Extracellular vesicle (EV) therapeutics: good things in small packages
Once considered simply as byproducts of cellular recycling and waste management, extracellular vesicles (EVs) have become the focus of translational research and industrial interest aimed at exploiting their potential in therapeutic drug delivery, and as a safer and flexible alternative to stem cell therapy.EV-Based Therapeutics: “Nature’s Nanotechnology”
EVs broadly fall into three main classes depending on size and origin: exosomes, which are small, virus-sized (40-120nm diameter) lipid bilayer-bound particles arising through the endocytic pathway; and macrovesicles and apoptotic bodies, which are much larger and originate from the plasma membrane. “Exosome” is widely, if inaccurately, used as a generic term in EV literature. In reality, most exosome preparations contain a variety of similarly sized nanoparticles. The International Society of Extracellular Vesicles (ISEV) is endeavoring to bring standardization to EV nomenclature, characterization, and define acceptable standards of content for in EV study reporting.1
The principal biological function of EVs is intracellular communication, allowing cells to exert regulatory effects on their near and distant neighbours through the transport and delivery of bioactive molecules. EVs also play a role in antigen presentation in both antibody and cellular immune responses.2 Clinical studies support a role for EVs in immune tolerance, offering the prospect of new treatments for autoimmune and inflammatory conditions. EVs mediate interplay between cancer, stromal and immune cells, contributing to the maintenance of a supportive tumor microenvironment and to metastatic spread.3
EVs make attractive drug delivery vehicles. Unlike man-made liposomal or polymeric nanoparticles, EVs have inherent tissue targeting capabilities mediated by integrins, cell-specific proteins and other binding moieties. Other desirable characteristics include compatibility with a variety of bioactive cargoes, including proteins, DNA, mRNA, small RNA species, lipids, and both hydrophobic and hydrophilic small molecule drugs; high biocompatibility; low immunogenicity; direct delivery of bioactive molecules into the cytoplasm, and the ability to cross the blood-brain barrier.
Unlike stem cell therapies, EVs do not trigger rejection by the host, cannot differentiate or proliferate, and are likely to have superior biodistribution and concentration in target tissue. EV preparations are amenable to long-term storage following cryopreservation, lyophilization, or spray-drying, offering simpler treatment logistics.
Into the clinic
Taking EVs into the clinic has proved challenging. Cultured stem cells, commonly bone marrow or adipose tissue-derived mesenchymal stem cells (MSC), and also other mammalian and plant cells, can serve as source material. Yields are notoriously low, typically 1µg EV protein, or roughly 10 raised to 10th power exosomes, per ml of culture medium at laboratory scale. Upscaling strategies combine hollow fibre and stirred-tank bioreactor culture, with chemical and/or physical stimulation (through shear stress or ultrasound), and cell membrane disruption4. MDimune Inc is applying serial extrusion which forces stem cells through membrane filters to produce “cell-derived vesicles”, with a claimed 10 to 100-fold increase in yield over secretion-dependent production.
EV isolation may involve ultrafiltration, ultracentrifugation, affinity and size-exclusion chromatography, and polymer precipitation, either alone or in combination, but no approach has yet fully resolved the issues of batch-to-batch variation, low yield, and low purity. EV characterization and validation requires determination of size, morphology, and cargo profiling, necessitating application of a battery of analytical methods including electron microscopy, particle diffusion or dynamic light scattering measurements, immunoassay and immunoblotting, mass spectroscopy and PCR.
EV engineering can enhance therapeutic efficacy over naïve EV preparations. Decoration with targeting peptides or proteins, single-domain antibodies, or even magnetic particles can be achieved through genetic modification of the parent cell line or post-isolation chemical modification5. Bioactive drug, nucleic acid or protein loading methods can be applied prior to EV production through transfection, electroporation, or co-incubation of parent cells, or by freeze-thawing, incubation, sonication, extrusion, and hypotonic dialysis of isolated EVs.
Despite the complexity of EV therapeutic manufacture, and yet to be defined regulatory requirements, candidate EV therapies aimed at a variety of disease indications are in academic and industry sponsored clinical development. Several companies are targeting SARS-COV-2 associated conditions. ExoFlo™ (Direct Biologics), a MSC EV preparation, significantly reduced 60-day mortality over placebo treatment in a Phase II study in patients with SARS-CoV-2 adult respiratory distress syndrome (ARDS). A Phase III study in patients with any cause ARDS is underway.
On the back of symptom reduction observed in mild-to-moderate SARS-CoV-2 infection, ZEO Scientifix (formerly Organicell) has expanded its evaluation of Zofin™, an amniotic fluid-derived EV preparation, into SARS-CoV-2 ARDS and post-SARS-CoV-2 syndrome (“long COVID”). Vitti Labs is aiming at the same indications using a combination product comprising umbilical cord-derived MSC and MSC-derived EVs.
Wound healing is considered another area of promise. ExoFlo™ is in early clinical evaluation for perianal fistula, while RION is applying platelet-derived EVs in the treatment of diabetic foot ulcers and skin graft site wounds, Early clinical data from a study of a naïve MSC EV topical preparation, AGLE-102 (Aegle Therapeutics) indicated near complete epithelialization of a second degree burn wound and significant clinical improvement after one week.
A solution still looking for a problem?
While the potential therapeutic scope of EVs ranges across cancer, autoimmune and inflammatory diseases, cardiovascular insult (myocardial infarction, ischaemic stroke), neurodegenerative conditions, and rare disease indications, most current development activity remains at discovery and preclinical phase within start-up and early-stage biopharma companies reliant on venture and stage non-dilutive sources of funding. There is only a handful of publicly listed EV players.
Despite the promise of EV therapies, few global pharmaceutical companies have taken an active interest in individual EV candidates or platform technologies. The publicly listed EV developers Codiak Biosciences, once a leader in cancer EV therapy development, and ReNeuron, an EV platform developer, both entered administration in 2023. Certain Codiak assets and its manufacturing site remain alive within Evox Therapeutics, a UK-listed company working on EV-mediated gene delivery, and Lonza, a contract development and manufacturing organisation (CDMO). ExoPharm, a clinical-stage, Australian-listed company is exiting the EV field through merger.
The relatively low level of investor and pharma interest in EV product development likely reflects their early-stage clinical development, the lack of obvious “blockbuster” products in the current EV therapeutic pipeline, along with unknowns around regulatory agency acceptable product standardization and characterization, and the feasibility of commercial-scale manufacture. The potential advantages of EV delivery over synthetic nanoparticle delivery have still to be established through clinical study.
Not surprisingly, several companies with historical expertise in stem cell banking and manufacture have leveraged their expertise into the EV field, fueling a “grey market”, with unlicensed EV products being touted directly to consumers as miracle cosmetics or regenerative medical treatments through private clinics, resulting in a slew of FDA Warning Letters, although the grey market is by no means confined to the US6. ExoCoBio, which claims to operate the world’s largest GMP-certified EV manufacturing facility hopes to sway Korean regulators through a focus on topical cosmetics, supported by extensive clinical trial data.
There are positives in the EV therapy landscape. A growing number of CDMOs offer EV-related services, including heavyweights such as Lonza, GBI (formerly Goodwin Biotechnology) and AGC Biologics, along with EV specialists RoosterBio and EXO Biologics, offering a clear path from concept to clinical trial supply and beyond. An “Exosome Industry Council” has been formed in South Korea, home to many EV-focused companies, with the objective of building an industrial ecosystem with international ties.
Innovation abounds within small companies when it comes to improving over nature with respect to EV targeting and loading: EVs may finally achieve breakthrough through enabling tissue-specific, highly efficient delivery of next generation gene and RNA therapies7.
References
[1] Samuels M & Giamas G. MISEV2023: Shaping the future of EV research by enhancing rigour, reproducibility, and transparency. Cancer Gene Therapy (2024).
[2] Kumari, P et al. Role of extracellular vesicles in immunity and host defense. Immunological Investigations, 53(1), 10–25 (2024).
[3] Wang X et al. Exosomes and cancer - Diagnostic and prognostic biomarkers and therapeutic vehicle. Oncogenesis 11, 54 (2022).
[4] Syromiatnikova V et L Methods of the large-scale production of extracellular vesicles. Int J Mol Sci. 10;23(18):10522 (2022).
[5] Frolova L & Li ITS. Targeting capabilities of native and bioengineered extracellular vesicles for drug delivery. Bioengineering (Basel). Sep 22;9(10):496 (2022).
[6] Asadpou A et al. Uncovering the gray zone: mapping the global landscape of direct-to-consumer businesses offering interventions based on secretomes, extracellular vesicles, and exosomes. Stem Cell Res Ther 14, 111 (2023).
[7] Sun M et al. Extracellular vesicles: a new star for gene drug delivery. Int J Nanomedicine 6; 19:2241-2264 (2024).