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Investigating Mechanisms and Models Predictive of Accessibility of Therapeutics (IM2PACT) Into The Brain

Periodic Reporting for period 2 - IM2PACT (Investigating Mechanisms and Models Predictive of Accessibility of Therapeutics (IM2PACT) Into The Brain)

Reporting period: 2020-01-01 to 2020-12-31

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.
The second year of IM2PACT has been significantly affected by the COVID-19 pandemic. Many institutions were closed or with with very restricted activity for about 6 months over this period. Nevertheless, the conssortium has made impressive progress with the programme of work and we have also adapted experiments to regain time of critical milestones and deliverables.

1. Enhanced protocol for extraction of microvessels from human postmortem brain.
The protocols for isolation of BBB endothelial cells have been radically improved with much better purity and improved viability for downstream omics analysis. This will likely have an important impact for the field as prior studies where the endothelial cell isolation was less pure may have resulted in significantly biased results. This protocol has been shared across the consortium and will allow completion of an important WP1 task to undertake RNA-seq and proteomics on AD and MS brain tissue.

2. A validated iPSC-BMEC differentiation model
An iPSC based brain endothelial differentiation protocol has been validated and adopted by the consortium. This is important because it will enable the consortium to develop functional assays that will be used to assess small molecule and biologic transport. A reference panel of compounds and benchmarking antibodies for these assays has been established in support of this.

3. The viral interactome of West Nile virus (WNV) and human endothelial cells.
The viral interactome of WNV has been established using hCMEC/D3 cells. This is an important dataset to help understand the mechanism of WNV engagement and entry into the brain through the vasculature. It will also provide candidate targets for development as therapeutic transport mechanisms.

4. Candidate blood-brain-barrier transport targets
Using a combination of data from IMI COMPACT and newly generated data from IM2PACT, a list of candidate targets expressed in brain endothelial cells in healthy control and Alzheimer's disease has been established. This has been through a process of validation to confirm expression at a protein level as well as the availability of tools to progress these targets for further development. This is a valuable dataset for the field to develop therapeutic transport mechanisms.
IM2PACT has already moved research ahead of the state of the art through identification of new BBB transporter candidates. Once these candidates are prioritised, further work will commence to experimentally validate their potential for improving therapeutic access into the brain. Over the next year, IM2PACT will generate a large body of de novo molecular data on the BBB that will add a significant body of knowledge to the field. We therefore expect to rapidly utilise this data to develop cellular and preclinical models for the duration of the IM2PACT project. In addition, IM2PACT researchers will continue to investigate better stem cell models of the BBB and several novel strategies are under investigation. We expect these will mark a watershed in the field in finally achieving a genuine brain capillary endothelial cell phenotype that will be highly valued by researchers in the BBB field. The potential impact on society of IM2PACT research is very significant as getting therapeutic into the brain remains a major challenge for drug development.
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