We are grateful to Dr Takashi Saito, Dr Tetsuya Nagata, Mr Satoshi Masuda, and Dr Eri Takeshita for scientific discussion.

The operational procedure for the investigator-initiated trial has been approved by the NCNP ethical committee (approval ID: A2013-019).
1. Preparation of muscle samples
NOTE: Biceps brachii or quadriceps muscles are often selected as biopsy sites. However, the tibialis anterior was used in the clinical trial.
<ol>
	<li>Muscle biopsy from patients
	<ol>
		<li>Mark the incision site before skin preparation.</li>
		<li>Prepare saline-dampened gauze for the biopsy specimen. Squeeze the gauze well.</li>
		<li>Inject local anaesthesia into skin and subcutaneous tissue, but not into muscle. Make sure of the depth of the anesthesia by the absence of cutaneous pain. 
		NOTE: General anaesthesia is generally used in paediatric patients under 15 years of age.</li>
		<li>Make an incision from the skin to the subcutaneous tissue. Use small retractors to open the incision and separate the subcutaneous fat. 
		NOTE: At this step, a portion of the skin can be cut for fibroblast culture.</li>
		<li>Make another small incision in the fascia and cut the fascia further. Clamp the edges using mosquito forceps to expose the muscle.</li>
		<li>Use a suture to pull up the selected portion of the muscle. Make a small tunnel using Iris scissors and insert mosquito forceps into the tunnel.</li>
		<li>Separate the muscle bundle using forceps and cut both ends of the target portion. 
		NOTE: Muscle biopsy specimens should be around the size of a pencil for adult patients (length 1.0-1.5 cm, diameter 0.8-1.0 cm). The specimen should be around 1 cm in length and 5 mm in diameter in paediatric patients.</li>
		<li>Wrap the specimen in saline-dampened gauze and transport the fresh muscle at room temperature (RT) immediately to a facility where the specimen can be prepared for further analysis. 
		NOTE: The specimens should be transported at 4 &#176;C if the duration of transportation exceeds 1 hour.</li>
	</ol>
	</li>
</ol>
2. Muscle sample preparation
<ol>
	<li>Mix equal volumes of tragacanth gum and water until the gum becomes soft and sticky. Load the mixture into 25 mL syringes. The syringes can be stored in a refrigerator.</li>
	<li>Place approximately 0.5 to 1 cm of tragacanth gum on cork discs. Label the discs on the opposite side.</li>
	<li>Place a container of isopentane in liquid nitrogen until some of the liquid freezes.</li>
	<li>Place the specimen in the tragacanth gum. The longitudinal axis of each muscle should be perpendicular to the cork. Place gum around the bottom of each muscle to help proper placement.</li>
	<li>Using tweezers, place the muscle/cork specimen in cold isopentane prepared in step 2.3 to freeze. Move the specimen constantly for 1 min or until completely frozen, and place on dry ice temporarily.</li>
	<li>Place the specimen in glass vials and store at -80 &#176;C.</li>
</ol>
3. Muscle sectioning
<ol>
	<li>Set up the cryostat for sectioning with a working temperature of -25 &#176;C.</li>
	<li>Mount the cork/muscle block and trim until flat sections are achieved.</li>
	<li>Use a section thickness of 10 &#181;m for RT-PCR and western blotting. Use a thickness of 6-8 &#181;m and 10-12 &#181;m for immunohistochemistry or haematoxylin and eosin staining, respectively. Put sliced sections in 2.0 mL tubes and store at -80 &#176;C for RT-PCR and western blotting.</li>
</ol>
4. RNA extraction and reverse transcription polymerase chain reaction (RT-PCR)
<ol>
	<li>Extract approximately 10 slices with 10 &#181;m thickness from frozen muscle by a cryostat. Collect the purified total RNA using the purification kit of total RNA according to the manufacturer&#39;s instructions.</li>
	<li>Measure the RNA concentration using a spectrophotometer.</li>
	<li>Combine the required reagents for a RT-PCR reaction in PCR tubes according to Table 1. See Table 2 for primer sequences. 
	NOTE: The forward primer was 44F for patient NS-03 and 46F for patient NS-07. The reverse primer was 54/55R as a default. If non-specific products were evident, 54R was used as an alternative.</li>
	<li>Place the PCR tubes containing the mixture in a thermo-cycler. Run the thermo-cycler according to Table 3.</li>
	<li>Store PCR product at 4 &#176;C for short-term storage or -20 &#176;C for long-term storage.</li>
</ol>
5. Microchip electrophoresis and exon skipping calculation
NOTE: A microchip electrophoresis (MCE) system is often selected to analyze exon skipping efficiency. In this protocol, we describe the steps necessary to analyze the exon skipping efficiency using a MCE system by manufacturer A as well as from manufacturer B, hereafter called system A and B. During the clinical trial, the exon skipping efficiency was analyzed on system A. However, system A is no longer for sale, and we recommend system B to analyze exon skipping efficiency. We have included protocols for both systems (see section 5.1 for system A and 5.2 for system B). See Table of Materials for information regarding system A and B. Furthermore, this step can also be performed with normal agarose gel electrophoresis, but the sensitivity markedly decreases.
<ol>
	<li>Microchip electrophoresis using System A 
	NOTE: System A has two types of chips. Here, we describe the steps for the DNA chip.
	<ol>
		<li>Equilibrate kit reagents (stain buffer, loading buffer, ladder, and gel buffer) to RT, vortex, and spin down.</li>
		<li>To prepare gel-stain buffer (GS), add 12.5 &#181;L of stain buffer to 250 &#181;L of gel buffer and vortex for 10 s. Move the solution to a spin filter tube and centrifuge at 2,400 x g for 15 min. Discard the filter. 
		NOTE: The filtered GS can be stored at 4 &#176;C for 1 month.</li>
		<li>If samples are highly concentrated, dilute to approximately 50 ng/&#181;L with TE buffer or DNase-free water. 
		NOTE: If samples are in a salt concentration &#8805; 200 mM KCl (or NaCl) and/or 15 mM MgCl2, exchange the buffer.</li>
		<li>Add 12 &#181;L of GS solution to the gel priming well (highlighted and labelled GS) of the DNA chip and place the chip in the priming station. 
		NOTE: To avoid air bubbles, insert the pipette tip vertically and to the bottom of the well when dispensing. Dispense slowly to the first stop on the pipette, and do not expel air at the end of the pipetting step.</li>
		<li>Select C3 mode and press the Start button. After priming is completed, remove the chip.</li>
		<li>Visually inspect the microchannels for trapped air bubbles or incomplete priming.</li>
		<li>Load the prepared samples and ladder onto the chip as below.
		<ol>
			<li>Pipette 9 &#181;L of GS solution into the other 3 GS wells.</li>
			<li>Pipette 5 &#181;L of loading buffer into the L well and each sample well (1-11).</li>
			<li>Pipette 1 &#181;L of ladder into the L well.</li>
			<li>Pipette 1 &#181;L of DNA sample into each of the 11 sample wells.</li>
			<li>Pipette 1 &#181;L of TE or DNase-free water into any unused wells. The kit quick guide shows a figure of the chip layout. 
			NOTE: Inspect all wells for air bubbles by holding the chip above a lightly colored background. Dislodge any trapped air bubbles at the bottom of a well with a clean pipette tip or by removing and reloading the solution.</li>
		</ol>
		</li>
		<li>Place the chip in the vortex station and press Mix. The vortex station automatically stops after 1 min.</li>
		<li>Run the chip in the electrophoresis station within 5 min of loading.</li>
		<li>Select New Run in the software toolbar. On the New Run screen, select DNA and DNA 1K from the Assay pull-down list.</li>
		<li>Either select a project folder for the run from the Project pull-down list or create a new project folder by entering a name in the Project field.</li>
		<li>Enter a name for the run in the Run Prefix field and click Start Run.</li>
		<li>The instrument beeps when the analysis is complete, and a window opens indicating the end of the run. Select OK and remove the chip from the chip platform.</li>
		<li>Select File | Export Data to export the data. Select the desired options in the Export dialogue.</li>
		<li>To clean the electrodes, fill a cleaning chip with 800 &#181;L of deionized water and place it on the chip platform, close the lid, and leave it closed for 1 min. After 1 min, open the lid, remove the cleaning chip, and allow the electrodes to dry for 1 min.</li>
	</ol>
	</li>
	<li>Microchip electrophoresis using system B
	<ol>
		<li>Open the operating software and enter the sample information. After entering the information, the software calculates the amount of separating buffer and marker solutions automatically.</li>
		<li>Prepare the necessary amount of separating buffer calculated by the software. First, prepare 100x nucleic acid gel stain solution by diluting 10,000x solution with the appropriate amount of TE buffer. The 100x solution can be stored at -20 &#176;C.</li>
		<li>Mix a suitable amount of 100x nucleic acid gel stain withseparation buffer in the specific tube provided by the company to prepare a 1x solution. Vortex the solution.</li>
		<li>Prepare the required amount of marker solution in the tube.</li>
		<li>Start the probe wash program.</li>
		<li>Start the microchip wash program when finished.</li>
		<li>Simultaneously, pipette the samples to a PCR multiplate (96-well, clear) and dilute 4 times with deionized water. The minimum volume the machine can handle is 6 &#181;L, and the maximum is 30 &#181;L. We suggest a total volume of 10 &#181;L (2.5 &#181;L of sample and 7.5 &#181;L of water/TE-buffer).</li>
		<li>Cover the plates with adhesive PCR sealing foil sheets and spin-down the samples.</li>
		<li>Set the plate, marker solution, and 1x separating buffer in the machine according to the placement shown by the machine. Push the Start button.</li>
		<li>When the run is finished, export the result data as a .csv file from the software and calculate the exon skipping efficiency using molar concentration as follows: 
		Exon skipping efficiency (%) = (Skipped band / (Skipped band + Non-skipped band) X 100</li>
	</ol>
	</li>
</ol>
6. Complementary DNA (cDNA) sequencing
<ol>
	<li>Agarose gel preparation
	<ol>
		<li>Measure 1.5 g of agarose powder and mix the powder with 100 mL of 1x TAE buffer in a microwavable flask (resulting in a 1.5% agarose gel).</li>
		<li>Microwave for 1-3 min until the agarose is completely dissolved. 
		NOTE: Do not overboil the solution, since some of the buffer will evaporate and alter the final agarose percentage of the gel.</li>
		<li>Let the agarose solution cool to about 50 &#176;C.</li>
		<li>Add 10 &#181;L 10,000x fluorescent nucleic acid dye to the agarose solution.</li>
		<li>Pour the agarose into a gel tray with the well comb in place. Leave at RT for 20-30 min until it has completely solidified.</li>
	</ol>
	</li>
	<li>Sequencing
	<ol>
		<li>Mix 5 &#181;L of RT-PCR product (from step 4.4) and 1 &#181;L of 6x loading buffer. Load them into the wells of the 1.5% agarose gel.</li>
		<li>Run the gel at 135 V for 5 min and 120 V for 20 min.</li>
		<li>Visualize the bands using a transilluminator according to the manufacturer&#39;s protocol.</li>
		<li>Excise bands of interest from the gel and use a gel and PCR cleanup kit to retrieve RT-PCR products.</li>
		<li>Measure the concentration using a spectrophotometer. 
		NOTE: The purified band can be sent for sequencing at a company or sequencing facility if no Sanger sequencing equipment is available in-house.</li>
		<li>Prepare the required reagents for a cycle sequencing kit and pipette into a multiplate PCR plate according to Table 4. See Table 2 for primer sequences.</li>
		<li>Seal the plate with clear adhesive film.</li>
		<li>Vortex the plate for 2 to 3 s. Centrifuge briefly in a swinging bucket centrifuge so that the contents settle to the bottom of the wells (5 to 10 s) at 1,000 x g. 
		NOTE: Air bubbles may be present in the wells, but they do not adversely affect the reaction.</li>
		<li>Place the mixture in a thermo-cycler and run according to Table 5.</li>
		<li>Purify the sequencing reactions with plates according to the manufacturer&#39;s instructions.</li>
		<li>Use an DNA analyzer to determine the sequences according to the manufacturer&#39;s protocol.</li>
		<li>Compare the sequence obtained to the patient&#39;s expected sequence.</li>
	</ol>
	</li>
</ol>
7. Western blotting
<ol>
	<li>Sample preparation
	<ol>
		<li>Prepare SDS sample buffer (4% SDS, 4 M urea, 10% 2-mercaptoethanol, 10% glycerol, 70 mM Tris-HCl pH 6.4, 0.001% bromophenol blue, and protease inhibitor).</li>
		<li>Place around 100 slices of the 10 &#181;m muscle sections collected from cryo-sectioning in 150 &#181;L of SDS buffer.</li>
		<li>Briefly homogenize the protein samples on ice for 30 s using a handy micro homogenizer.</li>
		<li>Centrifuge at 16,500 x g for 15 min, and transfer the supernatant to a fresh tube. Proceed with analysis or store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>SDS-PAGE for protein concentration measurement 
	NOTE: A fluorescent gel stain was used to determine the protein concentration.
	<ol>
		<li>Determine the normal healthy control sample weight and the concentration using the BCA protein assay kit according to the manufacturer&#39;s instructions.</li>
		<li>Prepare the samples for gel electrophoresis. Pipette 10 &#181;g of total protein of the healthy control and 5 &#181;L of the patient samples, 5 &#181;L of 4x sample buffer, 2 &#181;L of 10x sample reducing agent. Once these have been mixed, add deionized water to a final volume of 20 &#181;L in each sample.</li>
		<li>Heat samples at 70 &#176;C for 10 min.</li>
		<li>Prepare 2,000 mL of 1x SDS running buffer using 50 mL of 40x SDS running buffer. Dilute with 1,950 mL of deionized water. 
		NOTE: At this point, 1,000 mL of running buffer is enough. The remaining 1,000 mL buffer is used at step 7.3.1.</li>
		<li>Mix thoroughly and set aside 800 mL of the 1x SDS running buffer for use in the lower (outer) buffer chamber of the gel box.</li>
		<li>Immediately prior to electrophoresis, add 500 &#181;L of antioxidant buffer to 200 mL of 1x SDS running buffer to use in the upper (inner) buffer chamber of the gel box.</li>
		<li>Prepare for gel electrophoresis by setting up a 3-8% Tris-acetate gel according to the manufacturer&#39;s protocol. Load 20 &#181;L of each sample onto the gel.</li>
		<li>Perform electrophoresis at 150 V for 75 min.</li>
		<li>After electrophoresis, place the gel directly into a clean tray containing 50 mL of fluorescent gel stain solution.</li>
		<li>Cover the tray, place on a shaker, and agitate without splashing liquid or damaging the gel for 90 min.</li>
		<li>Transfer the gel into water before imaging in a fluorescence system at 312 nm illumination with ethidium bromide emission filter to detect fluorescence.</li>
		<li>Measure the concentrations of the patient samples based on an analytical curve constructed using the normal control lysate with known concentration.</li>
	</ol>
	</li>
	<li>SDS-PAGE for western blotting
	<ol>
		<li>Prepare the gel box according to steps 7.2.4-7.2.6.</li>
		<li>Based on the concentration measurement obtained in step 7.2.11, load 100 &#181;g per lane of healthy control lysate and 300 &#181;g per lane of the patient sample. Electrophorese using the same settings as step 7.2.7.</li>
	</ol>
	</li>
	<li>Protein transfer
	<ol>
		<li>Prepare the transfer buffer. Soak a PVDF membrane for 20 s in methanol. Move it to the blotting buffer B until use.</li>
		<li>Soak a extra-thick blotting paper in blotting buffer A, B, and C for at least 30 min per buffer.</li>
		<li>After electrophoresis, soak the gel in blotting buffer B for 5 min.</li>
		<li>Place blotting papers, PVDF membrane, and gel on the semi-dry transfer machine according to the drawing in Figure 1. Roll out air bubbles between the gel and membrane with a roller.</li>
		<li>Transfer at 2 mA/cm2 membrane for 1 h at RT. 
		NOTE: After transferring, it is recommended to perform a total protein stain similar to Ponceau staining to confirm successful transfer of the large molecular weight proteins.</li>
	</ol>
	</li>
	<li>Blocking and antibody staining
	<ol>
		<li>Rinse the membrane twice with PBST (1x PBS with 0.1% Tween 20).</li>
		<li>Place the membrane in 1% blocking agent, and rock gently for 1 h at RT to block.</li>
		<li>Incubate the membrane with anti-dystrophin antibody in blocking solution (1:125) and anti-spectrin antibody (1:25,000) for 1 h at RT or overnight at 4 &#176;C.</li>
		<li>Wash the membrane three times for 10 min each with PBST at RT.</li>
		<li>Incubate the membrane with horseradish peroxidase (HRP)-conjugated secondary anti-mouse/rabbit antibody in PBST (1:100) for 40 min at RT.</li>
		<li>Wash the membrane again three times for 10 min with PBST at RT.</li>
	</ol>
	</li>
	<li>Detection
	<ol>
		<li>Mix detection solutions A and B in a ratio of 1:1. The final volume of detection reagent required is 0.1 mL/cm2 membrane.</li>
		<li>Remove the excess PBST from the washed membrane and place it with the protein side up on a sheet of plastic wrap or other suitable clean surfaces. Add the mixed detection reagent onto the membrane and incubate for 5 minutes at RT.</li>
		<li>Remove the excess detection reagent by holding the membrane gently in forceps and touching the edge against a tissue.</li>
		<li>Place the blot protein side up on a sample tray. Operate the fluorescent system according to the user documentation. Set machine as follows. Exposure type: Increment, Interval time: 10 s, Sensitivity/Resolution: high. 
		NOTE: Measured areas were boxed with a blue rectangle (Figure 4a-b): BG: background; D: dystrophin 427 kDa, SL: spectrin beta long isoform (274 kDa); SS: short isoform (253 kDa). Dystrophin/spectrin signal ratio was calculated as (D-BG)/[(SL-BG) + (SS-BG)]. The ratio of normal control was set as 100%.</li>
	</ol>
	</li>
</ol>

<ol><li>Bushby, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diagnosis+and+management+of+Duchenne+muscular+dystrophy%2C+part+2%3A+implementation+of+multidisciplinary+care%5BTitle%5D)+AND+%22Lancet+Neurology%22%5BJournal%5D)">Diagnosis and management of Duchenne muscular dystrophy, part 2: implementation of multidisciplinary care.</a> Lancet Neurology. 9 (2), 177-189 (2010).</li>
<li>Hoffman, E. P., Brown, R. H. Jr, Kunkel, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dystrophin%3A+the+protein+product+of+the+Duchenne+muscular+dystrophy+locus%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Dystrophin: the protein product of the Duchenne muscular dystrophy locus.</a> Cell. 51 (6), 919-928 (1987).</li>
<li>Mendell, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence-based+path+to+newborn+screening+for+Duchenne+muscular+dystrophy%5BTitle%5D)+AND+%22Annals+of+Neurology%22%5BJournal%5D)">Evidence-based path to newborn screening for Duchenne muscular dystrophy.</a> Annals of Neurology. 71 (3), 304-313 (2012).</li>
<li>Moat, S. J., Bradley, D. M., Salmon, R., Clarke, A., Hartley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Newborn+bloodspot+screening+for+Duchenne+muscular+dystrophy%3A+21+years+experience+in+Wales+%28UK%29%5BTitle%5D)+AND+%22European+Journal+of+Human+Genetics%22%5BJournal%5D)">Newborn bloodspot screening for Duchenne muscular dystrophy: 21 years experience in Wales (UK).</a> European Journal of Human Genetics. 21 (10), 1049-1053 (2013).</li>
<li>Greenberg, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+studies+in+newborn+males+with+Duchenne+muscular+dystrophy+detected+by+neonatal+screening%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Gene studies in newborn males with Duchenne muscular dystrophy detected by neonatal screening.</a> Lancet. 2 (8608), 425-427 (1988).</li>
<li>Forrest, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Further+studies+of+gene+deletions+that+cause+Duchenne+and+Becker+muscular+dystrophies%5BTitle%5D)+AND+%22Genomics%22%5BJournal%5D)">Further studies of gene deletions that cause Duchenne and Becker muscular dystrophies.</a> Genomics. 2 (2), 109-114 (1988).</li>
<li>Monaco, A. P., Bertelson, C. J., Liechti-Gallati, S., Moser, H., Kunkel, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+explanation+for+the+phenotypic+differences+between+patients+bearing+partial+deletions+of+the+DMD+locus%5BTitle%5D)+AND+%22Genomics%22%5BJournal%5D)">An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus.</a> Genomics. 2 (1), 90-95 (1988).</li>
<li>Flanigan, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutational+spectrum+of+DMD+mutations+in+dystrophinopathy+patients%3A+application+of+modern+diagnostic+techniques+to+a+large+cohort%5BTitle%5D)+AND+%22Human+Mutation%22%5BJournal%5D)">Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort.</a> Human Mutation. 30 (12), 1657-1666 (2009).</li>
<li>Aoki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In-frame+dystrophin+following+exon+51-skipping+improves+muscle+pathology+and+function+in+the+exon+52-deficient+mdx+mouse%5BTitle%5D)+AND+%22Molecular+Therapy%22%5BJournal%5D)">In-frame dystrophin following exon 51-skipping improves muscle pathology and function in the exon 52-deficient mdx mouse.</a> Molecular Therapy. 18 (11), 1995-2005 (2010).</li>
<li>Lu, Q. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+amounts+of+dystrophin+produced+by+skipping+the+mutated+exon+in+the+mdx+dystrophic+mouse%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse.</a> Nature Medicine. 9 (8), 1009-1014 (2003).</li>
<li>Yokota, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficacy+of+systemic+morpholino+exon-skipping+in+Duchenne+dystrophy+dogs%5BTitle%5D)+AND+%22Annals+of+Neurology%22%5BJournal%5D)">Efficacy of systemic morpholino exon-skipping in Duchenne dystrophy dogs.</a> Annals of Neurology. 65 (6), 667-676 (2009).</li>
<li>Kesselheim, A. S., Avorn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Approving+a+Problematic+Muscular+Dystrophy+Drug%3A+Implications+for+FDA+Policy%5BTitle%5D)+AND+%22Journal+of+the+American+Medical+Association%22%5BJournal%5D)">Approving a Problematic Muscular Dystrophy Drug: Implications for FDA Policy.</a> Journal of the American Medical Association. 316 (22), 2357-2358 (2016).</li>
<li>Mendell, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Eteplirsen+for+the+treatment+of+Duchenne+muscular+dystrophy%5BTitle%5D)+AND+%22Annals+of+Neurology%22%5BJournal%5D)">Eteplirsen for the treatment of Duchenne muscular dystrophy.</a> Annals of Neurology. 74 (5), 637-647 (2013).</li>
<li>Komaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systemic+administration+of+the+antisense+oligonucleotide+NS-065%2FNCNP-01+for+skipping+of+exon+53+in+patients+with+Duchenne+muscular+dystrophy%5BTitle%5D)+AND+%22Science+Translational+Medicine%22%5BJournal%5D)">Systemic administration of the antisense oligonucleotide NS-065/NCNP-01 for skipping of exon 53 in patients with Duchenne muscular dystrophy.</a> Science Translational Medicine. 10 (437), (2018).</li>
<li>Verheul, R. C., van Deutekom, J. C., Datson, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Digital+Droplet+PCR+for+the+Absolute+Quantification+of+Exon+Skipping+Induced+by+Antisense+Oligonucleotides+in+%28Pre-%29Clinical+Development+for+Duchenne+Muscular+Dystrophy%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Digital Droplet PCR for the Absolute Quantification of Exon Skipping Induced by Antisense Oligonucleotides in (Pre-)Clinical Development for Duchenne Muscular Dystrophy.</a> PLoS One. 11 (9), e0162467(2016).</li>
<li>Takizawa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modelling+Duchenne+muscular+dystrophy+in+MYOD1-converted+urine-derived+cells+treated+with+3-deazaneplanocin+A+hydrochloride%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Modelling Duchenne muscular dystrophy in MYOD1-converted urine-derived cells treated with 3-deazaneplanocin A hydrochloride.</a> Scientific Reports. 9 (1), 3807(2019).</li>
</ol>

Clinical trials of DMD have produced both successes and failures in the last few years. Both RT-PCR and western blotting are common techniques to assess the skipping efficiency generated by exon-skipping compounds administered to the patients. However, RT-PCR has been reported to over-estimate the skipping efficiency compared to digital PCR15. Although this is due to a number of reasons, it is primarily caused by the more efficient amplification of the smaller skipped fragments during PCR cycles. It appears that RT-PCR used in this clinical trial also generated higher skipping efficiencies compared to the protein expression estimated by western blotting. According to the FDA, this is a more reliable way to quantify dystrophin restoration12. Hence, caution should be exercised when interpreting RT-PCR skipping results; however the samples can still be compared. Samples showing higher skipping efficiencies based on RT-PCR results commonly exhbit higher protein expression levels in western blot analyses.
Since all patients in DMD clinical trials do not have the same deletion pattern, it can be difficult to design primers and probes that are adequately specific to perform digital or qPCR on all samples. Hence, RT-PCR is still a good alternative for a first assessment of the skipping efficiency. Before the clinical trial of NS-065/NCNP-01 commenced, it was tedious to assess skipping efficiency for each patient in vitro since either a muscle or skin biopsy was mandatory to generate patient-specific myoblasts. However, we have recently published a novel technique to generate patient-specific MYOD1-converted, urine-derived cells (UDCs) as a novel DMD muscle cell model16. Thus, only urine collected from the patient is required to generate the myoblasts, and no invasive procedure is necessary. We believe that this method can be used to screen different AONs in patient-specific cells. Furthermore, different primers and probes can be tested before the patient starts any clinical trial. This can facilitate the use of qPCR or digital PCR for exon skipping measurement in DMD clinical trials in the future.
For performing western blot analysis in this clinical trial, a single antibody against dystrophin was used, and only one healthy control was used as a reference sample. Hence, the specificity of the antibody was not validated appropriately. This, together with the fact that the antibody only recognises the C-terminal domain of dystrophin, is a limitation of the protocol. More healthy controls and antibodies directed against different domains of the dystrophin molecule are advisable in the future.
Here, we summarized the protocols that were used in the recent exploratory investigator-initiated, first-in-human trial of NS-065/NCNP-01. NS-065/NCNP-01 is potentially applicable to 10.1% of patients with DMD.

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease causing progressive loss of muscle mass, respiratory failure, and cardiomyopathy, and it leads to premature death1. The disease is caused by a lack of the large structural muscle protein dystrophin2. Mutations in the DMD-gene on the X-chromosome are recessive, and the disease affects 1 in 3500-5000 new-born males3,4,5. The mutations are often large deletions in a hotspot region between exons 44 and 55 that lead to a distorted reading frame and premature stop codons, causing nonsense-mediated decay and an almost total lack of dystrophin protein6,7,8. The reading frame can be corrected using antisense oligonucleotides (AONs) that induce exon skipping and restore the reading frame, partially restoring dystrophin expression and delaying disease progression9,10,11. The morpholino AON eteplirsen, which was recently approved by the Federal Drug Agency (FDA), induces skipping of exon 51 and can restore the reading frame in patients with exon 52 deletions12,13. However, exon 53 skipping restores the reading frame for patients with exon 52 deletions, and it can potentially treat approximately 10% of DMD patients14. The morpholino AON drug NS-065/NCNP-01 has been shown to induce exon 53 skipping in human cells, and we recently administered NS-065/NCNP-01 to DMD patients in a phase 1 open-label dose-escalation clinical trial (hereinafter, referred to as &#34;the study&#34;) (registered as UMIN: 000010964 and ClinicalTrials.gov: NCT02081625)14. The study showed a dose-dependent increase of exon 53 skipping based on RT-PCR and dystrophin protein levels based on western blotting, and no severe adverse drug events or dropouts were observed14.
In all clinical trials, the analysis of the results are of paramount importance. For DMD clinical trials, a debate is still ongoing regarding the best method to show a treatment benefit. Clinical tests such as the 6-minute walk test have certain drawbacks. Molecular characterization of the dystrophin expression can be performed using several methods, such as RT-PCR, qPCR, digital-PCR, western blotting, and immunohistochemistry. However, the extent of protein expression restoration that is required to impart a clinical benefit remains unclear. In this methods article, we describe in detail the RT-PCR and western blotting methods used to determine the exon skipping and protein levels, respectively, in the phase 1 trial of the AON skipping drug NS-065/NCNP-0114.

<table><tbody><tr><td>100 bp DNA Ladder</td><td>Takara</td><td>3407A</td><td>marker solution for MultiNA</td></tr><tr><td>1mol/l-Tris-HCl Buffer Solution(pH 7.6)</td><td>Nacalai</td><td>35436-01</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Nacalai</td><td>21418-42</td><td></td></tr><tr><td>2-Methylbutane (Isopentane)</td><td>Sigma-Aldrich</td><td>15-2220</td><td></td></tr><tr><td>5 mL Round Bottom Polystyrene Test Tube</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>6&amp;times; Loading Buffer</td><td>Takara</td><td>9156</td><td></td></tr><tr><td>Agarose ME</td><td>Iwai chemical</td><td>50013R</td><td></td></tr><tr><td>Applied Biosystems 3730 DNA Analyzer</td><td>Applied Biosystems</td><td>A41046</td><td></td></tr><tr><td>BD Matrigel Matrix</td><td>BD Biosciences</td><td>356234</td><td></td></tr><tr><td>BigDye Terminator v3.1 Cycle Sequencing Kit</td><td>Applied Biosystems</td><td>4337455</td><td></td></tr><tr><td>Bovine Serum Albumin solution</td><td>Sigma-Aldrich</td><td>A8412</td><td></td></tr><tr><td>Bromophenol blue</td><td>Nacalai</td><td>05808-61</td><td></td></tr><tr><td>Centri-Sep 96-Well Plates</td><td>Applied Biosystems</td><td>4367819</td><td></td></tr><tr><td>cOmplete Protease Inhibitor Cocktail</td><td>Roche</td><td>4693116001</td><td></td></tr><tr><td>DMEM/F-12</td><td>Gibco</td><td>11320033</td><td></td></tr><tr><td>ECL Prime Blocking Reagent</td><td>GE healthcare</td><td>RPN418</td><td></td></tr><tr><td>ECL Prime Western Blotting Detection Reagent</td><td>GE healthcare</td><td>RPN2232</td><td></td></tr><tr><td>Endo-Porter</td><td>GeneTools</td><td>2922498000</td><td></td></tr><tr><td>Experion Automated Electrophoresis Station</td><td>Bio-Rad</td><td>7007010</td><td>Microchip electrophoresis system A</td></tr><tr><td>Experion DNA 1K Analysis Kit for 10 Chips</td><td>Bio-Rad</td><td>7007107JA</td><td></td></tr><tr><td>Experion Priming Station</td><td>Bio-Rad</td><td>7007030</td><td></td></tr><tr><td>Experion Vortex Station II</td><td>Bio-Rad</td><td>7007043</td><td></td></tr><tr><td>Extra Thick Blot Filter Paper</td><td>Bio-Rad</td><td>1703965</td><td></td></tr><tr><td>EzBlot</td><td>Atto</td><td>AE-1460</td><td></td></tr><tr><td>GelRed Nucleic Acid Gel Stain</td><td>Biotium</td><td>41003</td><td></td></tr><tr><td>glass vial</td><td>Iwaki</td><td>1880 SV20</td><td></td></tr><tr><td>Glycerol</td><td>Nacalai</td><td>17045-65</td><td></td></tr><tr><td>Histofine Simple Stain MAX PO</td><td>Nichirei</td><td>424151</td><td></td></tr><tr><td>Horse Serum</td><td>Gibco</td><td>16050122</td><td></td></tr><tr><td>Immobilon-P Transfer Membrane</td><td>Millipore</td><td>IPVH304F0</td><td></td></tr><tr><td>ITS Liquid Media Supplement</td><td>Sigma-Aldrich</td><td>I3146</td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>34860</td><td></td></tr><tr><td>MicroAmp Clear Adhesive Film</td><td>Applied Biosystems</td><td>4306311</td><td></td></tr><tr><td>MultiNA</td><td>SHIMADZU</td><td>MCE-202</td><td>Microchip electrophoresis system B</td></tr><tr><td>Multiplate 96-Well PCR Plates</td><td>Bio-Rad</td><td>mlp9601</td><td></td></tr><tr><td>NanoDrop One Microvolume UV-Vis Spectrophotometer with Wi-Fi</td><td>Thermo Scientific</td><td>ND-ONE-W</td><td></td></tr><tr><td>NovocastraTM Lyophilized Mouse Monoclonal Antibody Dystrophin (C-terminus)</td><td>Leica biosystems</td><td>NCL-DYS2</td><td></td></tr><tr><td>NovocastraTM Lyophilized Mouse Monoclonal Antibody Spectrin</td><td>Leica biosystems</td><td>NCL-SPEC1</td><td></td></tr><tr><td>NuPAGE 3-8% Tris-Acetate Protein Gels</td><td>Invitrogen</td><td>EA03785BOX</td><td></td></tr><tr><td>NuPAGE Antioxidant</td><td>Invitrogen</td><td>NP0005</td><td></td></tr><tr><td>NuPAGE LDS Sample Buffer</td><td>Invitrogen</td><td>NP0007</td><td></td></tr><tr><td>NuPAGE Sample Reducing Agent</td><td>Invitrogen</td><td>NP0009</td><td></td></tr><tr><td>NuPAGE Tris-Acetate SDS Running Buffer</td><td>Invitrogen</td><td>LA0041</td><td></td></tr><tr><td>Oriole Fluorescent Gel Stain</td><td>Bio-Rad</td><td>1610496</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution Stabilized</td><td>Sigma-Aldrich</td><td>P4458</td><td></td></tr><tr><td>Physcotron Handy micro-homogenizer</td><td>MICROTEC</td><td>NS-310E3</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23227</td><td></td></tr><tr><td>Polybrene Infection / Transfection Reagent</td><td>Sigma-Aldrich</td><td>TR-1003</td><td></td></tr><tr><td>Propidium Iodide Staining Solution</td><td>BD Pharmingen</td><td>556463</td><td></td></tr><tr><td>QIAGEN OneStep RT-PCR Kit</td><td>Qiagen</td><td>210212</td><td></td></tr><tr><td>RNeasy Fibrous Tissue Mini Kit</td><td>Qiagen</td><td>74704</td><td></td></tr><tr><td>SDS</td><td>Nacalai</td><td>31606-75</td><td></td></tr><tr><td>Semi-dry transfer machine</td><td>Bio Craft</td><td>41-1293</td><td></td></tr><tr><td>SYBR Gold Nucleic Acid Gel Stain</td><td>Invitrogen</td><td>S11494</td><td>for MultiNA</td></tr><tr><td>TAE Buffer</td><td>Nacalai</td><td>35430-61</td><td></td></tr><tr><td>Tragacanth Gum</td><td>Wako</td><td>200-02245</td><td></td></tr><tr><td>Tris-EDTA Buffer</td><td>Nippon Gene</td><td>316-90025</td><td>for MultiNA</td></tr><tr><td>Trypsin-EDTA (0.05%)</td><td>Gibco</td><td>25300054</td><td></td></tr><tr><td>TWEEN 20</td><td>Sigma-Aldrich</td><td>P9416</td><td></td></tr><tr><td>Urea</td><td>Nacalai</td><td>35907-15</td><td></td></tr><tr><td>Wizard SV Gel and PCR Clean-Up System</td><td>Promega</td><td>A9281</td><td></td></tr><tr><td>XCell SureLock Mini-Cell</td><td>Invitrogen</td><td>EI0001</td><td></td></tr></tbody></table>

characterizing exon skipping efficiency in dmd patient samples in clinical trials of antisense oligonucleotides

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease that causes progressive loss of muscle mass, leading to premature death. The mutations often cause a distorted reading frame and premature stop codons, resulting in an almost total lack of dystrophin protein. The reading frame can be corrected using antisense oligonucleotides (AONs) that induce exon skipping. The morpholino AON viltolarsen (code name: NS-065/NCNP-01) has been shown to induce exon 53 skipping, restoring the reading frame for patients with exon 52 deletions. We recently administered NS-065/NCNP-01 intravenously to DMD patients in an exploratory investigator-initiated, first-in-human trial of NS-065/NCNP-01. In this methods article, we present the molecular characterization of dystrophin expression using Sanger sequencing, RT-PCR, and western blotting in the clinical trial. The characterization of dystrophin expression was fundamental in the study for showing the efficacy since no functional outcome tests were performed.

To use RT-PCR to detect exon skipping, primers on either side of the exon that will be skipped were designed and evaluated to yield only specific bands (see Figure 2 for a schematic diagram of exon skipping and primer position). The primers should generate products that can easily be distinguished by size on a MCE system or normal agarose gel electrophoresis if MCE is not available. Figure 3a shows MCE system A gel images of RT-PCR reactions and the sequencing results of the skipped band from patients NS-03 and NS-07 before and after treatment with NS-065/NCNP-01 in the dose-escalation phase 1 trial. NS-03 and NS-07 harbor deletions that span exons 45-52 and 48-52, respectively. NS-03 received 5 mg/kg and NS-07 20 mg/kg of NS-065/NCNP-01 weekly for 12 weeks. 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 visualised for NS-07. However, it was still difficult to detect any skipped product for patient NS-03. The sequencing results showed a concatenation of exons 47 and 54 for NS-07, as well as exons 44 and 54 for NS-03. According to the sequence, these could theoretically produce a functional but shortened dystrophin isoform. To calculate the skipping percentage shown in Figure 3b, the molar concentration for the skipped band was divided by the skipped and un-skipped band. For patient NS-07, the percentage after treatment was 72%, and it was 3.4% for NS-03. Western blot data (in triplicate) from patients NS-03, NS-07, and a healthy control are shown in Figure 4a. Expectedly, no dystrophin band was detected before treatment. After treatment a band from patient NS-07 was detected with a lower molecular weight compared to healthy control (wild type dystrophin has a molecular weight of 427 kDa, and NS-07 dystrophin has a molecular weight of 389 kDa). Because of the exon deletion and skipping, the dystrophin isoform from patient NS-07 lacked several exons, and it was expected to have a lower molecular weight. Patient NS-03 showed no detectable levels of dystrophin after treatment. In Figure 4b, the amount of dystrophin compared to a healthy control for patient NS-07 is shown. The location where signal intensities were measured for the calculation of dystrophin restoration are indicated with blue squares. The dystrophin spectrin ratio was used to calculate the amount of dystrophin compared to that of the healthy control. To this end, the signal from the dystrophin minus background was divided by the sum of the two spectrin isoforms (long and short) minus background (see step 8.6.5). The ratio for healthy control was set to 100%. The amount of dystrophin compared to the healthy control in patient NS-07 after treatment was 9.1%. However, the percentage could not be calculated for patient NS-03 due to a weak dystrophin band, which similar to those obtained for prior treatment samples for both patients.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60672/60672fig1.jpg" /> 
Figure 1: Schematic diagram of the transfer stack for western blot analysis. Assembly of the western blot transfer stack with blotting paper soaked in blotting buffer A in the bottom followed by blotting paper soaked in blotting buffer B, PVDF membrane, the 3-8% Tris-Acetate Gel and on the top 2 blotting papers soaked in blotting buffer C is shown. <a href="https://www.jove.com/files/ftp_upload/60672/60672fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60672/60672fig2.jpg" /> 
Figure 2: Schematic drawing of exon skipping of exon 53 in a patient with exon 45-52 deletion in the DMD gene. For example, patient NS-03 in the dose-escalation clinical trial had this deletion. If exon 53 is retained, the mRNA will be out of frame since the exon-exon junction between exon 44 and 53 disrupts the reading frame, causing a stop codon in exon 53. The reading frame is restored when exon 53 is skipped, and a shorter isoform of dystrophin is produced. To detect exon 53-skipping by RT-PCR, primers in exon 44 and 54 are used so that the PCR-product between skipped and un-skipped mRNA can easily be detected. FWD: forward primer, REV: reverse primer, PMO: phosphorodiamidate morpholino oligomer. <a href="https://www.jove.com/files/ftp_upload/60672/60672fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60672/60672fig3.jpg" /> 
Figure 3: Skipping efficiency for patients NS-03 and NS-07 from tibialis anterior muscle biopsy samples. a) Gel image generated by electrophoresis system A of RT-PCR samples of untreated and treated samples from patients NS-03 and NS-07. Upper panel shows NS-03 and lower panel NS-07 in triplicate. For NS-03, the un-skipped band is 422 bp, and the skipped band is 212 bp. For NS-07, the bands are 836 bp and 624 bp, respectively. Sequence analysis showed a concatenation of exon 44 and exon 54 for NS-03 and exon 47 to exon 54 for NS-07. b) Skipping efficiency before and after treatment is shown for patients NS-03 and NS-07, calculated from the molar concentration of the two bands provided by system A as skipped band/(skipped band + un-skipped band) x 100. The skipping efficiency for NS-03 was very low after treatment; for NS-07, the skipping efficiency was over 70%. <a href="https://www.jove.com/files/ftp_upload/60672/60672fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60672/60672fig4.jpg" /> 
Figure 4: Protein quantification of Dystrophin isoforms before and after exon skipping therapy. a) Protein quantification of dystrophin isoforms before and after exon skipping therapy. Western blot of muscle biopsy from tibialis anterior from patients NS-03 and NS-07 before and after treatment and from healthy control in triplicate. Dystrophin can be seen at 427 kDa for the healthy control and at 389 kDa for NS-07 after treatment. No dystrophin could be detected for either patient before treatment, or neither could any be detected after treatment for patient NS-03. The area in the black box is shown in Figure 4b. To the left in each blot the marker is shown. b) The amount of dystrophin restored after treatment for patient NS-07. Dystrophin restoration was calculated based on the intensities of the bands for dystrophin, background, and the long and short isoform of spectrin according to the formula: (dystrophin - background)/(spectrin L -background) + (spectrin S - background) where the ratio for healthy control is set to 100%. The intensity of each band was measured inside the blue boxes shown in the figure. The dystrophin restoration for NS-07 was 9.1% after treatment. The dystrophin signal was too weak to measure in the untreated samples. Marker sizes are shown in kilodaltons. Dys: Dystrophin, BG: background, SL: Spectrin long isoform, SS: Spectrin short isoform, My: Myosin type 1. Marker sizes are shown in kilodaltons. <a href="https://www.jove.com/files/ftp_upload/60672/60672fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Volume/Reaction (&#956;l)</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>RNase-free water (provided)</td>
			<td>Variable</td>
			<td>-</td>
		</tr>
		<tr>
			<td>5x QIAGEN OneStep RT-PCR Buffer</td>
			<td>5.0</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>dNTP Mix (containing 10 mM of each dNTP)</td>
			<td>1.0</td>
			<td>400 &#956;M of each dNTP</td>
		</tr>
		<tr>
			<td>Forward Primer (10 &#956;M)</td>
			<td>1.5</td>
			<td>0.6 &#956;M</td>
		</tr>
		<tr>
			<td>Reverse Primer (10 &#956;M)</td>
			<td>1.5</td>
			<td>0.6 &#956;M</td>
		</tr>
		<tr>
			<td>QIAGEN OneStep RT-PCR Enzyme Mix</td>
			<td>1.0</td>
			<td>-</td>
		</tr>
		<tr>
			<td>RNase inhibitor (optional)</td>
			<td>Variable</td>
			<td>5&#8211;10 units/reaction</td>
		</tr>
		<tr>
			<td>Template RNA</td>
			<td>50 ng</td>
			<td></td>
		</tr>
		<tr>
			<td>Total</td>
			<td>25</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: RT-PCR reagents. The necessary compounds for one reaction of the RT-PCR.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Primer</td>
			<td>Sequence</td>
		</tr>
		<tr>
			<td>44F</td>
			<td>5&#39;-CCTGAGAATTGGGAACATGC-3&#39;</td>
		</tr>
		<tr>
			<td>46F</td>
			<td>5&#39;-AACCTGGAAAAGAGCAGCAA-3&#39;</td>
		</tr>
		<tr>
			<td>48F</td>
			<td>5&#39;-CCAAGAAGGACCATTTGACG-3&#39;</td>
		</tr>
		<tr>
			<td>54/55R</td>
			<td>5&#39;-TCTCGCTCACTCACCCTTTT-3&#39;</td>
		</tr>
		<tr>
			<td>54R</td>
			<td>5&#39;-GTGGACTTTTCTGGTATCAT-3&#39;</td>
		</tr>
	</tbody>
</table>
Table 2: Primer list. Sequences for the primers used in this study. F: Forward, R: Reverse.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 cycle</td>
			<td>Reverse transcription</td>
			<td>30 min</td>
			<td>50 &#176;C</td>
		</tr>
		<tr>
			<td>1 cycle</td>
			<td>Initial PCR activation step</td>
			<td>15 min</td>
			<td>95 &#176;C</td>
		</tr>
		<tr>
			<td rowspan="3">35 cycles</td>
			<td>Denaturation</td>
			<td>1 min</td>
			<td>94 &#176;C</td>
		</tr>
		<tr>
			<td>Annealing</td>
			<td>1 min</td>
			<td>60 &#176;C</td>
		</tr>
		<tr>
			<td>Extension</td>
			<td>1 min</td>
			<td>72 &#176;C</td>
		</tr>
		<tr>
			<td>1 cycle</td>
			<td>Final extension</td>
			<td>7 min</td>
			<td>72 &#176;C</td>
		</tr>
		<tr>
			<td>Hold</td>
			<td></td>
			<td>&#8734;</td>
			<td>4 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 3: PCR conditioned used. Show PCR conditions for the RT-PCR reaction.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Volume/Reaction (&#956;l)</td>
		</tr>
		<tr>
			<td>RNase-free water</td>
			<td>Variable</td>
		</tr>
		<tr>
			<td>BigDye Terminator 3.1 Ready Reaction Mix</td>
			<td>3.5</td>
		</tr>
		<tr>
			<td>Forward Primer/Reverse Primer (3.2 &#956;M)</td>
			<td>2.0</td>
		</tr>
		<tr>
			<td>Template RNA</td>
			<td>20 ng</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>20</td>
		</tr>
	</tbody>
</table>
Table 4: Reagents necessary for the sequencing reaction. Use either Forward or Reverse primer in the setup, not both at the same time.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 cycle</td>
			<td>1 min</td>
			<td>96 &#176;C</td>
		</tr>
		<tr>
			<td rowspan="3">25 cycles</td>
			<td>10 s</td>
			<td>96 &#176;C</td>
		</tr>
		<tr>
			<td>5 s</td>
			<td>50 &#176;C</td>
		</tr>
		<tr>
			<td>4 min</td>
			<td>60 &#176;C</td>
		</tr>
		<tr>
			<td>Hold</td>
			<td>&#8734;</td>
			<td>4 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 5: PCR conditions for the Sanger sequencing.

Watch this Scientific Journal Video about Characterizing Exon Skipping Efficiency in DMD Patient Samples in Clinical Trials of Antisense Oligonucleotides at JoVE.com

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

Here, we present the molecular characterization of dystrophin 38 expression using Sanger sequencing, RT-PCR, and western blotting in the clinical trial.

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease that causes progressive loss of muscle mass, leading to premature death. The mutations ...

characterizing-exon-skipping-efficiency-dmd-patient-samples-clinical

Research

JoVE Journal

Medicine

6.8K Views.  National Center of Neurology and Psychiatry. Here, we present the molecular characterization of dystrophin 38 expression using Sanger sequencing, RT-PCR, and western blotting in the clinical trial.

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

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

Cancer Research

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease.

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

Genetics

Exon Skipping in Directly Reprogrammed Myotubes Obtained from Human Urine-Derived Cells

Duchenne muscular dystrophy (DMD), a progressive and fatal muscle disease, is caused by mutations in the DMD gene that result in the absence of dystrophin protein. To date, we have completed an investigator-initiated first-in-human study at the National Center of Neurology and Psychiatry based on the systemic injection of the morpholino oligonucleotides which are prone to exon-53 skipping. For the effective treatment of DMD, in vitro testing with myoblasts derived from DMD patients to screen drugs and assess patient eligibility before undertaking clinical trials is thought to be essential. Very recently, we reported a new MYOD1-converted urine-derived cell (UDC) treated with the histone methyltransferase inhibitor (3-deazaneplanocin A hydrochloride), as a cellular model of DMD. The new autologous UDC might show phenocopy of the disease-specific phenotypes of DMD, leading to the application of precision medicine in a variety of muscle-related diseases. In this article, we describe a detailed protocol for efficient modelling of DMD muscle cells using MYOD1-converted UDCs along with reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry to evaluate the restoration of dystrophin mRNA and protein levels after exon skipping.

In this article, we describe a detailed protocol for efficient modelling of Duchenne muscular dystrophy muscle using MYOD1-converted urine-derived cells to evaluate the restoration of dystrophin mRNA and protein levels after exon skipping.

Duchenne muscular dystrophy (DMD), a progressive and fatal muscle disease, is caused by mutations in the DMD gene that result in the absence of dystrophin ...

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Antisense Oligonucleotides as a Tool for Prolonged Knockdown of Nuclear lncRNAs in Human Cell Lines

Long noncoding RNAs (lncRNAs) play key regulatory roles in gene expression at the transcriptional level. Experimental evidence has established that a substantial fraction of lncRNA preferentially accumulates in the nucleus. For analysis of the function of nuclear lncRNAs, it is important to achieve efficient knockdown of these transcripts inside the nucleus. In contrast to the use of RNA interference, a technology that depends on the cytoplasmic silencing machinery, an antisense oligonucleotide (ASO) technology can achieve RNA knockdown by recruiting RNase H to the RNA-DNA duplexes for nuclear RNA cleavage. Unlike the use of CRISPR-Cas tools for genome engineering, where possible alterations in the chromatin state can occur, ASOs allow the efficient knockdown of nuclear transcripts without modifying the genome. Nevertheless, one of the major obstacles to ASO-mediated knockdown is its transitory effect. For the study of long-lasting effects of lncRNA silencing, maintaining efficient knockdown for a long time is needed. In this study, a protocol was developed to achieve a knockdown effect for over 21 days. The purpose was to evaluate the cis-regulatory effects of lncRNA knockdown on the adjacent coding gene RFC4, which is related to chromosomal instability, a condition that is observed only through time and cell aging. Two different human cell lines were used: PrEC, normal primary prostate epithelial cells, and HCT116, an epithelial cell line isolated from colorectal carcinoma, achieving successful knockdown in the assayed cell lines.

To analyze the function of lncRNAs in time-dependent processes such as chromosomal instability, a prolonged knockdown effect must be achieved. To that purpose, presented here is a protocol that uses modified antisense oligonucleotides to achieve effective knockdown in cell lines for 21 days.

Long noncoding RNAs (lncRNAs) play key regulatory roles in gene expression at the transcriptional level. Experimental evidence has established that a ...

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

Biochemistry

Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype&#160;is&#160;more similar to human DMD patients, was&#160;used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented.

Exon skipping is currently a&#160;most promising therapeutic option for Duchenne muscular dystrophy (DMD). To expand the applicability for DMD patients and to optimize the stability/function of the resulting truncated dystrophin proteins, a multi-exon skipping approach using cocktail antisense oligonucleotides was developed and we demonstrated systemic dystrophin rescue in a dog model.

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon ...

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide (AON)-mediated therapy. While SMN1 mutations are responsible for the disease, AONs targeting intronic splice silencer (ISS) sites in SMN2, including FDA-approved nusinersen, have been shown to restore SMN expression and ameliorate the symptoms. Currently, many studies involving AON therapy for SMA focus on investigating novel AON chemistries targeting SMN2 that may be more effective and less toxic than nusinersen. Here, we describe a protocol for in vitro evaluation of exon inclusion using lipotransfection of AONs followed by reverse transcription polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), and Western blotting. This method can be employed for various types of AON chemistries. Using this method, we demonstrate that AONs composed of alternating locked nucleic acids (LNAs) and DNA nucleotides (LNA/DNA mixmers) lead to efficient SMN2 exon inclusion and restoration of SMN protein at a very low concentration, and therefore, LNA/DNA mixmer-based antisense oligonucleotides may be an attractive therapeutic strategy to treat splicing defects caused by genetic diseases. The in vitro evaluation method described here is fast, easy, and sensitive enough for the testing of various novel AONs.

Various antisense oligonucleotides (AONs) have been shown to induce exon inclusion (splice modulation) and rescue SMN expression for spinal muscular atrophy (SMA). Here, we describe a protocol for AON lipotransfection to induce exon inclusion in the SMN2 gene and the evaluation methods to determine the efficacy in SMA patient fibroblasts.

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

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

Summary

Explore More Videos

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Exon Skipping in Directly Reprogrammed Myotubes Obtained from Human Urine-Derived Cells

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Antisense Oligonucleotides as a Tool for Prolonged Knockdown of Nuclear lncRNAs in Human Cell Lines

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice