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Exosomes for Medicine

Babak Nami www.exosometherapy.org

What are exosomes?


Exosomes are small (30100 nm in diameter) virus-like, lipid-bilayer membrane vesicles of that are released from many types of cells into the extracellular environment on fusion of multivesicular bodies (MVB) with the plasma membrane.
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Structure of exosomes
Diameter of around 30 100 or 1000 nm. lipid-bilayer membrane which it inherits from the cell membrane. Contain protein, DNA, mRNA and miRNA that differ in its composition depending on the cell of origin. Can be contain many similarities in the protein composition, for example protein Hsc70 have been recognized in most exosomes. Density between 1.13 1.19 g/ml.
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Composition of exosomes
Varies depending on the cell type of origin. Contain a number of common protein components: adhesion molecules: intercellular adhesion molecule1, CD146, CD9, milk-fat-globule EGF-factor VIII (MFG-E8), CD18, CD11a, CD11b, CD11c, CD63, CD81, CD82, CD166, MHCl, MHCll and LFA- 3/CD58. heat- shock proteins: Hsp70 and Hsp90. proteins involved in apoptosis: thioredoxin peroxidase II, Alix, 14-3-3 and galectin 3. metabolic enzymes: peroxidases, pyruvate and lipid kinases and eno-lase-1.
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Exosomes biomaterial contents

Prof. Richard Simpson and Dr. Suresh Mathivanan from La Trobe University launched ExoCarta database serves exosomes biomaterial composions details in as free for public: www.exocarta.org

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Biogenesis of exosomes

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Functions of exosomes
Cell cell communication. Major function is exchange of materials between cells. Minor function will depend on the cell type from which they were derived and the composition of exosomes in terms of lipids, carbohydrates, proteins and nucleic acids.

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Functions of exosomes
Secretion of proteins: Tumor-derived exosomes contain cancer cells receptors. p53-regulater proteins. Tumor necrosis factor (TNF) receptor 1. Endothelial cell caspase-3 activation and apoptosis of vascular cells.

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Functions of exosomes
Antigen presentation:
Antigen cross-presentation. Transfer antigens from tumor cells to DCs. Tumor antigens to sensitized T cells and can promote tumor rejection in vivo (tumor immunity). Exosomes isolated from immature DCs treated with cytokines, such as IL-4 and IL-10, when injected into mice reduced the severity of established collagen-induced arthritis. Peche et al. showed exosomes involved in DCs-based immune response and rejection during bone marrow transplantation. But created an CD4+ T cell-based immunotolerance.
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Functions of exosomes

Shuttle for RNA: Valadi et al. showed that exosomes are enriched in messenger RNA and micro RNA. They proved for the first time that mast cell exosomes contain RNA. The exosomal RNA differs from the donor cell RNA. Exosomes contain very little or no ribosomal RNA but a substantial amount of small RNA. Ekstrm et al. propose that this RNA be called exosomal shuttle RNA (esRNA).

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Functions of exosomes

Shuttle for infectious agents: Viruses released have markers commonly found on exosomes. HIV utilizes MVBs as the major site for accumulation in human macrophages. Do viruses utilize exosomes to spread own genome to other cells? Does HIV goes into exosomes in order to be hide for immune system?
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Therapeutic potentials of exosomes


Diagnostic biomarkers Vaccine Cancer immunotherapy Tumour regression Cell and tissue engineering Gene therapy

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Diagnostic biomarker
Many type cells secrete exosomes in common body fluids include plasma, urine, lymph, cerebrospinal fluid, semen, saliva, sweat, tears, mucus, amniotic fluid, synovial fluid, breast milk, vitreous humour and aqueous humour. Exosomal cell/tissue-specific protein and RNA contents has potential diagnostic and prognostic values in various types of physiological conditions, cancer, tissue failure, infections and other physicochemical confusions.

Diagnostic biomarker 64 papers and a total of 2400 different proteins. Plasma-derived exosomes: Depended to cells that secreted. Infections: HIV, bacterial antigens Tissue failure Inflammatory mediators
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Diagnostic biomarker
Urine-derived exosomes: In 2004 exosomes isolated from fresh human urine by ultracentrifugation [Pisitkun et al., 2004]. Urinary exosomal Fetuin-A and Na+/H+ exchanger isoform 3 (NHE3) are increased in patients with acute kidney injury (AKI) [du Cheyron
et al., 2005].

Welton et al. published a report on the profiling of exosomes from a bladder cancer cell line
[Welton et al., 2010].
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Exosomal Vaccine

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Exosomal Vaccine
Current cancer vaccines: Cervarix is designed to prevent infection from human papillomavirus (HPV), types 16 and 18, that cause about 70% of cervical cancer cases and are responsible for most HPV-induced anal, vulvar, vaginal, and penile cancer cases. As of September 2009, Cervarix was shown to be effective 7 years after vaccination. FDA recommends vaccination before adolescence and potential sexual activity.
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Exosomal Vaccine
Gardasil is a vaccine approved June 8, 2006 by FDA for use in the prevention of certain types of human papillomavirus (HPV), specifically HPV types 6, 11, 16 and 18.

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On April 2010, Dendreon Corporation manufactured a new cancer vaccine for prostate cancer Provenge (sipuleucel-T). It was approved by FDA on April 29, 2010 to treat asymptomatic or minimally symptomatic metastatic prostate cancer. It costs $93,000 for a course of treatment. In metastatic prostate cancer, it has extended survival by about four months. It utilizes antigen-presenting cell technology involving dendritic cells in cancer immunotherapy.

Exosomal Vaccine

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Exosomal Vaccine
Esosome as cancer vaccine: Exosomes carry immunorelevant structures which play important roles in immune response, such as HSPs, MHC class I molecules, tetraspanins, and cytoskeletal proteins. Tumour-derived exosome contain cancer cells antigens and miRNA. immunomodulation, transferring tumor antigens to APCs, and triggering CTL responses. It have been used in phase I clinical trials [Andre et al,. 2004].

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Cancer immunotherapy
In 1996, seminal work by Raposo et al. and soon after by Zitvogel et al. demonstrated that exosomes isolated from antigen-presenting cells (APCs) can act essentially as miniature antigenpresenting cell surrogates, capable of activating T cells in vitro and importantly, also in vivo [Zitvogel
et al., 2000].

Maturing DCs in eliciting cytotoxic T-lymphocyte (CTL) responses associated with objective antitumor effects in patients [Zitvogel et al., 2000].
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Cancer immunotherapy
DC-derived exosome-based immunotherapy:
DC-derived exosomes analyzed systematic proteomic: contain anti-tomour molecules. Tumor-specific cytotoxic T lymphocytes were found in the spleen of exosome-treated nude mice, and the anti tumor effect of exosomes was sensitive to in vivo depletion of CD8+ T cells [Amigoren et al., 2000]. in implementation in larger populations in dening quality control parameters in storage over a long period.
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Cancer immunotherapy
Exosomes activate T cells: APC-derived exosomes can lead to CD4+ and CD8+ T-cell activation. Major histocompatibility complexs (MHCs) are main mediators in T-cell activation. In vivo, DC-exosomes may be further potentiated by also adding exogenous adjuvants [Chaput et al., 2004].

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Cancer immunotherapy
Tumour-derived exosomes: tumor-derived exosomes contain tumor-specific antigens expressed in the parental tumor cells. Enrichment of tumor antigens such as melan-A [Andre et al., 2002], Silv [Wolfers et al., 2001], carcinoembryonic antigen (CEA) [Clayton er al., 2007] and mesothelin [Dai et al., 2005] is observed in tumor- derived exosomes. tumour antigens to the immune system, assisting the immune response, majorly through the activities of DCs and T-cells. Immunosuppressive effects of tumor-derived exosomes have been identified. Directly suppress the activity of effector T cells.
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Tumour-derived exosomes:

Cancer immunotherapy

Exosomal FasL and TRAIL, both of which can trigger the apoptotic death of activated T cells. Tumor-derived exosomes have also been implicated in facilitating tumor invasion and metastasis. Khan et al. demonstrated Survivin is released from cancer cells via exosomes [Khan et al., 2011]. Contribute to the establishment of a pre-metastatic niche, generating a suitable microenvironment in distant metastatic sites [Peinado et al., 2011]. latest studies demonstrate that tumour-derived exosomes impair lymphocyte responses to interleukin-2 [Clayton et al., 2011]. As well as the exosomes express TGF-beta-1 that these factors are strongly implicated in immune evasion mechanisms and may be responsible for the antiproliferative (oranticytotoxic) effects [Clayton et al.,
2011].

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Cell and tissue engineering


Stem cell exosomes: Ratajczak and coworkers [Ratajczak et al., 2006] demonstrated that MVs derived from embryonic stem cells support self-renewal and expansion of the stem cells. Deregibus et al. demonstrated that MVs derived from endothelial progenitor cells may activate an angiogenic program in mature quiescent endothelial cells [Deregibus
et al,. 2007].

mRNA shuttled by exosomes derived from mesenchymal stem cells may induce repair of acute kidney injury [Bruno et al,. 2009].
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Cell and tissue engineering


Lai et al. suggest Mesenchymal stem cell exosomes are capable for treatment of cardiovascular disease [Lai et
al., 2011].

Tumour-derived exosomes: Cho et al. suggest xosomes from breast cancer cells induced adipose tissue-derived mesenchymal stem cells to myofibroblastic cells [Cho et al., 2011]. Van der Vos et al. demonstrate that brain tumour cells interfere in intercellular communication of nervous system by releasing exosomal miRNA and DNA [van der
Vos et al., 2011].
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Cell and tissue engineering


Do embryonic/fetal tissue- or fluid-derived exosomes in the organogenesis stages, carry some key factors in organogenesis? Can we use the exosomes derived from the fetal nervous system in order to neuroregeneration.

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Gene therapy

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Gene therapy

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Production of exosomes

Since exosomes are secreted to the extra-cellular space, they are usually produced from the supernatants of cell culture medium. Pelleting by centrifugation at around 100,000g for at least 1 h [Davis et al., 1986]. 1.13 g/ml (for B cell-derived exosomes) to 1.19 g/ml (for intestinal cell-derived exosomes). Under electron microscopy these vesicles have a characteristic cup-shaped morphology: a flattened sphere limited by a lipid bilayer, with a size range of about 30100 nm in diameter [Fevrier et al., 2005].
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Some molecules, such as MHC-I and MHC-II, are rich on the surfaces of these vesicles, as demonstrated by immunogold labeling, electron microscopy, Immunoblotting and mass spectrometry peptide mapping. Antibody-coated magnetic beads and a flow cytometry technique [Clayton et al., 2001] : 1. Magnetic beads are coated with monoclonal antibodies against molecules that are rich in exosomes. 2. The coated magnetic beads are incubated with culture supernatants that are isolated after a series of differential centrifugation, but without the ultracentrifugation step for exosome purification [Clayton et al., 2001].
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Production of exosomes