Research

What are Exosomes?

Extracellular vesicles (EVs), commonly known as exosomes, secreted by living cells mainly include exosomes (50-150 nm diameter) and microvesicles (100-1000 nm). Among stem cells, mesenchymal stem cells (MSCs) produce large numbers of EVs, with neuroprotective and regenerative capabilities. Although originally believed to be mediators of cellular homeostasis by secreting cellular waste, recent studies have highlighted important roles of EVs in intracellular communication and modulating cellular aspects of immunology, cancer biology, and regenerative medicine. The EV membrane’s lipid bilayer is a subset of the plasma membrane. EVs contain messenger RNA (mRNA), microRNA (miRNA), and cytosolic and trans-membrane proteins. Exosomes are the smallest subset of EVs (30 – 150nm diameter) and play a significant role in the transfer of biomolecules such as RNA, proteins, enzymes, and lipids in physiological and pathological conditions.

In addition to their nanoscale size, a defining characteristic of exosomes is their endosomal origins. The biogenesis of exosomes is complex; exosomes start with reverse membrane invagination and processing in multivesicular bodies (MVBs), followed by release into the intercellular fluid when MVBs fuse with the cell membrane. All exosomes share a common set of proteins, tetraspanins (CD9, CD63, CD81), Alix, and TSG101, but also contain cargo reflective of the molecular processes within the parent cell. When endocytosed by effector cells, EVs trigger a cellular response controlled by their cell of origin.

As a cell-free therapy, EVs have potentially significantly greater safety and specificity, can be safely administered cross-species without immunosuppression, and are efficient small molecule carriers that can deliver anti-inflammatory agents to the brain . EVs could easily be administered intranasally or into the vitreous to access RGCs and potentially the optic nerve. We have demonstrated that MSC - EV endocytosis is receptor mediated and endocytosed in retinal neurons by heparin sulfate peptidoglycan (HSPG) receptors via the caveolin pathway. Therefore, it is possible that cellular uptake of EVs can be enhanced by genetically engineering the parental cells to express ligands on their surface. These engineered ligands will be transferred to the vesicles surface and bind to specific receptors and cross the target cell membrane barriers.

Exosomes have a superior capacity for transporting various cargo due to their role as facilitators of cellular communication. EVs can pass through the blood-brain-barrier because of their bilayer lipid structure, making them the most suitable agent for CNS treatment. MSC-EVs’ stability, biocompatibility, and low toxicity make them a favorable direction for research in drug delivery and precision medicine.

Functionally Engineered Exosomes

In addition to proteins, exosomal cargo includes various nucleic acids including mRNAs, microRNAs (miRNAs), and other non-coding RNAs. The function of these RNAs is to be taken up by surrounding cells and allow exosomes to modulate recipient cell function. Although the function microRNA can be compared to that of synthetic short interfering RNA (silencing RNA) (siRNA) in terms of its role in decreasing mRNA expression, miRNA function is more complex. MicroRNAs mediate post-transcriptional gene silencing and are involved in cellular activities including proliferation, differentiation, and migration, as well as disease initiation and disease progression. It has been shown that white adipose tissue miRNAs decline as a result of the decrease in the miRNA processing enzyme Dicer. The role of miRNA in mediating exosome function is confirmed through observation of miRNA profiling and functionality tests. Additionally, the communication of exosomal miRNA down-regulated 19 genes involved in the maintenance of the blood-brain barrier. The profiles of exosomal miRNA differ depending on the parent cell from which the exosomes are derived and can be used to provide information about disease pathogenesis.

Modification of exosomal miRNA can promote regeneration processes. For example, miR-219 has specifically been identified for its ability to promote oligodendrocyte maturation and regeneration in the EAE MS model, and the ability of mir-146 to mediate crossing the blood brain barrier and improve functional recovery.

Functionally engineered EVs (FEEVs) are also emerging as a potential nano-therapeutic tool. Apart from engineering exosomes for the delivery of miRNAs, which have been proven to mediate various immune responses in the retina and brain, exosomes can function to carry various cargo as a novel drug delivery tool. Another modification involves priming receptors involved in exosomal endocytic pathways, as it can lead to an enhanced drug delivery process by facilitating effective endocytosis. Other methods of EV modification use cell specific receptor binding affinity towards specific surface proteins.

Ongoing Projects:

Therapeutic Exosomes for Treatment of Retinal Diseases

Glaucoma

Glaucoma, the 2nd leading cause of worldwide blindness, afflicting greater than 60M, is characterized by RGC loss and optic nerve defects. A principal unmet need in glaucoma is that medical or surgical intra-ocular pressure reduction is the only clinically-approved treatment. But vision cannot be restored because retinal ganglion cell (RGC) loss is irreversible. Knowledge gaps to improved therapy include how to achieve neuroprotection, i.e., preventing RGCs from dying, or, to use cell-based therapy to re-grow or replace. Administration route, dosage, and adverse effects limit clinical application of neuroprotection and cell transplantation.

Studying a new treatment using extracellular vesicles (EVs), biologically-active 50-150 nm diameter nanoparticles derived from stem cells, this study fills a gap and urgent need in the development of new treatments to prevent RGC death and vision loss for glaucoma patients. Their stability, bio-compatibility, biological barrier permeability, and low toxicity render EVs attractive vehicles as delivery therapies to the eye. EVs represent a potential clinically applicable means to prevent RGC, axonal, and visual functional loss and decreasing the excitotoxic and inflammatory component of glaucoma.

Our central hypothesis is that EVs can be designed and optimized to specifically target RGCs as a basis for precision treatment of glaucoma and, ultimately, other retinal diseases. We propose to test engineered EVs as a novel cell-free means to specifically target neuroprotection to RGCs and to fill the knowledge gaps that presently prevent clinical translation of EVs for retinal disease.

The study offers promise to save sight via development of safe, effective, and cost-efficient therapy to restore or prevent loss of sight in patients with glaucoma. This contribution is expected to be significant because these studies will provide a basis to develop EVs as a therapy for glaucoma, either primarily restoring RGC function and axonal growth, or optimizing existing therapies such as RGC transplants. Innovative features of this work are cell-free therapy of glaucoma, novel targeted EVs binding RGC-specific receptors for specific RGC action, and novel delivery materials for EVs. RO1 applications are expected to follow, to further examine mechanisms of EV actions and develop retinal cell-specific targeted, safe, low-cost therapeutics.

You can read more about the NoGo project and view the NIH grant here, and the BrightFocus grant here.

Multiple-Sclerosis Induced Optic Neuritis

Multiple sclerosis (MS) is a chronic autoimmune disease characterized by the inflammation and demyelination of the central nervous system (CNS), resulting in neuronal death and axon degeneration. Optic neuritis (ON), the inflammation to the optic nerve, is one of the most common presentations of MS, and appears as the first manifestation even before the detection of demyelination in approximately 20% of patients. Optic neuritis causes significant axonal damage and the loss of retinal ganglion cells (RGC) in the retina which leads to serious and often irreversible vision loss. To decrease the amount of neurological damage and promote neuroprotection in MS-induced optic neuritis, the attenuation of inflammatory response and the protection of retinal axons and RGCs are of utmost importance.

The process of immuno-regulated inflammation in MS and MS-induced ON makes cellular therapy a promising candidate for its treatment. Human mesenchymal stem cells (hMSCs) are multipotent cells found in bone marrow with neuroprotective properties and a strong presence in stem cell therapy. Not only do hMSCs have the capacity to produce large amounts of exosomes for drug delivery, but the notion of exosomes being a cell-free therapy for MS and MS-induced optic neuritis has numerous benefits compared to regular stem cell therapy. Being a means of cellular communication, they have a superior capacity for being loaded with various cargo and can be engineered to transport certain macromolecules between cells. In addition, as they are made of cell membrane, are able to be taken up directly and better tolerated by hosts. MSC-EVs’ exceptional stability, biocompatibility, and low toxicity thus make them a favorable direction of research in drug delivery and precision medicine.

Read more about EVs for MS-ON in our review paper here, and Alice's SFN poster presentation here

Age-Related Macular Degeneration

Glaucoma, the 2nd leading cause of worldwide blindness, afflicting greater than 60M, is characterized by RGC loss and optic nerve defects. A principal unmet need in glaucoma is that medical or surgical intra-ocular pressure reduction is the only clinically-approved treatment. But vision cannot be restored because retinal ganglion cell (RGC) loss is irreversible. Knowledge gaps to improved therapy include how to achieve neuroprotection, i.e., preventing RGCs from dying, or, to use cell-based therapy to re-grow or replace. Administration route, dosage, and adverse effects limit clinical application of neuroprotection and cell transplantation.

Studying a new treatment using extracellular vesicles (EVs), biologically-active 50-150 nm diameter nanoparticles derived from stem cells, this study fills a gap and urgent need in the development of new treatments to prevent RGC death and vision loss for glaucoma patients. Their stability, bio-compatibility, biological barrier permeability, and low toxicity render EVs attractive vehicles as delivery therapies to the eye. EVs represent a potential clinically applicable means to prevent RGC, axonal, and visual functional loss and decreasing the excitotoxic and inflammatory component of glaucoma.

Our central hypothesis is that EVs can be designed and optimized to specifically target RGCs as a basis for precision treatment of glaucoma and, ultimately, other retinal diseases. We propose to test engineered EVs as a novel cell-free means to specifically target neuroprotection to RGCs and to fill the knowledge gaps that presently prevent clinical translation of EVs for retinal disease.

Our specific aims are:

Aim 1: Determine the time course and factors regulating the distribution of EVs in the vitreous and retina, and optimize EV delivery to retina.

Aim 2: Develop and optimize novel engineered EVs to specifically target RGCs.

Successful completion of Aim 1 will optimize delivery of EVs to the retina following intravitreal injection. Aim 2 will guide administration of EVs to produce innovative, specific, targeted delivery into RGCs, allowing specificity for treatment at the major pathophysiological site of glaucoma. Fulfillment of these objectives will set the stage to develop glaucoma therapeutics using EVs by optimizing administration, and by specific RGC-targeted EVs.

The study offers promise to save sight via development of safe, effective, and cost-efficient therapy to restore or prevent loss of sight in patients with glaucoma. This contribution is expected to be significant because these studies will provide a basis to develop EVs as a therapy for glaucoma, either primarily restoring RGC function and axonal growth, or optimizing existing therapies such as RGC transplants. Innovative features of this work are cell-free therapy of glaucoma, novel targeted EVs binding RGC-specific receptors for specific RGC action, and novel delivery materials for EVs. RO1 applications are expected to follow, to further examine mechanisms of EV actions and develop retinal cell-specific targeted, safe, low-cost therapeutics.