Identification of druggable targets for C9ORF72-related toxicity via an innovative CRISPR/Cas9 kinome-wide screen

Amyotrophic lateral sclerosis, also known as motor neuron disease, (ALS/MND) is a fatal disease, usually of mid-adulthood affecting ~1/300 people. It is caused by the progressive death of motor nerve cells that allow us to move/swallow/breathe. Current treatments that impactfully modify the course of the disease are lacking, and we aim to contribute to a prompt reversal of this.

If we can uncover the reasons why motor nerve cells die, we can use that knowledge to find ways of preventing or even reversing this. The commonest known genetic cause of ALS is a fault in the C9ORF72 gene. We will use this gene as a starting point for our studies.

We will focus on a class of proteins called "kinases". Protein kinases are enzymes that catalyse the addition of a phosphate molecule to amino acids—the building blocks of proteins. This action is called phosphorylation and is involved in nearly all cell signalling processes.

These proteins 'signal' changes in cellular functions including cell division, cellular energetics, transport, secretory processes, and many others. Thus, kinases play pivotal roles in regulating cellular actions. In other areas of medicine, understanding their biology has led to the development of targeted treatments.

In ALS, however, there has been little dedicated research on kinases. We wish to change that. Our objective is to painstakingly test whether removal of any kinase prevents death of motor nerve cells.

This would then suggest that they play an important role in ALS and that drugs that target these kinases could have therapeutic potential. It could also suggest that monitoring of kinase activity might have therapeutic utility. During the accelerated 18-month timeframe of the project, we will:

Engineer a human stem-cell line derived from a person with C9ORF72-ALS that specifically allows us to assess the role of kinases in an ALS context compared to a healthy context;

Identify which of the >700 genes encoding protein kinases are involved in motor nerve cell death (called CRISPR-screening). We will use C9ORF72 motor nerve cells grown with the brain's immune cells (microglia). The microglia will be C9ORF72 defective and will be present in conjunction with a chemical stress (glutamate excitotoxicity) that C9ORF72 motor nerve cells have previously been shown to be vulnerable to.

This will create an environment for the motor nerve cells that will result in their death that more closely mirrors the environment present in the spinal cords of people with ALS/MND than has been modelled previously;

validate these 'in-a-dish' findings. We will focus on the top three targets identified from the 'long-list', to find kinases which, when not present or blocked from functioning, lead to increased motor nerve health. First, we will use already proven cellular measurements of motor nerve health.

These include survival, transport of cargo along the electrical nerve cell cables (axons) and protein clumping. Second, we will look for overlap in targets in human postmortem spinal cord tissue both from C9ORF72-ALS patients, and sporadic (more general) ALS cases, looking for converging mechanisms.

Ultimately, this groundbreaking foundational work will set the scene for the next stage of targeted drug discovery, aiming to uncover druggable kinase-based targets that prevent MN death. As there are ~134 approved kinase inhibitors (https://www.ppu.mrc.ac.uk/list-clinically-approved-kinase-inhibitors), it is possible that there are already clinically-approved inhibitors against the identified target(s), suggesting that these could enter trials rapidly.