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Extracellular Vesicle Workshop

Executive Summary

Date of Workshop: September 14, 2023

The National Eye Institute (NEI) hosted the Extracellular Vesicle Workshop virtually and brought together a group of multidisciplinary experts in extracellular vesicle (EV) research to review current EV studies, explore the utility of EVs, and identify the critical knowledge gaps, needs, and opportunities for studying EVs in eye health and disease.

Background

Extracellular vesicles (EVs) are heterogeneous lipid membrane-bound nanoparticles (e.g., exosomes, ectosomes, microvesicles, apoptotic bodies) secreted by cells that mediate cell-cell communication via diverse cargoes they carry, such as RNA, DNA, proteins, and lipids. EVs have been implicated in eye health and disease. Special interest has been given to EVs for their translational potential as a cell-free approach for regenerative medicine. However, the diagnostic and therapeutic potential of EVs for vision research has yet to be fully determined. In addition, basic EV biology is still poorly understood in the visual system. There is an opportunity for vision research to be a leading figure in the emerging EV field.

NEI published the National Eye Institute Strategic Plan: Vision for the Future (2021-2025) in 2021. EV research was identified as a top priority in Regenerative Medicine, one of seven cross-cutting areas of emphasis in the Strategic Plan. As part of the implementation efforts for EV research as a priority, NEI convened a workshop focused on understanding EV biology and the potential for diagnosis, therapy, and prognosis in the visual system.

Workshop goals

The workshop provided a platform for cross-disciplinary discussions with expectations to build a roadmap for advancing EV research in the visual system based on the recommendation published in the NEI Strategic Plan. The workshop goals were to:

  1. Identify gaps and challenges in understanding EV biology in the visual system and its translational potential for diagnosis, therapy, and prognosis;
     
  2. Explore opportunities for leveraging novel technologies and existing resources that have been implemented in other fields, and for innovation based on unique attributes of the visual system; and
     
  3. Discuss strategies for prioritizing the needs and opportunities identified for EV research in eye health and disease.

Participants

Twenty invited panelists participated in the workshop, each with EV expertise from diverse specialties including vision research (cornea, glaucoma, immunology, retina), aging, cancer, cardiology, engineering, immunology/periodontitis, neurobiology/drug addiction, neurodegenerative disease, radiology/imaging, and stroke. The NEI EV working group coordinated and organized the workshop. The final workshop was facilitated by NEI leadership and staff along with workshop leads with expertise within and outside of the visual system.

Summary of the workshop

Drs. Alissa Weaver and Ali Djalilian co-chaired this workshop, which comprised three main sessions with presentations and discussions including: 1) EVs: Biology to Biomarkers, 2) EVs for Regenerative Medicine, and 3) Tools and Approaches for the Rigorous Study of EVs. In addition, panelists also actively engaged in the final roundtable discussion to prioritize the challenges and opportunities of EV research, particularly in the visual system. The panelists shared research experience addressing key topics as detailed below with an emphasis on the scientific impact and provided insights on what could be applied to the visual system and how it could inform the gaps, needs, and opportunities for eye health and disease.

Key Topics

Understanding EV Biology and the Potential for EVs as Biomarkers

The current understanding of the basic EV biology was first discussed, covering: 1) the role of retinal pigment epithelium (RPE) EVs in retinal health and disease, 2) mechanisms regulating RNA cargo sorting in EV biogenesis, and 3) challenges and opportunities for studying EV signaling in vivo. Several panelists then presented work showcasing how EV assays could be applied to various sample types and demonstrating promise for EVs as disease biomarkers. Topics included: 1) characterization of EVs for biomarker discovery for cardiovascular disease, 2) tear exosomes as potential biomarkers for ocular and systemic diseases, and 3) EV-based assays for the diagnosis of disease in clinical practice.

Implications for EVs in Regenerative Medicine and Beyond

Panelists shared promising research results on 1) neuroprotective and pro-regenerative potential of EVs in models of glaucoma and traumatic optic neuropathy, 2) strategies of EV-based therapy for retinal diseases, 3) clinical studies of mesenchymal stem cell (MSC) and MSC-EVs for promoting corneal repair, 4) recovery of motor function by MSC-EVs after cortical injury, 5) therapeutic effects (anti-inflammatory, neuroprotective, cognition-enhancing) of stem cell derived EVs for neurodegenerative disease, 6) therapeutic potential (anti-coagulant, anti-inflammatory, pro-fibrinolytic) of vascular endothelial cell EVs in animal models of stroke and neurovascular diseases, and 7) EV-based immunotherapy for uveitis, Graft-Versus-Host Disease (GVHD), and periodontitis.

Novel Technologies and Considerations for the Rigorous Study of EVs

Panelists emphasized the importance of rigor and standardization in the EV field and introduced tools, techniques, and approaches for the study of EVs. Topics presented included: 1) EV isolation and characterization in glaucoma, 2) a new HiMEX (high-throughput integrated magneto-electrochemical extracellular vesicle) assay for high-throughput characterization of EVs, 3) digital Flow Cytometry (dFC) for single EV and particle analyses 4) non-invasive imaging for in vivo tracking of EVs, and 5) engineering functional EVs for regenerative medicine.

Gaps, needs, and opportunities for future EV research in the visual system

Panelists identified and discussed the following gaps, needs and opportunities on various aspects for future EV research in the visual system.

  1. Fundamental understanding of EV biology: Understand the EV biogenesis and regulation of cargo content. Understand the biological relevance of the heterogeneity of EV subpopulations.
     
  2. Characterization of EVs for biomarker discovery: Define EV molecular signatures for cellular origins and disease stages and develop biomarkers for cell type specific EVs. Compare EVs from samples collected from ocular fluids and/or blood to determine their potential utility in diagnosing or predicting ocular diseases.
     
  3. EV functional studies and opportunities for developing therapeutics: Characterize mechanisms of action of EV therapy including cargoes and membrane components of EVs. Identify endogenous EVs and their cargoes generated in the eye in response to disease/injury that may foster and perpetuate pathology. Customize EVs for specific indications in the eye (anti-inflammatory, regenerative, pro-survival and/or active targeting). Understand the pharmacokinetics/pharmacodynamics, immune responses, and safety profiles of EV-based therapeutics in translation-enabling models.
     
  4. Technologies, approaches and resources for the study of EVs: Establish standardized techniques capable of working with low volume samples to distinguish the differences in EV abundance, composition, and cargo. Explore novel approaches to define EV signaling within an organ system and organism, including leveraging new animal models for in vivo EV studies and a multi-omics approach for understanding disease. Develop tailored techniques and approaches for EV isolation and purification, characterization of EVs and their contents, EV labelling and quantification, in vivo tracking of EVs, multi-cargo detection, controlled or prolonged EV release, maintenance of EV integrity, and large-scale EV production. Generate an EV atlas to indicate the composition of EV subpopulations in various types of biological fluids/samples.
     
  5. Rigor and standardization of EV studies: Establish the rigor and standardization of EV studies by addressing issues such as incomplete separation methods, nonspecific methods of isolation and purification, “EV” attributes from bovine serum and other media additives in in vitro studies, unreliable literature results. Recognize limitations of approaches available to rigorously investigate EVs in vivo. Conduct cross-species and cross-platform validation of EV studies.
     
  6. Diverse collaborations: Create opportunities for collaborations between multiple disciplines (e.g., bioengineers and biologists) and between different labs for learning EV research from the fields outside of the eye, while acknowledging uniqueness of the visual system.

In conclusion, promising data across disciplines was presented regarding the therapeutic potential of stem cell and other EVs, including anti-inflammatory, regenerative, neuroprotective, and pro-survival effects of EVs. However, several barriers hindering the progress of the field have been identified. Foremost among these is a limited understanding of EV cargoes and regulatory mechanisms. The unique attributes of ocular samples necessitate improved and standardized methodologies. Nevertheless, there remains a dearth of fundamental knowledge about EV biology, especially within the context of ocular research. This gap presents an opportunity for fundamental biological studies that can drive translation and advance clinical applications centered around EVs. In parallel, the integration of novel technologies and resources developed in the broader EV field holds promise for application in ocular research which may catalyze innovations to address the distinctive fluids or cell types in the visual system. Thus, interdisciplinary collaborations create opportunities to propel the field forward, fostering synergy among diverse expertise and perspectives.

Publication Plans

Drs. Sun Young Lee and Mikael Klingeborn will be developing a workshop report for publication in a peer-reviewed journal. All workshop panelists will contribute to the report, covering the meeting’s presentations and discussions in more detail, while also outlining and prioritizing the gaps, needs, and research opportunities that were identified at the workshop.                                               


Agenda

Welcome by NEI Director

9:00 - 9:10 a.m.

  • Michael F. Chiang, MD, Director, National Eye Institute

Organizer’s Introductions

9:10-9:15

  • Ashley Fortress, PhD, Scientific Review Officer, National Eye Institute
  • Hongman Song, MD, PhD, Program Director for Glaucoma, National Eye Institute

Welcome and Logistics by Workshop Co‐Chairs

9:15 - 9:30 a.m.

  • Overview of Extracellular Vesicle (EV) Biology
    Alissa Weaver, MD, PhD, Vanderbilt University
  • Setting the Stage for EVs in the Eye
    Ali Djalilian, MD, University of Illinois Chicago

Session 1: EVs: From Biology to Biomarkers

9:30 - 11:00 a.m.
Moderators:

  • Sarah Hamm-Alvarez, PhD, University of Southern California
  • Saumya Das, MD, PhD, Harvard Medical School

Discussant:

  • Mikael Klingeborn, PhD, McLaughlin Research Institute

Speaker Presentations

  • EVs in Retinal Health and Disease
    Mikael Klingeborn, PhD, McLaughlin Research Institute
     
  • Exosome biogenesis and regulation of cargo content
    Alissa Weaver, MD, PhD, Vanderbilt University
     
  • EV-mediated Signaling in vivo: Challenges and Opportunities
    Christie Fowler, PhD, University of California Irvine
     
  • Characterizing EVs for Biomarker Discovery: Lessons from Cardiovascular Diseases
    Saumya Das, MD, PhD, Harvard Medical School
     
  • Tear Exosomes
    Sarah Hamm-Alvarez, PhD, University of Southern California
     
  • EV-based Assays for the Diagnosis of Disease – Translating Lab Findings into Clinical Practice
    Johan Skog, PhD, Founder, Exosome Diagnostics

30-minute Discussion

Session 2: EVs for Regenerative Medicine

11:10 am – 12:58 pm
Moderators

  • Ben Mead, PhD, Cardiff University
  • Tara Moore, PhD, Boston University

Discussant:

  • Sun Young Lee, MD, PhD, University of Southern California

Speaker Presentations

  • Extracellular Vesicles – A Novel Treatment Strategy for Glaucoma and Traumatic Optic Neuropathies
    Ben Mead, PhD, Cardiff University
     
  • Therapeutic Opportunities of EVs for Retinal Diseases
    Sun Young Lee, MD, PhD, University of Southern California
     
  • MSC and MSC-EV Therapies for Ocular Surface Injury
    Ali Djalilian, MD, University of Illinois Chicago
     
  • Extracellular Vesicles as a Restorative Treatment in a Monkey Model of Cortical Injury
    Tara Moore, PhD, Boston University
     
  • Intranasal Delivery of Stem-Cell Derived EVs for Neurodegenerative Disease
    Ashok Shetty, PhD, Texas A&M University
     
  • sEV Therapeutics for Stroke and Neurovascular Diseases
    Michael Chopp, PhD, Henry Ford Health
     
  • Exosomes: Emerging Immunotherapy for Uveitis and Graft-Versus-Host Disease (GVHD)
    Charles Egwuagu, PhD, MPH, National Eye Institute
     
  • Extracellular Vesicles: Lessons Learned for Immune Therapy
    Christopher Cutler, DDS, PhD, Augusta University

30-minute Discussion

Session 3: Tools and Approaches for the Rigorous Study of EVs

1:23 pm – 2:55 pm
Moderators

  • Sriram Ravindran, PhD University of Illinois Chicago
  • Ken Witwer, PhD, Johns Hopkins University

Discussants:

  • Mikael Klingeborn, PhD, McLaughlin Research Institute
  • Sun Young Lee, MD, PhD, University of Southern California

Speaker Presentations

  • Rigor and Standardization of EV Studies
    Ken Witwer, PhD, Johns Hopkins University
     
  • EV Isolation and Characterization in Glaucoma
    Yutao Liu, PhD , Augusta University
     
  • New Assay Systems for High-Throughput Characterization of EVs
    Hakho Lee, PhD, Harvard Medical School
     
  • Digital Flow Cytometry (dFC)
    Daniel Chiu, PhD, University of Washington
     
  • Non-invasive Imaging of EVs: Quo vaditis in vivo?
    Jeff Bulte, PhD, Johns Hopkins University
     
  • Functionally Engineered EVs (FEEs) for Regenerative Medicine and Eye Diseases
    Sriram Ravindran, PhD, University of Illinois Chicago

30-minute Discussion

Roundtable Discussion on Gaps, Needs, and Opportunities

3:05 pm – 3:45 p.m.

Moderators (Co-Chairs):

  • Alissa Weaver, MD, PhD, Vanderbilt University
  • Ali Djalilian, MD, University of Illinois Chicago

Summary by the Co-Chairs: Key Takeaways

3:45 pm – 3:55 p.m.      

  • Alissa Weaver, MD, PhD, Vanderbilt University
  • Ali Djalilian, MD, University of Illinois Chicago

Closing Remarks

3:55 pm – 4:00 p.m.

  • Kathleen Anderson, PhD
    Director, Division of Extramural Activities
    Acting Director, Division of Extramural Science Programs
    National Eye Institute

Workshop Participants

Jeff Bulte, PhD
Professor
Department of Radiology
Johns Hopkins University
jwmbulte@mri.jhu.edu

Daniel Chiu, PhD
Professor
Department of Bioengineering
University of Washington
chiu@uw.edu

Michael Chopp, PhD, FAFA, FESO
Vice Chairman,
Department of Neurology
Zolton J. Kovacs Chair in Neuroscience Research
Distinguished Professor, Physics,
Oakland University
Professor, Physiology,
Michigan State University
Henry Ford Health
mchopp1@hfhs.org

Christopher Cutler, DDS, PhD
Professor and Chair,
Department of Periodontics
Augusta University-Dental College of Georgia
CHCUTLER@Augusta.edu

Saumya Das, MD, PhD
Associate Professor
Department of Medicine
Harvard Medical School
SDAS@mgh.harvard.edu

Ali Djalilian, MD
Professor
Department of Ophthalmology
University of Illinois Chicago
adjalili@uic.edu

Charles Egwuagu, PhD, MPH
Chief, Molecular Immunology Section
National Eye Institute
National Institutes of Health
egwuaguc@nei.nih.gov

Christie Fowler, PhD
Professor
Department of Neurobiology and Behavior
University of California, Irvine
cdfowler@uci.edu

Sarah Hamm-Alvarez, PhD
Professor
Department of Ophthalmology

Department of Pharmaceutical Sciences
University of Southern California
sarah.hamm-alvarez@med.usc.edu

Mikael Klingeborn, PhD
Assistant Professor
McLaughlin Research Institute
mikael@mclaughlinresearch.org

Hakho Lee, PhD
Associate Professor
Department of Radiology
Massachusetts General Hospital
Harvard Medical School
HLEE@mgh.Harvard.edu

Sun Young Lee, MD, PhD
Assistant Professor
Department of Ophthalmology
Keck School of Medicine
University of Southern California
SunYoung.Lee@med.usc.edu

Yutao Liu, PhD
Professor
Department of Cellular Biology and Anatomy
Medical College of Georgia
Augusta University
YUTLIU@Augusta.edu

Ben Mead, PhD
Associate Professor
School of Optometry and Vision Sciences
Cardiff University, UK
MeadB@cardiff.ac.uk

Tara Moore, PhD
Associate Professor
Department of Anatomy and Neurobiology
Center for Systems Neuroscience
Boston University Chobanian & Avedisian School of Medicine
tlmoore@bu.edu

Sriram Ravindran, PhD
Associate Professor
Department of Oral Biology
University of Illinois Chicago
Sravin1@uic.edu

Ashok K. Shetty, PhD
Professor
Department of Cell Biology and Genetics
Associate Director
Institute for Regenerative Medicine
Texas A&M University
Ash.Shetty@tamu.edu

Johan Skog, PhD
Chief Scientific Officer
Vice President
Exosome Diagnostics, a Bio-techne brand
Johan.Skog@bio-techne.com

Alissa M. Weaver, MD, PhD
Professor
Department of Cell and Developmental Biology
Department of Pathology, Microbiology, and Immunology
Director, Center for Extracellular Vesicle Research
Vanderbilt University School of Medicine
alissa.weaver@vanderbilt.edu

Ken Witwer, PhD
Associate Professor

Department of Molecular and Comparative Pathobiology
Johns Hopkins University
kwitwer1@jhmi.edu

NEI Staff

Michael F. Chiang, MD
Director
National Eye Institute
National Institutes of Health
michael.chiang@nih.gov

Santa J. Tumminia, PhD
Deputy Director
National Eye Institute

National Institutes of Health
tumminias@nei.nih.gov

Kathleen Anderson, PhD
Director
Division of Extramural Activities
Acting Director
Division of Extramural Science Programs
National Eye Institute
National Institutes of Health
KAnders1@mail.nih.gov

Sangeeta Bhargava, PhD
Acting Deputy Director
Division of Extramural Science Programs
Program Director
Collaborative Clinical Research Program
National Eye Institute
National Institutes of Health
bhargavas@nei.nih.gov

Shefa Gordon, PhD
Associate Director for Science Policy and Legislation
Office of Program Planning and Analysis
National Eye Institute
National Institutes of Health
shefa@nei.nih.gov

Alicia Kerr, PhD
Health Science Specialist
Office of Program Planning and Analysis
National Eye Institute
National Institutes of Health
alicia.kerr@nih.gov

NEI Intramural Investigators

Patricia Becerra, PhD
Senior Investigator
Protein Structure and Function
Intramural Research Program
National Eye Institute
National Institutes of Health
becerras@nei.nih.gov

Kapil Bharti, PhD
Scientific Director
National Eye Institute
Intramural Research Program
Senior Investigator
Ocular and Stem Cell Translational Research Section
National Eye Institute
National Institutes of Health
Kapil.bharti@nih.gov

Stanislav Tomarev, PhD
Senior Investigator
Retinal Ganglion Cell Biology
Intramural Research Program
National Eye Institute
National Institutes of Health
TomarevS@nei.nih.gov

NEI Extracellular Vesicle Working Group Members

Ashley Fortress, PhD (Co-Lead)
Scientific Review Officer
Division of Extramural Activities
National Eye Institute
National Institutes of Health
ashley.fortress@nih.gov

Hongman Song, MD, PhD (Co-Lead)
Program Director
Glaucoma
Division of Extramural Science Programs
National Eye Institute
National Institutes of Health
Hongman.Song@nih.gov

Houmam Araj, PhD
Program Director
Ocular Pain
Lens and Cataract
Oculomotor Systems
Strabismus and Amblyopia
Conference Grants
Division of Extramural Science Programs
National Eye Institute
National Institutes of Health
arajh@nei.nih.gov

James Gao, PhD
Program Director
Informatics and Data Science
Division of Extramural Science Programs
National Eye Institute
National Institutes of Health
James.Gao@nih.gov

Nataliya Gordiyenko, PhD
Program Director
Angiogenesis and Immunology
Division of Extramural Science Programs
National Eye Institute
National Institutes of Health
gordiyenkon@nei.nih.gov

Tony D. Gover, PhD
Program Director
Corneal Injury and Repair
Glaucoma and Optic Neuropathy Bioengineering and Technology
Small Business, Anterior Segment
Division of Extramural Science Programs
National Eye Institute
National Institutes of Health
tony.gover@nih.gov

Tom Greenwell, PhD
Program Director
Development and Regeneration
Division of Extramural Science Programs
National Eye Institute
National Institutes of Health
greenwellt@nei.nih.gov

Paek Lee, PhD
Program Director
Retina Bioengineering and Technology
Small Business SBIR/STTR
Division of Extramural Science Programs
National Eye Institute
National Institutes of Health
paek.lee@nih.gov

George A. McKie, DVM, PhD
Program Director
Structure, Function, and Diseases of the Cornea
Division of Extramural Science Programs
National Eye Institute
National Institutes of Health
mckiegeo@nei.nih.gov

Lisa A. Neuhold, PhD
Program Director
Retina, Photoreceptor/RPE Biology
Cell and Molecular Technologies
Division of Extramural Science Programs
National Eye Institute
National Institutes of Health
lneuhold@nei.nih.gov

Jennifer Schiltz, PhD
Scientific Review Officer
Division of Extramural Activities
National Eye Institute
National Institutes of Health
jennifer.schiltz@nih.gov

Grace Shen, PhD
Program Director
Retinal Diseases
Division of Extramural Science Programs National Eye Institute
National Institutes of Health
sheng@mail.nih.gov

Charles B. Wright, PhD
Program Director
Retinal Circuitry
Division of Extramural Science Programs
National Eye Institute
National Institutes of Health
charles.wright@nih.gov

Last updated: December 21, 2023