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Developing novel tools and technologies to assess the safety and efficacy of cell-based regenerative medicine therapies, focusing on kidney disease

Periodic Reporting for period 1 - RenalToolBox (Developing novel tools and technologies to assess the safety and efficacy of cell-based regenerative medicine therapies, focusing on kidney disease)

Reporting period: 2018-11-01 to 2020-10-31

According to the WHO, chronic diseases are the leading cause of mortality worldwide. Chronic kidney disease (CKD) has a global prevalence of about 9.1% and in Europe, prevalence doubled in recent years. Advanced CKD requires renal replacement therapies (RRTs, dialysis or transplantation) and the number of patients needing RRTs is expected to reach 5.4 million by 2030. Existing RRTs have major drawbacks. Dialysis patients face a 5-year survival rate of only 35%, at annual costs >€40.000. Transplantation suffers from a lack of donors, with >50.000 patients on waiting lists in Europe, of which >6% are expected to die while waiting.

Advanced CKD usually develops following acute kidney injury and/or years of worsening CKD, presenting a window of opportunity to implement new therapies that delay or prevent progression to advanced stages. This has the potential to impact survival and quality of life of patients, and reduce the demand for RRTs. Therapies based on mesenchymal stromal cells (MSCs) are showing great promise in rodent models of kidney disease and have high potential for clinical translation. However, bottlenecks remain before their use for CKD. In particular, there is a need for (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 types of MSCs have greatest therapeutic potential and how they might act to prevent kidney disease.

RenalToolBox aims to train a new a cohort of scientists and equip them with the skills, tools and technologies to address these bottlenecks so that MSC therapies for CKD can be realised. Our three research objectives are:

1. Develop novel tools for diagnosing kidney disease and assessing renal function: a new device system to measure kidney function quickly and easily will be devised. This will be based on the excretion/absorption of tracers given intravenously, with the measurements obtained via a device attached to the skin. We also want to identify specific molecules (biomarkers) in urine that can indicate the health/disease state of the kidney, to monitor the progression and regression of renal disease more accurately. We will also develop a kidney-on-a-chip device for the testing of tracers and therapeutic potential of MSCs, allowing us to screen the most promising strategies.

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 will use complementary imaging technologies and probes to monitor the biodistribution and fate of MSCs in animals. Tissues will be subjected to a thorough safety evaluation at the study end-point. For efficacy, we will apply novel imaging strategies and analysis tools to monitor renal function and morphology longitudinally. Intact kidneys from sacrificed animals will be analysed by implementing novel ‘clearing’ techniques for making organs transparent to validate the in vivo biodistribution results and accurately quantify the extent of morphological damage and/or recovery following MSC therapies.

3. Establish the optimal source of MSCs, develop a matrix of potency assays and explore therapeutic mechanisms: we will compare the properties and therapeutic potential of MSCs derived from different tissues to identify which one has most potential to treat kidney injury. We will also look if extracellular vesicles (EVs), a product of MSC culture, has any therapeutic effect. To understand repair mechanisms, we will investigate secreted factors, miRNAs, immunomodulatory mechanisms and the role of mitochondrial transfer and correlate that with the imaging and functional data obtained in objective 2.
Objective 1: the design of a new transcutaneous device with the prospect of measuring not only glomerular filtration rate (current state-of-the-art) but also tubular function (secretion/re-absorption) is complete. A prototype has been built and is undergoing functional tests using phantoms and tests in rodents will start soon. Tracers for measuring tubular function have been designed and the chemical synthesis and characterisation is close to completion. The kidney-on-a-chip device has been built and shown to be functional and is now undergoing long-term functional tests. To further expand its utility, the tubular cells which currently can perform secretory function, are being genetically modified to be able to do re absorption. A liquid chromatography with tandem mass spectrometry method for the measurement of a panel of seven uremic biomarkers has been setup and validated. The levels of these biomarkers in samples from mice with different kidney pathologies is being measured with a view to determining their effectiveness to detect renal damage.

Objective 2: a mouse model for testing of the new tools and therapies, based on ischaemia-reperfusion injury, is set-up and has already been used for our first experiments on the efficacy of MSC-based therapies. Assessment of kidney function via in vivo imaging modalities is on-going and we are close to finalising the experimental protocols and mathematical models required for measuring renal clearance via multispectral optoacoustic tomography and magnetic resonance imaging. Safety of MSC therapies is being assessed by tracking the biodistribution and fate of these cells via bioluminescence imaging. Post-mortem imaging of kidneys and other organs to determine renal structure and the biodistribution of cell therapies is progressing well. For that, we are applying standard (histology) and advanced 3D methods based on: (i) ECi clearing in tandem with vascular perfusion with our own bespoke dyes, for microscopic mapping of vasculature, (ii) expansion microscopy for looking at ultrastructural details of the kidneys and (iii) CUBIC clearing, which is gentler than ECi and optimal for determining the distribution of fluorescently tagged MSCs.

Objective 3: MSCs derived from the umbilical-cord, bone marrow, adipose tissue and skin have been characterised and banked. For each of these sources, cells from multiple donors were obtained to ensure that any results are donor independent. To ensure reproducibility, cell culture reagents and protocols have been harmonised across all sites producing MSCs. We determined the properties of all MSCs in terms of morphology, growth rate and marker expression, and harvested and characterised their EVs. A panel of in vitro potency assays for measuring the cells’ metabolic state and their capacity to induce angiogenesis, modulate immune cells, or recover injured tubular cells is established, which will also be used for EVs. Mechanisms of action are being investigated in vitro by measuring the release of immunomodulatory factors and the response to signalling cytokines; in vivo by tracking the MSCs’ persistence in organs and their effects on renal function and vasculature; and post-mortem by assessing their effect on renal structure and interaction with the host’s immune cells.
At the conclusion of the project we expect to have achieved the following exploitable results, which have a direct socio-economic impact in healthcare: (a) a MSC therapy for kidney disease, (b) near-infrared dyes for measuring kidney function that are compatible multiple optical devices, (c) a clinical transcutaneous device for measurement of renal function, (d) image analysis tools and, (e) biomarkers for assessing kidney disease.
Summary of RenalToolBox's aims.