Emerging Exosome-based Therapies: Opportunities, Challenges and Coping Strategies

Exosomes are a subtype of extracellular vesicles, which are lipid bilayer closed structures derived from cells and secreted by almost all types of cells, including exosomes (30-150 nm), micro vesicles ( 150 nm to 1 μm) and apoptotic bodies (1-5 μm). These vesicles have long been thought to be a way to transport the waste products produced by the cell. It was not until the 1980s, when researchers were studying the development of sheep reticulocytes, that they initially determined the role of some 30-150 nm vesicles and named them exosomes. Observed under an electron microscope, exosomes are generally cup-shaped or spherical in shape. They protect and deliver functional macromolecules, including nucleic acids, proteins, lipids, and carbohydrates, between cells and transfer their "cargoes" to the recipient cells.

Based on years of research, the industry has recognized the potential of exosomes in a variety of applications. In current clinical trials, exosomes are used as biomarkers, cell-free therapies (exosome therapy), drug delivery systems, and anti-tumor vaccines. Their sources include mesenchymal cells, T cells and dendritic cells, as well as other engineered cell lines.

Exosomes have irreplaceable advantages as drug delivery vectors, including low immunogenicity, excellent biocompatibility and biostability. In addition to using natural exosomes without any genetic/chemical modification, there are two main ways to load payloads into exosomes: in the direct method, exosomes are loaded with therapeutic drugs after purification (exogenous loading), while in the indirect method, appropriate cells are genetically engineered or co-cultured with therapeutic drugs to produce engineered exosomes (endogenous loading).

For exogenous loading, the industry has explored various strategies to load drugs into exosomes and maximize their delivery potential, including simple incubation as well as electroporation, sonication, freeze-thaw, etc. There are usually some differences between studies, attributed to the biological behavior of different parental cells and reagent properties. In addition, exosomes are naturally loaded with natural proteins and nucleic acids, which greatly reduces the required load loading efficiency. The correct method to achieve optimal loading depends to some extent on the load molecule, which must be carefully selected in advance and should consider loading capacity, drug retention and potential effects on exosome properties. The limitations of direct loading strategies limit the use of exosome-based therapies in clinical trials.

The creation and use of rationally and purposefully designed, engineered exosomes with highly defined and reproducible properties and a known mechanism of action are a compelling alternative to naturally derived exosomes, which are often highly heterogeneous and have unclear mechanisms of action, and are a more viable basis for the development of important new drugs. However, engineering approaches require certain improvements in maintaining the ideal physicochemical properties of exosomes and improving loading efficiency. Another challenge is that most methods used for exosome engineering have difficulty finding a balance between stable loading of the desired cargo and surface modification vs. maintaining exosome biocompatibility.

Another bottleneck in scaling up exosome-based therapies to industrial-scale production and subsequently entering the clinic trial is the production of large-scale clinical-grade exosomes. The yield of exosomes is highly dependent on their parent cells, limited by the different abilities of cells to secrete exosomes and the high difficulty and high cost of large-scale cell culture. For the pharmaceutical exosome application, scaling up to industrial levels is still in its infancy, and it is most important to determine the methods that can produce the required number of exosomes containing therapeutic payloads as soon as possible.

The inefficiency of large-scale exosome isolation methods is another obstacle to the development of clinical-grade exosomes. The quantity, physicochemical characteristics, and composition of exosomes released by different cell types may vary. Currently, technologies based on different principles have been used for exosome isolation, including differential/ultracentrifugation, filtration, size exclusion chromatography, immunoaffinity-based capture, precipitation, etc. Although some exosome purification methods have been developed and optimized, it is still difficult to find a specific method to solve all related challenges, such as low separation efficiency, sample loss, low exosome recovery and purity, and batch-to-batch variability. Accordingly, it is also crucial to fully characterize exosomes, especially in terms of size, morphology, concentration, presence of exosomal markers/contents, and removal of contaminants.

Commonly used exosome isolation methods and their advantages and disadvantages

Although challenges and limitations remain, various pharmaceutical companies and startups have paved the way for the development of clinical-grade exosome therapeutics. An increasing number of companies are focusing on developing such exosome-based therapies to address drug delivery issues for a variety of therapies, including small molecules, RNA therapies, proteins, viral gene therapies, and even clustered regularly interspaced short palindromic repeats (CRISPR) gene editing tools. Some of these companies are also pursuing more innovative exosome engineering methods to design exosome-based therapeutics to increase drug loading and improve targeting capabilities.

Traditional methods of delivering RNA, proteins, and chemical molecules have shown some limitations, while exosomes as drug delivery vectors have great advantages such as low immunogenicity, long-term safety, and non-cytotoxicity. There are still challenges that must be overcome in the clinical translation, large-scale production, stable preparation, storage strategies, and quality control of exosome-based drugs. Further development of cell-derived engineered exosomes and their separation, purification, and drug loading technologies will help overcome these shortcomings. Engineered exosomes have significant commercial advantages in improving productivity. In addition, by anchoring specific surface molecules to exosomes, the local concentration of exosomes in target cells or target tissue can be increased, thereby reducing toxicity and adverse reactions and maximizing therapeutic effects. In the future, the industry will likely develop new multifunctional engineered exosomes to improve healthcare, so further research is needed to explore new strategies for exosome-mediated therapies.

Duoning Biotech is committed to be a one-stop bioprocess technology supplier to meet all unique needs of various emerging therapies at different stages, to help more therapeutics move from research to clinical trial and finally commercialization.