Periodic Reporting for period 2 - EXO-EYE (Stem Cell-Derived Exosomes for the treatment of Traumatic and Degenerative Eye Disease)
Okres sprawozdawczy: 2019-06-01 do 2020-05-31
Retinal ganglion cells (RGC) are the sole projection neuron of the retina and their axons make up the optic nerve. Damage to the optic nerve can come in many forms including neurodegenerative diseases like glaucoma, (affecting >7M EU citizens and increasing each year), as well as traumatic neuropathy. Traumatic injury affecting the optic nerve similarly leads to the death of RGC and blindness with an estimated 1-5% of head injuries resulting in optic nerve damage. The functional deficit and lack of recovery is because RGC and their axons are part of the central nervous system (CNS) and thus suffer from two unfortunate characteristics, they are irreplaceable, and their axons are incapable of regeneration.
Current research and ongoing clinical trials suggests mesenchymal stem cells (MSC), such as those isolated from bone marrow, are an effective treatment for injured RGC, despite the mechanisms being poorly understood. MSC are a self-replicating multipotent stromal cell isolated from mesenchymal tissues. I and others have demonstrated the therapeutic efficacy of MSC in models of traumatic neuropathy and glaucoma, in vitro and in vivo. In retinal cultures, MSC proved neuroprotective and neuritogenic for injured RGC. After ONC, MSC transplanted into the vitreous were able to promote significant neuroprotection of RGC and regeneration of their axons. In animal models of glaucoma, MSC promote the survival of RGC and their axons and preserve their function.
Although the efficacy is well established, the mechanisms by which MSCs protect RGC and promote regeneration of their axons is poorly understood, although strongly believed to be paracrine-mediated (through the secretion of factors). Mounting evidence exists for the potential of MSC to benefit nearby injured tissues through the secretion of exosomes. Exosomes are endocytic-derived structures composed of proteins, lipids and mRNA surrounded by a phospholipid bi-layer that are secreted into the extracellular space. Exosomes contain (along with proteins) mRNA and miRNA, which are both functional and, when delivered to another cell via fusion with the cell membrane, lead to the translation of new proteins or miRNA-mediated knockdown of genes. Various studies have shown that the protective effect MSC provide is indeed exosome-mediated. Currently MSC exosomes remain untested in the eye and exosomes in general have not been tested as a treatment for injured RGC. Equally their mechanism of action as a whole is poorly understood with very few miRNA candidates identified as the active components.
The global aim of this project is to utilize exosomes from various sources and under different isolation techniques to promote survival of RGC and regeneration of their axons.
Current research and ongoing clinical trials suggests mesenchymal stem cells (MSC), such as those isolated from bone marrow, are an effective treatment for injured RGC, despite the mechanisms being poorly understood. MSC are a self-replicating multipotent stromal cell isolated from mesenchymal tissues. I and others have demonstrated the therapeutic efficacy of MSC in models of traumatic neuropathy and glaucoma, in vitro and in vivo. In retinal cultures, MSC proved neuroprotective and neuritogenic for injured RGC. After ONC, MSC transplanted into the vitreous were able to promote significant neuroprotection of RGC and regeneration of their axons. In animal models of glaucoma, MSC promote the survival of RGC and their axons and preserve their function.
Although the efficacy is well established, the mechanisms by which MSCs protect RGC and promote regeneration of their axons is poorly understood, although strongly believed to be paracrine-mediated (through the secretion of factors). Mounting evidence exists for the potential of MSC to benefit nearby injured tissues through the secretion of exosomes. Exosomes are endocytic-derived structures composed of proteins, lipids and mRNA surrounded by a phospholipid bi-layer that are secreted into the extracellular space. Exosomes contain (along with proteins) mRNA and miRNA, which are both functional and, when delivered to another cell via fusion with the cell membrane, lead to the translation of new proteins or miRNA-mediated knockdown of genes. Various studies have shown that the protective effect MSC provide is indeed exosome-mediated. Currently MSC exosomes remain untested in the eye and exosomes in general have not been tested as a treatment for injured RGC. Equally their mechanism of action as a whole is poorly understood with very few miRNA candidates identified as the active components.
The global aim of this project is to utilize exosomes from various sources and under different isolation techniques to promote survival of RGC and regeneration of their axons.
The project is broken down into 4 Research Objectives.
RO1: The use of exosomes to treat RGC is currently untested. The in vitro model of RGC death and regenerative failure will be used to determine the therapeutic potential of BMSC-derived exosomes, optimal dosing and fate. We hypothesise that exosomes will promote significant neuroprotection/neurite regeneration of injured RGC.
- I have demonstrated great success using rat retinal culture systems to show MSC exosomes are significantly efficacious at promoting neuroprotection and neuritogenesis. (Data published). I have also collaborated with John Hopkins University and utilized human induced-pluripotent stem cell-derived RGC as a second culture system with great success. (Data Ready To Be Published)
RO2: No studies exist on the delivery of MSC exosomes into the eye. We aim to deliver an optimal dose of MSC exosomes into the eyes of animal models of traumatic and degenerative eye disease and assess their therapeutic efficacy. We hypothesise that MSC exosomes will protect RGC form death in a rodent model of glaucoma and traumatic optic neuropathy and promote regeneration in the latter.
- We have demonstrated that MSC exosomes elicit a strong neuroprotective effect on RGC after optic neuropathy and glaucoma models. (Data Published).
RO3: Characterization of the mRNA and miRNA cargo of MSC exosomes has not been performed. We aim to use next generation sequencing to identify and quantify candidate RNA sequences and cross-examine with known neuroprotective/axogenic targets as well as identify novel targets. We will also perform flow cytometry and correlate this data with their internal RNA cargo. We hypothesise that different exosome subtypes exists, identified by their epitopes and containing unique miRNA cargo.
- We have sequenced the miRNA profile of exosomes and demonstrated many candidate miRNA molecules that may explain the observed therapeutic effect. We are also sequencing RGC before and after injury/treatment to better understand these proposed mechanisms. (Data Collection Still Ongoing).
RO4: MSC from different sources are known to possess differing secretory potentials. We aim to test exosomes from different MSC including dental pulp- and adipose-derived MSC. We hypothesise that exosomes from DPSC will be more therapeutically efficacious.
- To be completed.
RO1: The use of exosomes to treat RGC is currently untested. The in vitro model of RGC death and regenerative failure will be used to determine the therapeutic potential of BMSC-derived exosomes, optimal dosing and fate. We hypothesise that exosomes will promote significant neuroprotection/neurite regeneration of injured RGC.
- I have demonstrated great success using rat retinal culture systems to show MSC exosomes are significantly efficacious at promoting neuroprotection and neuritogenesis. (Data published). I have also collaborated with John Hopkins University and utilized human induced-pluripotent stem cell-derived RGC as a second culture system with great success. (Data Ready To Be Published)
RO2: No studies exist on the delivery of MSC exosomes into the eye. We aim to deliver an optimal dose of MSC exosomes into the eyes of animal models of traumatic and degenerative eye disease and assess their therapeutic efficacy. We hypothesise that MSC exosomes will protect RGC form death in a rodent model of glaucoma and traumatic optic neuropathy and promote regeneration in the latter.
- We have demonstrated that MSC exosomes elicit a strong neuroprotective effect on RGC after optic neuropathy and glaucoma models. (Data Published).
RO3: Characterization of the mRNA and miRNA cargo of MSC exosomes has not been performed. We aim to use next generation sequencing to identify and quantify candidate RNA sequences and cross-examine with known neuroprotective/axogenic targets as well as identify novel targets. We will also perform flow cytometry and correlate this data with their internal RNA cargo. We hypothesise that different exosome subtypes exists, identified by their epitopes and containing unique miRNA cargo.
- We have sequenced the miRNA profile of exosomes and demonstrated many candidate miRNA molecules that may explain the observed therapeutic effect. We are also sequencing RGC before and after injury/treatment to better understand these proposed mechanisms. (Data Collection Still Ongoing).
RO4: MSC from different sources are known to possess differing secretory potentials. We aim to test exosomes from different MSC including dental pulp- and adipose-derived MSC. We hypothesise that exosomes from DPSC will be more therapeutically efficacious.
- To be completed.
The expected project outcomes and impacts for the final year are two-fold.
Firstly, following the completion and analysis of the remaining sequencing data, a clear mechanism of action for how exosomes elicit their neuroprotective effects will be revealed. This will build upon the consensus that exosomes have a strong potential at being the next breakthrough therapy for a variety of conditions, including neurodegeneration/neurotrauma. Knowing the exact mechanism of action will also allow the therapy to be improved significantly through purifying select subtypes or utilizing not the exosomes but the specific candidate miRNA of interest.
Secondly, it is also expected that comparisons between exosomes will reveal crucial information on how the therapy can be improved further through careful selection of the exosome source. Knowing the mechanism of action will also allow stronger comparisons. For example, knowing a particular miRNA is heavily involved in the therapeutic effect, exosomes from different sources can be screened to determine which has the highest abundance of said candidate exosome.
These results are expected to reveal the next phase of therapies beyond stem cell therapy. The utilization of exosomes offers a much safer and efficacious alternative to stem cell therapy. Researchers working on exosomes, which are still poorly understood, will also gain much from the data revealed during the final phase of this project. That data will directly inform their research and may push others to work in new and interesting directions in the hopes of exploiting exosomes as a therapy for a variety of conditions.
Firstly, following the completion and analysis of the remaining sequencing data, a clear mechanism of action for how exosomes elicit their neuroprotective effects will be revealed. This will build upon the consensus that exosomes have a strong potential at being the next breakthrough therapy for a variety of conditions, including neurodegeneration/neurotrauma. Knowing the exact mechanism of action will also allow the therapy to be improved significantly through purifying select subtypes or utilizing not the exosomes but the specific candidate miRNA of interest.
Secondly, it is also expected that comparisons between exosomes will reveal crucial information on how the therapy can be improved further through careful selection of the exosome source. Knowing the mechanism of action will also allow stronger comparisons. For example, knowing a particular miRNA is heavily involved in the therapeutic effect, exosomes from different sources can be screened to determine which has the highest abundance of said candidate exosome.
These results are expected to reveal the next phase of therapies beyond stem cell therapy. The utilization of exosomes offers a much safer and efficacious alternative to stem cell therapy. Researchers working on exosomes, which are still poorly understood, will also gain much from the data revealed during the final phase of this project. That data will directly inform their research and may push others to work in new and interesting directions in the hopes of exploiting exosomes as a therapy for a variety of conditions.