Exosomes - The Good, Bad, Ugly and Current State

Exosome Background and Overview

What is an exosome?

Exosomes are small membrane sacs or vesicles produced by cells. From a pharmaceutical scientist view, exosomes are endogenous lipid nanoparticles (LNPs). They are produced by all eukaryotic cells, surrounded by a bilayer lipid membrane, and typically range in size from 30 - 150 nm.1 Prior to 2005 many in the scientific community regarded exosomes as cell waste products, but today we know the contents of naturally produced exosomes include nucleic acids such as genomic DNA, and various forms of RNA, proteins, and lipids that carry information to other cells, sometimes to cells a great distance from the originating cell.2

One easy way to think about exosomes is as the natural Federal Express pathway of the body.3 Exosomes are nano-sized vesicles released and received by nearly all cells in the body and serve as a natural cellular communication system. Exosomes can be found in every bodily fluid: blood, serum and plasma, urine, cerebrospinal fluid (CSF), tears, milk, etc. Exosomes are also present in cell culture media used to grow various cell lines.

Within the realm of biology exosomes are one of three extracellular vesicles (EVs). Other EVs include microvesicles and apoptotic bodies depicted in Figure 1.4

Figure 1.

Due to the substantial size overlap among these membrane vesicles, confusion on the origin and nomenclature of EVs has spread through the field. In addition, exosomes are produced in different sizes, shapes, and types. Importantly, they have different surface properties and molecular components. Their outer membrane contains different surface markers, typically the tetraspanins CD9, CD63 and CD81.1 Isolating exosomes and purifying them in a functional form, free of other EVs and contaminants is a major roadblock to translating exosomes into viable therapeutics.

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Unique to cells and cell populations

Exosome science has evolved quickly and many scientific questions remain unanswered. For example, the human body contains some 35 trillion cells (3.5X1012). If one assumes each cell produces 1,000 exosomes in a day, this equates to 35 quintillion (1X1015) exosomes in our body. How can we make sense out of so many exosomes from so many cells?

Every cell produces unique exosomes with unique characteristics and contents. Exosomes produced from cancer cells are likely to lead to more cancer. Exosomes produced from stem cells are likely to promote anti-inflammatory and regenerative activity, like their originating cell.

Cancer focused scientists have been seeking biomarkers for early detection of cancers, and also for monitoring of cancer recurrence, for many years. Exosomes offer a potential solution because the exosomes produced by cancer cells are different from those of noncancerous host cells, and are thought to play a significant role in cancer growth and eventual metastases. The challenge is to find those exosomes, isolate them, and make sense of their contents.

Consider also that various conditions increase production of exosomes. For example, cells grown in culture produce more exosomes under certain conditions, typically under stress conditions. This is also true in our bodies, where stress increases exosome production.8 Furthermore, the contents of exosomes change when cells are stressed. For example, hypoxia, reduced nutrients, or other stress stimuli produce exosomes with different contents, and likely different cargo messages. Culture conditions and timing also impact type and function of exosomes produced by the cells.

Before getting too complicated, let’s understand the scope of the problem using an example. Assume a single cell type in cell culture, for example human mesenchymal stem cells (hMSCs). These cells have potential to treat many diseases, including numerous cancers, arthritis, graft versus host disease (GvHD), neurologic conditions such as Parkinson’s disease, stroke, Alzheimer’s, and many more. These cells can be isolated and grown in cell culture, and form the basis for numerous clinical trials. Typically, hMSCs are grown in several passages to increase the number of MSCs. Each passage has different numbers of exosomes, and perhaps different types of exosomes. The cell culture media in which the MSCs grow contains components such as glucose, amino acids, vitamins, inorganic salts, and serum as a source of growth factors, and hormones. There are many proprietary cell culture media products, which can influence exosome production and types of exosomes produced. From this simple example it is easy to recognize the complexity of the science of exosomes.

How do we identify exosomes from different cell types and different exosomes from the same cell?

Exosome subtypes

As the exosome industry evolves, the ability to isolate and study specific exosome subtypes is becoming increasingly important. Exosome subtypes are critical to research, diagnostic biomarker identification, and therapeutic development.

Analysis of exosomes from a single cell type can produce different exosomes as noted in Figure 2.

Figure 2. Exosomes are highly variable in size, content, function
and source. Raghu Kalluri, Valerie S. LeBleu, The biology,
function, and biomedical applications of exosomes, Science,
2020, 367, 640.

To make things more complex, exosomes purified from a single cell type have diverse morphologies. Exosomes can be subtyped in many ways: size, shape, density, membrane receptor, cargo load, and cell source. Perhaps the best way to subtype exosomes is based on their surface protein markers. Immune capture using antibodies to these surface markers allows the ability to capture specific sub-populations of exosomes. Using the immunocapture and release method described below, these subtypes of exosomes can be harvested and analyzed or further processed for diagnostic or therapeutic purposes.

Exosome content and type can be influenced by cell culture media. In addition, some cell culture media, which contains fetal bovine serum (FBS) may contain exosomes from the source, further complicating exosome analysis.

The implications of the different exosomes even within a single cell type creates endless scientific questions for an inquisitive mind. These questions provide fodder for additional studies in the fast-growing exosome scientific community and industry.

Why do we care about subtypes of exosomes?

Based on the previous information one can begin to appreciate the complexity of exosome study. Nevertheless, this new scientific frontier offers many opportunities, and differentiating exosome subtypes, their purpose, function and identity could help identify disease states early, before symptoms arise, and lead to potential treatments for numerous conditions. Although exosomes have much promise, they also provide much challenge, including devising consistent, reproducible methods within and among laboratories.
To summarize, the key characteristics of exosomes as they relate to diagnostic and therapeutic applications are:

  • Exosomes are nanometer sized, membrane bound vesicles released and received by nearly all cells in the body.
  • Exosomes play an integral role in cellular communication and regulation, of which we are just beginning to understand.
  • Exosome membranes contain cellular markers capable of targeting specific cell types.
  • Exosomes are not cells. Thus, they are non-immunogenic, more stable, and easier to handle from a pharmaceutical delivery view.
  • The contents of exosomes contain and deliver proteins, RNAs, DNAs and lipids which can be endocytosed by neighboring or distant cells, and modulate the recipient cells.

Exosome Purification

Why do we need pure exosomes?

For some applications the secretome, which represents all of the materials a group of cells (or organism) secretes into the extracellular space is acceptable for downstream activities. For a human cell line, such as hMSCs, the secretome includes all of the proteins, including cytokines, growth factors, extracellular matrix proteins and regulators, shed receptors, EVs including exosomes, microvesicles and apoptotic bodies, peptides, cell free DNA or other portions of nucleic acids, viral particles, and cell waste products. The secretome can be collected in whole and analyzed, or even administered as a treatment for research purposes.

However, for some applications a mixture of all of the components in the secretome are unacceptable. Some diagnostic applications require minimal background noise. For example, seeking exosomes specific to an asymptomatic form of cancer, such as early ovarian or GI cancers requires a more purified exosome population.

Pure exosomes are even more important when considering regulatory requirements for a therapeutic application of exosomes. FDA and other regulatory agencies throughout the world require purity, potency, safety, and efficacy to grant approval. A pure product without contaminants such as peptides, proteins, cell free DNA and other cell debris is critically important. It is also important for exosome therapeutics, specifically. Although exosomes can be dosed based on protein or nucleic acid content, the current state is to dose based on the number of exosomes (e.g., 1 X 1010). If foreign nanoparticles are present as contaminants, the dose could potentially contain a mixture of exosomes in addition to other nanoparticles which are not exosomes. In addition, unknown dilutions of the active ingredient create additional complexity.

Challenges, Problems and Methods of Purification.

Several excellent reviews have summarized the current challenges in harvesting pure exosomes and reviewed state-of-the-art methods.12 The exosome field is experiencing exponential growth due to increased interest and research into exosome roles in disease pathology and potential treatment; however, inconsistency in methodology for the collection, isolation, and analysis of exosomes has created a significant barrier to rapid advancement in the field. In fact, to address these issues, the International Society for Extracellular Vesicles (ISEV) has published a position statement offering guidelines to researchers in order to prevent variations across the studies of exosomes and EVs.13

Ultracentrifugation (UC)

Currently, the most-recognized and used isolation approach is ultracentrifugation (UC). UC is a specialized technique used to spin samples at exceptionally high speeds. Current ultracentrifuges can spin to as much as 150,000 rotations per minute (rpm). The basis of ultracentrifugation is the same as normal centrifugation: to separate the components of a solution based on their size and density, and the density/viscosity of the medium.

UC involves many different steps of centrifugation that remove components such as large vesicles, debris and cells. The UC process leads to marked heterogeneity in isolated exosomes with variable sizes and protein compositions. It is unable to differentiate exosome subpopulations and other vesicle types (e.g., microvesicles and apoptotic blebs). Because exosomes are sensitive to shear stresses, the UC method is also more likely to damage them. Finally, the isolation efficiency of ultracentrifugation is rather low (~10-25%) and the steps for repetitive centrifugation and filtration may exceed 10 hours, thus putting a toll on researchers.

UC has been the cornerstone of the industry for the last 10 years. It is slow, laborious, and produces a low yield. It does however, have a background of research and discoveries, and while it’s full of noise, it does afford exosomes.

Size Exclusion Chromatography (SEC)

Size exclusion chromatography (SEC) is used in numerous life science applications. It is a separation technique based on the molecular size of the components. Separation is achieved by the differential exclusion from the pores of packing material as the material passes through a bed of porous particles. The principal feature of SEC is its gentle, non- adsorptive interaction with the sample. SEC retains bioactivity and does not cause shear stresses like UC, but is affected by morphology of EVs and exosomes.

SEC methods are prone to dilution, contaminations, and pressure- caused damage. To date, there are no well-defined methods for exosome isolation in high-efficiency and high-throughput, which severely hinders the broad applications of exosomes in the biomedical field for large scale production. From a purity view, this method results in a product which also contains everything within the size filter range. There are companies that provide a method of filtration via chromatography and it can be combined with other techniques such as UC and Tangential Flow FIltration (TFF).

Polymer Precipitation (PP)

Precipitation of exosomes (and other EVs) using polymers is another method to harvest exosomes. Several kits are available. This process uses water-excluding polymers such as polyethylene glycol (PEG) which are super-soluble in water and can “tie up” water molecules forcing less soluble components, such as EV’s, exosomes, proteins, and other less soluble constituents out of solution. Then, the EVs can be isolated via centrifugation at low g-forces. This method is associated with less steps than UC, and typically is less time intensive.

The biggest challenge with PP methods, however, is this method suffers from low purity and moderate yield. The low-purity problem is the result of co-precipitation of proteins in a sample because PEG decreases the solubilities of EV’s, proteins, and other constituents present in the sample. In addition, it is possible that the presence of polymer material may be non-compatible with the downstream analysis, such as exosome protein or RNA analyses. Thus, the purity of exosomes delivered by the PP is not acceptable for therapeutic exosomes, though it may be suitable for various research and diagnostic applications.

Tangential Flow Filtration (TFF)

Tangential Flow Filtration is a method used in bioprocessing and purification of biomolecules, such as specific proteins. The reader is referred to an excellent two minute video describing the proces - https://www.youtube.com/watch?v=LkoQX7U4eeo - which uses an ultrafiltration membrane with pores (which can be modified) to filter the source of EVs and exosomes. TFF differs from conventional filtration because fluid flows tangentially across the surface, avoiding filter cake formation and clogging.

TFF has been compared to UC to harvest EVs, and showed several advantages.18 Because TFF has a history of use in biopharma it can be used at large scale volumes. It offers advantages of efficiency, flexibility, speed, and it self-cleans. However, like SEC, it allows similar size nanoparticles to flow through along with exosomes, other EV’s, nucleic acids and protein residues. For this reason, TFF is often paired with other techniques, such as SEC, ion-exchange chromatography or other chromatographic techniques.

Immunocapture and Immuno-Magnetic Beads

The process of immunocapture involves the use of tiny beads, typically about 1um, with an iron core, often called magnetic beads, which are coated with capture molecules that recognize exosome surface markers, such as CD9, CD63 and CD81. Capture molecules are typically antibodies, but could also be aptamers or nanobodies. Beads can be produced to capture on one surface marker, such as CD63, or use a pan-capture approach for all three of these surface markers.

Magnetic separation is much easier than UC, SEC and other methods. Tedious steps involved in centrifugation, precipitation, filtration, or columns are not required. Magnetic handling enables washing, separation, and concentration of the target. Vendors create beads to which antibodies can be conjugated and used to capture exosomes when mixed with solution. The major disadvantage of this method is that the captured exosomes cannot be dislodged from the capture molecules. Thus, the functional activity of isolated vesicles may be lost, and use for therapeutics is not possible.

Immunocapture and Release Beads

One new technology, which recently became available uses immunocapture as previously described, along with a release system. This new technology releases the exosomes from the beads using photo-release technology. In this process, an extra step after exosome capture involves shining a specific wavelength of light onto the bead-Ab-exosome complex, thus cleaving the bond and releasing the exosome.

This process produces pure exosomes based on the capture molecule used. Like traditional immunocapture, this process can be used to subtype specific exosome populations based on the capture molecule selected, e.g., CD63. The limitation of immunological separation is it has not been scaled for large volumes of sample yet.

Below is a summary chart comparing methods of purification and some advantages and disadvantages of each. The reader is referred to several excellent reviews for additional information.14-16

Table 1. Comparison of Exosome isolation
and purifi cation methods

Exosome methods of production, isolation, and purification have significant effects on yield, viability, and function. The exosome community is still defining proper standards for methods so research can be compared. The newest isolation technologies today are laying the groundwork for future exosome research, development, and commercialization.

Microfluidics

One additional process that incorporates features of one or several of the methods noted above is the use of microfluidic- based techniques. These methods are based on super small channels which can expose the fluid of interest to different properties of exosomes such as size, density, immunoaffinity or ionic behaviors. Microfluidics have the potential to provide fast, portable, low cost, and automation. However, to date, lack of method validation, lack of standardization, and moderate to low sample capacity limit microfluidic application to exosomes.

Current State - Exosome Purification

In a recent survey conducted by the International Society of Extracellular Vesicles (ISEV) separation methods such as UC and density gradients are still the most commonly used methods, the use of SEC has increased, and techniques based on TFF and microfluidics are now being used by more than 10% of respondents. The survey also reveals that most EV researchers still do not perform sample quality controls before or after isolation of EVs, thus creating some difficulty in comparing various results. Importantly, the survey found that most EV researchers believe that separation and characterization of EVs should receive more attention.11

Table 2. Distribution of Exosomes Isolation Procedures in 2019
(adapted from Royo)11

Manufacture of exosomes at scale

Isolation and purification of exosomes at lab scale is becoming common practice. Harvesting exosomes at commercial scale is still a work in progress. One issue often raised is the ability to isolate, purify, and potentially modify exosomes at a scale to support thousands or millions of diagnostics or therapeutics.

A number of exosome focused companies are using proprietary methods, which often combine two or more of the isolation/ purification features noted above, or add proprietary elements. One example of this technology is called LEAP - ligand-based exosome affinity purification, which has been described described as a combination of TFF and ion-exchange, and is fully scalable. Some companies are focused on developing immortal cell lines to achieve consistency in exosome production and to avoid replacing cell lines.

In addition, several major contract development and manufacturing (CDMOs) are focused on this task as well.

Exosome Characterization

Once exosomes and/or EVs are isolated and purified it is important to characterize them. These characterization studies are often called downstream processing. An entire review could be written about this topic. This is a short non-comprehensive summary.

Nanoparticle Tracking Analysis (NTA)

Nanoparticle Tracking Analysis (NTA) is a way to measure nanoparticles in a small volume. The system is based on nanoparticles moving rapidly in a liquid sample under Brownian motion under a laser beam. NTA determines particle size and uses particle numbers to determine concentration. Importantly, NTA measurements are not specific to exosomes or EVs, and instead, will quantify any particle within the size range of detection. Several vendors offer examples of commercially available NTA instruments, and there is debate in the scientific community regarding direct comparability. The result is a detailed differential particle size distribution graph which is produced using analytical software. An example is displayed in Figure 3.

Figure 3. Sample NTA report showing nanoparticle standard. Graph generated by Nanosight Software; image provided by Clara Biotech.
Figure 4. Example TEM showing isolated exosomes

In addition to NTA, a modification is fluorescence NTA or fl-NTA, which allows for accurate sizing, counting, and phenotyping of exosome subtypes. Fluorophores attached to antibodies or other biological probes bind only vesicles with the appropriate marker and are fluorescently labeled and tracked.

Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)

SEM is a form of microscopy which produces images of a sample by scanning it with a focused beam of electrons. TEM is a form of microscopy in which a beam of electrons is transmitted through an ultra-thin specimen. Both techniques have been used to view EVs and exosomes. It is with the use of these tools that one can actually view the wide range of particles and wide range of exosome morphologies.

Proteomics and Protein Analysis

As noted above, exosomes contain cargo consisting of RNA, DNA, proteins and other biomolecules. It is of scientific interest to identify the protein contained within exosomes. The protein composition of exosomes is studied via total protein analysis, Western blot analysis or other electrophoresis, mass spectrometry, enzyme- linked immunosorbent assay (ELISA) specific detection systems and other protein detection assays. Importantly, these assays do not discriminate between non-vesicular and vesicular proteins, so the sample must be free of non-vesicle proteins (or have consistent and controlled expression levels) for these measurements to be useful. Peptide fractionation and bioinformatics analyses may also be performed.

Exosomes as a source of biomarkers

Exosomes are a fascinating group of small vesicles with sophisticated cargo and multiple functions which are only partially understood. From our present and past work with serum, plasma, urine and CSF, we found that exosomes derived from these bodily fluids contain substantial amounts of different RNA species such as miRNA, mRNA, rRNA, tRNA, scaRNA, snoRNA, snRNA and piRNA.

RNA analysis

RNA analysis of exosomes becomes complex very quickly. In one example exosomes harvested from plasma and urine contained extremely diverse RNA cargo, including miRNA and mRNAs, but also ribosomal (rRNA), transfer RNA (tRNA) and many other RNA variants.19 Many commercial kits are now available with appropriate bioanalyzers and microarrays for RNA analyses.

DNA analysis

Exosomes also carry multiple forms of DNA, including double- stranded DNA (dsDNA), single-stranded DNA (ssDNA), mitochondrial DNA, and oncogene amplifications (i.e., c-myc).20 For this reason, exosomal DNA (exoDNA) is representative of the entire genome and reflects the mutational status of parental tumor cells. The potential to measure exoDNA from tumor-derived exosomes has significant potential as a circulating biomarker for the early detection of cancer and metastasis. DNA analysis of exosomes using polymerase chain reaction (PCR) analysis and next generation sequencing (NGS) is also becoming increasingly common.

Exosomes in Diagnostics

Applications of exosomes in diagnosing disease

Exosomes, by their nature, have great potential to be used as diagnostic tools and biomarkers for many diseases. Current evidence indicates that exosomal contents are altered during active disease, whether that be cancer, cardiovascular disease, infections, immunologic diseases, or CNS pathologies such as Alzheimer’s and Parkinson’s disease. In general, most literature indicates cells that are undergoing some inflammatory process exude more exosomes. Thus, exosome studies are part of a currently evolving and rising trend focused on liquid biopsies. Many molecular diagnostics rely on analyzing solid tumor cancer cells from a tissue biopsy by needle aspiration or surgery, which carries several significant limitations when compared to liquid biopsies. Liquid biopsies are more accessible, more generally representative of the tumor (rather than one section), and allow for serial monitoring.

Exosomes from cancer cells are released into all body fluids and contain double-stranded DNA (originated from nucleus and mitochondria), a variety of RNA species, and specific protein biomarkers that can be utilized as cancer biomarkers and therapeutic targets. Therefore, the specific exosomes secreted by tumor cells could be used to predict the existence of the presence of a tumor in cancer patients, and potentially status and progression.

Although additional research is required, exosomal membrane markers can potentially be used to identify their parent cell by subtyping exosomes. For example, L1CAM is a marker commonly found on exosomes of neuronal origin. Thus, exosomal membrane markers can potentially be used to identify their cellular origin. Once these subtyped exosomes can be isolated from bodily fluids they can be used for detection of various proteins, nucleic acids, or lipids that may be disease indicating. However, technical issues, validated and consistent methods, and better understanding of exosomes in disease are required before exosomes can be used in routine diagnostic tests.

Purity, reproducibility, time, background noise

Although not critical for every diagnostic application, purification of exosomes is important, especially if the signal (i.e., identification of a specific protein or segment of RNA) is in low concentration. When one considers a plasma sample, for which 1ml may contain 10’s of billions of exosomes, among 100’s of billions of other nanoparticles, this is a formidable task. A diagnostic test needs to be reproducible, amenable to time and labor constraints, and must show sensitivity and specificity commensurate with the task. Based on the methods of isolation and purification noted above, certain methods may or may not be suitable for a test.

One example of a diagnostic which uses exosomes is a prostate test. This exosome-based diagnostic is used to rule out patients who don’t need a biopsy of the prostate, and is performed in male patients with slightly elevated PSA. The test uses urine as the source material, captures exosomes from the urine using a method involving bind-wash-elute protocols to isolate exosomes and eventually conduct PCR to identify the genetic targets. The company utilizes a proprietary algorithm based on exosomal RNA contents such as ERG, PCA3, and SPDEF to derive a risk score. The score provides the clinician with risk-based guidance regarding the need for biopsy. In fact, the company has worked to develop the use of this test in the 2019 National Comprehensive Cancer Network (NCCN) guidelines.

The exosome diagnostic market is in early stages of development. It will be exciting to monitor progress as more exosome-based diagnostics are developed to diagnose and monitor many different disease states. This excitement will be balanced with the need for regulatory approvals, reimbursement requirements, and scientific and clinical superiority over existing products.

Exosomes as Therapeutics

More than 20 biopharma companies are pursuing exosomes as therapeutics. As noted above in the Exosome Purification section, purity is important to regulators. Companies that are developing exosome-based therapeutics must ensure lot-to-lot consistency of the product, and the cargo contained in the exosomes. In clinical research, exosomes derived from specific tissues or tumors can contain important circulating or excreted biomarkers, but the population of interest may only be a small percentage of the total.

Exosomes are master communicators in the body. They take on the properties of the cell from which they are derived. It has been suggested that nearly all stem cell activity is the result of the exosomes they produce.22 The proposed mechanism of exosomes derived from hMSC’s include:

  1. Reduced inflammation at least partly due to reduced proinflammatory T-cells and increased number of anti- inflammatory T-cells and anti-inflammatory macrophages.
  2. Regenerative and restorative action evidenced by cell proliferation and growth at injured sites.
  3. General cell protection by reducing cell death and apoptotic activity.

In a recent systematic review of more than 200 studies evaluating MSC derived exosomes for preclinical use, the exosomes demonstrated benefits in 72% of studies.21 However, only 60% of studies used nomenclature consistent with the size definitions of exosomes. The most common isolation techniques were UC (70%) and isolation kits (23%). Dosing was inconsistent and based on EV protein content or particle concentration. Approaches for determining size, protein markers, morphology, and other identifiers were highly heterogeneous, making scientific comparisons very difficult. This review points out multiple opportunities to improve study design and methodology in the rapidly growing field of EV therapeutics.

Using nanoparticle exosomes rather than cell therapies provides several advantages:

  1. Exosomes can be loaded to carry a variety of therapeutic agents for delivery to specific tissues within the body. These include small molecules, RNA types, antisense oligonucleotides (ASOs), genes, etc.
  2. Stem cell-derived exosomes are generally less immunogenic than cells. Thus, “off the shelf” products may readily be developed using exosomes with negligible immunogenicity.
  3. By changing the exosome producer cell type, you can alter the region to which the payload is delivered.
  4. Stem cells require special care and processing, which is much less rigorous for exosomes because they are not cells. Exosomes can be frozen and stored without cryo- preservatives with no loss in their biochemical activities. They can also be lyophilized.
  5. Stem cells have the potential to transform into other cells, including oncogenic cells. Exosomes lack this capacity, but may be modified in various ways to affect cargo and delivery.

Since exosomes and EVs are often compared to lipid nanoparticles (LNPs), which are well known in the drug delivery field, it is appropriate to share potential advantages of exosomes to LNPs:

  1. Exosomes are natural particles. As such, their likelihood to stimulate immune response is generally much less than LNPs.
  2. Lipid load and lipid composition of LNPs can lead to toxicities.
  3. In-vivo stability and distribution without destruction by the liver and reticuloendothelial system (RES) or other immuno-activity is viewed as an advantage of exosomes.
  4. Delivery efficiency of exosomes is currently viewed as superior to that of LNPs.

However, the choice to pursue delivery of a nucleic acid or ASO using an exosome or LNP ultimately depends on the application and use of the therapy. For example, the current Pfizer-BioNTech vaccine for SARS-CoV-2 utilizes an LNP formulation, and was used based on previous experience to hasten development.

Exosomes in the clinic

Exosomes have been purported as potential treatments for numerous conditions. In Table 3 is a summary of 112 studies in clinicaltrials.gov for exosomes as interventional treatments for the diseases noted. It is evident that therapeutic exosomes are being investigated for a wide variety of disease states.

Table 3. Exosome Therapeutics Target Indications

The majority of clinical trials with therapeutic exosome products are sponsored by individual institutions and investigators. However, the following biotech companies are notable for having exosome products in or near clinical development:

  • Aegle Therapeutics
  • Aethlon Medical
  • Aruna Bio
  • Capricor Therapeutics
  • Carmine Therapeutics
  • The Cell Factory - Esperite Group
  • Codiak BioSciences
  • Evox Therapeutics
  • Exopharm
  • Kimera Labs
  • ReNeuron
  • United Therapeutics

As with all new therapies, it is important to seek patient and caregiver perspective throughout product development. Exosomes are likely to be administered parenterally, but it is possible they could be administered orally since exosomes are bioavailable. For example, exosomes present in cows’ milk can be detected in blood after ingestion.23 Exosomes also have the potential to be used topically or incorporated into devices such as stents.

Naive Exosomes and Modified Exosomes

An analysis of the clinical studies and companies developing exosomes quickly highlights that therapeutic exosomes can be divided into two big buckets: naive exosomes and modified exosomes. Modified exosomes are also called engineered or targeted. Naive exosomes are unmodified exosomes naturally produced by cells. Exosome surface properties and cargo reflect their cell of origin. For example, exosomes from MSCs possess the inherent regenerative and anti-inflammatory activities of their parent cell. Although naive, the cells that produce them can be modified by different culture media contents and mechanisms of stress, such as hypoxia or cytokine introduction, to produce more and/or different exosomes. The “cell factory itself can also be modified or engineered to produce exosomes of specific type.

Modified exosomes are vesicles that have been modified to express a targeting molecule on their surface and/or to carry a specific drug cargo, e.g., miRNA, ASO or gene. Therefore, the exosomes can be designed as drug carriers to deliver small molecules, proteins, nucleic acids, ASOs, or genes into specified cells. The process of incorporating these specific cargos into exosomes is the subject of much research. To date, three general approaches are being investigated:

  1. Modifying host exosome producing cells to generate specific exosomes,
  2. Loading cargo (e.g., nucleic acids) into the exosome producing cells, or
  3. Loading cargo after exosome harvest.

Great interest and investment is being poured into modified exosomes as evidenced by the deals with Evox, Codiak, and Carmine noted below.

Although details and additional progress is required one big difference between naive and modified exosomes is how Regulatory Authorities view them. To date, naive exosomes are viewed much like their cells of origin. For example, if the cell of origin is an MSC, then the exosome is regulated as such. However, modified exosomes, by their nature of being modified are new biologics and require the more traditional biologics pathway.

Regulatory requirements - purity, potency, safety and more

Since there are no exosome products currently approved, the exact pathway is yet to be defined. Nevertheless, the Food and Drug Administration (FDA) and other Regulatory Authorities around the world have specific requirements for drugs and biologics used to mitigate, treat, cure, or prevent disease. The FDA provides guidance to inform sponsors how to provide sufficient Chemistry, Manufacturing and Control (CMC) information required to assure product safety, identity, quality, purity, and strength including potency (21 Code of Federal Regulations (CFR) 314). Isolation and purification of exosomes from cell culture media requires significant controls for quality, purity, potency, and reproducibility. Subsequent modifications to exosomes also require significant controls. Specifications for exosomes are likely to include specifications for their origin cells as well as their contents.

When considering the methods of purification summarized above, it is obvious that much work is required because sources of exosomes generally contain an unknown amount of exosome content, and are also contaminated with proteins, cell-free DNA, vesicles, potentially viruses, and other components at this size range. Purifying these samples is not trivial, and current methods will require more rigorous controls to become approved therapies.

Product identity, quality, and purity are important to ensure safety, but efficacy of the exosome product is critical for approval, clinical acceptance, and reimbursement for successful commercialization. FDA’s Center for Biologics Evaluation and Research (CBER), Office of Tissues and Advanced Therapies (OTAT) is the division responsible for managing stem cell and exosome products.

Exosomes and biopharma deals

The exosome business climate is beginning to be recognized by early adopters who believe in the technology. Table 5 summarizes five noteworthy deals in the exosome space within the last 2 years. All of these deals focus on targeting and loading exosomes, and provide the basis for the significant potential of exosomes to treat, mitigate, or cure several diseases. In addition, there are deals with undisclosed terms from other companies, such as ReNeuron and Aruna Bio.

Table 4. Exosome Market

Exosome Market

From a market definition view, exosomes have a role in scientific research and laboratory work, diagnostics and disease identification and monitoring, and therapeutics as naive or modified exosomes. Several exosome market reports are available from various vendors, but in the authors’ view, none of them accurately depict the future market potential for exosomes, especially related to therapeutic exosomes. One thing most of the market reports agree upon is the growth of the exosome markets, for which compound annual growth rate (CAGR) ranges from 25-50%.

The exosome market is evolving in the pharma/biotech industry. In general, it can be divided into three main categories: research/lab, diagnostics and disease monitoring, and therapeutics. Each of these sectors has its own specific trends, drivers, and challenges, yet they are interconnected and affect further market development. Worldwide market estimates range as follows:

Table 5. Recent Exosome Deals

Exosome Hype

Exosomes are not limited to the traditional science community. As with stem cells, there are many “clinics”, “practitioners”, and sources of exosomes being sold as anti-aging treatments, pain and arthritis treatments, and numerous other claims. The FDA recognizes this and has produced a public safety notification - (https://www.fda. gov/vaccines-blood-biologics/safety-availability-biologics/public- safety-notification-exosome-products#:~:text=There%20are%20 currently%20no%20FDA,offering%20exosome%20products%20 to%20patients) - “There are currently no FDA-approved exosome products. Certain clinics across the country, including some that manufacture or market “stem cell” products in violation of current law, are now also offering exosome products to patients.”The FDA regulates exosome products in the United States. Some clinics may falsely advertise that it is not necessary for FDA to review and approve their therapies. They may claim that their registration with FDA or an FDA inspection equates to FDA approval or a form of FDA endorsement. These claims are false.

Future of Exosomes

Exosomes were relatively unknown just 10 years ago. Today, interest in the use of exosomes is growing exponentially. Research and analyses of exosomes at the lab scale will continue to generate new, exciting data. Exosome utility for diagnostic applications will continue to increase. The potential for exosomes as therapeutics is very exciting. They have potential to treat new diseases, deliver new genes and potentially cure diseases previously thought incurable. To paraphrase the CEO of a leading exosome company: “Exosomes are going to do to biotechnology what antibodies once did. Antibody development started 25-30 years ago. The top 12 biopharma products in the world are antibodies” (Linda Marban, Capricor CEO, August 6, 2020 second quarter meeting).

Nevertheless, isolation and purification of exosomes using consistent and reproducible methods remain a challenge. The scientific community will continue to evolve such that our understanding of exosomes and methods of purification and analysis will lead to new knowledge, new diagnostics, and new therapeutics that will improve health broadly for ourselves and our loved ones.

References

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