Periodic Reporting for period 3 - IM2PACT (Investigating Mechanisms and Models Predictive of Accessibility of Therapeutics (IM2PACT) Into The Brain) Reporting period: 2021-01-01 to 2021-12-31 Summary of the context and overall objectives of the project The complex interplay between neurons and microvessels responsible for the coupling of brain activity and blood supply to the brain requires an integrated, functional unit, termed the ‘neurovascular unit’ (NVU). The composition of the NVU varies with vessel size, but typically includes capillary endothelial cells, neurons, astrocytes, pericytes and extracellular matrix components. In addition to ensuring neurovascular coupling, the NVU provides the first line of defence against the detrimental effects of potentially neurotoxic molecules and cells in the systemic circulation. The barrier between blood and nervous tissues is evident at multiple interfaces; the best described being the cerebrovascular blood-brain barrier (BBB). The barrier arises from the specialized capillary endothelial cells of the NVU, which differ from peripheral endothelial cells in that they are not fenestrated, they have minimal pinocytotic activity, very low rates of transcytosis, express drug efflux transporters and are coupled by tight junctions (TJ). The properties of the endothelium therefore also represent the primary barrier for the transport of drugs across the BBB, and is crucial in maintaining the homeostasis of the brain’s microenvironment. The other cells of the NVU may help maintain the barrier properties of endothelial cells and may also directly be involved in the barrier. Most molecules and especially biologics do not cross the BBB and this is therefore a critical factor limiting the future application of neurotherapeutics. However, the need to develop CNS-active agents for neurological disorders including dementia or multiple sclerosis is greater than ever. This will require a more comprehensive understanding of BBB transport mechanisms and targets that could facilitate brain delivery. Recently, new opportunities for brain specific delivery of biologics have arisen. Challenging Receptor Mediated Transcytosis (RMT) and Carrier Mediated Transcytosis (CMT) with specific antibodies and peptides along with several drug delivery systems (including liposomes, nanoparticles, exosomes) or viral delivery agents have shown to deliver biopharmaceuticals into the CNS. Due to the lack of understanding of fundamental biological processes enabling the translocation of macromolecules or viruses through the BBB, the breakthrough of clinically relevant brain delivery approaches is still waiting to arise. The overall goal of the IM2PACT consortium is to address critical gaps in the field by comprehensively investigating the molecular and cellular properties of the BBB in human patient material, cutting-edge human in vitro models and in vivo preclinical models in order to understand BBB transport mechanisms and identify new BBB targets for brain delivery in health and disease. IM2PACT will robustly validate the models by establishing the ability of these models to truly predict in vivo CNS exposures of therapeutics, through an EU network of BBB translational scientists. The synergistic expertise of the partners in cellular and molecular biology, neuroscience, pharmacology, virology, drug delivery and bioinformatics along with the chemical/analytical resources, powerful biologics production facilities and direct link to the clinic brought by the EFPIA partners will enable rapid identification of new target mechanisms for brain delivery of therapeutics to treat neurodegenerative diseases and potentially wider applications in other CNS disorders. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far IM2PACT has acheived important milestone and deliverables with exciting new methods developed and datasets generated, despite the COVID-19 pandemic, which is continuing to have impacts. We are delighted to share the following important outputs1. A comprehensive single cell transcriptomic dataset from Alzheimer’s Disease and control post-mortem brain using a novel isolation protocol. We developed a novel protocol for isolation of neurovascular cells from the human brain, which we find is superior to any other method currently available. We have used the unique opportunity provided by the protocol to understand the molecular networks in both vascular and parenchymal cells of the human brain for the first time. The comparison between Alzheimer’s Disease and control is indicating key disease associated changes that may change the field’s understanding of disease mechanism. The dataset will be made available following publication of key results, with manuscripts currently in preparation.2. Transport functionality of a validated iPSC-BMEC differentiation modelA major part of the work of the IM2PACT consortium was to develop robust protocols for the generation of endothelial-like cell monolayers from human induced pluripotent stem cells. This was done by a cross-collaborative effort by consortium partners. The phenotype and functionality screening has now confirmed a robust phenotype with tight barrier function and expression of BBB transporters and receptors. In particular it represents a reproducible model for screening for Prominin-1 and Tfr-1 receptor mediated uptake of antibodies. The manuscript describing the method for generating this model is being prepared to allow the international research community to utilise the model. This model is a step forward for the field although further work is required to improve the transport propoerties and this will be continued in IM2PACT.3. Implementation of a Spheroid Model of the Blood-Brain-BarrierThere are several advanced BBB iPSC models that have been proposed to investigate BBB transport function. Our consortium evaluated several candidate models and has established a spherid model as the preferred model for use in IM2PACT research. In this model, the cells are arranged with astrocytes and neurons forming an inner compact core, surrounded by pericytes and an outer dense layer of brain capillary-like endothelial cells (BCECs). Importantly key junctional proteins and transporters are expressed by the BCECs. Analysis with sodium fluorescein transport and rhodamine-123 efflux confirmed BBB functionality. This model is more representative of the physiological state than other models. Hence it will allow robust and relevant investigation of human BBB transport mechanisms. Furthermore IM2PACT partners contributed to the development of spheroid array platforms suitable for high throughput analysis. Further work with the spheroid model is planned for the remainder of IM2PACT and if this proves successful, we anticipate that the spheroid array may be adopted by academia and industry as a BBB transport assay method of choice. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) IM2PACT has substantial moved research ahead of the state of the art by significantly improving methods to isolate vascular cells from human post-mortem brain material and then undertaking a comprehensive single cell analysis. This has revealed BBB transporter molecules and also highlighted potential disease networks. We have also established human cellular models to interrogate BBB transport and disease mechanisms. We expect that these methods and data will now be taken forward to study BBB transport in detail in healthy and disease states. Our dataset require ongoing validation using in vitro models and in vivo models as well as state of the art pharmacokinetic analysis tools. The impacts to patients and society are in the long-term but they remain critical challenges for effectively treating brain disorders.