Report from the FSHD Society’s 29th annual International Research Congress
by Alexandra Belayew, PhD, Mons, Belgium
This year’s congress (June 16-17) opened with a keynote presentation by Lexi Pappas, who gave a very moving testimony of what it can be like to live with FSHD. She noted that the image of FSHD as a slowly progressing disease for older adults is not true for many. At 28 she has already experienced a rapid decline, as seen in videos taken now and five years ago. Developing effective therapies is an urgent matter for patients since they lose strength and independence with every passing year.
I then gave a talk to provide a historical perspective for the many researchers who recently joined the FSHD field. I described the issues encountered by the pioneers who discovered and characterized the DUX4 gene: the conditions in which FSHD muscle cell cultures express very low amounts of DUX4 mRNA and protein, the high toxicity of DUX4 protein, hundreds of similar genes that proved not to be linked to FSHD, and the then-widely accepted concept that repeat-DNA was “junk DNA” and could not harbor a functional gene like DUX4.
The other keynotes also engaged the audience around broader topics. Eva Chin, PhD, executive director of Solve FSHD, a new Canadian venture philanthropy with a $100 million budget, described the organization’s aims to facilitate the transition from fundamental research to drug development. Jane Larkindale, PhD, of Pepgen, pleaded for scientific and clinical data sharing in rare-disease research and presented an FDA-funded project, the RDC-DAPP, to standardize data from diverse sources and facilitate investigators’ navigation through such datasets.
DUX4 protein is normally only expressed for 24 hours in the four-cell human embryo and wakes up a large gene set to initiate its development. We already knew that in FSHD muscles, inappropriate DUX4 expression was activating the same gene set, as if to convert muscle to early embryo. Danielle Hamm, PhD, from the Stephen Tapscott lab at Fred Hutchinson Cancer Center, presented an additional DUX4 property: It only allows protein synthesis of the mRNAs expressed from the genes it activates and prevents protein synthesis from other mRNAs normally present in muscle (and needed for healthy muscle function).
Christopher Brennan and collaborators from several pharmaceutical companies (Pfizer, Entrada, Kymera, Sanofi) searched for ways in which DUX4 protein could convey toxic signals inside the muscle cell. They found it activates enzymes including p38 MAPK, which activates DUX4 expression which in turn further activates p38 MAPK, driving more DUX4. Losmapimod, the Fulcrum Therapeutics drug that is in a Phase 3 clinical trial for FSHD, appears to interfere with this vicious cycle.
Researchers of Davide Gabellini’s group at San Raffaele Scientific Institute, Milan, Italy, are developing new therapeutic strategies. Paola Ghezzi found a protein called MATRIN3 which prevents DUX4 from binding to its target genes. She cut MATRIN3 into pieces and identified the smallest fragment capable of inhibiting DUX4. This fragment has been synthesized by a collaborating biotech to be evaluated as a putative drug in mouse models. Emanuele Mocciario focuses on the mechanism by which the DBET long RNA, found several years ago by the group, activates DUX4. He has identified a protein that binds to DBET and is required for this activation, and is now developing molecules to inhibit this process.
Genetics and epigenetics
Epigenetics refers to the way DNA is packed with proteins to form chromatin. Small chemical marks called methylations can decorate the DNA. DNA hypermethylation causes compact chromatin and prevents gene activation, while hypomethylation is linked with open chromatin allowing for gene expression.
Anna Karpukhina from Yegor Vassetzky’s group (Institut Gustave Roussy, Villejuif, and Koltzov Institute, Moscow) presented her complex strategy to restore normal DNA/ chromatin loop organization in the nuclei of FSHD muscle cells with the aim of decreasing DUX4 gene expression.
Russell Butterfield (University of Utah) studies the large FSHD kindred with more than 2,000 descendants of a single gene carrier who arrived in Utah in the 19th century. Using Nanopore sequencing technology, he studied methylation of long DNA stretches with up to 12 D4Z4 repeats and found lower methylation of the last D4Z4 unit, which favors activation of its DUX4 gene. Mitsuru Sasaki-Honda (Kyoto University) received the Best Poster prize for his project of “hit and run” D4Z4 methylation to prevent DUX4 gene expression. With a modified CRISPR/Cas9 system he targeted the DUX4 gene with KRAB, a potent transcription inhibitor (similarly to C. Himeda and P. Jones, University of Nevada), combined with a DNA methylating enzyme to form compact chromatin. The inhibitor combination strongly decreased DUX4 expression. His therapeutic strategy is to use an engineered virus to administer these inhibitors multiple times as needed by patients.
Pathology and disease mechanism
Although the role of aberrant DUX4 expression in the pathophysiology of FSHD is undisputed, the mechanism by which DUX4 exhibits toxicity to muscles remains elusive. This session focused on the various mechanisms by which DUX4 can elicit toxicity in muscles. Philipp Heher (King’s College London, Prof. P. Zammit) focused on mitochondria, the small, energy-producing units in our cells. DUX4 causes oxidative stress by disturbing mitochondrial function, resulting in less effective metabolism. Use of antioxidants that enter mitochondria proved more efficient in rescuing cell metabolism.
Michael Kyba (University of Minnesota) discovered a new mechanism of DUX4 toxicity causing cell suicide. This process is normally counteracted by the FAIM2 protein, but DUX4 interferes in two ways: by inducing an enzyme that destroys FAIM2 and by miR-3202 microRNA, which blocks FAIM2 synthesis. Suppression of miR-3202 rescues FAIM2 and could constitute a new therapeutic strategy.
Prakash Kharel (Harvard Medical School, Prof. Pavel Ivanov) described his investigation of DUX4 mRNA toxicity linked to unusual G-rich structures (G-quadruplexes) it harbors that favor interaction with various proteins.
Tessa Arends (Fred Hutchinson Cancer Center, Prof. S. Tapscott) followed previous studies on DUX4 target genes in repeated DNA (satellite repeats) located in the central part of chromosomes. Accumulation of these repeated RNAs in muscle cells contributes to DUX4 toxicity by disturbing chromatin structure and DNA repair pathways.
These strategies address either DUX4 repression or improvement of muscle regeneration. Several groups are investigating mesenchymal stem cells (MSCs), usually derived from bone marrow, that can be injected to help muscle regeneration.
Nizar Saad (The Ohio State University) studies tiny bubbles (extracellular vesicles) which MSCs produce in the test tube and naturally contain therapeutic substances. He has optimized their purification and shown that injecting them into mouse models of FSHD strongly reduced DUX4- induced damage to muscles.
Barbora Malecova (Avidity Biosciences) presented very encouraging preclinical data about a small inhibitory RNA (siRNA) targeting the DUX4 mRNA for destruction. This agent is coupled to an antibody that recognizes transferrin receptor 1, which is found mostly on muscle membrane. The antibody delivers the siRNA to the muscle. Full muscle protection occurred when the drug was injected in mouse blood two weeks before DUX4 expression. However, additional mouse tests should now target muscles already expressing DUX4, which better mimics the situation in patients.
Afrooz Rashnonejad (The Ohio State University) is in the early stages of developing an AAV virus that expresses a CRISPR/Cas13 system to cut DUX4 mRNA, which would prevent DUX4 from being translated into the toxic protein.
Karim Azzag (University of Minnesota, Profs. R. Perlingeiro and M. Kyba) proposes to graft muscle progenitors to help mouse muscle regeneration. Interestingly, these cells engrafted more efficiently in DUX4-expressing muscles and formed new fibers that improved contraction.
Clinical studies and outcome measures
A major issue for clinical trials is the availability of biomarkers and imaging markers to evaluate disease progression and treatment efficacy despite the heterogeneity in FSHD presentation. A study named ReSolve has been following the evolution (natural history) and muscle strength/function of 240 patients over two years at nine US and two EU centers.
Sjan Teeselink (University Hospital Nijmegen, Prof. Baziel van Engelen) shared data on muscle imaging by easy and cheap ultrasound analysis to distinguish FSHD from healthy muscles.
Mauro Monforte (University Hospital A. Gemelli, Rome, Profs. E. Ricci and G. Tasca) received the Best Young Investigator award for his use of artificial intelligence to improve the accuracy of diagnosis with MRI (magnetic resonance imaging) by retrospective analysis of 300 patients with and without FSHD, and evaluation of 15 image parameters.
Jeffrey Statland (University of Kansas) presented on the reachable workspace measure (RWS), which was used for the losmapimod (Fulcrum Therapeutics) Phase 3 clinical trial. RWS correlates with patients’ everyday functions (see story on page 17). The test is easy to perform, with the person sitting on a chair and moving the arms as shown on a video screen, with automated movement measures.
I have attended every FSHD International Research Congress since 1997, and this year I was really impressed by the number of companies that joined the search for drugs against FSHD, and by the efficiency of researchers and clinicians networking in the quest for biomarkers that could reliably detect changes in disease progression or drug efficacy in the many clinical trials that are about to start.