Validation of neutrophil elastase as a target to improve muscle regeneration in Duchenne muscular dystrophy

In Duchenne muscular dystrophy (DMD), muscle cells are extremely fragile and break down easily as the muscles are used. Children diagnosed with DMD have a shorter life expectancy and will undergo progressive loss of muscle mass and strength that will lead them to be wheelchair bound by the age of 12 and often require external respiratory support by the age of 20. There is no cure for DMD. Although our body contains stem cells that can regenerate damaged muscle, in DMD these cells become prematurely exhausted and their lack of function leads to progressive muscle loss. Current standard of care based on the use of corticosteroids is mildly effective at slowing down disease progression but at the cost of significant adverse effects. We have studied muscular dystrophy in mice and we have discovered that a protein produced by white blood cells, called elastase, is increased in the muscles of dystrophic mice as compared to the muscles of non-affected mice. Elastase is a protease, which means is a protein that cleaves other proteins. We then went on to understand what this increase means and we found that elastase damages muscle stem cells, which are responsible for regenerating the muscle tissue when is damaged. Thus, we speculated that if we could block the activity of elastase in dystrophic muscle, we might be able to preserve muscle stem cell function and promote muscle regeneration in response to the continuous damage to muscle cells caused by the disease. To test this hypothesis we have carried out a preliminary experiment where we have inhibited elastase activity and indeed observed an improvement in muscle pathology in dystrophic mice. However the elastase inhibitor used in these experiments cannot be prescribed to patients because is not a synthetic compound but one purified from bacteria.

We now aim to test in mice drugs that can be prescribed to patients and that have been developed to inhibit elastase activity in a variety of diseases such as cystic fibrosis, bronchiectasis and chronic obstructive pulmonary disease. The project we propose to carry out will develop in two phases. Firstly, we will test a few of these elastase inhibitors on muscle stem cell cultures that have been derived from patients with DMD to find the one drug that is more potent at inhibiting the effects of elastase in cells. Secondly, we will treat mice that have muscular dystrophy with this drug and study whether they improve. During the course of the treatment we will measure their grip strength and also how much they voluntarily run on a wheel to see if their muscle function has improved. At the end of the treatment we will cull the mice, collect their muscles, make thin sections and stain them to see whether the treatment has improved regeneration, which will be shown as an increase in muscle mass and a decrease in scar tissue within the muscle. Moreover, we will find out whether inflammation has decreased in the muscles, by quantifying specific markers of inflammation such as the presence of inflammatory cells and the levels of pro-inflammatory molecules in the muscle. Lastly, we will quantify the levels in the blood of certain molecules that are indicators of either muscle damage or inflammation.

With this project we aim to develop a novel treatment for DMD based on elastase inhibition that would help maintain muscle mass and strength by promoting muscle regeneration.

Duchenne muscular dystrophy (DMD) is a genetic disorder caused by loss of function mutations in the gene that encodes the cytoskeletal protein dystrophin. In the absence of dystrophin muscle fibres become more prone to damage, which induces muscle fibre necrosis, and in turn inflammation and fibrosis. Muscle regeneration is impaired in DMD leading to loss of muscle mass and strength. Children diagnosed with DMD have shorter life expectancy and undergo progressive loss of muscle mass and strength that will lead them to be wheelchair bound by the age of 12 and often require external respiratory support by the age of 20. There is no cure for DMD and the treatments currently in place, which are mostly based on corticosteroids, can only mildly delay diseases progression but also produce significant adverse effects.

In a mouse model of DMD, the mdx4cv mouse, we have discovered that the chronic presence of neutrophils in the muscle leads to accumulation of neutrophil elastase, which impairs muscle stem cell proliferation and differentiation ex vivo. Thus, neutrophil elastase might play a key role in the impairment of muscle regeneration observed in DMD and therefore stands out as a potential therapeutic target to enhance muscle regeneration and prevent muscle loss in DMD.

The goal of this project is to validate neutrophil elastase as a therapeutic target for DMD by assessing muscle regeneration, muscle pathology and muscle function in dystrophic mice treated with a selective neutrophil elastase inhibitor. More specifically, we will first select a lead compound amongst three candidates by using an ex vivo assay that we have developed. For this purpose we will use myoblasts (which are the myogenic progeny of muscle stem cells) derived from patients with DMD. This compound will be then taken further into animals and tested for its efficacy at improving muscle regeneration, histopathology and function in dystrophic mdx4cv mice.

The proposed work will have scientific/academic, translational and societal impact.

In order to achieve scientific/academic impact we have set out two objectives:

Objective 1: To present our data at national and international meetings. The PI regularly attends both national and international meetings on muscle and DMD biology. The PDRA will be strongly encouraged to attend as well. They will both present data through oral presentations and posters.

Objective 2: To prepare, submit and publish two scientific papers containing the results of this research and one review on a topic that is closely related to the topic of this research (e.g. Extracellular matrix remodeling in Duchenne muscular dystrophy).

In order to achieve translational impact, we will seek the following objective:

Objective 3: Should the results of the proposed research successfully demonstrate a positive effect of neutrophil elastase inhibition at improving muscle regeneration and pathology in dystrophic mice, we will conduct clinical trials to test the efficacy and safety of neutrophil elastase inhibition in patients with Duchenne muscular dystrophy. For this purpose, we will engage with various partners including: clinicians, DMD charities and industries that produce elastase inhibitors. We already have a long-standing collaboration with clinicians and Alder Hey Children's Hospital in Liverpool, which is host to a state-of-the-art clinical research facility with extensive experience in DMD trials. Moreover, we have started early stage conversations with pharma companies (Astra Zeneca and Solid Biosciences) and charities (Charlie's Fund, Joining Jack and Duchenne UK).

Lastly, we will pursue societal impact with the following objective:

Objective 4: We will contribute to raising awareness about muscle biology and the need to develop effective treatments for DMD via collaborations DMD charities and professional societies. Moreover we will publish press releases together with our peer-reviewed papers to explain in lay terms the findings of the published studies and their importance to society.