Periodic Reporting for period 2 - RenalToolBox (Developing novel tools and technologies to assess the safety and efficacy of cell-based regenerative medicine therapies, focusing on kidney disease)
Période du rapport: 2020-11-01 au 2023-04-30
Chronic diseases are the leading cause of mortality, with chronic kidney disease (CKD) affecting >10% of the population worldwide. There is a need for therapies that can impact the survival and quality of life of CKD patients.
Therapies based on mesenchymal stromal cells (MSCs) are showing promise in rodent models of kidney disease but before they can be used for CKD we need: (i) cost-effective and convenient methods for diagnosing CKD, (ii) reliable data on the safety and efficacy of MSC therapies and (iii) a better understanding of which MSCs have greatest therapeutic potential and how they might act to prevent kidney disease.
Our three research objectives and conclusions were:
1. Develop novel tools for diagnosing kidney disease and assessing renal function: a new device to measure kidney function quickly and easily was devised. This was based on the excretion/absorption of tracers given intravenously, with the measurements obtained via a device attached to the skin. We also identified biomarkers that can indicate the health/disease state of the kidney, to monitor the progression and regression of renal disease more accurately. Finally, a kidney-on-a-chip device for the testing of tracers and MSC efficacy was developed.
2. Develop and apply cutting-edge imaging technologies to evaluate the safety and efficacy of MSCs in a rodent model of kidney disease: to assess safety, we applied complementary imaging technologies and probes to monitor the biodistribution and fate of MSCs in animals and, at study end-points, in tissues. For efficacy, we applied novel imaging strategies and analysis tools to monitor renal function and structure longitudinally in rodents with an ischaemia reperfusion injury (IRI). Intact organs were analysed post-mortem with novel microscopy ‘clearing’ techniques to validate the in vivo biodistribution results, accurately quantify the extent of morphological damage and determine potential mechanisms by which MSCs act.
3. Establish the optimal source of MSCs, develop a matrix of potency assays and explore therapeutic mechanisms: we compared the properties and therapeutic potential of MSCs derived from different tissues (bone marrow, adipose and umbilical cord) to identify differences in their potential to treat kidney injury. We also looked at the properties of extracellular vesicles (EVs), a product of MSC culture, and their therapeutic effects.
Therapies based on mesenchymal stromal cells (MSCs) are showing promise in rodent models of kidney disease but before they can be used for CKD we need: (i) cost-effective and convenient methods for diagnosing CKD, (ii) reliable data on the safety and efficacy of MSC therapies and (iii) a better understanding of which MSCs have greatest therapeutic potential and how they might act to prevent kidney disease.
Our three research objectives and conclusions were:
1. Develop novel tools for diagnosing kidney disease and assessing renal function: a new device to measure kidney function quickly and easily was devised. This was based on the excretion/absorption of tracers given intravenously, with the measurements obtained via a device attached to the skin. We also identified biomarkers that can indicate the health/disease state of the kidney, to monitor the progression and regression of renal disease more accurately. Finally, a kidney-on-a-chip device for the testing of tracers and MSC efficacy was developed.
2. Develop and apply cutting-edge imaging technologies to evaluate the safety and efficacy of MSCs in a rodent model of kidney disease: to assess safety, we applied complementary imaging technologies and probes to monitor the biodistribution and fate of MSCs in animals and, at study end-points, in tissues. For efficacy, we applied novel imaging strategies and analysis tools to monitor renal function and structure longitudinally in rodents with an ischaemia reperfusion injury (IRI). Intact organs were analysed post-mortem with novel microscopy ‘clearing’ techniques to validate the in vivo biodistribution results, accurately quantify the extent of morphological damage and determine potential mechanisms by which MSCs act.
3. Establish the optimal source of MSCs, develop a matrix of potency assays and explore therapeutic mechanisms: we compared the properties and therapeutic potential of MSCs derived from different tissues (bone marrow, adipose and umbilical cord) to identify differences in their potential to treat kidney injury. We also looked at the properties of extracellular vesicles (EVs), a product of MSC culture, and their therapeutic effects.
Obj. 1: we designed and built a transcutaneous diagnostic device capable of measuring three tracers simultaneously. This device was successfully tested in phantoms and other surrogates. Final tests are being finalised to enable a patent application. We have also developed a range of dyes suitable for determining glomerular filtration and tubular excretion/re-absorption using this device and other tools such as multispectral optoacoustic tomography (MSOT). A biomarker panel, which can inform renal function, was established based on the measurement of 7 protein bound uremic toxins in tissues (i.e. renal tissue, blood, urine).
Obj. 2: we explored a range of imaging tools to determine the biodistribution and fate of MSC therapies and renal function/structure in the course of IRI. In animal models, genetic reporters in combination with BLI was the most appropriate modality to track biodistribution and fate of the MSCs. Probe-based imaging provides higher spatial resolution to determine intra-organ distribution of MSCs, which we have shown via MRI and MSOT. These modalities, as well as ultra-fast ultrasound, were also able to inform kidney function/structure. MSOT, with dyes produced in our network, provided a safe, fast, radiation-free method to measure single kidney GFR in mice.
At the doses used, the safety profile of the MSCs was good. MSCs tended to die shortly after administration and we believe that the risks they pose are minimal. Post-mortem assessment showed their accumulation in the pulmonary microvasculature immediately after administration, and death in subsequent days. We did not observe significant efficacy in our animal model of renal IRI with any type of MSC, apart from a reduced serum creatinine following administration of bone marrow-derived MSCs. Our results do not support the translation of any of the MSC sources to clinical trials for the treatment of renal IRI at the present time.
Obj. 3: we established assays to characterise MSCs at multiple levels from purity to efficacy. Head-to-head comparison of different MSCs and their bioproducts shows that different MSC types have individual properties, suggesting that each of them might have specific benefits depending on the therapeutic setting.
Our potency assays involving an in-vitro model of IRI suggest that MSC bioproducts such as conditioned medium (CM) suffice to induce a recovery of renal cells that is comparable to, and in some cases, better than reperfusion, depending on the specific assay. This provides a different avenue as to what product to use as a therapy. Our matrix of potency assays does not suggest a specific regenerative role for EVs, with CM sufficing to induce beneficial effects.
MSC pulmonary entrapment and the rapid death soon after administration suggests that in vivo, any therapeutic mechanisms are likely due to paracrine factors derived from the cells. Our in vitro assays suggested an immunomodulatory capacity of MSCs and at a systemic level, MSCs triggered an innate immune response by the host and we found that pro-inflammatory molecules were reduced in the serum of animals that received MSCs, supporting their immunomodulatory MoA.
Exploitation and dissemination is ongoing, with 1 patent application and 1 in preparation. Over 50 scientific publications are expected, of which 32 have already been published. Our data was presented in over 30 conferences and workshops.
Obj. 2: we explored a range of imaging tools to determine the biodistribution and fate of MSC therapies and renal function/structure in the course of IRI. In animal models, genetic reporters in combination with BLI was the most appropriate modality to track biodistribution and fate of the MSCs. Probe-based imaging provides higher spatial resolution to determine intra-organ distribution of MSCs, which we have shown via MRI and MSOT. These modalities, as well as ultra-fast ultrasound, were also able to inform kidney function/structure. MSOT, with dyes produced in our network, provided a safe, fast, radiation-free method to measure single kidney GFR in mice.
At the doses used, the safety profile of the MSCs was good. MSCs tended to die shortly after administration and we believe that the risks they pose are minimal. Post-mortem assessment showed their accumulation in the pulmonary microvasculature immediately after administration, and death in subsequent days. We did not observe significant efficacy in our animal model of renal IRI with any type of MSC, apart from a reduced serum creatinine following administration of bone marrow-derived MSCs. Our results do not support the translation of any of the MSC sources to clinical trials for the treatment of renal IRI at the present time.
Obj. 3: we established assays to characterise MSCs at multiple levels from purity to efficacy. Head-to-head comparison of different MSCs and their bioproducts shows that different MSC types have individual properties, suggesting that each of them might have specific benefits depending on the therapeutic setting.
Our potency assays involving an in-vitro model of IRI suggest that MSC bioproducts such as conditioned medium (CM) suffice to induce a recovery of renal cells that is comparable to, and in some cases, better than reperfusion, depending on the specific assay. This provides a different avenue as to what product to use as a therapy. Our matrix of potency assays does not suggest a specific regenerative role for EVs, with CM sufficing to induce beneficial effects.
MSC pulmonary entrapment and the rapid death soon after administration suggests that in vivo, any therapeutic mechanisms are likely due to paracrine factors derived from the cells. Our in vitro assays suggested an immunomodulatory capacity of MSCs and at a systemic level, MSCs triggered an innate immune response by the host and we found that pro-inflammatory molecules were reduced in the serum of animals that received MSCs, supporting their immunomodulatory MoA.
Exploitation and dissemination is ongoing, with 1 patent application and 1 in preparation. Over 50 scientific publications are expected, of which 32 have already been published. Our data was presented in over 30 conferences and workshops.
• A new renal diagnostic device and the dyes for its operation were developed and will lead to a new product and patent. A new panel of biomarkers, which is reliable as an indicator of kidney disease, will be published with potential to change how kidney disease is monitored in different settings.
• New protocols and dyes for imaging the (micro)vasculature the kidneys and other organs, via microscopy, were developed. A patent application has been filed to protect this invention.
• Data on the properties of MSC bioproducts, obtained from MSCs from different sources, is available to the community, informing on their potential and benefits for different therapeutic settings.
• We have shown that MSC administration is safe. Although our results do not support the translation of any of the MSC sources to clinical trials for IRI at present, our data and results are extremely valuable in informing the (lack of) potential of these cells as therapies for kidney disease in specific settings, giving hints how to fine-tune clinical applications.
• We produced and published protocols on methods to monitor MSC biodistribution as well as on their potential mechanisms of action, expanding the body of knowledge in these areas.
• Novel imaging analyses tools were developed, bringing innovations on the application of imaging devices, in particular the use of MSOT and UFUS, for monitoring renal structure and renal disease.
• Fifteen researchers have been trained with interdisciplinary research and technical skills, most of whom will or have already joined the EU’s workforce and apply their knowledge in different sectors.
• New protocols and dyes for imaging the (micro)vasculature the kidneys and other organs, via microscopy, were developed. A patent application has been filed to protect this invention.
• Data on the properties of MSC bioproducts, obtained from MSCs from different sources, is available to the community, informing on their potential and benefits for different therapeutic settings.
• We have shown that MSC administration is safe. Although our results do not support the translation of any of the MSC sources to clinical trials for IRI at present, our data and results are extremely valuable in informing the (lack of) potential of these cells as therapies for kidney disease in specific settings, giving hints how to fine-tune clinical applications.
• We produced and published protocols on methods to monitor MSC biodistribution as well as on their potential mechanisms of action, expanding the body of knowledge in these areas.
• Novel imaging analyses tools were developed, bringing innovations on the application of imaging devices, in particular the use of MSOT and UFUS, for monitoring renal structure and renal disease.
• Fifteen researchers have been trained with interdisciplinary research and technical skills, most of whom will or have already joined the EU’s workforce and apply their knowledge in different sectors.