.1021/ja504845f, PMCID: PMC4280063, PMCID: PMC4280063.
15. Chinen, A. B.; Guan, C. M.; Mirkin, C. A. “Spherical Nucleic Acid Nanoparticle Conjugates Enhance G-Quadruplex Formation and Increase Serum Protein Interactions,” Angew Chem., 2014, 54, 527-531, doi: 10.1002/anie.201409211, PMCID: PMC4314381.
16. Radovic-Moreno, A. F.; Chernyak, N.; Mader, C. C.; Nallagatla, S.; Kang, R.; Hao, L.; Walker, D. A.; Halo, T. L.; Merkel, T. J.; Rische, C.; Ananatatmula, S.; Burkhart, M.; Mirkin, C. A.; Gryaznov, S. M. “Immunomodulatory Spherical Nucleic Acids,” Proc. Natl. Aca. Sci, 2015, 112, 3892-3897, doi: 10.1073/pnas.1502850112, PMCID: PMC4386353.
17. Narayan, S. P.; Choi, C. H. J.; Hao, L.; Calabrese, C. M.; Auyeung, E.; Zhang, C. Goor, O. J. G. M.; Mirkin, C. A. “The Sequence-Specific Cellular Uptake of Spherical Nucleic Acid Nanoparticle Conjugates,” Small, 2015, 11, 4173-4182, doi: 10.1002/smll.2015100027, PMCID: PMC4560454.
18. Zhang, C.; Hao, L.; Calabrese, C. M.; Zhou, Y.; Choi, C. H. J.; Xing, H.; Mirkin, C. A. “Biodegradable DNA-brush Block Copolymer Spherical Nucleic Acids Enable Transfection Agent-Free Intracellular Gene Regulation,” Small, 2015, doi 10.1002/smll.201501573.
19. Randeria, P. S.; Jones, M. R.; Kohlstedt, K. L.; Banga, R. J.; Olvera de la Cruz, M.; Schatz, G. C.; Mirkin, C. A. “What Controls the Hybridization Thermodynamics of Spherical Nucleic Acids?” JACS, 2015, 137, 3486-3489, doi: 10.1021/jacs.5b00670.
20. Randeria, P. S.; Seeger, M. A.; Wang, X. Q.; Wilson, H.; Shipp, D.; Mirkin, C. A.; Paller, A. S. “siRNA-based Spherical Nucleic Acids Reverse Impaired Wound Healing in Diabetic Mice by GM3 Synthase Knockdown,” PNAS, 2015, 112, 5573-5578, doi: 10.1073/pnas.1505951112, PMCID: PMC4426446.
21. Rouge, J. L.; Sita, T. L.; Hao, L.; Kouri, F. M.; Briley, W. E.; Stegh, A. H.; Mirkin, C. A. “Ribozyme-Spherical Nucleic Acids,” JACS, 2015, 137, 10528-10531, doi:10.1021/jacs.5b07104.
22. Barnaby, S. N.; Perelman, G. A.; Kohlstedt, K. L.; Chinen, A. B.; Schatz, G. C.; Mirkin, C. A. “Design Considerations for RNA Spherical Nucleic Acids (SNAs)” Bioconjugate Chemistry, 2016, 27, 2124-2131, doi: 10.1021/acs.bioconjchem.6b00350, PMCID: PMC5034328.
23. Wang, X.; Hao, L.; Bu, H.-F.; Scott A. W.; Tian K.; Liu, F.; DePlaen, I. G.; Liu, Y.; Mirkin, C. A.; Tan, X.-D. “Spherical Nucleic Acid Targeting MicroRNA-99b Enhances Intestinal MFG-E8 Gene Expression and Restores Enterocyte Migration in Lipopolysacchardie-induced Septic Mice,” Scientific Reports, 2016, 6, 31687 doi: 10.1038/srep31687.
24. Sprangers, A. J.; Hao, L.; Banga, R. J.; Mirkin, C. A. “Liposomal Spherical Nucleic Acids for Regulating Long Noncoding RNAs in the Nucleus,” Small, 2017, doi: 10.1002/smll.201602753.

About Dr. Chad Mirkin

Dr. Chad A. Mirkin is the Director of the International Institute for Nanotechnology and the George B. Rathmann Prof. of Chemistry, Chemical and Biological Engineering, Biomedical Engineering, Materials Science & Engineering, and Medicine at Northwestern University.

He is a chemist and a world-renowned nanoscience expert, who is known for his discovery and development of spherical nucleic acids (SNAs) and SNA-based biodetection and therapeutic schemes, Dip-Pen Nanolithography (DPN) and related cantilever-free nanopatterning methodologies, On-Wire Lithography (OWL), and Co-Axial Lithography (COAL), and contributions to supramolecular chemistry and nanoparticle synthesis.

He is the author of over 670 manuscripts and over 1,000 patent applications worldwide (290 issued), and he is the founder of multiple companies, including Nanosphere, AuraSense, and Exicure, which are commercializing nanotechnology applications in the life sciences and biomedicine.

Mirkin has been recognized with over 100 national and international awards, including the 2016 Dan David Prize and the inaugural Sackler Prize in Convergence Research. He was a member of the President’s Council of Advisors on Science & Technology (Obama Administration), and one of very few scientists to be elected to all three US National Academies. He is also a Fellow of the American Academy of Arts and Sciences and the National Academy of Inventors, among others.

Mirkin has served on the Editorial Advisory Boards of over 20 scholarly journals, including JACS, Angew. Chem., and Adv. Mater.; at present, he is an Associate Editor of JACS. He is the founding editor of the journal Small, and he has co-edited multiple bestselling books.

Mirkin holds a B.S. degree from Dickinson College (1986, elected into Phi Beta Kappa) and a Ph.D. degree from the Penn. State Univ. (1989). He was an NSF Postdoctoral Fellow at the MIT prior to becoming a professor at Northwestern Univ. in 1991.

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