Unlocking the
Mysteries of
Extracellular RNA
Communication

Once thought to exist only inside cells, RNA is
known to travel outside of cells and play a role in newly
discovered mechanisms of cell-to-cell communication.

Early detection of viral infections is extremely important for control of disease transmission, prompt initiation of treatment, and prevention of infection-related complications. Because of our hypothesis that viral DNA, messenger RNA, and proteins cannot be detected in all infected individuals, we wanted to determine whether detection of exogenous miRNAs encoded by viruses represents a more sensitive assay of the true prevalence of infection by viruses including latent Kaposi Sarcoma Herpes Virus (KSHV) and Epstein Barr Virus (EBV). Therefore, we measured plasma miRNAs using RT-qPCR and compared it to the current standard method for detection of viral infection, an enzyme-linked immunosorbent assay (ELISA) of blood plasma, which detects antibodies generated by the host against the infecting virus. Our study population was 214 Caucasian patients from the United States and Romania, separated into four independent patient cohorts. We examined a total of 300 plasma samples from this population. This study enabled us to develop an approach to detect infection by KSHV using multiplexed RT-qPCR of multiple viral miRNAs.

We found that our method had clear advantages over the current ELISA-based approach. It detected a significantly higher prevalence of KSHV infection than that determined by seropositivity, with the difference most pronounced in immuno-depressed patients. When applied to EBV, our new method based on plasma viral miRNA quantification proved that EBV infection is ubiquitous. This strategy has the potential to become a gold standard method in clinical practice to detect latency of viruses and viremia — viral infection of the bloodstream — in both general and immune-compromised populations.

Detecting viral infection by amplifying viral miRNA has clear advantages over the current ELISA-based approach.

Fuentes-Mattei E, Giza DE, Shimizu M, Ivan C, Manning JT, Tudor S, Ciccone M, Kargin OA, Zhang X, Mur P, do Amaral NS, Chen M, Tarrand JJ, Lupu F, Ferrajoli A, Keating MJ, Vasilescu C, Yeung SJ, Calin GA. Plasma viral miRNAs indicate a high prevalence of occult viral infections. EBioMedicine. (2017) 20:182-192. doi: 10.1016/j.ebiom.2017.04.018. Pubmed: 28465156.

Extracellular vesicles, such as exosomes and microvesicles, are small vesicular particles that are constantly being produced and shed by cells. Due to their natural origin, and their ability to efficiently deliver their cargo to target cells and alter biological functions, exosomes attracted researchers to study their potential use as drug delivery systems. In the past few years, numerous studies have reported the effective use of exosomes to deliver therapeutic cargo ranging from miRNA, siRNA and even small molecule drugs in both in vitro cell models and in vivo animal models. However, since exosomes are cell-derived vesicles, it is unclear how these natural carriers of biomolecules may induce immune responses or induce toxicity either in animal models of disease or eventually in humans as we progress toward clinical evaluation of exosomes in healthy volunteers or in patients. Furthermore, what can we conclude about the presence or lack of immunogenicity or toxicity in our animal models as we work toward delivery of exosomes in humans?

Our lab has been studying the production of therapeutic exosomes using genetically engineered HEK293T cells for treatment of hepatocellular carcinoma (HCC). We developed engineered HEK293T cells that endogenously package miR-199a-3p, a miR commonly downregulated in HCC, into exosomes, and we are evaluating these and also exosomes exogenously loaded with therapeutic miRs in vitro and in vivo. Although demonstrating in vivo efficacy is a major milestone for all drug development efforts, understanding the potential toxicities and immunogenic responses associated with exosome therapy is equally important. The ability to identify and characterize adverse responses in preclinical models is critical to the drug development process and a necessary component of an Investigational New Drug (IND) application. Therefore, approaches to characterizing potential toxicities and immune responses induced by exosomes will be a necessary component of any effort to develop therapeutic exosomes.

In our article, “Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells,” that was just published online in the Journal of Extracellular Vesicles, we provide a general template process for comprehensively evaluating toxicity and immunogenicity of therapeutic exosomes in preclinical animal models. We started by dosing mice with wild type or engineered HEK293T-derived exosomes over a period of three weeks. Mice received 10 doses via intraperitoneal and intravenous routes of injection, and blood samples were collected at various times throughout the 3-week study. Animals were euthanized 24 hours after the last dose, and blood and all organs were collected from each animal for gross necropsy and evaluation of various markers of immune response and potential exosome-induced toxicity markers.

This study demonstrates one approach to immunogenicity and toxicity evaluations of human-derived exosomes in mice, and it highlights some of the variables that must be considered during these evaluations. For example, what are appropriate animal models in which to study immunogenicity and toxicity? What doses and dose regimens should be evaluated? How might the cell type from which the exosomes were harvested impact immunogenicity and toxicity? Just as with efficacy evaluations in animals, comprehensive study of these other factors will be necessary for safely moving therapeutic exosomes into human trials.

Acute liver failure is a potentially fatal consequence of severe liver injury. Liver transplantation may be necessary for survival if the liver injury exceeds the ability of the liver to regenerate (also called fulminant liver failure). New therapeutic interventions are needed to enhance tissue regeneration and improve the outcome of acute or fulminant liver failure. Previous studies have reported that mesenchymal stem cell (MSC) transfusions can improve function in liver facing acute failure. Stem cells can grow into multiple cell types and may support the replacement of functional liver tissue. In addition, paracrine effects — signalling between nearby cells — resulting from the release of soluble factors and extracellular vesicles (EV) may contribute to some of the beneficial effects observed with stem cell therapies.

A new study by Haga et al. reports on the beneficial effects of EV derived from stem cells. EV were isolated using classical ultracentrifugation methods. To mimic liver injury, D-galactosamine and recombinant tumor necrosis factor-α were injected into male mice. Subsequently, systemic administration of EV was shown to result in a dramatic improvement in survival. Whereas control animals receiving placebo showed complete lethality within 12 hours of D-galactosamine/TNF-α injection, the mice injected with mouse stem-cell-derived EV had a 57% survival at 24 hrs. When human MSC-EV were administered, a 37.5% survival was noted. Also noteworthy, survival was observed even with EV that had been cryopreserved. The EV reduced hepatic inflammation, likely by protecting the hepatocytes from apoptosis and recruiting Kupffer cells that protect from liver injury. The figure shows an overview of this process. Some of the beneficial effects were shown to be mediated by Y-RNA-1, a long non-coding RNA that is enriched within MSC-EV.

Stem-cell-derived extracellular vesicles repair tissue after acute liver failure

The beneficial effects of stem cells, mediated through EV and their RNA content, in severe injury models provides new avenues for investigation of the pathophysiology of liver injury and inflammation. These observations provide a very compelling justification for the future use of MSC-EV as therapeutics for severe liver injury.

Reference:
Extracellular vesicles from bone marrow-derived mesenchymal stem cells improve survival from lethal hepatic failure in mice. Haga H, Yan IK, Takahashi K, Matsuda A, Patel T. Stem Cells Transl Med. (2017) 6:1262-1272. doi: 10.1002/sctm.16-0226.