#apaperaday: Quantitative Evaluation of Exon Skipping in Urine-Derived Cells for Duchenne Muscular Dystrophy

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: Quantitative Evaluation of Exon Skipping in Urine-Derived Cells for Duchenne Muscular Dystrophy

Today’s pick is from a methods and protocols book on muscular dystrophy therapeutics by Kunitake et al on using urine derived cells to evaluate exon skipping DOI: 10.1007/978-1-0716-2772-3_9

Method book papers have a different format than general scientific publications. They contain a protocol of how to do something, with instructions on the compounds used in each step and how to do it.

For other scientists these protocols are very useful as the methods section in scientific publication is usually rather limited and does not contain a note section while these methods papers do. Notes are the little things that make a difference when doing experiments.

Anyway, to the paper: it introduces that for exon skipping ideally you assess ASO efficiency also in patient-derived cells. However, doing this in myoblasts (muscle stem cells) requires a biopsy, which is invasive. You can do a skin biopsy – less invasive but still painful.

Authors here outline how to isolate stem cells from urine! Much less painful obviously. Doing this takes multiple steps: first the urine derived cells need to be isolated from the urine and cultured. Then they need to be told to differentiate to muscle cells.

Authors use a retroviral vector that expresses MyoD – MyoD is a protein that tells cells to become muscle cells. Then you need to wait for the urine cells to convert into muscle cells and only then can you treat them with ASOs to assess exon skipping and dystrophin restoration

The whole process takes week: first ~10 days to get enough urine derived cells, then 2 weeks for the cells to convert into muscle cells, then 3 days of ASO incubation and then probably up to a week for RT-PCR and western blotting.

However, this method is less invasive and does not require one to schedule a muscle or skin biopsy – from which you also need to isolate and grow cells. So that probably takes at least as much time and is more invasive.

I mention the notes section with little tricks and tips from the authors. One example: for cell isolation it is best to use midstream urine. This will reduce the chance of having contamination with a virus. Cells need to be isolated quickly as they lose viability <4 hrs

I refer the people interested in this technique to the methods paper itself. I will discuss a few more of these papers in the coming weeks.