How Professor Ken Witwer’s Lab at Johns Hopkins Is Revising Assumptions About Extracellular Vesicles

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An Interview with Dr. Kenneth Witwer, PhD | Associate Professor of Molecular and Comparative Pathobiology | Johns Hopkins University School of Medicine

Progress in cell and gene therapy is made by people who work across disciplines and build across constraints. RoosterBio’s AMA (Ask Me Anything) series brings to the page those exceptional scientists and engineers whose work sits at the frontier of advanced therapies, bioprocessing, and mechanistic biology. Our aim is simple: let their thinking speak for itself, and let it inform yours.

In this latest edition of RoosterBio’s AMA series, we welcome Professor Ken Witwer. Below, he shares why the field’s ‘sacred sentences’ deserve scrutiny, how EV surface biology may unlock a new dimension in vesicle therapeutics, and his advice for young scientists entering the field.

Kenneth Witwer

Bio:

Dr. Kenneth Witwer holds a joint appointment in molecular and comparative pathobiology and neurology at the Johns Hopkins University School of Medicine. His lab works at the intersection of extracellular vesicle biology, RNA-based regulatory mechanisms, and biomarker discovery, with an emphasis on how these pathways modulate innate and intrinsic immune defenses. Much of that work centers on neurodegenerative conditions, including HIV-associated neurocognitive disorders, Alzheimer’s, and Parkinson’s.

Witwer is the immediate past President of the International Society for Extracellular Vesicles (ISEV), the field’s leading professional organization, after previously holding the roles of Secretary General and Executive Chair of Science and Meetings. His advisory work spans two federal agencies: the NIH’s Extracellular RNA Communication Consortium (Stage 1) and the EPA’s FIFRA Scientific Advisory Panel. He also serves as an associate editor of the Journal of Extracellular Vesicles.

What motivated you to first dive into brain and retrovirus-related topics, and how did it lead to your current interest in EVs?

As a beginning graduate student with interests in HIV and its life cycle, I learned about EVs from another graduate student in the very first week of my first research rotation. My colleague told me that our virus preparations contain a large number of inactive viruses, but he also said that uninfected cell cultures seem to have many of those same particles. This intrigued me and led me to the understanding that enveloped viruses are a specialized type of extracellular vesicle, existing along a continuum. [1] Some EVs from infected cells have purely host-encoded components, while others have a mixture of host- and virus-encoded molecules. Replication-competent virions have all components needed for replication once they reach a host cell, but they also have host-encoded markers. I was fascinated by these relationships and began to wonder how non-viral EVs contribute to or oppose viral replication and host immune responses.

My focus on the brain grew out of my first PhD project, in which I examined the central nervous system effects of infection with Visna virus, a lentivirus that infects sheep. We used this model to understand more about how HIV affects the brain. [2] These studies and the results that arose from them eventually led us to an interest in other neurodegenerative diseases like Alzheimer’s and Parkinson’s.

What would you like to tell us about the exciting work your lab is doing in the last year or two?

I’m very proud of my group members and what they have accomplished recently. I would like to share two broad areas of progress.

The first is on the biodistribution, pharmacokinetics, and immunogenicity of native and engineered EVs. [3, 4, 5, 6, 7] Our data suggest that the half-life of exogenous EVs in blood circulation is usually much longer than in rodents. We also learned that, in contrast with many claims in the literature, exogenous EVs have the potential to elicit immune responses. This has implications for EV therapeutics: necessitating a careful choice of EV source cell, possible use of a patient’s own cells for EV production, or implementation of strategies to minimize immunogenicity. Finally, although unmodified EVs are rapidly cleared by the liver, we have learned that specific surface modifications may allow EVs to accumulate in other organs and also avoid immunogenicity or at least slow immune responses. We are very excited by these results and their ramifications for EV therapies.

The second area has to do with the modes of action of therapeutic EVs. With justification, the field has focused almost exclusively on EVs as a cargo delivery vehicle. That is, EVs are thought to fuse with a recipient cell and deliver active cargo into the cytoplasm to effect phenotypic change. However, the universality and efficiency of cargo delivery has been challenged by multiple rigorous studies in recent years. It’s not that cargo delivery never occurs, just that it’s more of a challenge than most of us had originally assumed. In a productive collaboration with Matias Ostrowski’s lab, [8] we have shown that the surface of EVs can be a major site of action. EVs can influence target cells dramatically, especially immune cells, by displaying signaling ligands and enzymes that modify inflammatory mediators in the extracellular environment. [9] Since these activities do not require cellular uptake or fusion, they bypass several major hurdles to development of EV therapeutics. Of course, EV surface activity is not something we discovered. It’s been known for a long time. The very first use of EVs in the clinic, as a vaccine in the 1980s, relied on surface antigenicity of EVs. However, the surface has remained relatively unexploited amid the strong focus on luminal cargo. We are enthused by the prospects of EV surface technologies and are currently working to make the EV surface even more potent through engineering.

Could your lab’s recent publication about EVs and their affinity for B cells provide insights into novel vaccine (or antigen tolerization) platforms?

We believe so. And not just for vaccines: also for using cargo delivery or surface activity to treat B cell diseases.

What advice might you have for a young graduate student who directs their thesis work toward EV related studies?

  1. Follow your interests–that’s the most important.
  2. Never assume the veracity of anything that your data don’t tell you. The EV field has several of these “sacred sentences” that appear in every review and paper introduction, but how correct are they? “There are three types of EVs.” “Exosomes are the most functional EVs.” “EVs are non-immunogenic.” “EVs function by delivering miRNAs to recipient cells.” “EVs readily cross bodily barriers.” And so on. Take pronouncements from authority with a grain of salt and challenge assumptions.
  3. Don’t be discouraged by negative results. In fact, don’t think of them as negative or indicative of a failed experiment. If you’ve set up your experiment with the proper controls and readouts, any reliable result is useful, especially in a relatively young field like EVs. Aim to test your hypothesis, not prove it, and do your best to report real results, positive or “negative.”
  4. Be your own biggest critic.
  5. Network and have fun! Science can be frustrating at times, but it’s an amazing opportunity to expand the boundaries of human knowledge, and we’re privileged to get to do it. Science is best done with friends, so reach out to others and keep in touch.

As a thought leader in the EV research community, what product, service, or application would you or your colleagues find indispensable if it could be developed?

  • More automated, hands-off, scalable options for EV separation.
  • Standardized and “dummy-proof” EV characterization products.
  • Some of these are available or under development, but it never hurts to have more and better options!

What are some online resources linking to useful organizations you would like to see supported by our readers?

  • With the caveat that I’m the current ISEV President, the International Society for EVs has developed a wide range of useful resources for EV research and applications.
  • The MISEV recommendations, with input from more than 1500 professionals over the years, take you through every step of an EV experiment or development, from nomenclature to functional testing: https://www.isev.org/misev
  • ISEV’s journals, the Journal of Extracellular Vesicles and the Journal of Extracellular Biology, have published some of the most impactful and useful papers in the field. They’re also a great outlet for your EV and EV-related research: https://www.isev.org/journals
  • In addition to the three MISEV publications, here are some other ISEV papers, most but not all appearing in ISEV journals: https://www.isev.org/position-papers-and-guidelines
  • Educational offerings include three Massive Online Open Courses: https://www.isev.org/education ISEV also has a new informal mentorship program.
  • The EVClub is an online journal and discussion forum that I founded at the start of the pandemic. Live events are recorded and placed on a YouTube channel https://www.youtube.com/c/extracellularvesicleclub along with various ISEV talks and educational videos.
  • EV-TRACK, developed by An Hendrix and colleagues, is a useful resource to assess quality and help you improve your methods reporting: https://evtrack.org/

 

Suggested References

  1. Witwer, K. W. Enveloped viruses are EVs by Kenneth W. Witwer. WebEV Talk 202; Available from: https://youtu.be/7UWmvaXJ7CA?si=LbJEzf0yJvkVs-T3.
  2. Witwer, K. W., et al., Coordinated regulation of SIV replication and immune responses in the CNS. PLoS One, 2009. 4(12): p. e8129. 10.1371/journal.pone.0008129
  3. Arifin, D. R., K. W. Witwer, and J. W. M. Bulte, Non-Invasive imaging of extracellular vesicles: Quo vaditis in vivo? J Extracell Vesicles, 2022. 11(7): p. e12241. 10.1002/jev2.12241
  4. Driedonks, T., et al., Pharmacokinetics and biodistribution of extracellular vesicles administered intravenously and intranasally to Macaca nemestrina. J Extracell Biol, 2022. 1(10). 10.1002/jex2.59
  5. Rodriguez, B. V., et al., An ex vivo model of interactions between extracellular vesicles and peripheral mononuclear blood cells in whole blood. J Extracell Vesicles, 2023. 12(12): p. e12368. 10.1002/jev2.12368
  6. Driedonks, T. A. P., et al., ELISA-based detection of immunoglobulins against extracellular vesicles in blood plasma. J Extracell Biol, 2024. 3(3): p. e129. 10.1002/jex2.129
  7. Troyer, Z., et al., Human endogenous retrovirus envelope proteins alter extracellular vesicle cellular interactions and biodistribution. bioRxiv, 2026. 10.64898/2026.04.30.722014
  8. Fabiano, M. P., et al., Plasma extracellular vesicle surface-located GAS6/PROS1 and CD39/CD73 attenuate inflammation. Cell Rep, 2025. 44(8): p. 116096. 10.1016/j.celrep.2025.116096
  9. Tan, T. T., et al., Enhancing EV-cell communication through “External Modulation of Cell by EV” (EMCEV). Cytotherapy, 2025. 27(1): p. 1-6. 10.1016/j.jcyt.2024.07.014

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