Q&A with Dr. Juliane Nguyen: Exosome Profiling with miRNA and Biotech’s Next Breakthrough
Dr. Juliane Nguyen is a professor in the Department of Pharmaceutical Sciences at the University of Buffalo. Her Therapeutic Biomaterials Laboratory investigates using exosomes and other nanoscale carriers for the treatment of diseases including cancer and ischemic heart disease.
NanoString (NS): Tell us about the research conducted in your lab and how exosome profiling applies to your work.
Juliane Nguyen (JN): In my lab, we develop platforms comprised of proteins to use as a biochemical tool to better understand biological systems. We also use these platforms for the delivery of therapeutic macromolecules for treating myocardial infarction or cancer. One focus of our research is the development of biomaterials that can also function as “molecular zip codes”. These zip codes can be used to actively sort macromolecules to multivesicular bodies of cells which can be repackaged into exosomes and then used for therapeutic applications.
NS: What kind of information do you get from exosome profiling that is unique?
JN: One of the unique properties of exosomes is that they carry a combination of molecular information. They contain a variety of lipids, proteins, and nucleic acids. In many cases, the molecular content is highly reflective of the cells that secreted the exosome. When you consider all these details in the analysis, the resulting molecular fingerprint has more relevant information than could be gained just by analyzing the upregulation of a protein or RNA. We are still in the process of optimizing the technology that will let us collect and isolate a pure population of exosomes from a biofluid.
In addition, I see a very important role in diagnosing cancer and other diseases because of the variety of molecular information exosomes contain. And remember, exosome secretion is not limited to eukaryotic cells. Bacteria and pathogenic parasites secrete small extracellular vesicles for host cell infection and pathogenesis. Potentially we can use the molecular fingerprints from these pathogens to diagnose diseases and follow disease progression.
NS: What are some specific ways in which exosomes derived from different cell types function differently?
JN: Depending on the cell sources, the exosomes can exert either beneficial or detrimental phenotypic effects. For example, exosomes derived from mesenchymal cells have intrinsic tissue regenerative effects—they induce angiogenesis, cell proliferation, and inhibit fibrosis. On the other hand, exosomes secreted from cancer cells can contribute to cancer growth by inducing macrophages to pro-malignant phenotypes and enhancing cell migration. How exosomes impact recipient cells depends on the interactions between the exosomal surface and the receptors that are present on the surface of the recipient cell –and different recipient cells may react differently to the same exosome.
NS: Are exosomes potentially a better drug delivery tool because they are already biologically present and physiologically “known” to the recipient?
JN: Absolutely. I think that their potential as drug carriers is why a lot of people have gotten interested in this field. There has been a lot of work on liposomes as drug carriers, but they lack the membrane proteins found in exosomes that mediate uptake into recipient cells. It’s very challenging to incorporate these proteins into a lipid bilayer while keeping the overall liposome size small. In a sense, exosomes are nature’s drug carriers and vehicles for communication so they are a smart option to explore. They are small, making them ideal for deep tissue penetration. They are also highly stable in the presence of enzymes, so they can protect their cargo from degradation. This is especially true for milk-derived exosomes; if we can better understand what makes them so stable we can use this to devise better drug carriers.
NS: What are some of the biggest challenges in using exosomes as a therapeutic treatment?
JN: Because exosomes contain so many different components (lipids, proteins, RNA) it is very important that they are thoroughly characterized prior to use as a drug carrier. We need this information to understand and then predict potential side effects. If we want to capitalize on the intrinsic properties of exosomes for therapeutic applications, then we must know the combination of “active ingredients” that will result in the desired effect. We also need to ensure that each batch of exosomes produced will have the same therapeutic efficacy. Exosome compositions can change even with small changes in culturing conditions such as the number of times cells are passaged or the culture media. All these variables need to be characterized before we can use exosomes in a clinical application.
NS: Are microRNAs (miRNAs) a significant portion of exosomal content?
JN: Several studies on exosomal content showed that while miRNAs and mRNAs are not the most abundant component they are key components, particularly in exosome-mediated communication. Exosomes are often enriched in non-coding RNAs such as snoRNA, scaRNA, tRNA, and rRNA. The biological contribution of these RNAs is less well understood but because they are so prevalent it is highly likely that they play an important role in cell-cell communication.
NS: Do you envision miRNAs being used as a therapy?
JN: It has been shown that the expression of miRNAs can be altered in the treatment of many diseases including cancer and ischemic heart disease. If we can identify the signature of these miRNAs, how they change during disease progression, and their mechanism of action then we can develop therapies that influence their expression. The advantage of miRNAs is that they can have many different mRNA targets and therefore many different gene targets. This also makes it a challenge to work with them because we do not yet understand all their downstream effects. I see a potential for using miRNAs as therapies across most types of diseases once we better understand these pathways and can predict the outcomes and side effects.
NS: Customizing synthetic exosomes seems like a logical next step. What is your lab doing in this area?
JN: There is so much exciting work going on around engineering exosomes with a variety of cargoes and decorating them with targeting ligands to enhance delivery specificity. These are being used to treat neurological disorders, cancer, and facilitate tissue regeneration. In my lab we use exosomes derived from mesenchymal stem cells to regenerate tissue after acute myocardial infarction. We are also interested in reprogramming pathological exosomes secreted from cancer cells to make them non-tumorigenic and then repackaging anti-metastatic miRNAs into them. We want to reintroduce them into cancer cells and see if they can decrease metastatic cancer growth.
NS: It sounds like you envision a personalized, therapeutic workflow using exosomes.
JN: Yes, we could do personalized medicine based on patient-derived exosomes. The advantage of exosomes over other cell-based therapies is that exosomes can be manufactured in advance, freeze-dried, and then stored for later use. This eliminates the time pressure associated with other cell-based therapies. Another option would be to produce exosomes from specific cellular sources independent of the patient that could have a broader application to multiple patients. Cell-derived exosomes require particular care in preventing the induction of an immune response. Mesenchymal stem cells could be used in exosome production because they have very low immunogenicity, making them a potential option for allogenic-based therapies.
NS: What advice do you have for people struggling with the preparation of exosomes?
JN: There is a lot of information out there about the best practices in purifying exosome samples and experts have still not yet reached a consensus. Differential centrifugation and density centrifugation are widely accepted techniques for exosome isolation. For larger batches, you can use tangential flow filtration. Other methods include microfluidic-based isolation, immuno-affinity capture, or precipitation-based methods. Bear in mind that no matter what type of isolation method you choose, the exosome population and purity can change based on the purification method. So be aware of the limitations of the purification method, the mix of exosome population created, and how that mix will influence the downstream effects in your assay.
NS: Why did you choose the NanoString® platform for miRNA profiling over qPCR or RNA sequencing?
JN: We saw a lot of publications that mentioned how sensitive, robust, and reproducible the NanoString platform is. NanoString was the perfect choice for the questions we were asking about the miRNA composition of the exosomes. Not only do we want to examine miRNAs with known biological function, we also want to better understand how the miRNA composition changes when they are derived from different cellular sources and how that, in turn, can impact their biological function on the recipient cell. The assays NanoString have are focused, allowing us to do our experiments quickly. When we first started using the platform we naturally had a lot of questions. Your customer service was really awesome! Anytime we emailed tech support with questions we got answers within minutes.
NS: Who and what excites and inspires you about this field?
JN: I work in a highly interdisciplinary field and there are many scientists that have made strong contributions to the field. I am inspired by scientist who think outside the box and use that creativity to advance the field. I look at the work done by my post-doctoral advisor, Dr. Francis Szoka at UCSF. He pioneered the field of using liposomes as drug delivery agents and now that is a clinical reality—he is a great inspiration to me. If we can do with exosomes what he has done with liposomes then we will have made a significant contribution to science and medicine.
We are only just starting to understand exosomes, the role they play under normal physiological and disease conditions, and their potential. It is a rapidly growing field—exosomes could be the next major breakthrough in biotech. My students and I recently surveyed the number of publications referring to exosome profiling. In 2006, there were about 70 articles that appeared using our search criteria in PubMed. In 2018, that number jumped to over 8,000 articles. There is a huge interest in understanding exosomes and the important role they play in the body, in diagnostics, and as a potential therapeutic tool. We’re looking forward to what we can do next with NanoString.
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