Precision control of protein dosage in vivo

The ability to control the dose of specific gene products within tissues underpins much of modern biomedical science, ranging from fundamental studies of biological pathways and disease mechanisms to gene and cell therapies, vaccines and pharmaceuticals. Experiments using human tissues are complicated by ethical and technical concerns, and hence rodent models have been used to advance our understanding of normal and abnormal tissue function, and for testing new therapeutic approaches. Although mice remain at the forefront of preclinical studies, new innovations in mouse genetics are needed to advance our understanding of human disease mechanisms, to identify new therapeutic targets, and to more accurately model therapeutic interventions.

Proteins carry out most biological functions; however, protein function is typically studied by introducing mutations into DNA. This has several drawbacks. It is not possible to study the immediate consequences of protein loss by mutating DNA, because it can take up to several days for the pre-existing pool of non-mutated gene-products to expire. Current approaches are not optimal to study the impact of reduced protein dose, which underlies many human disease states, or to allow protein function to be removed and then reinstated to model therapeutic intervention. More direct ways to rapidly change protein dose in living mice, in a manner that can be toggled on and off, could have a transformative impact on how mice are used for preclinical modelling.

This cluster will combine recent technological developments in the engineering of genomes, proteins and small molecules to address this unmet need. Our approach will use existing genetic engineering approaches to add short 'tags', termed 'degrons', onto proteins of interest. Degron-tagged proteins can then be targeted by small drug-like molecules that can enter cells and hijack their natural protein destruction pathways to facilitate rapid, tuneable and reversible degradation. Degron tags are already widely and successfully used in cultured mammalian cells, where they are providing unprecedented insight into how cells work. However, research using degrons in mouse models remains in its infancy and investment is urgently needed to set standards for their use. For example, several different degrons have been developed in cells, yet it is unclear which will work best in different mouse tissues and disease models, or to model particular therapeutic interventions. Different small molecules are available to degrade proteins with each degron, but how these molecules will behave in the more complex in vivo environment is unclear.

We believe that the MRC National Mouse Genetics Network is the ideal forum to translate degron technologies into mouse models of human disease. Our cluster brings together experts in mouse transgenics, protein degradation, structural biology, medicinal chemistry and pharmacology, and we have already shown that our approach works in two pilot projects. As we develop the core technology further, we will use the network to team up with national leaders in disease modelling under the auspices of the Mary Lyon Centre to improve the way that human disease is modelled in mice. Together, we aim to set standards for the use of degron technologies in living mammals and to reach out to industry and other national stakeholders who stand to benefit from their use.

Genome editing has transformed our ability to make human disease models. However, genetic manipulation occurs at the DNA level, whereas proteins mediate most biological functions. This limits the ability to understand disease mechanisms, identify therapeutic targets and model therapeutic interventions. This cluster will address this problem using degron-tagging technology to degrade target proteins in a rapid, tuneable and reversible manner. Although several degrons have been developed in cultured mammalian cells, how they will behave in the more complex in vivo environment is unclear.

Our workplan will proceed in two phases. During Phase 1, we will build genetic reporters to evaluate and compare three degron systems. We will test existing ligands and synthesise novel derivatives to identify compounds with favourable pharmacokinetic and pharmacodynamic properties in vivo. Our aim is to develop protocols for depletion across tissues, diseases and time-scales in line with network priorities. During Phase 2, we will work with the MLC to establish a national hub for degron technologies, disseminating knowledge from Phase 1 across the network via exemplar projects, and to the wider community via training courses and an online information portal.

Targeted protein degradation is an area of intense research. A cluster investment would enable the latest advances to be rapidly translated into advanced human disease models. The speed of degradation opens up a new acute temporal scale for downstream phenotypic analysis which is inaccessible using existing approaches, and will better model the effects of protein degrader drugs. Reversibility will determine whether and at what critical time-points therapeutic interventions can achieve clinical benefit. Our vision is that degron technologies will complement, extend, and in many cases replace recombinase approaches, simultaneously reducing and refining animal use while producing data that are easier to interpret.