Characterizing Exon Skipping Efficiency in DMD Patient Samples in Clinical Trials of Antisense Oligonucleotides

This protocol provides a standardized methodology for preparing and handling muscle tissue in pre-clinical settings and clinical trials, which is important for obtaining reliable results within the DMD field. The techniques are relevant for exon skipping tryouts for DMD. And the main advantage is that, they are well established and reproducible if carried out collectively.

These techniques are mainly focused on exon skipping in DMD, but with a change of primers, the same techniques can be applied to other diseases treated with splicing therapies. These methods can be used to achieve exon skipping efficiency both and RNA and protein. To prepare a sample for analysis, first mix equal volumes of tragacanth gum and water until the gum becomes soft and sticky.

Load this mixture into 25 milliliter syringes and dispense 0.5 to one centimeter of gum onto individual cork discs. Label the discs on the opposite side and place a container of isopentane in liquid nitrogen until some of the liquid freezes. Place each specimen into the tragacanth gum on the disks with a longitudinal axis of each muscle perpendicular to the cork.

Place gum around to the bottom of each muscle to help hold the muscle in place, and use tweezers to freeze the specimens in the cold isopentane. Move the specimens continuously for one minute or until completely frozen before placing them temporarily on dry ice. Then, place the specimens in glass vials for 80 degrees Celsius storage.

To section the frozen muscle tissue for analysis, set a cryostat to a working temperature of 25 degrees Celsius, and to mount the muscle block onto the holder. Trim the tissue block until flat sections are achieved. For reverse transcriptase PCR and Western blot analysis, obtain 10 micrometer thick slices.

For immunohistochemical analysis, obtain six to eight micrometer thick slices, and for hematoxylin and eosin staining, obtain 10 to 12 micrometer thick slices. After their acquisition, place the sliced sections in two milliliter tubes. Store the samples for PCR and western blotting at 80 degrees Celsius.

Here, microchip electrophoresis system, a gel images of reverse transcriptase PCR reactions, and the sequencing results of the skipped band from two patients before and after treatment with an antisense oligonucleotide in a dose escalation phase one trial are shown. Both patients harbor deletions. As expected, before treatment, both patients showed no skipping and only a non-skipped band could be visualized.

After 12 weeks of treatment, a clear skipped lower band was visualized for the second patient, however, it was still difficult to detect any skipped product for the first patient. The sequencing results showed a concatenation of exons 47 and 54 for the second patient, and exons, 44 and 54 for the first patient. To calculate the skipped percentage, the molar concentration for the skipped band was divided by the skipped and unskipped bands.

As expected, no dystrophin band was detected by Western blot before treatment. After treatment, a band was detected from the second patient with a lower molecular weight compared to that observed in the healthy control. The first patient, however, showed no detectable levels of dystrophin after treatment.

It is critical to handle the muscle tissue correctly, and to place each specimen on the tragacanth gum in consistent manner. The tissue sections can be analyzed in a number of different ways, including artificial to PCR, digital PCR, Western blot, immunohistochemistry, and with different types of omics. These techniques can be accelerate, the development, additional exon skipping drugs targeting different exons, and can be applied to the development of viral based and again in editing for DMD.