It is important to realize that pursuing the discovery of compounds that repress DUX4 (like any other drug discovery endeavor) comes with a risk of technical failure. Because the medical need of people with FSHD is both high and urgent, we have developed the following tools to maximize the chance of success early on.
First, as a discovery method we chose so-called phenotypic screening for the following reasons.
• Screening is a well-established, automated method for testing a large number of compounds for their desired effect in a suitable test system.
• Phenotypic screening is the most efficient form of screening because it is primarily designed to identify compounds that work in a biological, cell-based system. After identifying the most promising compounds, we look at how they work. In other words, phenotypic screening is not biased by assumptions – which may or may not prove to be correct – about how DUX4 repression might be brought about.
• Phenotypic screening is the most informative form of screening because it also reveals if compounds found to repress DUX4 have toxic side effects in the test system. This method thus enables gathering information on the effects of compounds on the biology of the test system – its phenotype, in other words. Hence the name.
Second, we chose to screen so-called small-molecule compounds because they are, by far, the best characterized class of therapeutic compounds; over 90% of marketed therapeutic drugs are small molecules. In addition, they are relatively simple compounds that can be manufactured by means of industrial chemical synthesis.
Third, we acknowledged that any compound purported to repress DUX4 must be shown to do exactly that in a test system that captures human FSHD biology as much as possible. Obvious though that may sound, the odds were that it couldn’t be done:
• Showing DUX4 repression presumes the ability to quantify DUX4 levels, but DUX4 is an extremely elusive protein. Its production is concentrated in only a tiny fraction of FSHD-affected muscle cells, and, to complicate things further, occurs in a burst-wise fashion.
• Capturing human FSHD biology in a test system fit for screening requires culturing human FSHD-affected muscle cells, but those cells have a very limited life span because they are killed by the DUX4 they produce.
But we did beat these odds. In fact, we remain the only entity in the FSHD field with a screening platform that enables reliable quantification of natural DUX4 protein levels in unadulterated (or “primary”) human FSHD-affected muscle cells.
Facio’s screening platform is dynamic. Basically, the system starts with precursor muscle cells called “myoblasts”, which have one nucleus (the cell nucleus is where almost all DNA, including the DUX4 gene, resides). Over a few days, myoblasts fuse to form mature muscle cells, or “myotubes”, which have multiple nuclei.
In sum, in the pursuit of our first goal we successfully built the fundamental ability to
• subject a well-established class of compounds (small molecules) to
• a well-established discovery method (phenotypic screening) in order to efficiently identify small molecules that
• reliably show meaningful effects (repression of human DUX4 protein production alongside minimal side effects) in
• a meaningful system (cultured primary human FSHD-affected muscle cells).
In an important advance, we successfully developed a mouse model for the purpose of testing DUX4 repressors in a living organism (“in vivo”). We based our model on engrafting human FSHD-affected muscle precursor cells (called “myoblasts”) onto a mouse thigh muscle. These human FSHD myoblasts then fuse and develop into mature muscle cells (called “myotubes”), which produce DUX4. Our model represents an improvement over similar models, mainly because it uses unadulterated (or “primary”) FSHD-affected muscle cells and therefore approximates natural FSHD biology as much as possible. Primary FSHD muscle cells are also the basis of our unique DUX4 screening platform. Achieving stability was the main challenge, but after about six months we were able to stably engraft human FSHD myoblasts in one mouse thigh and healthy human myoblasts in the other thigh, so that each mouse serves as its own control. DUX4 expression does occur in FSHD myotubes but not in healthy myotubes, which validates the model.