Challenges and Opportunities in Translating Extracellular Vesicles into Clinical Applications

The article by Wen et al., ¹ published in this issue, extends previous observations of the protective effect of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) on irradiation injury with two new major findings: improved survival after lethal total body irradiation and enhanced hematopoietic stem cell engraftment. These observations suggest potential clinical applications.

For over two decades, EVs, including ectosomes (microvesicles, exospheres, oncosomes, and apoptotic bodies) and exosomes, have garnered significant interest from researchers, ² leading to the notion that EVs could hold great promise for clinical applications in diagnostics, therapeutics, and drug delivery. ^2,3 EVs carry proteins, lipids, and nucleic acids that can modulate recipient cell behavior and mediate autocrine and paracrine effects. In many regenerative contexts, EVs are thought to recapitulate the beneficial effects of stem cells without the need for cell transplantation. Numerous studies have highlighted the relevance of miRNAs and ncRNAs as EV payloads, consequently increasing attention to their known/unknown bioactivity and their role in health and disease. ⁴ The realization that EV cargo is richly heterogeneous and that specific factors are therapeutically active has further shifted the focus from EVs to defined molecular entities. RNA sequencing-based inventories of therapeutic EV contents allow deciphering and exploiting the complex biology of EVs. Increasing knowledge of EV nucleic acid payloads provides extraordinary potential and has spurred new approaches for their use, both in their native form and as templates for new formulations with longer half-lives and increased biological effects. All Food and Drug Administration (FDA)-approved noncoding RNA (ncRNA) drugs (n ≈ 20) target known disease-causing molecular pathways by mechanisms such as antisense. ⁵

Despite the enthusiasm surrounding EVs’ potential curative effects in various organ and systemic disorders, significant uncertainties still obstruct their translation into clinical applications. This section aims to summarize the major hurdles in translation. Safe and sound translation into clinical applications requires standardization and product development. After more than two decades since the original description of biological effects, the fulfillment of the classical pathway for EVs as a “medicinal product” is still far from complete: identifying the most appropriate clinical applications (e.g., unmet needs in health care, biotech, or engineering); conducting research; developing a product (storage stability, quality control/quality assurance); iterating and refining (based on lab results and user feedback); and finally, regulatory approval. At present EVs are variably classified as biologics, cell-based therapies, or drug products, leading to unclear regulatory pathways.

Specification of products should reflect the cell source, stage of development, and clinical target of EVs, including pathology, dosing, and storage, among other variables. Many variables across different studies still exist or, in some cases, are not described in due detail, such as tissue source, isolation protocol, processing, expansion, number of passages, number of batches produced (relative parameters) used, as well as the methods used to elicit EV release.

Researchers and regulators are becoming aware of major inconsistencies in the world of EVs: differences in isolation, characterization, scalability, recognition of contaminants (non-vesicular vesicles, membrane blebs, proteins such as lipoproteins), rising concerns of immunogenicity associated with repeated dose regimens, and remarkable uncertainties in manufacturing and regulatory issues (Good Manufacturing Practice) compliance. Several clinical trials have been registered by ClinicalTrial.gov using MSC-derived EVs to treat patients with different diseases, but only a few reported clinical results. ^6,7 Unfortunately, most clinical trials with MSC-EVs fail in design by not being double-blind and placebo-controlled, which is crucial for concluding whether the regimen tested is effective. ⁸

The translation of EVs into clinical applications faces significant challenges due to their heterogeneous nature and the lack of standardized isolation and characterization methods.

Isolation methods used now are not EV-specific, and biogenic EV products isolated from tissue or fluids may contain other non-EV nanoparticles. Even if uniform-diameter EVs could be isolated (currently impossible), they may still contain nanoparticles with varying morphology, content, and even membrane composition, all of which can influence their efficacy. Different isolation techniques may yield different EV populations, and the source tissues from donors of different ages, sexes, and races may also contribute to variations in the EV populations obtained.

From its inception, the FDA has placed utmost importance on thorough characterization and purity concerns for drugs. However, as EVs carry differing functional biomolecules that may coordinate to effect their functions, and the products are heterogeneous mixtures, such determinations are challenging with the available methods and technology. Regulators recognize that EVs are a heterogeneous population, but their composition and associated contaminants must be fully characterized. Until 2020, there were no regulatory documents specific to EVs. In response to clinics advertising EV products as therapies, the FDA released a safety notification. Despite efforts by the FDA and the International Society for Extracellular Vesicles to provide guidelines, there are still no established standards to follow. ^9,10 Another crucial need is the establishment of appropriately validated potency tests. Potency assays should be diligently validated for specificity, accuracy, precision, linearity, range, limit of detection, and limit of quantification and should be quantifiable, reproducible, and relevant to the intended therapeutic function in each clinical application. For clinical applications, regulatory agencies require potency tests to ensure batch-to-batch consistency. In vitro potency tests and animal studies, along with toxicology assessments, should be conducted with the understanding that all data will form the basis of the Investigational Medicinal Product Dossier (IMPD) required for clinical trial applications. The IMPD provides comprehensive information on the investigational drug, including quality (CMC—chemistry, manufacturing, and controls): details on the drug’s composition, manufacturing process, and quality control; nonclinical data: preclinical studies evaluating pharmacology and toxicology; and clinical data: previous clinical trial results, safety, and efficacy data. It is therefore imperative that researchers actively engage with regulatory agencies (European Medicines Agency; FDA) at the earliest stage of their research to ensure successful translation into clinical applications.

It is not unexpected that EVs are only in the early phases of regulation, with most products still at a basic science or early translational stage of production, despite the substantial amount of published in vitro and in vivo studies. ⁶

Historically dismissed as mere cellular debris, EVs have now attained the status of being potent therapeutics in several diseases and injuries. However, significant technical and regulatory challenges remain. Whether EVs will become a medical breakthrough or remain an overhyped concept will largely depend on advances in isolation, characterization, and clinical validation. It is a call for all actors involved (public and private researchers, scientific communities, developers, and regulators) to work together to transform EVs from today’s mirages into tomorrow’s potent diagnostics and therapeutics for the ultimate safety and benefit of patients with unmet needs.