Extracellular Vesicles in Gene Therapy at this year’s ASGCT

The American Society of Gene and Cell Therapy (ASGCT) celebrated its 25th year at their annual meeting at the Walter E. Washington Convention Center in Washington, DC last week and a full agenda is available on their site [ 1 ]. Originally designed for academic researchers to share their work, the annual meeting has grown to serve clinicians, bio-industry development, regulatory agencies, equipment manufacturers, patient advocates, and more. A truly remarkable number of activities and topics were addressed in the 4-day meeting. But from the scientific symposia and education sessions to the industry sponsored symposia and technology forums, it is really impossible to even begin to describe the topics and activities offered. There were over 6000 attendees participating in an excess of 80 unique sessions, covering many distinct cell gene technologies and addressing over 15 tissues, organs, and disease indications.  However, beyond the science and technology, there were many networking events, both formal and informal. Just one was a catered tour of the International Spy Museum, which illustrates how much fun the event was.

A number of papers were presented describing the latest developments in approaches to nucleic acid delivery, such as electroporation, microbubble disruption, particle bombardment, microinjection, and nanoparticle fusion. This report reviews those papers examining the use of exosomes and other extracellular vesicles (EV) in cargo delivery. I produced this review contemporaneously with the lectures, and so it is a record of my understanding of the salient points at the time.

EVs are naturally phospholipid-enclosed nanovesicles released by many cells in the body. They can carry multiple functionally active biological molecules, including oligonucleotides and polypeptides and can bear targeting features on their surface. EVs play a vital role in normal physiology and cellular communication, are stable in circulation, and have low immunogenicity. They are produced by most every cell type, have been reported to exist in nearly every bodily fluid. and have been implicated in numerus natural functions. They are growing in popularity as noninvasive diagnostic, prognostic, and therapeutic tools [ 2 ]. The papers below address the natural limitations in EVs engineering, targeting, and loading.

The first paper on the topic, scheduled for the second day of the meeting, titled Exosomes in Nucleic Acid Delivery, was unfortunately withdrawn. However, that evening, in the session titled Physical Methods and Extracellular Vesicle-Based Gene Transfer, there were three papers in the field. It began with Kari Heck, University of Nebraska-Lincoln who on Assessment of Commensal E. coli Outer Membrane Vesicles for Application in a Novel Oral Delivery System. She spoke of the increasing need for oral gene delivery methods, which included vaccines and therapies for intestinal disease. She then introduced a particular type of nanovesicle: OMVs (outer membrane vesicles). They are produced by blebbing in many prokaryotes, and she noted that their safety in general is established by the fact that we actually already eat millions of them ever day in various foods. Notably, both benefit and harm for humans have been established from the activities of natural OMV. This U of N group examined OMV from 30 different bacterial strains for physical characteristics and plasmid delivery potential. Dr. Heck reported on work examining the relative delivery of a florescence marker, DIO, as well as of a standard marker of inflammatory response, within the various recipient cells. This was all in the context of designing OMV as a potential gene therapy, DNA vaccination, and an immune modulatory therapeutic.

Dr. Boya Peng then presented on Vesicle-mediated Therapeutic Delivery of RIG-I Agonists for Immunotherapy Against Breast Cancer (authored by Minh T.N. Le and Yong Loo of the National University of Singapore). She began with an exposition of breast cancer demographics, and the existing attempts at the use of RIG-I in diagnosis and therapy. She noted that a polymer-delivered RIG-I recently failed in clinical trial, due to immunogenicity and toxicity. She then provided a review of the use of EV as delivery vehicles, as well as the features and benefits of EVs from red blood cells (RBCEV). RBCEV mediated delivery of immunomodulatory RNA (immRNA) to breast cancer cells suppresses tumor growth by triggering RIG-I mediated immune responses. She showed that bi-functional antisense RNA activated RIG-1 pathways and promoted cell death. Cancer cell suppression was demonstrated by targeting EGFR and 4T1-EGRF. This was accomplished through streptavidin conjugation of biotinylated probes to the surface of RBCEVs loaded with immRNA. Finally, dendritic cell autoactivation was demonstrated by immRNA delivery to CD8+ cells. All-in all, target RBCEV-mediated delivery of RIG-1 agonists were demonstrated to confer anti-cancer activity through multiple avenues.

Andrew Hamann, University of Nebraska-Lincoln presented on Engineering Cells to Produce miRNA-loaded Exosomes for Potential Biotherapeutics. He began with a review of the nature and function of exosomes. He then described their popularity as both a therapeutic themselves (e.g., from MSCs), as well as vehicles for the delivery of therapeutic agents (e.g., mRNAs). Following a review of the use of mRNAs (or their knockdown) in various therapies, he outlined the limitations of, e.g., lipid nanoparticles (LNPs), as systemic delivery agents. After extoling the features of exosomes in such a capacity, he did review their limitations− such as that few exosomes are found that naturally include mRNAs, and existing loading techniques (e.g., using aptamers) have proved rather inefficient.  He then defined an optimized “active” loading system they have developed. They began by transforming cells to produce a VSVG fusion protein that will insert into exosome membranes. They have exploited this and MS2 aptamers to generate selectively targeted loading and delivery vehicles. Fluorescent markers bound to the HIS-tagged membrane protein demonstrated that it was exposed for potential targeting molecule binding.

On Wednesday morning Sriram Sathy of Codiak Biosciences spoke on the topic of Engineered Exosomes as a Delivery System from Bench to Bedside. He began by revealing that exosomes exist as a result of millions of years evolving as a natural communication mechanism. He noted that every blood transfusion permits the transmission of millions of exosomes between people. He described how loading of mRNA into exosomes is a topic of much current activity and many report that they can drag mRNA into the lumen− but he noted that much more work on this  is required. Co-loading is a concern, e.g., of host cell protein, HCP. It has been shown that intercellular adhesion molecules such as ICAM-1, as well as  other targets, can increase the selectivity of loading. The outside loading is the most difficult, while co-loading the inside is actually less of an issue. Analytical methods demonstrate that challenges include the fact that natural exosomes are rather heterogenous and carry a diversity of payloads. So, they needed to engineer a synthetic exosome that would be homogeneous. They also needed to design reproducible methods of scalable production.

Prostaglandin F2 receptor negative regulator (PTGFRN), designated CD315, has been shown to interact with CD9 and CD81. So, PTGFRM was used to enable adding multiple targeting moieties on the outside, while BASP-1 was embedded on the inside to orient payload. This complex construct supports multiple administration mechanisms and types of cargo. Sriram then presented a number of target cells they’d had success with, as well as many cargos, including such nucleic acids as siRNA, ASOs, and small molecules. Cells were then engineered to produce exosomes harboring PTGFRN. As tumor associated macrophages have become popular, Codiak progressed, and the FDA has cleared, the company’s Investigational New Drug Application (IND) for exoASOTM-STAT6. It’s their third engineered exosome therapeutic candidate to be cleared for clinical evaluation, and the first intended for systemic (intravenous) administration. It is designed to silence the transcription factor STAT6 selectively in tumor associated macrophages (TAMs).

Preclinical studies of exoASO-STAT6 showed single agent anti-tumor activity in models of aggressive hepatocellular carcinoma. It is rapidly accumulated in tumor associated macrophages in the liver, and mouse work shows a single agent reduction in tumors. Results include that macrophages initiate production of the enzyme inducible nitric oxide synthase (iNOS) and have increased CD8 infiltration. Now, they look for patients having tumors with elevated STAT6 levels. The encapsulated AAV within an exosome provides safer, targeted potency in getting more virus to cells− in fact natural AAV are already (although to a low frequency) encapsulated in exosomes! Codiak has measured increased transduction rates using such systems. Functionality was demonstrated in retinal ganglia that were specifically transduced using the exoAAV marker system. They have carefully compared unloaded, generic exosomes and see very little effect on the receptor cells, arguing that it the functional payload that produces the desired effect. They then tried to actively load off-the-shelf antibody to AAV expressed into the lumen of the exosome, but they couldn’t transduce their producer cells to produce the loaded exosomes. So, they made their own antibodies that would load, and demonstrated a 100-fold increase in efficiency. In summary, exosome associated AAV demonstrated resistance to neutralization as well as improved potency in vivo. Codiak as engineered a novel engineered exosome-based platform to deliver AAV.

Later that afternoon, Casey Maguire, Massachusetts General Hospital spoke on the topic of Selection of Engineered AAV Capsids with Enhanced Incorporation into Extracellular Vesicles and Stable Liver Transduction in vivo. He described the blurring of the distinction between viral-based transduction and EV-based transduction (as was described above) and reported what an exciting field this is. Exo-AAV envelopment vectors have now become a published phenomenon, and they have described how purified exo-AAV1 are more resistant to neutralizing antibodies than other AAV preparations. In the media of 293 cells producing AAV, one may observe many single and multiple encapsulated AAV within EVs. They also identified membrane-associated accessory proteins by mass spectrometry of purified exo-AAV1 preparations and showed that it is involved in not only genome packaging, but in stimulating EV release as well.  They have also produced a scalable method, size-exclusion chromatography, to isolate exo-AAV1− and demonstrated functional transduction in cultured cells as well as increased antibody resistance. This suggests that higher purity exo-AAV will have beneficial characteristics for gene delivery and also may lead to mechanistic insights into the incorporation of AAV into EVs.

They employed an AAV capsid 7-mer peptide display to discover best candidates for AAV enrichment. They showed that selected peptides enriched over 200-fold from background. This excoCAP variant vector is more resistant to intravenous immunoglobulin (IVIg) than native AAV9. exoCAP vectors have more genomes/EV than AAV9 and exoCAP mediates the stable transduction of liver in Balb/c mice,  it also maintained expression longer than that of AAV9. ExoCap A was also much more efficient than native CAPA in neuropeptide dosing. In questioning, Dr. Sathy acknowledged that it may be that increased efficiency in transduction is due to increased copy number within the EV encapsulated AAV.

All in all, these sessions presented significant data on both the power, and the advanced state of development, of natural, engineered, and reconstituted exosomes. This success has been demonstrated in both encapsulating a variety of therapeutically active cargo, as well as selectively targeting them to identified tissues.


  1. https://annualmeeting.asgct.org/
  2. Sai Priyanka Kodam and Mujib Ullah, Diagnostic and Therapeutic Potential of Extracellular Vesicles, Review Article, Technology in Cancer Research & Treatment Volume 20: 1-10, 2021 sagepub.com/journals-permissions DOI: 10.1177/15330338211041203 journals.sagepub.com/home/tct, https://doi.org/10.1177/15330338211041203


Tuesday the 17th

8:50–9:15 AM ROOM 201 Therapeutic Applications of RNA Therapy Strategies, Exosomes in Nucleic Acid Delivery Susmita Sahoo, PhD, Icahn School of Medicine

4:45–5:00 PM SALON H Physical Methods and Extracellular Vesicle-Based Gene Transfer, Assessment of Commensal E. coli Outer Membrane, Vesicles for Application in a Novel Oral Delivery System, Kari Heck, University of Nebraska-Lincoln

5:00–5:15 PM SALON H Physical Methods and Extracellular Vesicle-Based Gene Transfer, Extracellular Vesicle-mediated Therapeutic Delivery of RIG-I Agonists for Immunotherapy Against Breast Cancer, Minh T.N. Le, PhD, Department of Pharmacology, Yong Loo, Lin School of Medicine, National University of Singapore

5:15–5:30 PM SALON H Physical Methods and Extracellular Vesicle-Based Gene Transfer, Engineering Cells to Produce miRNA-loaded Exosomes for Potential Biotherapeutics Andrew Hamann, PhD, University of Nebraska-Lincoln

Wednesday, the 18th

8:00–8:25 AM ROOM 207 Non-viral Delivery: A Diverse Toolbox Comes of Age, Engineered Exosomes as a Delivery System from Bench to Bedside Sriram Sathy, PhD, Codiak Biosciences Inc

4:30–4:45 PM BALLROOM A AAV Developments in Liver, T-cells, and Toxicity, Selection of Engineered AAV Capsids with Enhanced Incorporation into Extracellular Vesicles and Stable Liver Transduction in vivo Casey Maguire, PhD, The Massachusetts General Hospital.

About the Author

William Whitford, Life Science Strategic Solutions Leader, DPS Group

Bill Whitford has recently joined DPS Group as the Life Science Strategic Solutions Leader. Here he will assist in developing creative strategies supporting the manufacturing of both classical and innovative biotherapeutics.

Bill began his carrier as an R&D Leader, commercializing over 40 distinct products supporting biomedicine and biomanufacturing. Applications ranged from assisted reproduction to the culture of animal cells in protein biological and vaccine production.

Most recently Bill has been a thought leader identifying burgeoning biomedical products and processes. An invited lecturer at international conferences, he has published over 300 articles, book chapters, and patents in bioproduction; is a regular presenter at international conventions; and is an instructor in biomanufacturing.

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