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disorder</span></div></div></header><div class="site-inner"><div class="content-sidebar-wrap"><main class="content"><article class="post-18475 post type-post status-publish format-standard has-post-thumbnail category-gene-therapy tag-applied-biosystems tag-digital-pcr tag-dpcr entry" aria-label="Digital and quantitative PCR research approaches to study Duchenne muscular dystrophy, a rare muscular disorder"><header class="entry-header"><h1 class="entry-title">Digital and quantitative PCR research approaches to study Duchenne muscular dystrophy, a rare muscular disorder</h1> <span class="post-info-author"> By <a href="https://www.thermofisher.com/blog/author/behind-the-bench-staff/" title="Posts by Behind The Bench Staff" rel="author">Behind The Bench Staff</a> </span> <span class="post-info-date"> 09.05.2024 </span> </header><div class="entry-content"><p><img decoding="async" class="size-full wp-image-18477 aligncenter" src="https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/09/DMDpic1.png" alt="" width="701" height="395" srcset="https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/09/DMDpic1.png 701w, https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/09/DMDpic1-300x169.png 300w" sizes="(max-width: 701px) 100vw, 701px" /></p> <p>Duchenne muscular dystrophy (DMD), a rare disease (1) affecting about 1 in 5,000 boys worldwide, is a severe X-linked recessive condition caused by mutations in the dystrophin gene. A large number of variable mutations, including deletions, duplications, or small mutations, have been identified in the <em>Dmd</em> coding sequence and splice sites. This variability, high mutation rate, and the large size of the gene make it challenging to develop effective gene therapies targeting DMD (2,3).</p> <p>Patients with DMD have a progressive disease that causes muscle wasting, respiratory insufficiency, and cardiomyopathy, starting from early childhood. They usually lose the ability to walk on their own around 12 years old, and death may occur in the late twenties, usually from cardiorespiratory failure (2,3).</p> <p>Current research into therapeutic strategies for DMD either aim to restore dystrophin protein function or target downstream effects of dystrophin deficiency, like muscle mass loss, inflammation, and fibrosis. Strategies to restore protein function include gene therapy and manipulation of cellular machinery for transcription, mRNA processing, and translation. These strategies are in various stages of pre-clinical and clinical research, with some focusing on CRISPR/Cas9-mediated gene editing or systemic delivery of functional dystrophin using viral vectors like adeno-associated viruses (AAVs) (2,3). In 2023, AAV-based delandistrogene moxeparvovec-rokl (Elevidys<sup>®</sup>) was brought to market as the first gene therapy for DMD. This therapy introduces a gene that produces a shortened version of the normal dystrophin protein, which is expected to improve muscle function (4).</p> <p>In this article, we discuss various genetic analysis tools used in DMD research, using recent examples from the literature.<strong> </strong></p> <p><strong>Evaluating the efficacy of exon-skipping approaches for DMD using quantitative real-time PCR (qPCR) and digital PCR (dPCR)</strong></p> <p>Research into exon-skipping approaches for mediating DMD have not shown significant benefit due to inconsistent dystrophin protein restoration. In a study aimed at understanding the turnover dynamics of dystrophin and its related proteins, researchers measured protein stability and turnover in the DMD mouse models (<em>mdx</em>) after treatment using a mass spectrometry approach. Findings indicate that treated <em>mdx</em> muscle shows slower dystrophin turnover and extended protein half-life, suggesting that these therapies stabilize the protein differently than in normal muscle (5).</p> <p>Quantitative real-time PCR using Applied Biosystems<sup>™</sup> TaqMan<sup>™</sup> assays and TaqMan<sup>™ </sup>Universal PCR MasterMix on an Applied Biosystems<sup>™ </sup>QuantStudio<sup>™ </sup>7 Real-Time PCR cycler, and digital PCR using the Applied Biosystems<sup>™ </sup>QuantStudio<sup>™ </sup>3D Digital-PCR System were used to evaluate the efficacy of the exon-skipping approaches (5).</p> <p> </p> <ul> <li>qPCR measured the levels of exon-skipped <em>Dmd</em> transcript relative to the total <em>Dmd </em> It provided a percentage of skipped mRNA at various time points post-treatment in different muscle tissues.</li> <li>dPCR was used for absolute quantification of skipped Dmd transcript levels within the tissue. This method allowed for the determination of the stability of the skipped Dmd transcript over time, confirming the sudden decline in skipped mRNA levels observed with qRT-PCR.</li> </ul> <p><img decoding="async" class="size-full wp-image-18476 aligncenter" src="https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/09/DMDPic2.jpg" alt="" width="1000" height="184" srcset="https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/09/DMDPic2.jpg 1000w, https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/09/DMDPic2-300x55.jpg 300w, https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/09/DMDPic2-768x141.jpg 768w" sizes="(max-width: 1000px) 100vw, 1000px" /></p> <p><strong>Figure 1: A simple research workflow for absolute transcript quantification.</strong> The QuantStudio Absolute Q Digital PCR System is an all-in-one instrument that integrates all dPCR steps in a single instrument. Pipette the reaction mixture into the MAP plate, just like in real-time PCR, and let the platform take care of the rest.</p> <p>The measurement of relative and absolute quantities of exon-skipped <em>Dmd</em> transcripts and the correlation of the presence of skipped transcripts with the expression and localization of dystrophin protein in muscle tissue was critical in assessing the therapy’s longitudinal efficacy (5).</p> <p><strong>Research into verifying differentiation of genome-edited DMD patient-derived stem cells using qPCR</strong></p> <p>Utrophin, a dystrophin-like protein deficient in DMD research patients, can functionally compensate for the absence of dystrophin when expressed at increased levels in the muscle fibers. Therefore, strategies to upregulate utrophin are considered promising approaches (6).</p> <p>In a research study using CRISPR/Cas9 genome editing to upregulate utrophin in DMD patient-derived human induced pluripotent stem cells (DMD-hiPSCs), miRNA binding sites in the utrophin gene’s 3′ UTR were deleted to alleviate miRNA repression and thereby, increase utrophin expression. This approach represents a promising avenue for possible future DMD treatments (6).</p> <p>To verify the differentiation of wild-type, DMD, and genome-edited hiPSC clones into the myogenic lineage, qPCR was performed using the PowerTrack SYBR Green PCR master mix and QuantStudio 3 Real-Time PCR System. The expression levels of myogenic markers (<em>MyoD1, Myogenin</em>, and endogenous <em>MyoD1</em>) and the pluripotency marker <em>Nanog</em> were measured. The increase in myogenic markers and decrease in the pluripotency marker post-tamoxifen induction confirmed the differentiation process (6).</p> <p><strong>Digital PCR assessment of gene editing efficiency in the dystrophin gene</strong></p> <p>A study investigating the long-term effectiveness of a gene editing approach for DMD (deletion of exons 52 and 53 in the dystrophin gene) in <em>mdx </em>mice found that low levels of dystrophin persisted in cardiomyocytes but not in skeletal muscles, where cells continued to be prone to damage and regeneration. Increasing the ratio of guide RNA to nuclease vectors improved gene-editing efficiency in both muscle types, although achieving high dystrophin levels in skeletal muscles remained challenging. The study suggests that improving gene editing efficiency is required for stable dystrophin expression (7).</p> <p>dPCR using the QuantStudio 3D Digital-PCR System quantified the percentage of genomes that had undergone successful deletion of dystrophin exons 52-53 in various tissues, including the heart, diaphragm, and gastrocnemius muscles. In addition, the percentage of dystrophin transcripts lacking exons 52-53 was also quantified using dPCR, providing insights into the efficiency of gene editing at the mRNA level (7).</p> <p><strong>See how you can use the Absolute Q Digital PCR System in your research. </strong><a href="https://www.thermofisher.com/de/en/home/life-science/pcr/digital-pcr/quantstudio-absolute-q-system.html"><strong>Learn more</strong></a><strong>.</strong></p> <p>For Research Use Only. Not for use in diagnostic procedures.</p> <p>© 2024 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a trademark of Roche Diagnostics, Inc.</p> <p>References:</p> <ol> <li>Crisafulli S, Sultana J, Fontana A, Salvo F, Messina S, Trifirò G. Global epidemiology of Duchenne muscular dystrophy: an updated systematic review and meta-analysis. Orphanet J Rare Dis. 2020 Jun 5;15(1):141. doi: 10.1186/s13023-020-01430-8. PMID: 32503598; PMCID: PMC7275323.</li> <li>Bez Batti Angulski A, Hosny N, Cohen H, Martin AA, Hahn D, Bauer J, Metzger JM. Duchenne muscular dystrophy: disease mechanism and therapeutic strategies. Front Physiol. 2023 Jun 26;14:1183101. doi: 10.3389/fphys.2023.1183101. PMID: 37435300; PMCID: PMC10330733.</li> <li>Fortunato F, Farnè M, Ferlini A. The DMD gene and therapeutic approaches to restore dystrophin. Neuromuscul Disord. 2021 Oct;31(10):1013-1020. doi: 10.1016/j.nmd.2021.08.004. PMID: 34736624.</li> <li>Press release: <a href="https://www.fda.gov/news-events/press-announcements/fda-approves-first-gene-therapy-treatment-certain-patients-duchenne-muscular-dystrophy">FDA Approves First Gene Therapy for Treatment of Certain Patients with Duchenne Muscular Dystrophy | FDA</a></li> <li>Novak JS, Spathis R, Dang UJ, Fiorillo AA, Hindupur R, Tully CB, Mázala DAG, Canessa E, Brown KJ, Partridge TA, Hathout Y, Nagaraju K. Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy. J Neuromuscul Dis. 2021;8(s2):S383-S402. doi: 10.3233/JND-210696. PMID: 34569969; PMCID: PMC8673539.</li> <li>Sengupta K, Mishra MK, Loro E, Spencer MJ, Pyle AD, Khurana TS. Genome Editing-Mediated Utrophin Upregulation in Duchenne Muscular Dystrophy Stem Cells. Mol Ther Nucleic Acids. 2020 Aug 29;22:500-509. doi: 10.1016/j.omtn.2020.08.031. PMID: 33230452; PMCID: PMC7554652</li> <li>Bengtsson NE, Tasfaout H, Hauschka SD, Chamberlain JS. Dystrophin Gene-Editing Stability Is Dependent on Dystrophin Levels in Skeletal but Not Cardiac Muscles. Mol Ther. 2021 Mar 3;29(3):1070-1085. doi: 10.1016/j.ymthe.2020.11.003. Epub 2020 Nov 5. 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