(541g) Invited Talk: Engineered Extracellular Vesicles for Gene Therapy Applications

Extracellular vesicles (EVs) are nanoscale, membrane-bound vesicles (50-200 nm in
diameter) naturally secreted by cells. They carry proteins, miRNAs, and metabolites,
mediating intercellular communication in both physiological and pathological conditions.
Clinical evidence strongly supports their safety: numerous trials report minimal
immunogenicity for autologous and allogeneic EVs, and plasma transfusions—routinely
performed in hospitals—transfer trillions of allogeneic EVs with negligible adverse effects.
These findings underscore EVs as well-tolerated carriers for therapeutic delivery.
Furthermore, EVs can be engineered to express specific surface ligands and encapsulate
gene-editing cargo, enabling targeted delivery with reduced risk of immune-related
complications. Collectively, EVs represent a compelling alternative to synthetic
nanoparticles, particularly for chronic inflammatory conditions such as sickle cell disease
(SCD).
Despite these advantages, two major challenges hinder EV-based therapeutics:
processing time and production yield. Conventional isolation methods—ultracentrifugation or
size-exclusion chromatography—require hours and often result in significant EV loss. To
overcome this, we have developed an automated, high-throughput isolation platform
integrating 3D-printed filters with a custom-designed tangential flow filtration system. This
technology is able to process >300 mL of conditioned media within one hour, eliminating
centrifugation steps and improving recovery efficiency.
The second challenge is EV biogenesis. Physical or chemical stimulation (e.g.,
sonication, ethanol) can boost EV release but often induces cellular stress, accelerates
senescence, and generates pro-inflammatory EVs. Our solution leverages metabolic
engineering: by rewiring pathways linked to EV biogenesis, we achieved >10-fold increases
in EV yield while reducing metabolic stress in producer cells such as mesenchymal stem
cells, thereby preserving cell health and longevity.
For SCD therapy, we have engineered EVs to express ligands targeting
hematopoietic stem/progenitor cells (HSPCs) and loaded them with CRISPR/Cas9
ribonucleoprotein complexes. Unlike current FDA-approved gene therapies, which require ex
vivo HSPC modification and intensive conditioning, our approach will enable in vivo delivery
of gene-editing machinery via EVs, offering a safer, more accessible, and non-viral
alternative.
In summary, our integrated strategies—automated isolation, metabolic engineering,
and ligand-directed targeting—position EVs as a transformative platform for gene therapy,
addressing critical bottlenecks in scalability, safety, and accessibility