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Finding new therapies for genetic diseases using ECSTASY

“Biology is the most powerful technology ever created. DNA is software, protein are hardware, cells are factories.” That is a quote from Arvind Gupta, one of our early investors. Arvind’s metaphor portrays the human body quite nicely..DNA is the set of genetic instructions inherited through generations and one of its tasks is to “code” for proteins, which are responsible for nearly every single event that happens inside a cell.

DNA software, however, is far from flawless. Every day people are born with DNA software that does not “code” for all the hardware our body needs to be healthy. These individuals are diagnosed with genetic disorders. Lysosomal storage disorder (LSD), for example, is a rare genetic disorder that affects how cells (the factory) break down waste. Under normal circumstances, cells can break down and recycle materials for new purposes. However, individuals with LSD cannot break down waste inside cell factories, leading to the accumulation of toxic waste and and premature factory shutdown.

One way to fix this issue is to reintroduce the missing hardware. This is a commonly used option and is known as enzyme replacement therapy (ERT). Doctors inject protein hardware into the body that carry out the function for which the DNA software is faulty for. While this is a viable option for some patients, it does not always work. That is because the body has security guards (our immune system) that are highly trained at identifying and preventing the entry of foreign substances. It is actually beneficial for our bodies to have this 24/7 security guard in place, as it helps protects us from infections and colds. For instance, ~91% of mucopolysaccharidosis (MPS) type I patients are resistant to ERT and this calls for new options and therapies for this unmet medical need.

Towards this end, researchers from the National Defense Medical Centre in Taiwan recently came up with an innovative idea to bypass the immune system. The concept is to “teach” pre-existing hardware inside the cells of an MPS I patient to break down the accumulated toxic waste. Any pre-existing hardware would not be detected and removed by the immune system security team.

MPS I patients are missing DNA software that ‘code’ for the alpha-iduronidase protein, a piece of hardware that removes toxic buildup of chemicals called glycosaminoglycans (GAG). MPS I patients, however, have another piece of software/hardware call beta-glucuronidase, an enzyme that carries out tasks that are very similar to the ones done by alpha-iduronidase. Like two chefs working on different recipes.

The theory was to “teach” beta-glucuronidase how to break down GAG, which in turn, would stabilize the cell factories. The method by which researchers “teach” hardware how to perform new functions is call protein engineering. Simply put, the researchers identify which parts of beta-glucuronidase that, when changed, could “convince” beta-glucuronidase to break down GAG. The protein engineers generate as many changes on those targeted parts as possible and test all of the possible changes in a competition to break down GAG.

The group ultimately focused on 19 parts (19 amino acids) that might convert beta-glucuronidase into a GAG destroyer. By mixing and matching all possible changes at the 19 parts, the researchers came up with a total of 3x10^9 variations of beta-glucuronidase (this is known as a combinatorial library), a small proportion of which might exhibit alpha-iduronidase-like functions. To identify beta-glucuronidase variants that might help MPS I patients, the Taiwan research group used an experimental system called ECSTASY (Enzyme Cleaveable Surface-Tethered All purpose Screen sYstem). The system enabled researchers to make tens of thousands of cell factories, each containing a single beta-glucuronidase variant that is exposed on the cell surface. They then isolate beta-glucuronidase from each cell surface, provided them with GAG and monitor whether GAG gets decomposed. The advantage to this system is that researchers can create high volumes of unique variants and test them separately on pure GAG.

The group found 73 beta-glucuronidase variants that were able to break down GAG. The best performing beta-glucuronidase variant that was identified had 1% of activity compared to alpha-iduronidase. While not a high percentage, it is a promising start. They were able to teach beta-glucuronidase an entire new function that nature did not intend.

To further validate this novel concept, Chuang and colleagues isolated cells from MPS I patients, which have toxic GAG build up, and tested whether the beta-glucuronidase variants could lower toxic GAG levels inside MPS I cells. In agreement with their ECSTASY results, they demonstrated that the beta-glucuronidase variants were able to clear away toxic GAG in actual cells similarly to alpha-iduronidase, albeit at a lower level/ efficiency.

Overall, this study provides a promising avenue for LSD patients where ERT is not a viable option. The ability to bypass the immune system bottleneck by teaching hardware to carry out new functions opens the doors to treating many genetic diseases. Of course, there are still years of optimization to be done before treating MPS I with beta-glucuronidase variants becomes a clinical option. There is a long road of protein engineering that is required to get GAG breakdown efficiency from 1% to >90%. Re-iterative cycles of combinatorial libraries combined with ECSTACY screening will be needed to find those improved beta-glucuronidase variants worth pursuing in the clinic. Furthermore, experiments would need to move beyond cells and into animal models to fully test the theory of whether beta-glucuronidase variants can bypass the immune system. We hope that one day beta-glucuronidase variants can push through and become a viable treatment option for MPS I patients worldwide.


Chuang HY, Suen CS, Hwang MJ, Roffler SR. Toward reducing immunogenicity of enzyme replacement therapy: altering the specificity of human β-glucuronidase to compensate for α-iduronidase deficiency. Protein Eng Des Sel. 2015;28(11):519-29.


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