Targeted delivery and sustained release of the therapeutically active substance is a major goal of drug developers today. Many novel drug delivery systems are being explored to enable delivery of the right dose in the right place at the right time. Exosomes have received significant attention due to the biocompatibility, minimal immunogenicity, ready ability to pass through cellular membranes and other biological barriers, and capacity for drug loading and surface modification. Key challenges relate to the development of robust, efficient, large-scale manufacture. Promising early preclinical and clinical data are driving the development of numerous exosome-based therapies and innovative solutions focused on enabling GMP production suitable for commercialization.
A Natural Drug Delivery Vehicle
To be maximally effective and safe, drugs must reach the right tissues/cells in the right format at the right concentration for the right amount of time. The limited solubility and permeability of small molecule drug substances, the aggregation propensity of many biologics, and the sensitivity to degradation and transduction challenges of oligonucleotides require the use of novel formulation strategies to achieve these goals. As a result, drug development today requires that as much attention is paid to the drug delivery vehicle as the drug substance.
Nanoscale carriers have received growing attention in recent years owing to the unique physical, chemical, and biologic properties imparted by their high surface area/volume ratios (enhanced stability, ease at entering cells and crossing biological barriers, etc.) and ability to achieve targeted and sustained delivery through surface functionalization.1–5
However, there are limitations to the use of some nanoparticle formulations. They can present toxicity concerns, cause unwanted immunogenic responses, and/or have limited targeting capabilities. If properly designed, exosomes can overcome these challenges.6–10
Exosomes are a type of nanoscale (30–150 nm in diameter) extracellular vesicle (EV) produced by most mammalian cells. They begin as multivesicular bodies within early endosomes and contain many of the biologic materials found in their parent cells. Other EVs include ectosomes, or microvesicles (100–1000 nm in diameter), released from plasma membranes and enveloped viral particles (100–200 nm).
The main role of exosomes is in intracellular communication and signaling, but they are also known to play a role in other cellular processes, including proliferation, growth, development, angiogenesis, and immunomodulation, as well as in disease pathogenesis (infection and metastasis). These functions are achieved through the delivery of many types of bioactive molecules (e.g., proteins, protein markers, RNA, DNA, metabolites, lipids) involved in key cellular processes. For instance, proteins on the surfaces of exosomes include tetraspanins, antigen-presenting complexes, and adhesion molecules, while those carried internally include heat shock proteins, cytokines and chemokines, and membrane transporters, among others.
They are present in most human cells, tissues, and bodily fluids and exhibit characteristics and behavior derived from their parent cells.
The biomimetic nature of exosomes makes them highly attractive as nanoscale drug delivery vehicles. They are not only biocompatible but also immune silent, readily interact with various cell types, and have the ability to cross biological barriers, including the blood–brain barrier. They also lack the safety risks posed by cellular therapies while offering many of their advantages, such as anti-inflammation, immunomodulation, and tissue regeneration properties for exosomes derived from stem cells. The surfaces of exosomes can be modified to support cell/tissue targeting, and drug substances can be loaded internally (in the lumen) or attached to the surface.
Engineering Exosomes for Drug Delivery
Engineering of exosomes to support their use as drug delivery vehicles can take various forms, including modification of both their lumens (interiors) and surfaces. Interior modifications support drug substance loading, while exterior functionalization typically is performed to allow attachment of ligands that enable binding to targeted cells/tissues.8
Loading of drug substances can be achieved using both passive and active approaches.10,11 Passive encapsulation is achieved by incubating exosomes in the presence of high concentrations of the drug substance to encourage diffusion of the active ingredient into the interior of the exosome. Cells can also be cultured in the presence of the drug substance, during which the active compound is taken up by the cells and packaged into exosomes. However, both approaches are inefficient and difficult to control. Active loading is achieved using either physical (e.g., electroporation, sonication, freeze-thaw cycling pores, extrusion) or chemical (e.g., treatment with surfactants or other agents that cause membrane disruption) methods designed to increase the permeability/porosity of the exosome membrane to facilitate incorporation of the drug substance. One downside of these methods is the increased risk for exosome damage.
For protein and other drug substances that can be produced by cells, there is growing interest in approaches that generate pre-loaded exosomes. Cells are engineered to overexpress both the desired exosomes and therapeutic active, leading to natural incorporation of the drug substance and higher yields of loaded exosomes.11 This strategy has been used to produce exosomes encapsulating IL-27, cytokines and other signaling proteins, microRINA, and many other payloads.
Engineering techniques have also been used to generate high yields of exosomes with certain proteins overexpressed in the lumen and/or on the surface to support loading and ligand attachment, respectively. The latter can be used to modify the surfaces of exosomes for targeted delivery and to facilitate exosome purification.
The EngEX® platform from Lonza is one example of an approach for production of engineered enzymes.12 With this technology, cells are engineered to express exosomes with high levels of the specific scaffold proteins, PTGFRN , which is in the EWI immunoglobulin superfamily, on the surface and/or BASP1 (in the MARCKS protein family) in the lumen. While HEK293 are most commonly employed, the technology has also been successfully demonstrated for CHO and many other cell types. To attach small molecule payloads, which is not possible during production of loaded exosomes via cell culture, Lonza developed an azo-based linker that does not impact exosome attributes but allows loading of nucleotides and other similar APIs. EngEX® technology is also used in Lonza’s exoVACC® system for production of exosomes that display antigens on their surfaces or in their lumens in combination, if necessary, with inclusion of adjuvants.
Many Potential Applications
Given the properties of exosomes and the recent advances in technologies for loading them with many different types of drug payloads and modifying their surfaces to support targeted delivery, their use as drug delivery vehicles is being investigated in many applications.
Some of the diseases receiving particular interest include neurodegenerative disorders, infectious diseases, musculoskeletal conditions, cardiovascular diseases, and cancer.7,9 The potential of exosomes derived from stem cells (e.g., embryonic, induced pluripotent, hematopoietic, mesenchymal, neural, and endothelial stem cells) to be effective in regenerative medicine applications is also being explored.8
In addition, exosomes are being investigated for the delivery of many different types of drug substances and therapeutic modalities, including not only traditional small molecules like anticancer agents but also various forms of RNA, DNA, peptides, and different classes of proteins. Selected examples include doxorubicin and paclitaxel, cisplatin, atorvastatin, curcumin, the proteins TRP2 and hMUC1, Let-7a miRNA. circIFNGR2, and BCL6- siRNA. These molecules only represent a fraction of the drug substances for which exosomes are being investigated as an effective nanoscale delivery vehicle.
In addition to their value as drug delivery vehicles, exosomes are proving to have significant use in diagnostic applications.7 There are several reasons. First, exosomes are present in nearly every bodily fluid and produced by most cell/tissue types. Second, the composition of biomolecules within exosomes often provides indications about disease states and progression. Diagnostics leveraging exosome detection and analysis have thus been explored for many different diseases including those involving the central nervous system, kidney, liver, and, lungs, as well as for various cancers.
Early Research Promising
Early preclinical (in vitro and animal) studies and clinical trial data with exosome-based therapeutic candidates has been quite promising. A few examples are listed below:6,7,8,10,12,13
Loading specific DNA and RNA molecules, proteins, and other actives into exosomes derived from various types of cardiac cells has been shown to achieve effective repair of damage to relevant cells (cardiomyocytes, vascular smooth muscle cells, endothelial cells, cardiac fibroblasts, and inflammatory cells).
Exosomes encapsulating CRISPR/Cas9 gene-editing component have been shown to reduce tumor angiogenesis.
Exosomes containing STING (Stimulator of Interferon Genes) have been shown to boost the immune response of immunotherapies.
Exosomes have been shown to modulate the activity of various cell types involved in different disease mechanisms, including macrophages, dendritic cells, and natural killer cells.
Exosomes containing the HuR-Lamp2b fusion protein and a CRISPR/Cas9 system were developed as treatments for liver fibrosis.
Exosomes derived from various stem cells show promise as treatments for Parkinson’s disease
In vitro studies of exosomes containing a wide variety of drug substances have shown their potential in orthopedic applications, particularly for repair and regeneration, inflammation, and immunomodulation.
Exosomes derived from various cancer cell types were shown to selectively penetrate target tumors and impact tumor growth and metastasis, increase immune system activity, and/or increase the activity of other cancer treatments.
Encapsulation in exosome delivery systems has been shown to increase the safety and efficacy of several different pharmaceutical actives through prevention of degradation, reduction of off-target effects and immunogenic responses, and greater solubility, bioavailability, and potency.
Large-Scale Manufacturing and Fit-for-Purpose Analytical Solutions Essential
One of the biggest hurdles to commercialization of exosome-based therapeutics is their cost-effective manufacture at scale.6–9,12,15 Efficient production, isolation, purification, and characterization are all challenging. The low concentration of exosomes in cells, their heterogeneity in terms of their size, content, surface markers, and source, and batch-to-batch variability are key issues. Achieving purification without impacting exosome properties is difficult.
At research scale, exosomes are isolated from plasma (or other bodily fluids) or cell culture supernatants (produced in flasks or flatware). For large-scale manufacture, exosomes are typically produced via cell culture using cell lines engineered to produce higher quantities of the exosomes, and for biologic actives, encapsulating the drug substance. Most of these processes involve adherent cell culture, which is typically performed in fixed-bed bioreactors, hollow-fiber bioreactors, or stirred tank bioreactors using microcarriers.15
Not surprisingly, best results to date have been obtained with HEK293-derived cell lines, which are widely used for biotherapeutic production. Processes have been implemented in single-use systems to at least the 2,000-L scale.15 When stem cells are used, in addition to genetic engineering, preconditioning using mechanical and chemical methods can boost exosome production.16 Using specialized media has also been shown to improve exosome yields in general.
At lab scale, desired exosomes are usually separated from these complex process fluids using ultracentrifugation. This technique is not practically scalable, however, and thus large-scale downstream processing typically involves concertation via tangential-flow filtration (TFF) followed by chromatography, which may include size-exclusion chromatography (SEC), anion exchange chromatography (AEX) and/or affinity capture chromtography.15 Precipitation methods employed specialize polymeric reagents may also be used. Commercial kits are available for some of these methods. For personalized exosome therapies produced at the point of care, small-scale processes are practical. In fact, microfluidic devices may be used for effective exosome purification and isolation.10
Various analytical techniques are used to characterize (e.g., size, morphology, contents) and quantify exosomes and their many bioactive components.7,8 Examples include nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), tunable resistive pulse sensing (TRPS), transmission electron microscopy (TEM),cryo-electron microscopy(Cryo-EM), fluorescence spectroscopy, western blot, flow cytometry, mass spectrometry, scanning electron microscopy (SEM), next-generation sequencing, polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), flow cytometry, and others.
While advances in exome manufacturing, purification, and analysis have been made, large-scale processes remain fairly inefficient, and development of fit-for-purpose analytical methods continues to be needed. As candidates progress into the clinic and toward commercialization, further progress will be needed to ensure cost-effective, robust production of GMP-compliant exosome therapeutics that support commercialization of safe and effective products.
Commercial Pursuit Increasing
A growing number of companies are focused on achieving that goal. As of late 2024, Delve Insights reported more than 70 firms developing exosome-based therapies, with more than 80 candidates targeting numerous indications in the clinical pipeline.17 The value of the global exosome therapeutics market was predicted to be expanding at a compound annual growth rate of 16.19% and thus to grow by $234.7 million.
Companies receiving significant attention include Aegle Therapeutics, Aruna Bio, Capricor Therapeutics, Evox Therapeutics, ILIAS Biologics, Direct Biologics, and Brexogen.17,18 Aegle announced positive results for the first patent dosed in its phase I/II trial of AGLE-102 for treatment of severe second-degree burns in early 2024 and started a second phase I/II trial in patients with dystrophic epidermolysis bullosa, a rare pediatric skin blistering disease.
Aruna is focused on exosomes derived from neural stem cells, with its allogeneic lead candidate AB126 targeting several challenging CNS conditions. It has received approval for a phase Ib/II trial in acute ischemic stroke. Capricor’s lead candidate StealthX is a multivalent vaccine for the prevention of COVID-19 and included in the U.S. government’s Project NexGen.
EXOB-001 from EXO Biologics is an exosome-based therapy in a phase I/II trial for bronchopulmonary dysplasia, a severe lung disease that is the most common cause of death in preterm newborns. It is the first European Medicines Agency (EMA)-authorized study with mesenchymal stromal cell (MSC)-based exosomes. ILB-202 from ILIAS Biologics leverages the company’s EXPLOR platform technology for engineered exosomes and delivers the anti-inflammatory protein super-repressor IκB (srIκB). Its phase I trial completed in Australia in 2023 was the first trial to involve systemic administration of an engineered exosome therapeutic.
Brexogen’s BRE-AD01 is therapy using exosomes derived from BxC stem cells and being investigated in a phase I trial for atopic dermatitis. Direct Biologics has one of the most advanced clinical candidates — ExoFlow, which is derived from human bone marrow mesenchymal stem cells and rich in growth factors and EVs, including exosomes. It is being investigated in a phase III trial for treatment of hospitalized adults with moderate to severe acute respiratory distress syndrome and phase I trials for ulcerative colitis and Crohn’s disease.
Notably, various classes of exosomes are being developed by these and other companies.19 Naïve exosomes are being leveraged by companies such as AGS Therapeutics, Avalon, Aegle Therapeutics, ExoCoBio, AgeX Therapeutics, Kimera Labs, and United Therapeutics, while Capricor, Evox, Ilias, Carmine Therapeutics, ReNeuron, Anjarium, Adipomics, Brainstorm Cell Therapeutics, Exocure Biosciences, and TriArm Therapeutics are focused on engineered exosomes. Others, including Aruna Bio, and Avalon use both approaches for different candidates.
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